Retrospective Theses and Dissertations
1972
Isozymes and subunits of porcine pancreatic αamylase
Catherine Ting Lee
Iowa State University
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73-3904
LEE, Catherine Ting, 1943ISOZYMES AND SUBUNITS OF PORCINE PANCREATIC
0( -AMYLASE.
Iowa State University, Ph.D., 1972
Chemistry, biological
University Microfilms, A XEROX Company, Ann Arbor, Michigan
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED.
Isozymes and subunits of porcine pancreatic a-araylase
by
Catherine Ting Lee
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of
The Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Department:
Major:
Biochemistry and Biophysics
Biochemistry
Approved:
Signature was redacted for privacy.
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the Major Départent
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For the Graduate College
Iowa State University
Ames, Iowa
1972
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ii
TABLE OF CONTENTS
Page
ABBREVIATIONS
I.
II.
INTRODUCTION
1
EXPERIMENTAL INVESTIGATION
6
A.
Materials
6
B.
Methods
^
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
III.
iv
Analytical methods
Amylase assay
Gel electrophoresis
Isoelectric focusing
Gel filtration chromatography
Ion exchange chromatography and isolation of isozymes
Preparation of the subunits
SDS-gel electrophoresis for molecular weight
determinations
Modification of protein sulfhydryl and disulfide
groups
Peptide mapping
Determination of the number of sulfhydiryl groups
and disulfide linkages
Test for disulfide conformers in subunit A and
subunit B
Amino acid composition analysis
Action pattern and product specificity studies
Multiple attack studies
Ultracentrifugation studies
Calcium analysis
7
8
9
9
9
10
10
11
11
12
13
13
14
15
15
16
16
RESULTS
17
A.
Gel Filtration of Crystalline PPA on Sephadex G-25
17
B.
Multiple Forms in Crystalline PPA
17
1.
2,
17
22
Polyacrylamide gel electrophoresis
Isoelectric focusing
C.
Isolation of Isozymes by Ion Exchange Chromatography
22
D.
Molecular Weight Determination of Isozymes
22
iii
Page
E.
F.
Isolation of Subunits by Reducing-DEAE-Cellulose
Chromatography
27
Study of the Nature of the Subunits
34
1.
2.
3.
34
34
4.
5.
G.
I.
IV.
V.
VI.
38
39
40
Study of the Quaternary Structure of PPA
43
1.
2.
43
3.
4.
H.
Peptide mapping of subunits A and B
Amino acid composition analysis of subunits A and B
Determination of the number of sulfhydryl and
disulfide groups in isozymes and subunits A and B
Test for disulfide conformera in subunits A and B
Calcium determination of subunits A and B
Study of the nature of the isozymes
Reduction and alkylation, performlc acid oxidation
and molecular weight determination of PPA
Sedimentation studies
Amino acid composition of the crystalline and
oxidized PPA
43
48
51
Product Specificity and Action Patterns of the
Isozymes and the Subunits
35
1.
2.
55
55
Product specificity
Blue value vs reducing value studies
Resistance of Isozymes and Subunits to Trypsin
Hydrolysis
55
DISCUSSION AND CONCLUSIONS
61
LITERATURE CITED
76
ACKNOWLEDGMENTS
82
iv
ABBREVIATIONS
DEAE - Cellulose
diethylaminoethyl-cellulose
DTT
dithiothreitol
DTNB
5,5'-dithio bis (2-nitro benzoic acid)
CNT
3-carboxylato-4-nitrothiophenolate
EDTA
ethylenediaminetetraacetic acid
DFP
diisopropylflourophosphate
PPA
porcine pancreatic a-amylase
SDS
sodium dodecyl sulfate
TCA
trichloroacetic acid
1
I.
INTRODUCTION
In the mammalian system, the a-amylase is of great biological
importance.
It hydrolyzes the a1-4 glycosidic linkages of polysalcharides
to yield much smaller oligosaccharides such as maltose and maltotriose
which can be further degradated to glucose by other enzymes and be
absorbed and utilized for energy.
The a-amylase from swine pancreas was isolated and crystallized by
Meyer, Fischer and Bernfeld in 1947 (1).
It had a reported molecular
weight of 45,000 by sedimentation studies (2, 3) and amino acid
composition analysis gave a minimum molecular weight of 51,300 + 450 (4).
It is a calcium metalo enzyme.
Emission spectrographic analysis of
crystalline porcine pancreatic Oramylase demonstrated that it contains at
least 1 gram atom of very firmly bound calcium per mole of enzyme (5).
It
was proposed that calcium maintains the protein in the proper configuration
for biological activity and stabilizes the secondary and tertiary structure
(6, 7).
No nonprotein organic material was found in the enzyme (3, 8, 9).
The specific action of this enzyme has been well studied and a hypothesis
for the mode of hydrolytic attack has been presented (10, 11).
It was reported previously that porcine pancreatic a^amylase was homo­
geneous by free boundary electrophoresis, by paper electrophoresis, and by
ultracentrifugation studies (1, 12, 13).
However, investigators in two
laboratories independently isolated two isozymes by DEAE-cellulose chroma­
tography and gel electrophoresis (14, 15).
Equilibrium sedimentation gave
identical molecular weight of 52,600 + 1200 for each isozyme.
Rowe et.
(14) indicated the multiple forms of porcine a-amylase are present in
2
the crystalline preparations and are not artifacts of the fractionation
procedures, nor do they result from interconversion of the two forms.
However, they are physically distinct but closely related proteins.
terminal residue analysis and dissociation studies, Cozzone et
From
(15)
concluded that the two isozymes they isolated, amylase I and amylase II,
were monomeric enzymes consisting of single polypeptide chains.
The first recognized case of isozymes was that of lactic dehydrogenase
(16).
This enzyme has been shown to be a tetramer made of two types of sub-
units, M and H.
Various combinations of the two subunits account for the
formation of five structurally distinct lactic dehydrogenase isozymes, (M^,
and H^).
different genes.
Formation of H and M subunits are controlled by
Several chymotrypsins (3-, 5-, ir- etc.) are obtained from
chymotrypsinogen by selective trypsin and chymotrypsin catalyzed cleavages
of peptide linkages (17).
isozymes.
Many amylases are also known to be composed of
Kauffman et al. (18) showed that five isozymic forms were found
to account for most of the a-amylase present in the human saliva of one
individual, with at least two additional forms present in trace amounts.
Differences in carbohydrate content and the degree of deamination is
attributed to the existence of these isozymes.
Malacinski and Rutter (19)
have demonstrated the presence of multiple molecular forms of o-amylases
from the rabbit pancreas and parotid gland.
They speculated that these
isozymes may be the products of separate genes.
Multiple forms of
Drosophila amylase are known to be under genetic control (20).
In contrast to the observations of Cozzone
al.. (15), McGeachin and
Brown (21) reported that there were three N-terminal amino acids in porcine
3
pancreatic a-amylase, phenylalanine, alanine, and glycine.
Straub et al.
(22) isolated two subunit chains with different sizes after the native
enzyme was reduced and carboxymethylated.
Further indication that PPÂ may
be composed of subunits was provided by the observations of Levitzki et al.
(23) that the enzyme formed multimolecular complexes with glycogen,
analogous to the antibody-antigen reaction.
This phenomenon demands that
the enzyme has at least two glycogen binding sites per molecule.
Loyter
and Schramm (24) showed that PPA has two maltotriose binding sites per
50,000 daltons.
Two enzymatically active and separable subunits of 25,000
were first isolated by Robyt ^ £l. (25).
It is common for proteins to contain subunits.
Hemoglobin is made of
four polypeptides, 2 a chains and 2 3 chains, held together through
hydrophobic and hydrophilic interactions (26).
made of 12 subunits (27).
Aspartyl transcarbamylase is
Glutamine synthetase is composed of 6 subunits
arranged in a hexagon, with association of two hexagons to give the whole
protein (28).
In certain proteins, the subunits are linked together by
disulfide linkages, such as chymotrypsin, insulin, and immunoglobulin G
(17, 29, 30).
The subunits in chymotrypsin and insulin are derived from a
single polypeptide chain, chymotrypsinogen and proinsulin respectively, by
proteolytic digestions (17, 29).
Subunits in immunoglobulin G are coded by
different genes and synthesized separately (31, 32, 33).
Most enzymes
composed of subunits exhibit cooperative effects and regulatory properties
(34).
As indicated by Robyt e]t
(25) additional treatment of the two
25,000 dalton subunits of PPA with EDTA and DTT, and iodoacetamide gave
derivatives that emerged in identical positions from DEAE-cellulose
4
columns and gave identical mobilities on gel-electrophoresis.
A similar
primary structure for these two subunits of PPA was proposed.
Proteins
with the same primary structure may still be different from each other in
many ways.
Disulfide conformera are known to exist in bovine plasma
albumin (35).
Prosthetic groups are found to be responsible for the many
forms of human parotid amylase (18).
It is also possible for a single
polypeptide to exist in more than one stable conformation (36, 37).
This study is concerned with the structural aspects of porcine
pancreatic a-amylase, the nature of the isozymes, and their relationship
to the subunits.
The structural differences between the two subunits was
further studied, and a hypothesis is proposed for the quaternary structure
of this enzyme.
In the present study, four active forms of PPA were obtained from
DEAE-cellulose column.
trace amounts.
The fourth form was found to be present only in
The molecular weight of each form was determined by two
independent methods.
The three major isozymes were each reduced and the
subunits were isolated.
To test the hypothesis that the two subunits of
PPA have the same primary structure, amino acid composition analysis and
peptide maps were made.
Additional experiments were performed to establish
the structural differences in the two subunits.
Under certain experimental conditions, a subunit of 12,000 molecular
weight was isolated from PPA.
Treatment of the native enzyme with
performic acid reduced the weight of the enzyme exclusively to 12,500.
A model for the quaternary structure of this enzyme is thus proposed.
5
Some catalytic properties of the Isozymes and subunits were studied
and compared.
It appears that there are at least two active sites on the
crystalline 50,000 dalton enzyme which are Independent.
The slight
deviation In activities might reflect small differences In structure.
6
II. EXPERIMENTAL INVESTIGATION
A.
Materials
The following is a list of the materials used in this study and the
chemical supply houses from whom they were obtained;
Porcine pancreatic a-amylase, 3 x crys., Worthington Biochemicals,
Freehold, New Jersey
TPCK-trypsin, ibid.
Bacillus subtilis a-amylase dimer, crystallized by Robyt* from HTconcentrate. Miles Laboratory, Elkhart, Indiana
Bovine serum albumin, Pentex, Kankakee, Illinois
Pepsin, 2 x crys., Sigma, St. Louis, Missouri
Aldolase, ibid.
Alcohol dehydrogenase, ibid.
Cytochrome C, Type II, ibid.
Aspergillus oryzae a-amylase, Calbiochem, Los Angeles, Calif.
Dithiothreitol, ibid.
DTNB, ibid.
lodoacetamide, Sigma, St. Louis, Missouri
14
C-labeled iodoacetamide. New England Nuclear Corp., Boston, Mass.
DEAE-cellulose, medium mesh 0.87 meq/gm. Sigma.
Biogel P-150, Bio-Rad Laboratories, Richmond, California
Sephadex G-25, Pharmacia, Uppsala, Sweden
Robyt, J. F. Unpublished work. Department of Biochemistry and
Biophysics, Iowa State University, Ames, Iowa. 1968.
7
B.
1.
Methods
Analytical methods
a.
Reducing values were measured by the alkaline ferricyanide-
cyanide procedure with the Technicon Auto Analyzer (38a, 38b).
b.
Blue values (absorbance at 620 nm of the starch-iodine-iodide
complex) were determined by adapting the conditions of McCready and Hassid
(39) for use with the Technicon Auto Analyzer (40).
c.
Total carbohydrate was determined by the phenol-sufuric acid
procedure adapted for use with the Technicon Auto Analyzer (40).
d.
Protein concentrations were determined by the method of Lowry e^ al.
(41) with the Technicon Auto Analyzer using various concentrations of serum
albumin as standards.
The amount of porcine pancreatic a-amylase in
solution was determined by measuring the absorbance at 280 nm using the
extinction coefficient (E 1%/cm) of 22.9 (42).
The absorbance was deter­
mined by using a Beckman model DU spectrophotometer with a Gilford Instru­
ments digital readout absorbance meter attachment.
Protein concentrations
of isozymes and subunits were also determined by absorbance at 280 nm.
The
extinction coefficients of the individual isozymes and subunits were
obtained from absorbance measurements at 280 nm and protein concentration
determinations by Lowry*s method on the Technicon Auto Analyzer.
From the
extinction coefficient for the crystalline PPA at 280 nm (E 1%/nm = 22.9),
the actual protein concentration of crystalline PPA in solution was obtained.
Comparing the value obtained by the spectrophotometric method and that
obtained from the Auto Analyzer analysis of the crystalline PPA, a con­
version factor was obtained for the subunits and the individual isozymes.
8
Protein concentrations of the Isozymes and subunits determined by Dowry's
method were then adjusted by this correction factor.
This conversion
factor can be applied to the isozymes and subunits because they have very
similar amino acid compositions (see Results: section F-2 and section 6-4).
From these studies, the extinction coefficients (E 1%/cm) for the isozymes
and subunits were obtained as follows:
isozyme 1:
22.88; isozyme II:
27.76; isozyme III: 21.91; subunit A: 28.07, subunit B: 26.17.
2.
Amylase assay
The enzyme solution (1 ml) was added to a 2% starch solution (1 ml)
which was 40 mM in sodium glycerophosphate (pH 7) and 20 mM in KCl.
After
10 minutes of incubation time, aliquots of 0.5 ml were diluted 1:10 with
water to which 0.1 ml 5 M trichloroacetic acid was added; after 10 minutes,
the solution was neutralized with 5 M sodium hydroxide.
The amount of
reducing sugars produced was determined by the alkaline ferricyanide method
on the Technicon Auto Analyzer.
continuous assay method (43).
Enzyme activity was also determined by a
Small volumes of enzyme solution (10 - 20 y 1)
were added to 10 ml of a 1% starch solution or to 10 ml of a 0.1% amylase
solution (both were 20 mM in sodium glycerophosphate, pH 7, and 10 mM in
KCl) and mixed quickly.
The mixture was sampled continuously by the Auto
Analyzer sample line for reducing value determination.
The reaction mixture
was kept in a 40°C water bath during the assay period.
The reducing value
was determined as an initial velocity.
defined as:
One unit of enzyme activity is
1 y mole of apparent maltose produced per minute.
9
3.
Gel electrophoresis
Polyacrylamide gel electrophoresis was performed using a Canalco
apparatus according to the procedures of Davis (44) and as modified in the
Canalco formulation sheet of 1965.
Gels were cast in tubes of 90 x 6 mm
with 2 ml separating gel and 0.2 ml each of spacer and sample gels.
Approximately 50 pg of protein were applied onto the top of each gel.
Protein bands were fixed and stained by aniline black or by the method of
Dunker and Rueckert (45) using Coomassie blue dye.
4.
Isoelectric focusing
LKB electrofocusing column and LKB ampholine carrier ampholytes were
used, according to the techniques described by Haglund (46) and as outlined
in a LKB instruction sheet (47).
A pH range of 5-8 was employed.
of 6 mg of protein was incorporated into the ampholine solution.
A sample
The
experiment was run for 42 hours at 600 volts; the column was cooled by
running tap water which was approximately 18°C.
After fractionation, the
protein peaks were located by absorbance measurements at 280 nm.
5.
Gel filtration chromatography
Bio-gel columns (1,5 x 60 cm) were packed with gel that had been washed
and swelled first in water, then equilibrated with 20 oM sodium glycero-4
phosphate buffer (pH 8.5), 10 M CaCl^, 10 mM KCl.
sample was applied for each run.
About 1-2 mg of protein
The Sephadex G-25 columns (1.6 x 30 cm)
were packed with gel that had been equilibrated in 20 mM sodium glycero­
phosphate buffer (pH 7.0), 10
placed onto the column.
CaClg, 10 mM KCl.
Enzyme (5 mg) was
10
6.
Ion exchange chromatography and isolation of isozymes
DEAE-cellulose was washed with 0.5 N HCl - 0.5 N NaOH for 2-3 times
and was degassed before it was equilibrated with 20 nM sodium glycero­
phosphate buffer (pH 8.5), 10
CaCl^, 10 mM KCl.
The column (1.5 x 30
cm) was packed and further equilibrated with the same buffer until the
eluent reached the same pH value as that of the buffer.
Enzyme (5 mg in
2 ml buffer) was placed onto the column and eluted at room temperature.
Fractions of 2.5 - 3 ml were collected and the protein was located by
measuring the absorbance of individual fractions at 280 nm.
7.
Preparation of the subunits
A specially packed, reduced - DEAE-cellulose column was used to
isolate the subunits from PPA (48).
Reduced - DEAE-cellulose was prepared
by incorporating 50 mM mercaptoethanol in the DEAE-cellulose slurry which
had been equilibrated with 20 mM sodium glycerophosphate buffer (pH 8.5),
10
M CaClg, 10 mM KCl.
for at least 20 hours.
The slurry was further stirred at room temperature
The columns were packed and eluted with buffer
containing no mercaptoethanol until the eluent was free of mercaptoethanol
order and did not give any absorbance at 280 nm.
Enzyme (5 mg) was reduced
with 10 mM DTT (dithiothreitol) in 2 ml buffer, pH 8.5, overnight at 4°C
before being placed onto the top of the column.
280 nm to locate the protein peak.
Fractions were read at
When working with small quantities, the
subunits were located by taking aliquots of each fraction and determining
the enzyme activities by measuring the reducing value of a starch digest.
11
8.
SDS-gel electrophoresis for molecular weight determinations
SDS-polyacrylamide gel electrophoresis was performed following the
method of Weber and Osborn (49), except that 0.2% EDTA was added to the gel
and the buffer system.
PPA was also incubated with 0.2% EDTA, 1%
mercaptoethanol (v/v), 4 M urea and 1% SDS, at 45°C for 30 to 60 minutes
prior to being applied to the gel.
Before applying the protein samples, the
gels were sometimes pre-electrophorized for one hour to remove the excess
persulfate ion which is used to catalyze the gel formation.
Protein bands
were fixed and stained by the method of Dunker and Rueckert (45).
9.
Modification of protein sulfhydryl and disulfide groups
a.
Derivatization with iodoacetamide
lodoacetamide was used to alter
the sulfhydryl groups and thereby prevent their oxidation to disulfide bonds.
Crystalline PPA (1 mg/ml) in 20 tnM sodium glycerophosphate buffer (pH 8.5)
and 10 mM EDTA was first reduced with 10 mM DTT for 16-20 hours.
The
solution was then made 100 mM in iodoacetamide for 4 hours at room
temperature.
Derivatized protein was kept at 4°C until it was subjected to
molecular weight determination by SDS-gel electrophoresis.
b.
used.
Oxidation with performic acid
The method given by Hirs (50) was
Performic acid was freshly prepared by adding 0.5 ml of 30%
4.5 ml of 90% formic acid at 50°C.
to
After 3 minutes, 1 ml was transferred to
a test tube containing 2-5 mg crystalline PPA in less than 0.2 ml volume.
The solution was kept at 50°C for 15 minutes and then diluted with
approximately 10 volumes of water; this was followed by lyophilization.
The
oxidized PPA was dissolved in water by adding 1 M ammonium hydroxide dropwise until the pH was 9 as judged by pH-paper.
This sample was then
12
subjected to molecular weight determination on SDS-gel electrophoresis.
Oxidized PPA was dissolved and dialyzed against 0.1 M (NH^^2C02 (pH 8.8)
for sedimentation velocity studies in the ultracentrifuge.
Lyophilized
dry samples were used for amino acid analyses.
10.
Peptide mapping
Pooled fractions of subunits A and B which were isolated from a reduced
DEAE-cellulose column were first dialyzed extensively against 10 mM NH^HCO^
(pH 8.5).
Samples were lyophilized and oxidized by perforraic acid.
performic acid was removed by a second lyophilization.
The
Trypsin hydrolysis
of the oxidized subunits was carried out at 25°C in 2 ml of 0.1 M NH^HCO^
buffer (pH 8.0).
of 1:100.
TPCK-trypsin was added to the amylase in a weight ratio
After 8 hours of stirring at room temperature, a second 1:100
aliquot of trypsin was added and stirring was continued for 4 additional
hours.
The lyophilized digests were dissolved in a very small amount of
water and were applied to a Whatman No. 3 MM paper (46 x 56.5 cm).
High
voltage electrophoresis was run at 2000 volts for 1 hour in a buffer system
of pyridine: acetic acid: water (1 ; 3.4 : 409, v/v, pH 4.0) followed by
decending paper chromatography for 16 - 20 hours with butanol-1: pyridine:
acetic acid: water (90 : 60 : 18 : 72, v/v).
Peptides were located by
spraying the dried paper with nonhydrin: collidine solution prepared by
mixing 10 ml 0.1% ninhydrin in ethanol with 1.3 ml collidine and 3 ml
glacial acetic acid.
The papers were heated in an oven at 100°C until
faint blue spots appeared.
13
11.
Determination of the number of sulfhydryl groups and disulfide
linkages
The DTNB method described by Robyt et al. was used (51).
Isozymes and
subunits were previously reacted with 10 mM EDTA at 5Q°C for approximately
20 hours.
DTNB was allowed to react with the protein sample at pH 8.1 for
30 minutes at room temperature.
The number of sulfhydryl groups per
molecule of protein was computed from the absorbance measurements and
extinction coefficients of protein and CNT at 280 and 412 nm respectively.
For determination of disulfide groups, the pH of the reaction solution
from the determination of free sulfhydryl groups was increased from 8.1 to
10.5 by carefully adding 0.1 M sodium hydroxide and allowing the reaction
to proceed for 1-2 minutes.
The pH was readjusted to 8.1 by adding 0.1 N
hydrochloric acid and the increase in absorbance was measured at 412 nm.
A DTNB reagent blank was prepared to correct for the formation of CNT from
the alkaline cleavage of DTNB.
The number of disulfide groups was
computed by the following formula:
disulfide = (No. of CNT after pH 10.5) - 2(No. of CNT before pH 10.5)
groups
12.
^
Test for disulfide conformers in subunit A and subunit B
The difference between subunits A and B may be in the tertiary struc­
ture involving the pairing of sulfhydryl groups to form disulfide linkages.
To test this hypothesis, subunits A and B were derivatized with
14
C-
iodoacetamide followed by hydrolysis with pepsin and peptide mapping.
Radioautograms were made to reveal the location of cysteine peptides.
14
Fractions of subunit A and B (about 0.75 mg in 2 ml) were dialyzed
against 0.01 M ammonium bicarbonate (pH 8.5) to remove the excess reducing
agent, which was used in the isolation of the subunits.
EDTA (100 mM) and
urea (8 M) solutions were added to make the final concentration of 10 mM
and 4 M respectively.
above 8.0.
The pH of the solution was checked to maintain it
Radioactive ^^C-iodoacetamide (30 yC^, 0.17 mg in 25 yl of HgO)
was added to each fraction followed by addition of cold iodoacetamide (IM)
to make the final concentration 0.04 M.
for 4 hours.
acid.
The reaction proceeded in the dark
The samples were then dialyzed against 5 liters of 5% formic
Pepsin equivalent to
of the weight of subunit sample was added.
The digestion was carried out for 24 hours at room temperature (52).
The
final products were lyophilized and a small amount of H^O was added to each
sample.
The samples were then applied to the paper for high voltage
electrophoresis and paper chromatography as was done for regular peptide
mapping.
An estimation of the radioactivity on the maps was made by a
Geiger survey meter.
The maps were then exposed to X-ray films for a
proper length of time (7 days) for obtaining the radioautograms.
13.
Amino acid composition analysis
For the analysis of the amino acid compositions in subunits A and B,
pooled fractions were first dialyzed against 0.01 M pyridine-acetate buffer
(pH 5.0) and lyophilized.
Protein samples were hydrolyzed in 6 N HCl in
sealed evacuated tubes normally for 20 hours at 110°C.
A Beckman 120-B
amino acid analyzer was used to determine the composition of the hydrolyzates.
15
14.
Action pattern and product specificity studies
The various amylase fractions, e.g., isozymes, and subunits, were
added (2.5 mU/ml) to a solution of amylose (1 mg/ml) which was buffered with
20 mM sodium glycerophosphate (pH 6.9) containing 10 nM KCl ; 2 ml aliquots
were taken at various times and transferred to test tubes containing 0.1 ml
of 5 M TCA.
After neutralization with sodium hydroxide, the samples were
desalted with ion exchange resin, Amberlite MB-3, and 200 yl of each sample
were placed on a Whatman No. 3 MM paper for ascending chromatography.
The
chromatograms were obtained by the techniques of French £t al^. (53).
15.
Multiple attack studies
The degree of multiple attack for enzymic action on polymeric
substrate may be defined as the average number of catalytic events,
following the first, during the lifetime of an individual E-S complex.
In amylolysis. Cue differences in the degree of multiple attack can be
expressed by the differences in the curves which are obtained by plotting
the drop in the absorbance (620 nm) of the amylose-iodine color to the
increase in reducing value as measured by the alkaline ferricyanide method.
The experimental method devised by Robyt and French (54) was followed.
Amylose (120 mg) was dissolved in 2 ml of dimethyIsulfoxide; this was
diluted with 20 mM sodium glycerophosphate buffer (pH 6.9) and 10 mM KCl
solution to 100 ml.
About 300 mU of enzyme was added to initiate the
reaction; the digests were incubated at 40°C, and 10 ml aliquots were
taken at various times over a 2 hour period and added to 0.1 ml of 5 M
TCA.
After 30 minutes, the samples were neutralized with 5 M sodium
hydroxide to pH 6.
Each sample was then subjected to reducing value and
16
blue value determinations.
Curves relating the drop in blue value to the
increase in reducing value for the action of isozymes and subunits were
obtained.
16.
Ultracentrifugation studies
Performic acid oxidized PPA (2 mg/ml), which was dissolved in 0.1 M
ammonium carbonate (pH 8.8), was subjected to sedimentation velocity
studies at 21°G and 52,000 rpm in a Spinco, Model E, analytical ultracentrifuge.
Sedimentation velocity studies of crystalline PPA (4 mg/ml) were run
at 21°C and 52,000 rpm under four solvent conditions:
(a) 20 mM sodium
glycerophosphate (pH 8.5); (b) 20 mM sodium glycerophosphate and 20 mM
DTT (pH 8.5); (c) 20 mM sodium glycerophosphate and 5 M guanidine HCl
(pH 8.5) and (d) 20 mM sodium glycerophosphate, 5 M guanidine HCl, and
20 mM DTT (pH 8.5).
17.
Calcium analysis
For the calcium analysis of subunits A and B, pooled fractions of
each subunit were dialyzed against more than 100 volumes of redistilled
water.
The glassware and dialysis tubing were presoaked and washed with
0.01 M EDTA and redistilled water.
After dialysis, the protein concentra­
tion in solution was determined by Lowry's method on the Auto Analyzer and
adjusted by the correction factor as indicated in section 1-d.
calcium content was determined by flame emission spectrometry by
Mr. Richard Kniseley of the Ames Laboratory, Ames, Iowa.
The
17
III.
A.
RESULTS
Gel Filtration of Crystalline PPA on Sephadex G-25
It was reported by Rowe e^ al. (14) that PPA after standing 24 to 48
hours in solution undergoes degradation by traces of contaminating
proteolytic enzymes and yields inactive peptides which can be separated on
G-25 Sephadex column.
Therefore it was necessary to obtain conditions
under which proteolytic hydrolysis can be prevented.
PPA is known to be
stabilized by calcium ion against proteolytic attack (6) and is most
stable in the presence of chloride ion (1).
When the native enzyme was
chromatographed on Sephadex G-25 in sodium glycerophosphate buffer (pH 7.0)
-4
-3
with 10
M CaClg, 10
M KCl, a major peak (enzymically active) emerged
first, followed by one minor inactive peptide peak.
When the active
fraction was rechromatographed on the same column after refrigeration for
48 hours, it re-emerged as a single peak (Fig. 1).
This result indicates
-4
that with the incorporation of 10
M calcium ion and 10 mM chloride ion,
the degradation of PPA by proteolytic enzymes is prevented.
Therefore,
-4
10
M CaClg and 10 mM KCl were incorporated into all the buffer systems
for the PPA studies.
B,
1.
Multiple Forms in Crystalline PPA
Polyacrylamide gel electrophoresis
Crystalline PPA was fractionated into four distinct bands (Fig. 2)
under the gel-electrophoretic conditions.
than the other two.
Two bands were more predominant
Figure 1.
Gel filtration of porcine pancreatic amylase:
(A) Elution profile of crystalline enzyme on Sephadex G-25
(B) Elution profile of major peak after standing 48 hours
at 2 - 5°C.
Buffer system contained: 20 mM sodium
-4
glycerophosphate (pH 7.0); 10
M CaCl^; 10 miM KCl
a:
b:
major protein peak, enzymatically active
peptide peak, enzymatically inactive
1.00
0.50
20
40
60
80
40
60
80
1.00
280
0.50
20
Eluent (ml)
Figure 2. Fractionation of porcine pancreatic a-amylase on
polyacrylamide gel-electrophoresis
A:
crystalline PPA in 10 mM CaCl^
B:
crystalline PPA in water
22
2.
Isoelectric focusing
At the pH range of 5 to 8, crystalline PPA was fractionated into four
species (Fig. 3) with their isoelectric points at pH 6.9, 6.2, 5.6 and 5.2.
Using a starch-iodine test*, all of the species were found to be
enzymatically active.
C.
Isolation of Isozymes by Ion Exchange Chromatography
Chromatography of the crystalline enzyme on DEAE-cellulose at pH 8.5
with 10 ^ M CaClg and 10 mM KCl gave three fractions (Fig. 4) with a protein
recovery of 90-95%.
found.
Occasionally a fourth and very small peak could be
They were designated as isozymes:
I, II, III and IV (Fig. 4).
They were considered isozymes because they had the same molecular weights
and very similar catalytic properties (see section D and section H).
The
separation was pH dependent; chromatography at or lower than pH 8.0 failed
to fractionate the species.
poor resolution.
Chromatography between pH 8.0 and 8.5 gave
When the isozymes were assayed for enzyme activity by the
alkaline ferricyanide method, the specific activity of each peak was
obtained as follows:
D.
I: 450 U/mg, II; 288 U/mg, III: 450 U/mg, IV: 91 U/mg.
Molecular Weight Determination of Isozymes
In order to understand the nature of the multiple forms of PPA
isolated from DEAE-cellulose column, each form was subjected to molecular
weight determination by two independent methods; namely, gel filtration
and SDS-gel electrophoresis.
*
Protein solution 1 ml was added to 9 ml of starch solution (1%)
buffered at pH 6.9. After a period of time, one drop of the starch digest
was stained with a 2% KI, 0.2% Ig solution containing 0.1 ml concentrated
HCl per 100 ml of the Ig" solution. The time required to change the starchiodine color from blue to brown or achroic gave an estimation of the
enzyme activity.
Figure 3.
Isoelectric focusing profile of porcine pancreatic amylase
pH range:
5-8;
x
x
A 280;
o
o
pH value
12
-
10
pH
6
-
4
_
2
-
tsJ
- 0.10
12
16
20
Fraction Number (3 ml)
Figure 4.
Chromatography and preparation of isozymes of porcine pancreatic amylase on DEAEcellulose
Buffer system contained:
20 mM sodium glycerophosphate
(pH 8.5); 10"^ M CaCl^; 10 mM KCl
0.60
0.50
0.40
0.30
0.20
0.10
0
20
Fraction Number (3 ml)
27
Fractions of isozymes I, II, and III were collected from DEÂEcellulose columns and chromatographed directly on Bio-gel P-150.
Bacillus subtilis var. amyloliquefaciens a-amylase, bovine serum albumin,
Aspergillus oryzae a-amylase, and horse heart cytochrome c were used as
standards.
As indicated in Fig. 5, the three isozymes possessed molecular
weights of I = 50,000, II = 53,000, and III = 55,000.
The amount of
isozyme IV isolated was not enough for this study.
For the molecular weight studies of the isozymes by SDS-gel
electrophoresis, it was necessary to incorporate 0.2% EDTA into the
conventional buffer and gel system as described by Weber and Osborn (49).
EDTA (10 mM) was also used in addition to urea and SDS in denaturing the
enzyme.
It is known that PPA binds calcium tightly (5).
The removal of
calcium by EDTA is necessary for the interruption of the amylase tertiary
structure (7).
Bovine serum albumin, aldolase, yeast alcohol dehydrogenase,
and cytochrome c were used as standards.
The mobilities of protein bands
were plotted against the logarithm of the molecular weights.
From Fig. 6,
isozyme I has a molecular weight of 54,000; isozyme II = 54,000; isozyme
III = 51,000; isozyme IV = 50,000.
E.
Isolation of Subunits by Reducing-DEAE-Cellulose Chromatography
Subunits of PPA were formed after PPA was treated with the reducing
agent, DTT (25).
For the isolation of subunits, DEAE-cellulose was treated
with mercaptoethanol before the packing of the column to prevent reoxidation
and dimerization.
Fig. 7 shows the formation of subunit A and subunit B
when PPA was treated with DTT prior to elution from the reducing-DEAEcellulose column.
They were considered subunits because the molecular
Figure 5.
Determination of molecular weight of the isozymes by gel filtration chromatography
on Bio-gel P-150
0
0 Standards; x
x Isozymes
Bacillus subtilis a-amylase, 96,000; Bovine serum albumin, 67,000;
Aspergillus oryzae a-amylase, 51,000; Cytochrome (, 12,500
10
9
8
Bovine Serum Albumin
III
A.£. a-Amylase
Cytochrome c
1
1
20
40
Elution Volume (ml)
60
Figure 6.
Determination of molecular weight of the isozymes by SDS-polyacrylamide gel
electrophoresis
0
0 Standards; x-—x Isozymes
Bovine serum albumin, 67,000; Aldolase, 40,000;
Alcohol dehydrogenase, 37,000; Cytochrome c, 12,500
Bovine Serum Albumin
Aldolase
&. Alcohol dehydrogenase
Cytochrome c
Mobility
Figure 7.
Chromatography and preparation of subunits A and B of porcine pancreatic amylase
on reducing DEAE-cellulose
Buffer system contained:
20 mM sodium glycerophosphate
(pH 8.5); 10"^ M CaClgi 10 mM KCl
0.60
0.50
280
0.40
0.30
0.20
0.10
20
30
40
Fraction Number (3 ml)
50
34
weight of each fraction was determined to be around 25,000 (25) which is
half of the molecular weight of the isozymes.
each subunit was obtained as follows:
F.
1.
The specific activity of
A: 490 U/mg, B: 540 U/mg.
Study of the Nature of the Subunits
Peptide mapping of subunits A and B
Peptide mapping was employed to study the structural difference of
subunits A and B.
8.
Finger prints of the two subunits are presented in Fig.
A great similarity between these two maps were observed.
The two sub-
units also gave the same chromatographic properties on DEAE-cellulose
columns and the same electrophoretic mobilities on gels after the disulfide
linkages in them were disrupted by reduction with DTT and S-carboxymethylamidation.
From these studies, it was concluded that subunits A and B
have very similar, if not the same, primary structures.
2.
Amino acid composition analysis of subunits A and B
The hypothesis that subunits A and B may have similar primary structure
was further tested by amino acid composition analysis of each form.
results are given in Table 1.
The
A high degree of similarity in amino acid
compositions of subunits A and B is observed. The largest variance in
amino acid content is that of aspartic acid + amide (18 residues for A and
25 residues for B).
The amide residues were not determined separately.
Thus, if the difference in the number of aspartic acid and asparagine was
mainly due to the difference in acidic groups, this might serve to explain
the differences in mobilities in electrophoresis.
The number of half
cystine is relatively low compared to the value obtained by other methods
35
Table 1.
Amino acid compositions of subunits A and B of porcine
pancreatic a-amylase
Subunit A^
Subunit
15
12
5
5
Arginine
12
12
Aapartic
18
25
Threonine
11
11
Serine
13
12
Glutamic
15
15
Proline
7
9
Glycine
29
28
Alanine
19
16
Lysine
Histidine
Half Cystine
Valine
6^
6»
16
17
Methionine
4
4
Isoleucine
9
9
13
12
8
8
10
10
Leucine
Tyrosine
Phenylalanine
Tryptophan
8=
8"=
dumber based on a molecular weight of 25,000.
^Data obtained by DTNB method (see section F-3).
^Value obtained from Cozzone et al. (15) and Romano and Strumeyer (55).
Figure 8,
Peptide maps of porcine pancreatic a-amylase subunits A and B. Both subunits were
oxidized with performic acid prior to hydrolysis with trypsin. Direction 1 was
high-vjltage electrophoresis (2000 V for 1 hr) using pH 4.0 pyridine: acetic acid:
water buffer (1:3.4:409 parts by volume) in the Gilson high-voltage electrophoresis
apparatus; direction 2 was descending paper chromatography using butanol-lr pyridine:
acetic acid: water (90:60:18:72 parts by volume) as irrigation solvent
Subunit A •
O"
Subynit B + Trypsin
»-2
>-2
%
i
0:
e
#
38
(see section F-3).
However, it is known that cystine is sometimes destroyed
by acid hydrolysis and tends to give lower values when it is determined by
the amino acid analyzer (56).
3«
Determination of the number of sulfhydryl and disulfide groups in
isozymes and subunits A and B
From amino acid analysis, peptide mapping, electrophoretic and
chromatographic (25) studies, there are good evidences that the primary
structures of subunits A and B are very similar.
Then, the structural
difference between A and B must be at the tertiary structure level.
Disulfide linkages play important roles in maintaining the tertiary
structure of proteins.
In native PPA, there are 10-12 half cystine as
reported by different research groups (15, 55).
sulfhydryl groups (7, 15).
There were only two free
Since subunits A and B were isolated under
reduced conditions, one possibility for the differences between A and B
was in the degree of reduction, that is in the number of sulfhydryl and
disulfide groups present in each of the subunits.
The number of sulfhydryl groups and disulfide linkages in the
isozymes and subunits was determined by the method devised by Robyt e^ al.
(51) using Ellman's reagent.
The isozymes and the subunits were incubated
with 10 mM CaNa^ EDTA at 50°C overnight to ensure full exposure of the
sulfhydryl groups.
The results obtained are presented in Table 2.
These
data show that there are two sulfhydryl groups in each of the isozymes
and in each of the subunits; this, thus, indicates that the differences
between the subunits are not in the number of disulfide linkages due to
the differences in the degree of reduction.
The results also demonstrate
39
Table 2.
Sulfhydryl groups and disulfide linkages analysis of the subunita
and isozymes of porcine pancreatic oamylase
No. of
-SH groups
No. of
disulfide linkages
Subunit A
1.89*
2^
1.81*
2^
Subunit B
2.10
2
1.79
2
Isozyme I
2.01
2
4,40
5
Isozyme II
1.98
2
5.24
5
Isozyme III
1.92
2
4.43
5
^Experimental data.
^Number assumed to the nearest integral.
that the subunits were formed by the reduction of a single disulfide
linkage.
This linkage holds the two subunits together.
12 half cystine was obtained by this determination.
A total number of
This data is in good
agreement with the results obtained by Romano and Strumeyer (55).
Cozzone
et al. (15) indicated there were only 10 half cystine residues from their
analysis.
The number of sulfhydryl groups in the isozymes is also very
close to the value 1.7 obtained by Schramm (7).
4.
Test for disulfide conformers in subunits A and B
When there are more than two sulfhydryl groups in a protein, conformers
can be present due to the formation of different disulfide linkages (57).
If during the process of obtaining the tertiary structure of subunits A
and B, the sulfhydryl groups involved in forming disulfide bonds in A are
not the same ones found in B, it is possible that the free sulfhydryl
40
groups in these two subunits will be distinguishable from each other.
sulfhydryl groups of A and B were labeled by derivatization with
iodoacetamide.
The
14
C-
The labeled proteins were then hydrolyzed with pepsin
followed by peptide mapping.
Oligopeptides originally containing sulfhydryl
groups were identified by the spots on autoradlograms which were made from
the peptide maps.
As shown in Fig. 9, there are two spots on each map
corresponding to the two sulfhydryl peptides in each subunit; the location
of the sulfhydryl peptides were identical in both A and B.
However, this
evidence could not in itself rule out the possibility that the sulfhydryl
groups involved in disulfide bond formation were the same in subunits A as
in B, but a cross pairing could occur in A and B which did not involve the
two free sulfhydryl groups; the remaining four sulfhydryl groups could be
paired differently to give rise to differences between the tertiary
structures.
5.
Calcium determination of subunits A and B
PFA was known to bind calcium ion (5).
The amount of calcium bound
to subunits may contribute to their differences in DEAE-cellulose
chromatographic and electrophoretic behavior.
From flame emission
spectrometry, it was found that subunit A contained 9 gram atoms of
calcium per 25,000 daltons and subunit B contained 10 gram atoms of
calcium per 25,000 daltons.
It was concluded that the difference of the
calcium content in subunits A and B is identical within experimental error
and probably is not sufficient to give rise to the differences in the
chromatographic and electrophoretic behavior of subunits A and B.
Figure 9.
Autoradiogram of the peptide maps of porcine pancreatic a-amylase subunits labeled
with ^^C-iodoacetamide.
The sulfhydryl groups in each subunit was first modified
with ^^C-iodoacetamide. The subunits were then subjected to pepsin hydrolysis.
Peptide maps were made by the same procedure as described in Figure 8. Direction 1
was high-voltage electrophoresis; direction 2 was descending chromatography
Subunlt
A
Subiuit
B
43
G.
1.
Study of the Quaternary Structure of PPA
Study of the nature of the isozymes
Since the native PPA is made of two kinds of subunits, it was
postulated that the differences in these isozymes were in the composition
of their subunits as in the case of lactic dehydrogenase.
was tested by isolating subunits from each isozyme.
This possibility
The isozymes were
treated with DTT and were rechromatographed on reducing-DEAE-cellulose
columns.
Due to the low protein concentration, the subunits were
located by measuring amylolytic activity of each fraction.
As shown in
Fig. 10, isozyme I was dissociated into subunits A and B, isozyme II was
dissociated into only subunit A and isozyme III into only subunit B.
These experiments demonstrated that isozyme I consisted of A-B, isozyme II,
A-A and isozyme III, B-B.
The crystalline PPA preparation was a mixture
of isozymes I, II and III.
Therefore, two kinds of subunits (A and B)
could be obtained from the native enzyme.
The nature of isozyme IV was
not studied because not enough material was isolated.
2.
Reduction and alkylation, performic acid oxidation and molecular weight
determination of PPA
Since the two subunits in PPA were linked together by disulfide
linkages, disruption of the disulfide bonds would reduce the molecular
weight of the protein.
Crystalline PPA was reduced and derivatized with
iodoacetamide before being subjected to molecular weight determinations on
SDS-gel electrophoresis.
After electrophoresis and staining, four protein
bands were observed (Fig. 11).
They corresponded to molecular weight
species of 50,000; 38,000; 25,000 and 12,000.
This result indicated that
Figure 10.
Isolation of subunits from isozymes of porcine pancreatic
a-amylase
(a) Chromatography of crystalline porcine pancreatic
a-amylase on DEAE-cellulose. j Indicates the fractions
taken for rechromatography on reducing-DEAE-cellulose
(b) Subunits isolated from each isozyme on reducingDEAE-cellulose.
bj, subunits A and B from isozyme I; b^, subunit A
from isozyme II; b^, subunit B from isozyme III
Arbitrary Unit of Activity
r
to
o
.
p
»
O
N
•^280
i
s
5
^
o
>
o
hO
o
o
^
o
o
ON
o
to
o
ro
Ln
M
to
Ln
W
to
O
O
LO
Ln
Ln
KJI
o
O
U)
lO
CO
U>
LO
Ln
Figure 11.
Determination of the molecular weight of modified porcine
pancreatic «-amylase on SDS polyacrylamide gelelectrophoresis:
(A) Porcine pancreatic a-amylase was reduced with DTT and
modified with iodoacetamide. The four protein bands
corresponded to molecular weights of: A = 50,000;
B = 38,000; C = 25,000 and D = 12,000, as judged by a
standard curve using bovine serum albumin, 67,000;
A. o. a-amy]ase, 51,000; pepsin, 35,000; and
Cytochrome c, 12,500.
(B) Porcine pancreatic amylase was oxidized with
performic acid. The protein band corresponded to
molecular weight of 12,000.
47
A
A
6
C
W'
B
48
the native enzyme was actually composed of 4 monomers of about 12,500
daltons.
These monomers were linked together through disulfide bonds.
When the disulfide linkages were disrupted by DTT, followed by Scarboxymethylamidation, the four possible molecular weight species were
formed.
These four forms represented varying degrees of reduction to give
a tetramer, a trimer, a dimer, and a monomer.
The fact that the unit peptide chain in making PPA had a molecular
weight of about 12,500 was further substantiated by the following finding.
The molecular weight of performic acid oxidized protein was around 12,000
(Fig. 11) when determined by SDS-gel electrophoresis.
was relatively diffuse.
and diffused quickly.
12,000.
The protein band
Probably it was because the molecule was small
However, it gave an average molecular weight of
From this result it can be deduced that the original molecule
was composed of four subunits which were linked together by disulfide
bonds.
After oxidation of the sulfhydryl groups and the disulfide bonds,
the protein was broken down entirely to the monomer form.
This form did
not recombine by oxidation because the sulfhydryl and disulfide groups
were converted to cysteic acid.
3.
Sedimentation studies
The molecular weight of reduced PPA and oxidized PPA was further
examined by sedimentation velocity studies in the ultracentrifuge.
As
shown in Fig. 12, PPA sedimented equally fast in sodium glycerophosphate
buffer solution with or without the presence of 20 mM DTT.
However, when
6 M guanidine HCl was incorporated, the sample with the presence of 20 mM
DTT moved slower than the sample without DTT.
This result indicated that
PPA was dissociated into subunits by disruption of disulfide bonds and the
non-covalent interactions between subunits.
The reduction of the disulfide
Figure 12.
Sedimentation patterns of porcine pancreatic ot-amylase at
52,000 rpm. Pictures were taken with 16 minute intervals
(a)
(b)
(c)
(d)
PPA in sodium
PPA in sodium
PPA in sodium
PPA in sodium
+ 20 mM DTT
glycerophosphate
glycerophosphate
glycerophosphate
glycerophosphate
buffer
buffer + 20 mM DTT
buffer + Guanidine HCl
buffer + Guanidine HCl
50
51
bonds was not sufficient to dissociate the subunits in the ultracentrifuge;
however, it is sufficient to dissociate the subunits on reducing DEAEcellulose column due to the interaction between the protein molecule and
DEAE-cellulose.
Performic acid oxidized PPA was too small to observe its
sedimentation pattern by the conventional method.
However, by using the
special technique of synthetic boundary, a sedimentation pattern was
obtained as shown in Fig. 13.
oxidized PPA was very small.
diffused considerably.
1.46 S.
From this study, it is apparent that the
In 20 minutes, the protein molecules had
The sedimentation constant was estimated to be
Assuming that the oxidized PPA remained as a spherical structure,
a sedimentation constant of 1.46 corresponds to a molecular weight
between 11,000 and 12,000 (58).
This value coincided well with that
obtained from SDS-gel electrophoresis for the oxidized PPA.
4.
Amino acid composition of the crystalline and oxidized PPA
The amino acid compositions of the native enzyme as well as of the
oxidized PPA are presented in Table 3.
The results are in good agreement
with those obtained by Romano and Strumeyer (55) and Cozzone et al. (15).
Except 12 half cystine residues were found in the present study and by
Romano and Strumeyer (55).
Cozzone ejt al^, (15).
Only 10 half cystine residues were found by
Discrepancies were observed between the data
obtained by this study and those published by Caldwell ^ al^. (4).
Figure 13.
Sedimentation pattern of oxidized PPA in ammonium carbonate buffer at 52,000 rpm.
Pictures were taken with 4 minute intervals
53
54
Table 3.
Amino acid composition of porcine pancreatic a-amylase
g
Oxidized
PPA
Crystal
PPA
Romano
and
^
Strumeyer
Cozzone
Amylase I
Cozzone
Amylase II
17
20
18
20
20
6
8
8
9
9
Arginine
25
27
25
27
27
Aspartic
79
69
62
66-67
61-62
Threonine
21
19
19
23
23
Serine
29
28
30
32
32
Glutamic
37
34
34
38
38
Proline
26
22
19
21
21
Glycine
60
55
49
51
51
Alanine
31
30
29
30
30
Half Cystine
12^
12^^
12
10
10
Valine
38
38
39
37
37
8
8
8
8
Lysine
Histidine
Methionine
8®
Isoleucine
22
21
23
21
21
Leucine
25
25
23
23
23
Tyrosine
18
18
17
18
18
Phenylalanine
23
24
22
23
23
Tryptophan
15^
15^
15
15
15
^Numbers based on a molecular weight of 50,000.
^Data obtained from Romano and Strumeyer (55).
^Data obtained from Cozzone ^
M wt = 53,132 for II.
(15), M wt = 53,811 for I,
^Number obtained by DTNB method (see section F-3).
e
Value obtained from Cozzone ^
(15) and Romano and Strumeyer (55).
55
H.
Product Specificity and Action Patterns
of the Isozymes and the Subunits
1.
Product specificity
Both subunits as well as the three isozymes were enzymatically
active.
It was of interest to compare their enzymatic properties.
A time-
sequence of the products formed from amylose by the three isozymes and the
two subunits were obtained by paper chromatographic analysis.
As shown in
Fig. 14, the action patterns of these digests were identical.
Obviously
the differences in structure did not effect the catalytic properties to
an observable extent.
Also, the whole enzyme had at least two active
sites which were independent of each other.
2.
Blue value vs reducing value studies
The blue value vs reducing value curves (Fig. 15) showed that for an
equal drop in blue value the isozymes have lower percentage conversions to
apparent maltose than the subunits.
Although the product specificities of
these isozymes and subunits were the same, these differences in the blue
value-reducing value curves indicated that the degree the multiple attack
was slightly different.
However, the differences among the isozymes and
subunits were relatively small compared to the differences of these enzyme
preparations and the blue value-reducing value curves for other ot-amylases
(54).
I.
Resistance of Isozymes and Subunits to Trypsin Hydrolysis
Native PPA was known to resist proteolytic hydrolysis in the presence
of calcium ion (6).
It would be important to know whether the subunits were
as resistant as the dimers.
It was found that after 24 hours of trypsin
Figure 14.
Time sequence chromatographic analysis of porcine pancreatic
a-amylase isozymes and subunits action on amylose. The
conditions of the digests were: Amylose (1 mg/ml); 20 mM,
pH 6.9 sodium glycerophosphate buffer; 10 mM KCl; 2.5 mU of
enzyme/ml. Aliquots of 200 yl were placed onto the
chromatogram
56b
Isozjpe I
G
O
5 15 30 45 60 75 90 105 liOtaln.
ImO
Imozyme II
G, ^ 0
5 15 30 45 60 75 90 105 12Ctaln. Gj»
Isozyme III
'1^8
1-8
Figure 14.
(Continued)
57b
Subunit A
G, a 0
5 15 30 W> 60 75 90 105 12CBdn, G.
Subunit B
G..0
5 15 30 45 60 75 90 105 120toin. G.
Figure 15.
Comparison of the drop in iodine color (blue value) with the
increase in reducing value for the hydrolysis of amylose by
isozymes and subunits. Blue value is defined as (At/Ao) x
100, where Ao and At are the absorbancies (620 my) of the
iodine complex of the digest at zero time and at t minutes
of hydrolysis
X
X subunit A;
0
0 isozyme II;
#-
9 subunit B;
A
isozyme III
é isozyme I;
59
100
II
90
III
m
D
*—4
PQ
40
4
8
12
16
24
20
Reducing value as % apparent maltose
28
32
36
60
(~ of the weight of amylase) digestion at room temperature, all of the
forms of PPA still remained very active.
The remaining activities in
each sample are presented in Table 4.
Table 4,
Measurements on the resistance of subunits and isozymes to
trypsin hydrolysis
Percentage of enzymic activity remained (%)
after 24 hours hydrolysis by trypsin
Subunit A
82
Subunit B
71
Isozyme I
81
Isozyme II
70
Isozyme III
88
61
IV.
DISCUSSION AND CONCLUSIONS
It is well known that many enzymes exist in multiple molecular forms.
The heterogeneity of an enzyme may be due to several mechanisms (59):
1) genetically independent proteins; 2) heteropolymers (hybrids) of two or
more polypeptide chains; 3) genetic variants (allelic); 4) proteins
conjugated with other groups; 5) proteins derived from one polypeptide
chain; 6) polymers of a single subunit; and 7) conformâtionally different
forms.
Despite the fact that the presence of multiple forms of PPA has been
observed for seven years (42) the nature of this multiplicity has remained
unknown.
In the present study, gel electrophoresis, isoelectric focusing, and
ion exchange chromatography, showed that four forms of porcine pancreatic
«-amylase were present in the crystalline preparation (see Figs. 2, 3, 4).
One of the forms was only present in trace amounts.
The molecular weights
of these forms were determined to be around 50,000 by two independent
methods (see Figs. 5, 6).
The product specificities of these forms with
amylose were identical (see Fig. 14).
These findings indicated that these
multiple molecular forms were isozymic in nature.
They were not derived
from polymerization nor were they due to contamination by other types of
a-amylases.
Two subunits of identical molecular weight, A and B, were isolated
from crystalline PPA after reduction with DTT (Fig. 7).
When the isozymes
prepared from DEAE-cellulose chromatography were subjected to reduction,
both subunits A and B were isolated from isozyme I; only subunit A was
62
obtained from Isozyme II; and only subunit B was obtained from Isozyme III
(Fig. 10).
It was evident that the heterogeneity of PPA is due to the
existence of hybrids of two subunlts.
A-A; and isozyme III was B-B.
Isozyme I was A-B; Isozyme II was
These subunlts were linked together by
disulfide bonds.
In 1970, Cozzone et al. (15) also Isolated three active forms of
porcine pancreatic a-amylase by DEAE-cellulose chromatography.
not find any structural differences among these forms.
termlnal group analysis, Cozzone e^
From their C-
(15) indicated that only a single
C-terminal leucine was obtained from each Isozyme.
obtain subunlts from PPA.
They did
They also failed to
They therefore concluded that the amylase
Isozymes had only a single peptide chain.
The discrepancies between their
findings and the present study can be explained as due to the differences
in experimental conditions used.
Cozzone et al. (15) initially treated
the Isozymes with 2-mercaptoethanol at pH 7.1.
SDS-gel electrophoresis of
the reduced Isozymes gave molecular weights of around 50,000 and did not
Indicate the presence of any lower molecular weight fractions.
present study, PPA was reduced with DTT at pH 8.5.
In the
It is known that the
reducing efficiency of mercaptans is greater at pH 8.5 than at pH 7.1 and
that DTT is a much more efficient reducing agent than 2-mercaptoethanol
(60).
For the molecular weight determinations in polyacrylamide gels, it
was also found that the SDS-gels had to be pre-electrophorized to remove
the excess oxidizing agent before applying the protein samples.
Szabo and Straub (61) reported that they separated two to four active
fractions on DEAE-cellulose.
They also suggested the heterogeneity of PPA
was formed by sulfhydryl-disulflde interchanges in the protein molecules.
63
Under the experimental conditions they used, one or more of the different
conformations might become stabilized.
Straub et al. also isolated the
smaller chains, A = 32,000 and B = 21,000, from crystalline enzyme under
reduced conditions (22).
From their amino acid analysis, they concluded
that PPA was composed of two chains of similar but not identical
compositions; no correlation between the subunits and isozymes was made.
In this study, efforts were made to establish the structural
differences between subunits A and B of PPA.
There were several
possibilities which would make subunit A different from subunit B:
1) A and B had different primary structures; 2) A and B had the same
primary structures but had different number of disulfide bonds; 3) A and
B were different in intramolecular disulfide bonds; 4) different amounts
of non-protein groups were bound to A and B; and 5) A and B had different
conformations or tertiary structures.
From earlier work by Chittenden (48), it was shown that treatment of
A and B with DTT in the presence of EDTA gave forms that migrated very
rapidly and identically on electrophoresis gels.
Reaction of these forms
with iodoacetamide gave derivatives that behaved identically on electro­
phoresis gels and on DEAE-cellulose.
Even though subunits A and B were
isolated under reducing conditions, apparently all of the intramolecular
disulfide bonds of A and B were not initially reduced by DTT, possibly
because of structural burying.
The disulfide masking might be due to a
tight protein structure formed by calcium chelation (5, 6, 7).
Presumably, the EDTA removed the calcium and opened the structure so that
the DTT was able to reduce the buried disulfide linkages, and thus allowed
64
them to react with iodoacetamide.
As a result, identical forms were
produced as judged by electrophoresis and chromatography.
These findings
suggested that A and B might have similar primary structures.
This notion
was further substantiated by the following evidence obtained in the present
study:
1) the amino acid analysis indicated that the amino acid composi­
tions of subunits A and B are very similar (Table 1); 2) after hydrolysis
by trypsin, identical peptide maps were obtained from subunits A and B
(Fig. 8); and 3) identical
14
C-labeled S-carboxymethylamido cysteine
peptides resulted from the pepsin digestion of A and B (Fig. 9).
These
results strongly indicated that subunits A and B had the same primary
structures.
Amino acid analysis of isozymes (amylase I and amylase II) by Cozzone
et al. (15) showed that these isozymes also had very similar amino acid
compositions.
Since the isozymes were shown to be composed of the different
subunits, Cozzone's results also strongly suggested that the amino acid
contents of subunits A and B are the same.
The determination of the number of sulfhydryl groups and disulfide
bonds in subunits A and B and in the isozymes indicated that there were a
total of 12 half cystine residues per isozyme and 6 per subunit (Table 2).
There were two sulfhydryl groups in each isozyme and in each subunit.
Therefore, after reduction and isolation of subunits, there were four
sulfhydryl groups per 50,000.
These results demonstrated that the sub-
units A and B are formed by selective cleavage of one disulfide bond.
The number of sulfhydryl and disulfide groups in both subunits was the same
(2 sulfhydryl and 2 disulfides).
Therefore it can be concluded that A and
B do not differ from each other in the extent of reduction.
65
There are two possible ways that disulfide linkages in a protein can
produce differences in the tertiary structure.
As illustrated in Fig. 16,
different conformations of a protein can be present due to:
a) the
cysteine residues involved in forming disulfide bonds are in different
positions in the primary sequence, and b) the cysteine groups involved in
forming the disulfide bonds are the same, but they are paired together
differently.
The results from the peptide mapping studies after the
sulfhydryl groups in subunits A and B were labeled with
14
C-iodoacetamide
excluded possibility "a" as the reason for the structural difference
(Fig. 9).
Additional studies are necessary to confirm whether A and B
are indeed disulfide conformers as proposed in possibility "b".
It has
been suggested that these kinds of disulfide conformers were responsible
for the presence of isomers in plasma albumin (35).
Some amylases do contain carbohydrate moieties (26, 62).
So, there
remained the possibility that the subunits A and B have different amounts
of carbohydrate moitiés.
From various studies (3, 8, 9), non-protein
groups have not been observed as prosthetic groups in PPA.
Calcium
analysis gave slightly different values for A and B (9 gram atoms for one
mole of subunit A and 10 gram atoms for one mole of subunit B).
This
difference, however, is probably too small to count for the cause of
structural differences between A and B.
It is then concluded that subunits
A and B could be disulfide conformers (mechanism "b" of Fig. 16), or A and
B have different conformations but the disulfide linkages are not
responsible for their differences.
Since A and B could be changed to the
same form after modification of the disulfide bonds, it is most probable
that A and B are disulfide conformers.
Figure 16.
Different possible protein conformations controlled by
sulfhydryl and disulfide linkages
A; Different sulfhydryl groups are involved in disulfide
bond formation in a and a*
B: The same sulfhydryl groups are involved in disulfide
bond formation but they are linked differently in b and b'
67
A
\rSH
riS
I
B
'6H
H5
•SH
3
HS
b
b'
68
As indicated in Fig. 11 and Fig. 13, four molecular weight species
(50,000; 38,000; 25,000; 12,000) were formed after PPA was partially
reduced and alkylated; one molecular weight species of 12,000 was formed
after PPA was oxidized with performic acid.
These molecular weight studies
indicate that PPA is actually a tetramer and subunits A and B were dimers,
composed of two monomers of 12,500 daltons held together by disulfide
bonds.
From these findings, the quaternary structure of PPA is proposed
in Fig. 17.
PPA contains four monomers; two disulfide bonds link two
monomers to form subunits A and B and one disulfide bond links these two
subunits to form the amylase molecule of 50,000 daltons.
The disulfide
bonds in subunits A and B are linked differently to give two kinds of
tertiary structure.
This model for quaternary structure of PPA can explain
many of the experimental results obtained in the present studies as well
as the results obtained by other investigators.
As summarized in Fig. 18, the three isozymes (I, II, and III) were
found to be hybrids of subunits A and B.
There are 12 half cystine in
each isozyme giving 2 sulfhydryl and 5 disulfide groups.
There is one
disulfide linkage between the two submits A and B, and two disulfide
linkages between the two monomers comprising A and B.
After reduction of
the 50,000 amylase molecule with DTT, subunits A and B are primarily formed
with only traces of the 12,500 monomers.
However, when PPA was oxidized
with performic acid, all of the disulfide bonds were cleaved and PPA was
dissociated completely into monomers having a molecular weight of
approximately 12,500.
Partial reduction, followed by derivatization of
sulfhydryl groups also dissociated PPA into trimers, dimers and monomers.
It is difficult to completely convert PPA into the monomer form by reduction
Figure 17.
Proposed quaternary structure for PPA and subunits A and B
70
I
i
3
-5H
3
H5-I
-SH
HS-
-s—s5
X
5—5—
B
HS"
5
—5—5 —
1
J
-5 — 5 — a
2
—5 —5— 3
3
AB
hSH
3
X
Figure 18.
Summary and illustration of the experimental observations on the PPA structure which
has been obtained in this study
a:
Reduction Products of PPA
b:
Reduction and S-carboxymethylamidation Products of PPA
R = -CHgCONHg
c:
Performic acid oxidation product of PPA
I, II, III represent the three observed isozymes, and A and B represent the two
observed subunits
â
sH
SH
I
il
X
I
°
T"
-|
SH
rSH
SH
Subunit B
Subunit A
SH
1
SH
I
Monomer
SR
_i
iH
_J
SR
_j
SR
SR
5R
S
s
-1
SH
D
1—
SR
Trimer
n—
SR
SR
Subunits (dimer)
JH
SH
SR
SOj
m
Isozymes
Monome r
SO3
Monomer
73
and alkylation because the tertiary and quaternary structure of PPA is
stabilized and protected by chelation of calcium ions.
This model also
explains the fact that various isozymes of PPA could be interconvertible
as observed by Szabo and Straub (61).
Subunits A and B are both enzymatically active.
From the end product
specificity analysis, A and B were found to have the same action patterns
as those of the three isozymes (Fig. 14).
It appeared that there are at
least two active sites in the native enzyme and that these two active sites
are independent of each other for their catalytic abilities.
The isozymes
and subunits have slightly different degrees of multiple attack (Fig. 15).
These differences might be due to the differences in the protein
conformations.
The presence of two active sites has been inferred by two other
studies.
In 1966, Loyter and Schramm (24) demonstrated there were two
active sites in native PPA by equilibrium dialysis studies with maltotriose
at pH 10.
Levitzki e^ al. (23) observed that the enzyme formed multi-
molecular complexes with glycogen.
Their experiments indicated that the
formation of the insoluble complex was analogous to the antibody-antigen
reaction.
If the enzyme was associating with the glycogen exclusively at
the active sites, a basic principle of polymer chemistry would demand that
the enzyme had at least two active sites per molecule.
It is generally recognized that the association of enzyme subunits
sometimes produce cooperative effects which would make the enzymes sensitive
to regulatory control and responsive to changes in metabolite concentrations
of an organism.
It has been shown that ribonucleoside diphosphate reductase
is composed of two subunits,
and B^.
Separately they are inactive;
together in the presence of Mg^ they form a catalytically active complex.
The B^ protein binds nucleoside triphosphates known to act as allosteric
effectors (63).
B.
Tryptophan synthetase is composed of two subunits, A and
Isolated A or B protein is relatively inactive and only partially
catalyzes the reactions which are catalyzed by the whole enzyme (64).
It
was also found that the subunits of Bacillus subtilis ct-amylase are more
susceptible to proteolytic hydrolysis than the native enzyme (65).
In the
present study, it has been found that the specific activities of the subunits, A and B, of porcine pancreatic a-amylase are in the same range as
the specific activities of the three isozymes (see Results, section C and
section E).
Further, subunits A and B, as well as the isozymes, are also
resistant to trypsin digestion.
Therefore, the biological significance for
PPA to exist as a tetramer composed of two active subunits is not selfevident.
Three isozymes were found in PPA which was isolated from a single pig
pancreas which had been treated with DFP in each purification step to
prevent proteolytic hydrolysis (66).
this enzyme preparation (67).
Subunits were also isolated from
As indicated earlier, subunits A and B have
similar sizes, amino acid compositions, and primary structures.
Therefore,
it is very unlikely that PPA existed as a single peptide that has under­
gone limited hydrolysis to give 4 peptides connected by intermolecular
disulfide bonds analogous to the chymotrypsinogen-chymotrypsin conversion.
It, however, is probable that the subunits or monomers are coded by a
single gene.
The subunits or monomers could then be assembled into the
75
three isozymes.
two steps:
It has been proposed that PPA biosynthesis proceeds in
1) de novo synthesis of an amylase precursor, and 2) accumula­
tion and conversion of the precursor into the active amylase (3).
The
assembly of the subunits of immunoglobulin G has been shown to be necessary
for the secretion of this protein (32, 33).
It has also been suggested
that some subunits may act as inducers for the synthesis of other subunits
of the same protein (68, 69).
The presence of subunits and monomers in
PPA may also have important biological roles yet unrecognized.
76
V.
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VI.
ACKNOWLEDGMENTS
The author wishes to express her sincere gratitude to her major
professor John F. Robyt for his guidance, encouragement and kindness
throughout the course of this work.
She acknowledges Dr. Dexter French for his helpful suggestions and
discussions. Dr. Malcolm A. Rougvie for his special technical guidance in
using ultracentrifuge.
She also wishes to acknowledge the valuable discussion, technical
assistance and warm friendship of Mrs. Rosalie J. Ackerman.
She appreciates the understanding and support provided by her
husband, Charles C. Lee.