CLIN. CF#{128}M.
21/3, 343-346(1975)
Comparison of Human Pancreatic and Parotid
Amylase Activities on Different Substrates
Doris J. Stiefel and Patricia J. Keller
The specific activities of highly purified
preparations
of
human parotid and pancreatic amylase on several different soluble and insoluble starches were compared.
The ratio of parotid to pancreatic activity varied with the
physical nature of the substrate. With all soluble starch-
lasesthat are present in other body fluids, notably
serum and urine (2-6). The possible contributionof
additional amylases, from fallopian tube and liver,
has not been completely established, but their chromatographic similarity to the pancreatic and salivary
es, irrespectiveof source, parotidamylase exhibiteda
enzymes, respectively, has been reported (6). General
higher specific activity than did pancreatic amylase; the
uncertainty remains with respect to the function and
reverse was true for an insoluble chromogenic starch
significance of serum and urinary amylases, their
(Amylose Azure). The variation in enzyme-substrate innormal activities, and alterations in these activities,
teraction supports previous indications of configuraboth nonspecific and individual, in diverse conditions
tional differences between the two amylases. The oband disease states (1, 5-9). For these reasons, more
served organ-specific
characteristics
according to
organ of origin may have value in determining relative
precise methods of differentiating
the amylases have
parotid and pancreatic amylase activities in body fluids been sought.
under normal and pathological conditions, thereby helpThe nonidentity of human salivary and pancreatic
ingto clarify
theirfunctional
and clinical
significance.
amylase has become increasingly evident on the basis
of a number of molecular and enzymatic findings,.
AddItIonal Keyphrases:
soluble and insoluble starches
#{149}
notably differences in electrophoretic
mobility
(3,
enzyme activity
10-12); chromatographicand gel filtration profile (6,
11); molecular weight, carbohydrate content, and the
The enzyme a-amylase (a-1,4-glucan-4-glucanaccessibility of reactive sulfhydryl groups (13). An
ohydrolase, EC 3.2.1.1) has been extensively studied
additional
approach to establishing organ-specific
not only for reasons of its role in starch digestion in
differences has been made by comparing the ability
various mammalian species, including man, but also
of the enzymes to digest different starch substrates.
for its potential as an indicator of pathological condiMeites and Rogols (14) reported that the amylases
tions (1). Amylase is synthesized primarily by the
salivary glands and the pancreas, and these organs
in human biological fluids can be divided into two
claasa. Group A, tha amylasa pr..nt
in sacratin.
nra conid.rod to ha th. principal iourcai of tha amy
stimulatad duodanal fluid, pancraat!c axtract, and
DepArtment or Ore! Biology, Univerlty of Weehtngton, Seettle,
anrum from patianta with pancroatitis, diastad corn=
Weeh UM1U
starch fastar than potato starch; Group B, tho amy.
Reelved ept fl, lU74 epted
Dec f, 1U74
CLINIOAL0HaMINTBY,Vet 21, Ne 3 1975 aa
lase present in saliva, normal urine, and serum, usuenzyme concentration. The latter was calculated
from the absorbance at 280 nm, by use of the value
ally digested potato starch faster than cornstarch.
Because the samples testedwere not pure,the validi- AIcm = 23.3 (17).
Activitytoward an insolublechromogenic
starch
ty of such differences is uncertain. Although noting
was determined by the method of Hall et
some exceptions, Hall et al.(8) also observed a rela- derivative
al. (18). After the reaction
was stopped, the undigesttively greater activity by pancreatic amylase than salivary amylase toward cornstarch but concluded that
ed substrate
was removed
by centrifugation
at 7000
X g for 12 mm. Enzyme activity was measured
specpancreatic amylase is the predominant type in nortrophotometrically
at 595 nm.
mal serum. However, in the latter study an insoluble
cornstarch was compared with a soluble potato starch
Starch Substrates
and the findings therefore did not resolvethe quesThe followingsubstrateswere used:
tionof whether the reversalin activityratiowas due
Cornstarches
to variation in plant source or to difference in the sol(a) The commerical food product “Argo Cornubility of the substrate.
starch” (CPC International
Inc., Englewood
Cliffs,
In the present study we have used highly purified
preparations of human parotid amylase and human
N.J.06632),as representative
of a natural cornstarch.
(b) A commercially modified, phosphate-derivapancreatic
amylase, to determine
whether
these
could be differentiated
on the basis of their activity
tized,and cross-bonded waxy maize starch, “VersaStabe” No. 4832 (CPC International,Inc., Argo, Ill.
toward various starches and to clarifythe nature of
60501).
such differences where present.
Materials and Methods
Amylase
Samples
Crystalline human parotid amylase was prepared
by the method of Kauffman et al. (15). Pancreatic
amylase was purified from lyophilizedhuman pancreatic juice as reported previously by us (13).
Amylase
Assays
Amylase activity toward soluble starches was assayed by the dinitrosalicylic
acid method of Fischer
and Stein (16), except that the assay was done at 30
#{176}C
and a buffer consisting of 20 mmol of sodium
phosphate and 10 mmol of NaC1 per liter, adjusted to
pH 6.9, was used. The amylase reaction was stopped
at the end of the usual assay period of 3 mm, during
which it is linear with time. Specific amylase activity
was defined as the ratio of absorbance at 540 nm to
Both of the above two soluble cornstarches
were
mildly
acid treated according to the method of
Meites and Rogols (14).
(c) An insoluble
Remazol
brilliant
blue starch derivative, “Amylose
Azure,” B grade (Calbiochem,
San
Diego, Calif. 92112), as used by Hall etal.(18).
Potato starches
(a) “Noredux”
starch
(Gane’s
Chemical
Works,
Inc., New York, N.Y. 10017). This product
was used
without further modification.
(b) “Aroostoocrat”
starch
(A. E. Staley Manufacturing Co., Decatur,
Ill. 62525). This was the same
material
as used in the investigations
of Meites and
Rogols (14) and was donated
by these authors.
This
starch was acid treated
in the same manner
as the
cornstarches.
Glycogen.
This substrate,
ofshellfish
origin(Mann
Research Laboratories,
Orangeburg,N.Y. 10962),was
modified as for cornstarchesexcept that the acid
treatment
was for 18 h at 25 #{176}C.
All substrate
solutions were freshly prepared
in a
concentration of 10 g/liter
of assay buffer.The potato and cornstarches
were dissolved by heating; glycogen was dissolved
at 25 #{176}C.
Specific
activity
of the enzymes
on “Noredux”
starch was used as the standard
for comparison.
C
Results
In Figure1 the specific
activities
ofthe two human
amylases on the natural,solublestarchesare com-
pared. Except for glycogen, which was less readily hydrolyzed,
negligible
differences
in amylolysis rates
were observed
between the various
natural
starch
products.
Similar
activities
were also noted toward
0.6
0.8
0.2
the commercially
modified
phosphate-derivatized
Enzyme
(pg/mi)
cornstarch,
“Versa-Stabe.”
For both enzymes
there
was only a small difference
between
their respective
Fig. 1. Comparison of thespecific
activity
of human pancreabilities
to digest potato starch as opposed to cornatIc() and human parotid amylases (0) with respectto varistarch, with slightly more activity
displayed
by each
ous solublesubstrates
Experimental details are presented In the text
enzyme
toward the potato starch. At no time was a
344
CLINICAL CHEMISTRY, Vol. 21, No.3, 1975
0.7
Discussion
os
05
04
‘
03
0
02
01
10
20
Ti,,,.
30
40
I,,,,,i
FIg. 2. Comparison of the activity of human pancreatic (A)
and human parotid (0) amylases, both at 2.0 g/ml concentration, toward an Insoluble substrate, Amylose Azure
Experimental details are presented In the text
Table 1. Effect of Variation in Source of Substrate
on Specific Activity of Human Amylases
SpecIficactivity
Substrate
Soluble
Parotld
Pancreatlc
ancreatlc
starch
Potato, “Noredux”
Potato, “Aroostoocrat”
0.93
0.62
1.50
0.92
0.57
1.61
Corn, “Argo”
0.83
0.55
1.51
Corn, “Versa.Stabe”
0.84
0.57
1.46
0.48
0.38
1.27
0.58
0.86
0.68
Glycogen
Insoluble
starch
Corn, “Amylose Azure”
reversalof the parotid/pancreaticactivityratiofound
for any of the soluble substrates;parotid amylase always exhibited a higher specificactivitythan did the
pancreatic enzyme.
In contrast, Figure 2 shows that when assays were
done with an insoluble chromogenic starch, Amylose
Azure, such a reversalin activityratiodid occur,with
relativelygreater activitybeing displayed by pancre-
atic amylase. Although the substrate-to-enzyme
ratio
in the reaction mixture was about 10-fold greater for
the insoluble starch than for the soluble starch, in
both cases activitywas measured under conditions
such that it was linearlyrelated to time. Substrate
concentration thereforewas not a limiting factor, and
variation in quantity of substrate was not responsible
for the difference in activity ratio.
The ratios of parotid to pancreatic amylase activities observed with the soluble and insoluble starches
are shown in Table 1. The reversal in activity of the
two enzymes toward the insoluble substrate is in
agreement with the findings of Hall et al. (8).
Our experiments on the highly purified enzymes
demonstrate a relatively
higher specificactivityby
human parotid amylase on all soluble substrates tested; in contrast, human pancreatic amylase has relatively more activity on an insoluble starch. Apparently the determining factor in substrate preference
by the two amylases is the physical nature of the substrata rather than the plant source per se.
In previous studies with the same highly purified
enzyme preparations, we observed certain physicochemical differences between the human amylases
(13). Thus, at both high and low temperature
extremes pancreatic amylase was more labile than the
parotid enzyme. Furthermore, the single free sulfhydryl group appeared to be more accessible for reaction in pancreatic amylase than in parotid amylase.
These findings suggest that the pancreatic amylase
molecule is less compact than the parotid amylase
molecule.
The variation in enzyme-substrate
interaction we
report here further substantiates
the concept of con-
figurational differences between the enzymes. The
presumably more compact structure of parotid amylasemay tend to hinder the binding of this enzyme
with an insolublesubstrate,
whereas such steric hindrance might not be encountered with the presumably more loosely structured pancreatic amylase molecule. The present indications of functional heterogeneity of the human axnylases, reflecting differences
at the structural level, are consistent with findings of
other investigators. Aw and Hobbs (19) observed a
greater inhibition of human pancreatic than parotid
amylase by a given antiserum. Fridhandler et al. (20)
noted that a binding substance isolated from human
serum displayed greater affinity for salivary amylase
than for pancreatic amylase.
In conjunction with suitable controls, it would appear productive to use the origin-specific differences
in the amylases with respect to specific activity
toward different substrates as the basis for relatively
simple diagnostic assays of the principal amylase
types present in a given biological sample. Where inconsistencies are observed it might be fruitful to examine the relationship to other parameters, both molecular and clinical. We do not know whether the differences we observed between parotid and pancreatic
amylase pertain equally to their individual
isoenzymes. If these can be isolated in adequate quantities, similar tests might be of value in making such
determinations and, should differences exist, in probing their clinical significance.
We thank Drs. S. Meites and S. Rogols for their gift of potato
starch.
We also express our gratitude
for the valuable
assistance
and technical advice of Ms. B. J. Allan, Ms. E. Cohen, and Ms. D.
L. Kauffman. This research was supported by NIDR Research
Training Grant DE-00102-1O and by USPHS DE-02600.05 from
the NIDR to the Center for Research in Oral Biology at the University of Washington. The work is taken in part from a thesis subCLINICAL CHEMISTRY, Vol. 21, No.3, 1975
345
by Dr. D. J. Stiefel in November 1971, in partial fulfillment
of the requirements
for a Master of Science degree from the University of Washington.
It was presented in part at the 50th annual
meeting
of the International
Association for Dental Research in
1972.
mitted
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1975
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