ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 288, No. 2, August 1, pp. 621-633, 1991
Isolation and Characterization
of a Homogeneous
lsoenzyme of Wheat Germ Acid Phosphatase
Parvin
P. Waymack
Department
and Robert
L. Van Ettenl
of Chemistry, Purdue University,
Received November
West Lafayette, Indiana
47907-1393
27, 1990, and in revised form April 17, 1991
An acid phosphatase
(orthophosphoric
monoester
phosphohydrolase,
acid optimum; EC 3.1.3.2) isoenzyme
from wheat germ was purified 7000-fold
to homogeneity.
The effect of wheat germ sources and their relationship
to the isoenzyme content and purification
behavior of acid
phosphatases
was investigated.
Extensive
information
about the purification
and stabilization
of the enzyme is
provided.
The instability
of isoenzymes
in the latter
stages of purification
appeared to be the result of surface
inactivation
together
with a sensitivity
to dilution
that
could be partially
offset by addition of Triton X- 100 during chromatographic
procedures.
Added sulfhydryl
protecting reagents
had no effect on activity
or stability,
which was greatest
in the pH range 4-7. The purified
isoenzyme was homogeneous
by polyacrylamide
gel electrophoresis
and exhibited
the highest specific activity
and
turnover
number reported for any acid phosphatase.
The
molecular
weights of the pure isoenzyme
and of related
isoenzymes
from wheat germ were found to be identical
(58,000).
The pure isoenzyme
contained
a single polypeptide chain and had a negligible
carbohydrate
content.
The amino acid composition
was determined.
Of the various reasons that were considered
to explain
isoenzyme
occurrence,
a genetic basis was considered
most likely.
The enzyme was found to exhibit
substrate
inhibition
with some substrates
below pH 6, while above pH 8 it
exhibited
downwardly
curving
Lineweaver-Burk
plots
of the type that are generally
described
as “substrate
activation.”
The observation
of a phosphotransferase
activity
was consistent
with the formation
of a covalent
phosphoenzyme
intermediate,
while inactivation
by diethyl pyrocarbonate
was consistent with the presence of
an active site histidine.
0 1991 Academic
Press, Inc.
The ubiquitous distribution and general characteristics
of acid phosphatase (orthophosphoric
monoesterphos1 To whom correspondence
should be addressed.
0003.9861/91$3.00
Copyright 0 1991 by Academic Press, Inc.
All rights of reproduction in any form reserved.
phohydrolase, acid optimum; EC 3.1.3.2) have been described in extensive reviews (1, 2). Most studies of acid
phosphatase, particularly
those from plant sources, are
largely of a descriptive nature. With a few exceptions (notably, human prostatic acid phosphatase) the acid phosphatases occur in very small quantities, are unstable in
dilute solution, and are subject to surface denaturation
when purified. These factors, together with a tendency to
occur in multiple forms or as isoenzymes, makes the isolation of highly purified acid phosphatases difficult.
Studies of acid phosphatases from various sources suggest a role in phosphate mobilization for the enzyme (35). The lysosomal location of the high molecular weight
mammalian liver acid phosphatases (6) can be compared
to the location of seedling acid phosphatase activity in
sphereosomes, which have been described as analogous
to lysosomes (7).
Wheat germ acid phosphatase (WGAP)’ exists in multiple chromatographic
and electrophoretic
forms which
have been called isoenzymes. Chromatographic
behavior
on DEAE-cellulose
under varied conditions have shown
multiple peaks of activity (8-10). Multiple electrophoretic
forms have been found by starch and polyacrylamide gel
electrophoresis (10, 11).
Acid phosphatase zymograms of extracts from wheat
plant tops appear to contain a distinct isoenzyme under
field conditions of phosphorus deficiency (12). Changes
of isoenzyme patterns in the various stages of germination
of hard red wheat have been studied (5). Polyacrylamide
gel electrophoresis and activity staining were used to detect changes in the number of multiple forms and changing mobilities in the dormant seed, germinated seed, coleoptile, and first leaf tissue. In seed, there were five bands
which contained most of the activity in the more mobile
bands. In contrast, the first leaves contained four bands
with most of the activity distributed among the less mobile
* Abbreviations
used: WGAP, wheat germ acid phosphatase;
diethyl pyrocarbonate; SDS, sodium dodecyl sulfate.
DEP,
621
622
WAYMACK
AND
bands. It was proposed that the combination of distinct
subunits controlled by different genes could give rise to
hybrid enzymes with different electrophoretic
mobilities (5).
The observation of increased acid phosphatase activity
during germination, and of greater isoenzyme mobility as
germination progressed, suggests that the possible synthesis, activation, or conversion to new forms of the enzyme occurs during germination. However studies of seed
germination under conditions designed to preclude de nouo
synthesis of active enzyme showed the typical increases
in acid phosphatase production (13). It was concluded
that the acid phosphatase activity arose by activation of
preformed macromolecules, i.e., zymogens. These observations suggest that the interconversion
of isoenzymes
may occur.
Interchangeable
subunit structures, if present, could
explain some of the multiple electrophoretic
forms of
wheat acid phosphatase. Studies of the acid phosphatase
of Drosophilia melanogaster showed the presence of AA,
BB, and AB electrophoretic variants (14). Sunflower seed
acid phosphatase has been shown to consist of two subunits (15). Because of the existence of acid phosphatases
with molecular sizes as low as 18 kDa (16), it was conceivable that two or even four subunits could be present
in wheat germ acid phosphatase.
The interaction of subunits is the primary mechanism
which has been implicated in the property of negative
cooperativity. This type of kinetic behavior has been observed at the pH optima of other acid phosphatases containing subunits, including rat liver (6), human prostate
(17), and human liver at pH 7.3 (18). Additionally,
rat
mammary carcinoma acid phosphatase (19) can be surmised (20) to contain subunits. Since the present report
revealed a type of negative cooperativity by wheat germ
acid phosphatase at pH 7.8 and above, the studies of subunit structure and enzyme purity were of additional interest.
In characterizing wheat germ acid phosphatase, some
uncertainty exists as to what constitutes a nonspecific
acid phosphatase. This is a somewhat loosely defined enzyme classification based on an acid pH optimum for
phosphomonoesterase
hydrolysis. While acid phosphatases typically have broad substrate specificity, the reported polyphosphatase and phosphodiesterase activities
of many such enzymes raise questions of purity of the
preparations involved. One frequently cited work (8) reported confirmation of an earlier report (21) of the presence of iron in four yellow isoenzymes which also hydrolyzed carboxylesterase substrates.
These questions of enzyme structure, substrate specificity, and unusual kinetic behavior are best answered with
studies using the highest purity enzyme. Therefore, purification to homogeneity was undertaken to allow reliable
characterization
of wheat germ acid phosphatase and to
VAN
ETTEN
clarify earlier reports of its properties. The present work
describes in detail the purification to homogeneity of an
isoenzyme of wheat germ acid phosphatase, as well as a
characterization
of some of its most interesting physical
and kinetic properties.
EXPERIMENTAL
Purification
PROCEDURES
of WGAP
All steps were carried out at 4°C except the initial acetone precipitation, which was performed in a fume hood at room temperature.
Step 1. Initial extraction.
Dry wheat germ was finely ground in a
Waring blender and extracted with 0.3 M pH 4.0 acetate buffer (10 ml
per gram of wheat germ) by stirring at lo-min intervals for 1 h.
Step 2. Acetone precipitation.
At timed intervals, cold acetone
(-ZO’C) was slowly added with stirring to batches of the extract from
Step 1, to give a final concentration
of 55% (v/v). After resuspending
the initial precipitate several times with gentle swirling and shaking,
the precipitate cleanly settled after 30-60 min, leaving a clear supernatant
that was decanted and discarded. The remaining solution containing
the precipitate was centrifuged at 159Og for 5 min. The precipitates
from several successive batches were collected in the same set of centrifuge bottles. The final centrifugation was extended to 15 min at 6370g
to minimize acetone carry-over in the pellet and to allow rinsing out of
acetone with cold water. The largely insoluble pellet was suspended by
gentle mechanical stirring in a volume of cold distilled water equal to
about one-fiftieth the final Step 1 volume. If the volume was adequate,
centrifugation
of the suspension at 13,300g for 20 min yielded a clear
amber supernatant. (A clear supernatant indicates the volume used to
dissolve the pellet was adequate.)
Step 3. Ammonium sulfate precipitation and acetone remoual. To the
clear supernatant fraction from Step 2 was added 432 g ammonium
sulfate/liter.
It was convenient to use the nomogram of Dixon (22) using
the 65% saturation point although the procedure was carried out at 4°C.
The mixture was allowed to stand for 30 min and then centrifuged at
4440g for 10 min. Acetone was discarded with the supernatant fraction.
After rinsing with cold water, the pellet was dissolved in a volume of
0.3 M pH 4.0 acetate buffer equal to one-half of the final Step 2 volume
and dialyzed against the same buffer for 8 h with several changes. The
dialysate was centrifuged at 11,300g for 15 min.
Step 4. Ammonium sulfate fractionation.
The dialysate from Step 3
was diluted to 8-10 mg/ml (protein assay) with 0.3 M acetate buffer, pH
4.0, and ammonium sulfate (210 g/liter) was added. The ammonium
sulfate solution was allowed to stand for 30 min and centrifuged at
11,300g for 15 min. The pellets were discarded and the clear supernatant
fractions pooled. Ammonium sulfate (91 g/liter) was added to the supernatant and after 30 min the solution was centrifuged as above. The
centrifuge bottle walls and pellet surface were rinsed with cold water to
remove supernatant ammonium sulfate solution which would interfere
with the next step. The pellet was dissolved in cold water, assayed for
protein concentration,
and diluted to 4-6 mg/ml (The protein concentration is critical for Step 5).
Step 5. Methanol precipitation.
The concentration of methanol was
brought to 23% (v/v) by slow addition with stirring of 0.3 ml cold (-2O’C)
methanol per milliliter of Step 4 material. A precipitate formed during
centrifugation
at 15,960g for 60 min. The largely insoluble pellet was
dissolved in the starting Step 5 volume of cold water and centrifuged at
48,000g for 45 min. The supernatant was used for the next step.
Step 6. G-75 Sephadex gel filtration.
The Step 5 fraction was concentrated and equilibrated with 0.01 M acetate buffer, pH 5.2, with 0.1
M NaCl and 0.1% Triton
X-100 added (G-75 buffer) by ultrafiltration
with an Amicon PM-10 membrane. Alternatively
it was precipitated by
55% acetone as previously described and then dissolved in G-75 buffer.
The sample was cleared by centrifugation
at 48,OOOgfor 20 min prior
HOMOGENEOUS
ISOENZYME
OF WHEAT
to application to a 5 X 100 cm G-75 Sephadex column equilibrated with
G-75 buffer. Separation at a flow rate of 35 ml/h gave a single activity
peak which corresponded to a shoulder on the trailing edge of a large
280-nm absorbance peak. Fractions containing two-thirds of the activity
were combined and concentrated by ultrafiltration
to enhance stability.
Step 7. SP-Sephadex chromatography.
The concentrate from Step
6 was applied to a 1.6 X 15 column of SP-Sephadex equilibrated in G75 buffer. The column was eluted at 12 ml/h with a linear NaCl gradient.
The G-75 buffer was in the mixing chamber and G-75 buffer containing
0.5 M NaCl in the reservoir. The fractions were monitored for protein
by a modified Folin method (23). The elution profile had two main activity peaks following a small void volume activity peak (~1% of total
activity). The center of the first peak (-9% of total activity) was eluted
at 0.14 M NaCl while the second peak (-90% of total activity) was
centered at 0.23 M NaCl. The protein assay indicated a correspondence
of activity and protein in the main WGAP peak.
Step 8. DEAE-cellulose
chromatography.
The combined, concentrated (Amicon PM-lo) main peak WGAP fractions from Step 7 were
dialyzed against 6 liters of 5 mM, pH 7.4, Tris-HCl
containing 0.1%
Triton X-100 (pH 7.4 buffer) with two buffer changes during 9 h of
dialysis. In order to minimize nonspecific protein adsorption, DEAEcellulose was treated by successively washing with 1 M HCl, 1 M NaOH,
pH 7.4, buffer, 1 mg/ml bovine serum, and pH 7.4 buffer containing 1
M NaCl before final equilibration
with pH 7.4 buffer. The enzyme sample
was applied to a 0.9 X 10 cm column and washed with 25 ml pH 7.4
buffer. The enzyme was eluted at 15 ml/h with a linear NaCl gradient
generated with 75 ml of pH 7.4 buffer containing 0.3 M NaCl and 0.1%
Triton X-100 in the reservoir and 75 ml of pH 7.4 buffer in the mixing
chamber. The elution profile showed a protein peak corresponding to
the single activity peak. The fractions with the highest specific activity
were combined, the pH adjusted to 5.2 with 0.5 M pH 4.0 acetate buffer
and concentrated by ultrafiltration.
The steep gradient and rapid flow
rate of this column are designed to minimize the length of time the
enzyme remains at high pH and to minimize dilution of protein, since
both factors reduce enzyme stability.
Step 9. Rechromatogrophy on SP-Sephadex.
The concentrated fractions from Step 8 were dialyzed against the pH 5.2 G-75 buffer. The
sample was applied to a 0.9 X 10 cm column as in Step 7 and rinsed
with 25 ml of G-75 buffer. The column was eluted at 8 ml per hour by
a linear NaCl gradient with 50 ml G-75 buffer in the mixing flask and
50 ml of G-75 buffer containing 0.5 M NaCl in the reservoir. The fractions
with highest specific activity were concentrated as soon as possible.
Specific Activity
Assay
The standard condition for measuring activity was assay in 0.1 M
acetate, pH 4.6, using 5 mM p-nitrophenyl
phosphate at 25’C. In a
typical assay, 2.00 ml was incubated 2-5 min and quenched with 1.0 ml
of 0.4 M NaOH and the 400-nm absorbance was read versus a blank in
which NaOH was added before enzyme. One unit (U) of enzyme is that
required to release 1 pmol p-nitrophenol
per min.
Molecular
Weight Measurements
A 2.5 X 100~cm column of Sephadex G-200 was calibrated with 0.05
pH 7.0 phosphate buffer using blue dextran and standard proteins
(catalase, aldolase, bovine serum albumin, soybean trypsin inhibitor
and cytochrome c) applied in a volume of 1.0 ml. A linear least squares
program was used to determine the linear relationship between log molecular weight and elution volume. Sodium dodecyl sulfate-gel electrophoresis for polypeptide molecular weight was conducted under denaturing and reducing conditions (24). Electrophoresis
was performed at
8 mA per gel for 8 hr followed by Coomassie blue staining. Mobilities
were calculated as recommended by Weber and Osborne (24) and were
the average of at least two determinations.
Samples containing IO-43
pg WGAP at a specific activity of 433 unit/mg were applied to each gel.
M
GERM
ACID
623
PHOSPHATASE
Carbohydrate Determination
A phenol-sulfuric
acid method (26) was used for carbohydrate determination in the early stages of purification.
A more sensitive method
(27), verified by ovalbumin and lectin from Bandeiruea simpZic$oZia, was
used in the latter stages of purification.
Sialic Acid Assay and Neuraminidase
Treatment
The mixture of WGAP isoenzymes found in the Step 4 ammonium
sulfate fraction was treated with neuraminidase from Clostridium perfringens (l-4 units/ml) at pH 5.0 in acetate buffer at 25’C. Aliquots
were withdrawn after incubation for 24, 48, and 72 hr, electrophoresis
was performed at pH 4.4, and the gels were stained for acid phosphatase
activity. Electrophoretically
homogeneous isoenzymes were similarly
treated.
Carbohydrate Detection after Polyacrylamide
Electrophoresis
Gel
Following polyacrylamide
gel electrophoresis, localization of carbohydrate was attempted using a sensitive fluorescence method (25) with
glycoprotein standards which allowed the detection of as little as 40 ng
of carbohydrate under uv illumination.
Amino Acid Composition
Homogeneous WGAP (605 U/mg) was absorbed on SP Sephadex C50 at pH 5.2, washed to remove Triton X-100, and eluted with 0.1 M
pH 7.0 ammonium carbonate. Samples (0.2 mg) were desalted on G-25
Sephadex and analyzed according to Ref. (15).
Enzyme Stability
The stability of WGAP at the concentration and conditions employed
in the continuous rate assays at high pH was investigated using the
same enzyme solution and concentration throughout the pH range 2.45
to 10.0. To 2.40 ml of each buffer at 25.0°C, 50 ~1 of solution containing
0.8 units of WGAP was added. Aliquots of 50 ~1 were withdrawn and
analyzed by the standard activity assay. The half-life of the enzyme was
estimated from the linear portion of log activity versus time. The storage
stability of partially purified WGAP (Step 5, methanol precipitation)
was also monitored as a function of pH at 4°C for 6 months. Samples
were incubated at 4.0 mg/ml in 0.025 M buffers as follows: glycine, pH
2.93; acetate, 3.9 and 5.0; maleate, 5.95; Tris, 7.4; barbital, 8.0 and 9.0.
Metal Ion Effects
The effect of 0.1 to 10 mM added metal ions upon activity in 0.1 M
pH 5.0 acetate was studied. Concentratedp-nitrophenyl
phosphate was
added to 2.00 ml of the metal ion solutions to give a final concentration
of 5.0 mMp-nitrophenyl
phosphate. After 2.0 min incubation at 25.O”C,
the reaction was quenched and read at 400 nm, after removal of any
precipitates by centrifugation.
The effect of preincubation
of the enzyme with metal ions in 0.1 M
pH 5.0 acetate was studied by addition of 0.050 ml of WGAP containing
175 U/ml (218 U/mg) to 1.0 ml of 1.05 mM metal ion solution at 25°C.
Aliquots of the mixture were withdrawn and assayed along with a control
experiment with no metal ions. Control experiments showed that the
effect of metal ions carried over into the assay mixture was negligible.
The effects of two chelating agents upon activity were investigated.
At pH 5.0, 0.1 M acetate contained 5 mM p-nitrophenyl
phosphate and
either 10 mM EDTA or 5 mM o-phenanthroline. At pH 7.0,0.03 M barbital
containing 0.1 M NaCl was used with 10 mM EDTA or 5 mM o-phenanthroline added. Assay was as above, using control experiments without
chelators. The effects of added EDTA upon the stimulatory effect of 1.0
624
WAYMACK
AND
mM Mg+‘, 1.0 mM Co+*, and 0.05 mM NazMoO, were investigated
with
the above conditions.
The effect of preincubation
in 25 mM EDTA and 5 mM o-phenanthroline was studied in pH 5.0, 0.1 M acetate at 4 and 25“C. To 0.50 ml
of these mixtures (and controls without chelating agents), 0.050 ml of
WGAP containing 216 U/ml (308 U/mg) was added and aliquots were
withdrawn at 24-hr intervals for assay.
Steady-State Kinetics
Substrate solutions were prepared by dilution of concentrated solutions
with the respective buffers. The concentration
of p-nitrophenyl
phosphate was varied from 0.1 to 10 mM, using 0.05 M acetate, pH 5.0, with
0.1 M NaCl added. At pH 6.0, 0.05 M 3,3-dimethylglutarate
buffer containing 0.1 M NaCl was used. To 2.00 ml of substrate solutions at 25’C,
25-50 PL of WGAP was added and quenched with 0.4 or 1.0 ml of 1.0
M NaOH. At pH 7.0 and above, direct continuous assays were employed
(25-50 ~1 WGAP added to 2.40 ml of substrate thermostatted at 25°C).
At pH 7.0, 0.05 M 3,3-dimethylglutarate
buffer containing 0.1 M NaCl
and 0.05 to 20.0 mM was used. pH values 7.0, 7.5 (0.05 M 3,3-dimethylglutarate buffer with 0.1 M NaCl), 8.0,8.5, and 9.0 (0.05 M barbital with
0.1 M NaCl) were used with the range of 0.1 to 10 mM substrate.
Heat Treatment and Specificity
Partially purified WGAP (Step 7) having a specific activity of 308
mg/dl was inactivated by heating 0.2 ml samples (in pH 5.2 0.1 M acetate)
for 2 min at water bath temperatures from 35 to 75°C and then rapidly
cooling them (ice water). Activity remaining was assayed in duplicate
using the standardpH 4.6 assay and directly at pH 8.5 in 0.05 M barbital
phosphate or
containing 0.1 M NaCl and either 10 mM p-nitrophenyl
10 mM (his)-p-nitrophenyl
phosphate. The same buffer was used in a
single determination withp-nitrophenyl
phenylphosphonate as substrate.
pH Dependence of the Activation
Energy
For the determination
of V,,,,,., five concentrations
of p-nitrophenyl
phosphate were prepared by the addition of 0.10 ml substrate solutions
to 2.00 ml of 0.055 M buffer containing 0.11 M NaCl. The reaction was
initiated by the addition of 50 pl of a WGAP solution and quenched
with 1.0 ml of 1.0 N NaOH. At low pH, the use of saturating substrate
conditions (50 X K,J was used to give a good approximation
to V,,,.
For this, 5 mM p-nitrophenyl
phosphate was used, since little or no
substrate inhibition is observed at this concentration
in the pH range
3-5. This facilitated testing the Arrhenius equation over a wide temperature range.
The temperature range lo-40°C was investigated at pH 5.0. The reaction in a thermostated sample was initiated by the addition of 50 ~1
of WGAP enzyme solution to 2.0 ml of 5 mM p-nitrophenyl
phosphate
in 0.05 M acetate buffer with 0.1 M NaCl added. The reaction was
quenched by the addition of 1.0 ml of 1.0 N NaOH and the absorbance
at 400 nm was determined. An identical procedure was employed with
pH 3.00,0.05 M glycine at 15,25, and 37.5”C, and pH 4.0,0.05 M acetate
buffer at 15.0 and 37.5”C. In each case an identically treated spectrophotometric blank was prepared in which the enzyme was added after
the reaction was quenched.
Transphosphorylation
Incubation mixtures containing 50% methanol and 10 mM p-nitrophenyl phosphate were prepared by adding 0.10 ml concentrated substrate solution to 1.90 ml of buffer solutions (pH 3.5 to 9.2) and 2.0 ml
of methanol. The pH of the mixture was that measured with a glass
electrode. The incubation time and amount of enzyme added were adjusted to give less than 2% total hydrolysis before the reaction was
quenched with 0.50 ml of 3% molybdate in 0.5 M pH 4.0 acetate buffer.
VAN
ETTEN
The quenched mixture was assayed for p-nitrophenol
by the addition
of 1.00 ml of 1 M NaOH to a 2.00-ml portion and the absorbance was
measured at 400 nm. The inorganic phosphate content of the same
quenched mixture was determined by the Lowry phosphate determination method. The slight effect of methanol upon the extinction coefficient at 700 nm (I& = 4600 M-’ cm-‘)
was determined
in separate
experiments where equal amounts ofp-nitrophenol
and inorganic phosphate were produced enzymatically
in the absence of methanol. The
amount of methyl phosphate ester that was produced was taken to be
the difference between the moles of p-nitrophenol
and inorganic phosphate produced.
Inactivation
by Diethyl Pyrocarbonate
Protein modification experiments with diethyl pyrocarbonate (DEP;
ethoxyformic anhydride) were conducted in 0.50 ml of 0.050 M dimethyl
glutarate or barbital buffers in the pH range 6.0-8.5 to which was added
10 ~1 of WGAP solution and 25 al of an ethanol solution of DEP. Solutions of DEP for the inactivation experiments were prepared by dilution
of commercial 7.0 M DEP with absolute ethanol. Control experiments
contained added ethanol without DEP. In experiments where phosphate
was added as an enzyme protecting agent, NaCl was added to the phosphate-free inactivation mixture to keep the ionic strength constant. Aliquots (25 ~1) were removed from the 25°C incubation mixture and assayed for activity for 2-4 min by the standard activity assay procedure
except that 5 mM EDTA was added to allow accurate assay of molybdatecontaining aliquots. The reaction was stopped by addition of 1.0 ml of
1.0 N NaOH and the absorbance at 400 nM was measured. Pseudo-firstorder rate constants for the initial loss of activity were evaluated from
the linear portion of plots of log activity remaining versus time. Reactivation with hydroxylamine
was carried out by adding equal volumes
of the DEP-inactivated
sample and 0.2 M hydroxylamine
adjusted to
pH 7.0 in 0.1 M pH 7.0 dimethylglutarate
(thus giving 0.1 M hydroxylamine in the reactivation mixture). The reactivation mixture was incubated at 25°C and aliquots were assayed for activity as described
above.
RESULTS
The purification procedure summarized in Table I provided a reliable method for obtaining homogeneous
WGAP. It was reproducible with every lot of wheat germ
from soft red wheat cultivar Arthur (supplied by AcmeEvans, Indianapolis, IN). The high specific activity going
into the column chromatographic
steps increased the
problem of activity loss from dilution and nonspecific adsorption. The addition of 0.1% Triton X-100 helped stabilize activity during G-75 Sephadex chromatography and
subsequent steps. The best results were obtained when
several preparations were pooled before the chromatographic steps, thus maximizing protein concentration
during chromatography. Pretreatment of the DEAE-cellulose column and the steep gradient were necessary to
avoid extensive activity loss. The procedure resulted in
an at least 7000-fold purification
of WGAP with a final
specific activity of 605 U/mg when assayed at pH 4.6 in
0.11 M sodium acetate buffer with 5 mM p-nitrophenyl
phosphate as a substrate. WGAP appeared homogeneous
by polyacrylamide gel electrophoresis at pH 8.0 and 4.4
and gave a single band upon SDS gel electrophoresis. For
two chromatographically
distinct isoenzymes (the two
activity peaks in Fig. 1) average molecular weights of
HOMOGENEOUS
ISOENZYME
OF WHEAT
60,000 and 58,000 were calculated for the native proteins
on Sephadex G-200 chromatography. On the basis of such
experiments, it is thought that these and other isoenzymes
from wheat germ have indistinguishable
molecular
weights. Lower molecular weight species or higher molecular weight aggregates were not observed.
From SDS-gel electrophoresis, a plot of mobility versus
log molecular weight exhibited a linear relationship from
which the molecular weight calculated for WGAP was
56,000 + 3000. This value is in agreement with the value
determined by gel filtration, and amino acid analysis (Table II). The failure of a mixture of WGAP isoenzymes to
bind to a concanavalin A-Sepharose column suggested
that none of the WGAP isoenzymes contain carbohydrate
chains with significant amounts of terminal mannose or
galactose residues.
The inability of high concentrations of neuraminidase
and long incubation times to generate new electrophoretic
forms is consistent with the absence of neuraminic acid
in the acid phosphatase isoenzymes. The progressive removal of carbohydrate contamination
with purification
(results not shown) indicated that pure WGAP contains
<l% carbohydrate. This conclusion is also consistent with
results obtained by the dansyl hydrazine staining procedure. A faint fluorescence was detectable only at the 43pg WGAP protein level in gels that had been periodateoxidized as well as in those which had not. If we assume
1% carbohydrate in WGAP, the calculated amount of
carbohydrate in such a sample would be 430 ng, which is
more than 10 times the minimum detection level. The
sensitivity
was confirmed with the standard proteins,
ovalbumin, and Bandeiraea simplicifolia lectin.
The results of amino acid analysis of the isoenzyme
WGAP are summarized in Table II. The residues per
molecule were calculated by an integer fit method (29).
A plot of the sum of the relative residuals versus total
residues showed a distinct minimum at 521 residues, cor-
TABLE
GERM
%
0.4
2
0.3
LT
::
i
ACID
625
PHOSPHATASE
0.2
0.1
IO
20
30
FRACTION
40
50
NUMBER
60
70
80
FIG. 1. SP-Sephadex chromatography during Step 7 of the purification
of wheat germ acid phosphatase. A 1.6 X 10 cm column was eluted with
a linear NaCl gradient as described under Experimental
Procedures.
The activity (0) was measured by the standard assay method and the
protein was measured by the Folin assay as 750 nm absorbance (0).
The separate activity peaks of fractions 23-33, and 40-55 were collected.
responding to a molecular weight of 54,950 g/mol. This
value (with the addition of a likely tryptophan contribution) agrees well with the value obtained by gel filtration
and SDS-gel electrophoresis.
The relative content of
acidic and basic residues is consistent with the observed
chromatographic and electrophoretic behavior of WGAP
as a slightly acidic protein. Attempts to obtain sufficient
quantities of a second homogeneous isoenzyme from
wheat germ strain Arthur were unsuccessful due to the
low content of any other individual isoenzyme.
Studies of the stability of WGAP at 25°C showed a
dramatic loss of stability above pH 7.5 and below pH 3.
Besides pH, the most serious stability problem appears
to be the interrelated effects of protein concentration and
surface denaturation that were evident in chromatography
steps involving highly purified enzyme. In the pH range
of maximal stability, pH 4 to 7, the half-life of partially
purified enzyme solutions stored at 4°C at a protein con-
I
Purification of Wheat Germ Acid Phosphatase
Volume
(ml)
step
1
2
3
4
5
6
7
8
9
Initial extract
Acetone precipitation
O-65% (NH,),SO,;
pH 4.0 dialysate
(NH,),SO,
Fractionation
23% Methanol precipitation
G-75 Sephadex chromatography
SP-Sephadex
DEAE-cellulose
2nd SP-Sephadex
’ Micromoles
p-nitrophenol
64,000
1,320
1,080
184
41.0
75.0
22.5
18.0
20.0
Protein
btdml)
Activity
(units/ml)
10.4
26.1
15.2
22.1
4.51
0.365
0.273
0.111
0.070
0.90
35.3
36.5
139
231
62.0
108
64.4
42.5
released per minute per milligram
Total
units
Specific
activity”
57,600
46,600
39,400
25,600
9,470
4,650
2,430
1,160
850
0.086
1.35
2.40
6.29
51.2
170.
396.
580.
605.
Recovery
(%)
(100)
81.0
68.5
44.6
16.5
8.1
4.2
2.0
1.5
of protein when measured at 29”C, pH 4.6, using 5 mM p-nitrophenyl
Purification
factor
(n-fold)
(1)
16
28
73
595
1980
4600
6740
7030
phosphate.
626
WAYMACK
AND
TABLE
VAN
ETTEN
II
Amino Acid Composition of the Wheat Germ Acid Phosphatase Isoenzyme
Amino acid
Quantity”
Aspartic acid
Threonine*
Serine*
Glutamic acid
Proline
Glycine
Alanine
Half-cystine* + cysteic acid
Valine’
Methionine + sulfoxide
Isoleucine’
Leucine
Tyrosine*
Phenylalanine
Histidine
Lysine
Arginine
Tryptophan
949.6
732.2
178.3
854.3
567.8
1319.4
872.3
583.6
491.0
151.6
245.1
359.1
495.9
291.1
360.8
338.2
470.1
Not determined
Residuesper molecule
Nearestinteger
Mole %
50.2
38.7
41.1
45.1
30.0
69.7
46.1
30.8
25.9
8.0
13.0
19.0
26.2
15.4
19.1
17.9
24.8
-
50
39
41
45
30
70
46
31
26
8
13
19
26
15
19
18
25
9.63
7.43
7.89
8.66
5.76
13.38
8.85
5.92
4.98
1.54
2.49
3.64
5.03
2.95
3.66
3.43
4.77
Total number of amino acid residues
521
54,950 g/m01
Calculatedmolecularweight
’ Output from Durrum 500 amino acid analyzer; average value.
* Correctedfor decompositionby extrapolation of 24- and 4%hr
’ 48-hr values.
valuesto zerotime.
centration of 1 mg/ml or greater was found to be approximately 3 months, while at room temperature the halflife was only 3-4 days. Extended storage requires that the
samples be sterilized by filtration.
Attempts were made to lyophilize or freeze aqueous
WGAP samples for storage. Activity losses (approximately 30%) were sustained during the initial freezing
step. When portions of highly purified (specific activity
> 200 U/mg) samples of WGAP were lyophilized, the
samples lost 70-80% of their activity. Lyophilized samples
at the methanol precipitate stage (Step 5) were found to
retain about 50% of initial activity immediately after lyophilization and to lose half of the remaining activity in
3-4 months when stored at -20°C. The half-life of
aqueous frozen samples (Step 5) was estimated to be about
2 weeks.
Protein concentrations below 1 mg/ml led to a rapid
loss of stability, with storage in glass, polyethylene, or
polypropylene vessels giving essentially the same results.
The nonionic detergent, Triton X-100 and crystalline bovine serum albumin were investigated
as stabilizing
agents. A concentrated enzyme sample (Step 6) was diluted with 0.01 M pH 5.2 acetate buffer containing 0.1 M
NaCl and either Triton X-100 or bovine serum albumin
was added. The diluted samples were assayed immediately
and at 24-hr intervals. The results indicate that added 1
mg/ml bovine serum albumin and 0.1% Triton X-100 are
comparable in their ability to stabilize the enzyme. These
studies suggested the usefulness of adding 0.1% Triton
X-100 to the chromatography buffers used in purification
and the buffers used for enzyme storage. Highly purified
samples (specific activity > 200 U/mg) that had a protein
concentration
greater than 0.5 mg/ml and which contained 0.1% Triton X-100 at pH 5.2 were generally found
to have half-lives of about 6 months when stored at 4°C.
At pH 5.0 the half-life of WGAP at 4°C was 2-6
months. At pH 8.0 the enzyme was very unstable; the
half-life at 25°C was approximately 40 min, which made
chromatography,
kinetic studies, and chemical modification experiments in this pH range difficult and indicated
the need for stabilizing conditions. The possible effect of
sulfhydryl
group protecting reagents on stability was
studied at two pH values. The effects of added 2-mercaptoethanol upon storage at 4°C at pH 5.0 and at pH 8.0
were small. At the end of a 4-week period, samples incubated with 10 mM 2-mercaptoethanol
contained about
20% less activity than the control sample, while samples
containing added 4.5 mM dithiothreitol
lost activity at
the same rate as the control sample. We conclude from
these studies that neither the storage instability nor the
high pH instability of WGAP is primarily due to an oxidation of sulfhydryl groups.
The effect of prolonged dialysis against Mgt2 and
EDTA at 4°C was investigated. When WGAP was dialyzed for 5 days against separate 2.0-liter portions of 0.1
M pH 5.0 acetate buffer containing either 10 mM EDTA,
HOMOGENEOUS
ISOENZYME
OF WHEAT
9
8
7
L
2r
6
5
/
-10-8-6-4-2
4
0
2
4
6
8 10
I/CSl
FIG. 2. Lineweaver-Burk
plot illustrating
substrate inhibition
of
WGAP-catalyzed
hydrolysis of p-nitrophenyl
phosphate at pH 5.0 (0);
K,,, = 0.09 ? .Ol mM, V,,, = 823 f 30 mmol mg-’ min-’ and pH 6.0 (0);
K,,, = 0.20 f 0.01 mM, V,,, = 956 f 12 mmol min-’ mg-‘.
10 mM Mgf2, or no addition, there was no change in the
specific activity. The inability of EDTA to inactivate or
of Mg+” to activate is in contrast to the acid phosphatase
of wheat leaf (30). Only a slight (10 to 20%) stimulation
of WGAP activity occurred in the presence of millimolar
concentrations of Mg+” and Co+‘.
Molybdate at a concentration of 0.05 mM caused complete apparent inactivation.
The heavy metal ions Hgt2
and Ag+ caused complete inactivation. Almost total (82%)
inactivation was caused by Cu+‘, while Mnf2, Fe+2, Caf2,
Fef3, and Zn+’ had little or no effect.
Additional
experiments with added chelating agents
also indicate that loosely bound endogeneous metal ions
are not required for activity. Incubation at 25 or 4°C in
the presence of EDTA and/or o-phenanthroline
did not
cause significantly
more rapid inactivation
than that
caused in the absence of the chelating agents. In contrast,
similar conditions could be used to inactivate sweet potato
acid phosphatase with o-phenanthroline
and subsequently
to reactivate it with metal ions (31). We were unable to
show any reactivation of the inactivated enzyme in this
experiment either by added Mg+2, Mn+’ or by dialysis
against Co+2. The slight stimulatory effect of Mg+2 and
CO+~ and the strong inhibitory
effect of molybdate are
reversed upon addition of EDTA.
Steady-state kinetic studies of WGAP revealed unexpected complexities. The nature of the reciprocal plots
observed with homogeneous WGAP change as a function
of pH. Substrate inhibition
is observed at low pH and
gives way to Michaelis-Menten
kinetics near neutrality;
finally, at high pH, apparent substrate activation is observed. Upward-curving
reciprocal plots characteristic of
substrate inhibition were observed at pH 5.0 and 6.0 with
p-nitrophenyl
phosphate as substrate (Fig. 2). Similar re-
GERM
ACID
PHOSPHATASE
627
sults (not shown) were obtained at pH 4.6 with glucose
6-phosphate or pyrophosphate as substrates but not with
P-glycerophosphate
as a substrate. At both pH 5.0 and
6.0, substrate inhibition
became clearly evident only at
substrate concentrations greater than 10 X K,,, and therefore might not be detected in a typical Lineweaver-Burk
plot. However, a consequence of this behavior is the potential for serious errors in V,,, estimations when using
a single high substrate concentration
in an attempt to
saturate the enzyme. Although such clearly evident substrate inhibition has not been reported as a kinetic property of other acid phosphatases, the determination
of
substrate “specificity”
using a single, fixed substrate concentration is a common practice.
At pH 7.0 and 7.5 withp-nitrophenyl
phosphate as the
substrate, WGAP gives linear Lineweaver-Burk
plots
over a wide substrate concentration range (up to 400-fold
at pH 7.0). Neither substrate inhibition nor substrate activation can be detected.
In the pH range of 8.0 and above, the phenomenon of
substrate activation is observable (Fig. 3), with a readily
apparent, downwardly curving region at high substrate
concentration. At pH 8.5 and 9.0, the nonlinearity is similarly prominent.
Concomitant
loss of activity toward
monoester (p-nitrophenyl
phosphate) and diester (bis-pnitrophenyl
phosphate) substrates as well as p-nitrophenyl phenylphosphonate
during the course of extensive
(80%) thermal denaturation was found at pH 4.6 and 8.5.
These results are consistent with activity of a single enzyme species toward all of the substrates.
The temperature dependence of the WGAP-catalyzed
hydrolysis of p-nitrophenyl
phosphate was investigated
to obtain information
about the mechanism of the enzyme. The apparent activation energy was estimated at
unit pH intervals from pH 3 to 8. V,,, was determined
by varying the substrate concentration
at two or three
temperatures. These values of V,,, were used to estimate
the apparent activation energy from the slope of the plot
of log vnmx versus l/T. The data at pH 5.0 show excellent
linearity from 10 to 40°C (Fig. 4). At pH 3.0 (Fig. 4) and
FIG. 3. Lineweaver-Burk
plot of WGAP-catalyzed
hydrolysis of pnitrophenyl
phosphate at pH 8.0; Km = 0.99 _+ 0.07 mM, V,,, = 144
& 1.2 pm01 min-’ mg ‘.
628
WAYMACK
l/T
AND
X IO3
FIG. 4. Arrhenius plot of the WGAP-catalyzed
hydrolysis ofp-nitrophenyl phosphate. The values of k,., were determined at pH 3 (m), pH
5 (0) and pH 8 (A) as described in the text.
at pH 4.0 (data not shown) the applicability
of the Arrhenius equation is also indicated by the linearity of the
Arrhenius plot in the temperature range 15 to 37.5”C.
The activation energies are given in Table III. These results indicate that at pH 5 and below the apparent activation energy is approximately constant. The linearity of
the Arrhenius plot is consistent either with single rate
process, or with a combination of rate constants that is
independent of pH.
However, above pH 5, the apparent activation energy
decreases. In Fig. 4 the three Michaelis V,,, values at pH
8.0 indicate a nonlinear dependence of log V,,, on l/T.
The range of the apparent activation energies at pH 8.0
in Table III are therefore the values estimated with each
extreme pair of temperatures. The values of the apparent
activation energies at pH 6.0 and 7.0 are intermediate
values representing estimates from data at two temperatures. A marked decrease in the apparent activation energy as the pH is increased is clearly indicated by these
data. The nonlinear Arrhenius plots at high pH indicate
that more than one temperature-dependent
rate constant
contributes to the V,,, expression (32).
Phospho group transfer is catalyzed by various acid
phosphatases (2). Using highly purified WGAP, transphosphorylation
was investigated because it provides a
way to obtain important information about the existence
and properties of a postulated covalent phosphoenzyme
intermediate (33, 34).
The results in Table IV show that at high concentrations, methanol can act as a phospho group acceptor in
reactions catalyzed by WGAP. Additional experiments
with pH 8.5 barbital buffer in 50% methanol using 2.5,
5.0, and 10 mMp-nitrophenyl
phosphate also showed that
the percentage of phospho transfer varied in the range of
lo-20%. Both enzymatic activity and phospho transfer
were inhibited by 1.0 mM molybdate.
VAN
ETTEN
WGAP was inactivated by DEP over the pH range 6.0
to 8.5 (Fig. 5). To determine if inactivation led to a modification of a residue(s) at or near the active site, competitive inhibitors were added to the modification reaction
mixture. The competitive inhibitors molybdate and inorganic phosphate were found to protect WGAP to some
extent from inactivation
by DEP. Molybdate at about
500 X Ki decreased the rate of inactivation about threefold
while inorganic phosphate (50 X Ki) decreased the rate
by fivefold at pH 7.0 (Fig. 5). These results suggest that
the inactivation reaction involves residues at or near the
active site. The initial pseudo-first-order
rate constant
was found to be independent of enzyme concentration
(0.11 to 5.5 PM) but was linearly dependent on the DEP
concentration at pH 7.4 (data not shown). This result is
consistent with a bimolecular reaction between WGAP
and DEP as indicated by the initial rate of inactivation,
before DEP depletion.
WGAP that was substantially inactivated by treatment
with DEP could be partially reactivated by 0.1 M hydroxylamine. At pH 7.4 75% inactivation was followed by 61%
reactivation (Fig. 6A) while in a similar experiment at
pH 7.6,91% inactivation was followed by only 39% reactivation. In an experiment at pH 8.5, 90% inactivation
by DEP was rapidly achieved (2.5 min) and 65% reactivation was obtained (Fig. 6B).
The pH dependence of the rate of modification is consistent with the modification of histidyl residues. Assuming that DEP reacts only with the unprotonated form of
histidine, then the measured rate of inactivation should
depend on the state of protonation of the presumed histidine, which should vary with pH. The measured rate
constant for inactivation,
Kapp, should be related to the
second-order rate constant, k2, for modification of unprotonated histidine, the dissociation constant of the histidine, K,, and the hydrogen ion concentration by the
The intercept and
equation l/kapp = (l/k2 + [H+]/k,KJ.
TABLE
III
Apparent Activation Energy of the Hydrolysis of
p-Nitrophenyl Phosphate by Wheat Germ Acid Phosphatase
PH
Activation energy”
(kcal/mol)
3.00
4.01
5.00
6.02
7.00
8.00
12.6
12.5
12.3
10.7*
8.3’
1.3-4.3’
a Determined from Michaelis V,,,, values.
*Estimated from data at 25.0 and 37.5”C.
’ Estimates from nonlinear Arrhenius plots; the numerical
included for comparative purposes only.
values are
629
HOMOGENEOUS ISOENZYME OF WHEAT GERM ACID PHOSPHATASE
TABLE IV
Transphosphorylation
Catalyzed by Wheat Germ Acid Phosphatase”
Buffer (pH)
pH of mixture *
p-Nitrophenol liberated
( wol)
Phosphateliberated
( gmol)
Phosphotransfer
(%)
Acetate (3.5)
Acetate (5.0)
3,3-Dimethylglutarate (7.0)
Barbital (8.5)
5.0
6.2
8.6
0.384
0.288
0.388
9.4
0.292
0.310
0.224
0.333
0.254
19.3
22.2
13.9
13.0
DTransfer to methanol in 50% methanol.
* Apparent pH as measuredusing a glasselectrodewith the 50% methanol solution.
slope obtained from a plot of l/kapp versus [H+] (Fig. 7)
was used to calculate a pK, of approximately 7.6, a value
consistent with histidine.
DISCUSSION
Critical examination
of previous attempts to purify
WGAP suggested likely explanations of the unreproducibility of earlier procedures (B-10). Comparison of the
chromatographic
behavior on DEAE-cellulose
at pH 7.4
of a crude enzyme from Sigma Biochemical Co., an enzyme from Nutritional
Biochemical Co. and the pure enzyme from Acme-Evans wheat germ as obtained here
(WGAP) revealed that the relative amounts and elution
positions of the phosphatase isoenzymes varies with the
source. This suggested that each source may have contained the same or similar isoenzymes but in different
amounts. Joyce and Grisolia (9) eluted their highly purified enzyme from pH 7.4 DEAE-cellulose at a lower ionic
strength, thus distinguishing
their isoenzyme from the
present WGAP. Verjee (10) eluted three WGAP isoenzymes but observed no significant void volume (unre-
tained) peak. Under identical initial conditions, about 95%
of the WGAP activity from Acme-Evans soft red wheat
germ was not bound to DEAE-cellulose,
indicating more
anionic isoenzymes.
In order to develop a new method, samples were surveyed as possible starting sources. From those samples,
wheat germ from Indiana grown soft red wheat supplied
by Acme-Evans (Indianapolis,
Indiana) was chosen because of the presence of a dominant activity band in the
sample and the increased control of the source that a
local supplier afforded. The strategy in the isolation of
WGAP was to select a wheat germ that contained large
amounts of one or two isoenzymes. The inclusion of significant details of the purification in this paper should be
helpful to future workers attempting to use wheat germ
as an enzyme source. The purification
procedure which
evolved has been thoroughly tested, but it is still more
complicated than the one used to purify sunflower seed
acid phosphatase to homogeneity (15). Intensive breeding
of wheat and the frequent introduction
of new varieties
may affect the details of the purification procedure.
The purification factor in Table I is 7030. A purification
factor of 11,000 would have been computed if the low
too
t go
I- 00
z 70
z
60
Li
w 50
0
6ot
\
NH20H/
5 40
TIME,
min
FIG. 6. Inactivation of WGAP by diethyl pyrocarbonate
and protection of the enzyme by competitive inhibitors. The rate of inactivation
of WGAP by diethyl pyrocarbonatewasdeterminedasdescribedin the
text: no inhibitor added (0); 50 mM inorganic phosphate added (0); and
1.0 mM molybdate added (A).
-
OLdI
2 45
152535455565
TIME,
min
I
2 IO 2030405060
TIME
, min
FIG. 6. Hydroxylamine
reactivation of WGAP that had been inactivated by treatment with diethyl pyrocarbonate.
At the arrow, the solutions were made 0.1 M in hydroxylamine.
630
WAYMACK
AND
[HI x IO’
FIG. 7. pH dependence of the inactivation
carbonate.
of WGAP by diethy
pyro-
specific activity water extract had been used as a basis,
as was the case for the earlier procedures (8-10). Reported
purification
factors ranged from 15.4-fold for the most
highly purified of three “isoenzymes” (10) or an estimated
lo-fold purification (8), to a 3500-fold purification
(9).
Comparison of specific activities is more complicated
due to the different assay conditions. However, approximate conversions show that two of the preparations had
specific activities of less than 1 U/mg (8, 10). Using the
present experimental
data on the pH dependence of
WGAP turnover in conjunction with the relative substrate
turnover of Joyce and Grisolia (9) enables a comparison
of the reported turnover of their enzyme with respect to
that of WGAP. This gives comparative specific activities
of 83 U/mg for the Joyce and Grisolia (9) enzyme at 25°C
and pH 5.7 as compared to 600 U/mg calculated for
WGAP at its pH optimum. No evidence for protein or
even activity homogeneity was presented by Joyce and
Grisolia (9). The specific activity of their enzyme can
however be compared to that of 40 U/mg for an isoenzyme isolated earlier by us from another wheat germ
source (35).
The molecular weight data indicate that WGAP occurs
as a monomer with a molecular weight 58,000. This is
similar to the soybean enzyme (36). Interestingly,
sunflower seed acid phosphatase consists of two polypeptide
chains (15), each of which is similar in size to the one
that is present in the wheat germ enzyme. The finding
that WGAP contains only a single polypeptide chain
eliminates recombination of electrophoretically
dissimilar
monomer units as a structural source of some of the
WGAP isoenzymes (5, 8).
The molecular weights of glycoproteins as determined
by gel filtration and SDS-gel electrophoresis are not reliable if the carbohydrate content is high (37,38). Of the
purified acid phosphatases that have been examined for
carbohydrate content, a significant number of them have
been found to be glycoproteins, including acid phosphatases from yeast, fungi, plants, and animal tissues. This
widespread occurrence of carbohydrate in acid phosphatases made the carbohydrate content of WGAP of fun-
VAN
ETTEN
damental interest. The use of two sensitive analytical
methods showed that, within the limits of experimental
error, no carbohydrate was present in the pure enzyme.
The size and lack of carbohydrate also distinguish this
enzyme from a 35-kDa, carbohydrate-containing
phosphotyrosyl-protein
phosphatase from wheat seedlings that
was recently described by Cheng and Tao (39).
The presence of a single polypeptide chain in WGAP,
and the absence of neuraminic acid or significant amounts
of other carbohydrates, serves to eliminate several of the
most likely origins of some of the multiple forms of wheat
germ acid phosphatase. Differences in amino acid composition and sequence represent another likely source of
the multiple forms. Bread wheats are hexaploidal and are
believed to contain three genomes that can be distinguished by the electrophoretic mobility of the proteins of
hexaploidal wheat seeds and the seeds of their postulated
genomic origin (40). The finding that different types of
wheat germs exhibited different isoenzyme patterns upon
electrophoresis may reflect diverse genetic composition
giving rise to different WGAP phenotypes.
WGAP, although exhibiting only mild sensitivity toward other thiol group reagents, is sensitive to Hg+’ (Ref.
(9) and present results). This suggested that some degree
of stability might be afforded by sulfhydryl-protecting
reagents. The use of 2-mercaptoethanol
as a stabilizing or
protecting agent has been successful with placental acid
phosphatase III (41), bovine brain acidphosphatase (42),
and isoenzymes of human liver and placenta acid phosphatase (43,44). These enzymes are very sensitive to Hg+’
and thiol group reagents.
The effect of added metal ions and the metalloenzyme
character of acid phosphatases has been of continual interest in the literature. There have been reports that wheat
germ acidphosphatase isoenzymes contained iron (8,21).
Other studies have indicated that a component of a wheat
leaf acid phosphatase extract was inactivated by dialysis
and reactivated by Mg+’ and other divalent metal ions
(30). This indicated the need for studies on the effects of
metal ions and chelating agents on the activity of purified
WGAP. The rapid inactivation of WGAP by EDTA that
was reported (21) and that was taken as evidence that
WGAP is a metalloenzyme was not observed with the
homogenous enzyme. The present results also show that
the conclusion in Ref. (21) which states that molybdate
irreversibly inactivates WGAP was simply due to a carryover of molybdate into the assay mixture.
Metallo-acid
phosphatases have been isolated from
sweet potato, soybeans, spinach, rice, and red kidney
beans (31,45). A unique property of these enzymes is the
characteristic violet color attributed to the presence of
binuclear iron, iron-zinc, or possibly manganese centers,
and which is directly correlated with activity (46). Previous workers suggested that a yellow or yellow-brown
color exhibited by wheat germ acid phosphatase solutions
HOMOGENEOUS
ISOENZYME
OF WHEAT
was due to iron (8). However, during the extensive purification which was carried out here with WGAP, the
brown coloration observed in crude preparations
was
eliminated. No violet coloration, which seems to be characteristic of the iron or manganese in the other acid phosphatases, is exhibited by homogeneous WGAP. In fact,
the spectral properties of the enzyme are similar to that
of human prostatic acid phosphatase, which has been
demonstrated to be a nonmetalloenzyme
(47).
The nonlinear nature of the Lineweaver-Burk
plots at
high pH (Fig. 3) was of fundamental interest. Although
many origins of this behavior are conceivable, one due to
enzyme heterogeneity would represent a serious challenge
to any other kinetic or enzyme specificity studies. Of the
kinetic origins, those involving more than one active site
would be in conflict with physical studies (present work)
and stoichiometric
covalent labeling of WGAP isoenzymes (48) which indicated a single polypeptide chain
with one active site.
The phenomenon of downwardly curving LineweaverBurk plots such as that seen in Fig. 3 is widely referred
to as “substrate activation.” The term implies something
unusual or anomalous about the high substrate range and
thus seems to suggest that the almost linear low substrate
data should be used in the determination
of Michaelis
constants as an interference-avoidance
strategy similar
to that employed with substrate inhibition.
It has generally been an accepted strategy to determine Michaelis
constants from the unaffected low substrate range when
substrate inhibition
is characteristic of an enzyme system (49).
Four broad categories for the origin of apparent substrate activation were considered. (i) Artifacts of the
methods or conditions of assay give apparent substrate
activation (50). (ii) Two enzymes are present, both of
which exhibit activity toward the substrate under consideration. (iii) A single enzyme is present which contains
two active sites exhibiting negative homotropic interaction (or without interaction). (iv) A single enzyme species
is present which is responsible for activity toward all of
the substrates but has more than one pathway for breakdown of enzyme intermediate to product.
We have carefully considered the sources of errors in
assays and procedures that are enumerated in Ref. (50)
and which can erroneously lead to nonlinear LineweaverBurk plots and concluded that the phenomenon is an innate, reproducible property of the WGAP enzyme.
Consideration was given to the possibility of phosphodiesterase activity at pH 8.5 which utilizedp-nitrophenyl
phosphate as a substrate. Alkaline phosphodiesterases
which require divalent metal ions for activity have been
found in plant sources and are inactived by EDTA (51),
but the addition of 1 mM EDTA did not affect the substrate activation phenomenon. The addition of 1 mM Mgt2
to p-nitrophenyl
phosphate substrate in the range l-10
GERM
ACID
PHOSPHATASE
631
mM also had no effect. The possibility
of more than one
enzyme activity (especially diesterase activity) being responsible for apparent non-Michaelis-Menten
kinetics
at high pH was further investigated. Concomitant loss of
activity toward monoester (p-nitrophenyl
phosphate) and
diester (bis-p-nitrophenyl
phosphate) substrates as well
as p-nitrophenyl
phenylphosphonate
during the course of
extensive (80%) thermal denaturation was found at pH
4.6 and 8.5. These results are consistent with the presence
of a single enzyme species that possesses activity toward
all of the substrates.
The experimental phenomenon of substrate activation
has been frequently observed in phosphatases with multiple equivalent subunits, containing one active site per
subunit, including enzymes that purportedly exhibit “half
of the sites reactivity”-an
extreme case of negative
cooperativity.
A similar behavior in adenosine triphosphatase from rat liver and acid phosphodiesterase from
tobacco has been taken as evidence for two substrate
binding sites in these enzymes (52, 53).
Theoretical treatments seem to preclude the observation of both substrate inhibition and substrate activation
in an enzyme with two independent sites (54,55). Therefore, if the low pH substrate inhibition and high pH substrate activation exhibited by WGAP are phenomena with
a common kinetic origin, either case B or case C of Harper
(54,55) should apply. Case B involves two initially equivalent interacting active sites while case C includes a single
active site and a single modifier site with the substrate
acting as the modifier. Both of these models can exhibit
either substrate activation or substrate inhibition. In the
case of WGAP, the presence of only one active site is
consistent with our finding of a single peptide chain per
molecule and with the results of stoichiometric covalent
incorporation from 32P-labeled substrate (48) so that case
C appears most likely. Thus, the kinetic behavior observed
at high pH with WGAP does not theoretically require two
active sites. Similar kinetic behavior is exhibited by trypsin (56,57) and carboxypeptidase A (58), which have single active sites. Although generally considered unusual,
it has been proposed that the property may be widespread
(59,60). The present results suggest the need to examine
a wide substrate range with other acid phosphatases to
determine if such behavior is a general property of acid
phosphatases.
Activation energies of enzyme catalyzed reactions may
be complicated and difficult to interpret (32,49). However,
when a single rate constant is rate limiting, a linear Arrhenius plot is predicted, while nonlinear Arrhenius plots
typically result when two or more steps in a two-intermediate scheme have rate constants of similar magnitude
but differing activation energies. The curvature would
thus be analogous to that seen in mechanistic studies on
prostatic phosphatase where pH variation
leads to
changes in the rate determining step (61). The activation
632
WAYMACK
AND
energy reported for an iron-containing
porcine uterine
acid phosphatase was 11.5 kcal/mol at pH 4.9 (62), while
12.5 kcal/mol was reported for prostatic acid phosphatase
at pH 5.6 (63) and 10.5 kcal/mol was determined for an
acid phosphatase from orange juice (64). The similarity
of these values to that observed for WGAP (Table III) is
interesting but probably fortuitous.
Earlier studies demonstrated the transphosphorylation
capability of acid phosphatases as well as the differing
specificities of various alcohols as phosphate acceptors
(2). The present studies establish that homogeneous
WGAP can carry out such a transphosphorylation.
The
results are consistent with the formation of a phosphoenzyme intermediate that can break down to products by
alternate pathways utilizing either water or alcohols (here,
methanol) as a phospho group acceptor. This interpretation is also consistent with the inactivation of the enzyme by DEP, and with the pH dependence of that inactivation. These results support the conclusion that
phosphoenzyme intermediate is involved and is in fact a
phosphohistidine
(48).
Finally, of the several possible origins of the observed
WGAP chromatographic
and electrophoretic
“isoenzymes” that can be considered: (i) electrophoretic or ion
exchange artifacts; (ii) subunit structure with electrophoretically
dissimilar subunits; (iii) varied content of
neuraminic acid, (iv) genetic differences reflected in amino
acid composition; (v) artifactual modification
(such as
partial proteolysis) during isolation; and (vi) proteolytic
action prior to storage in the seed (germ), the present
study provides evidence that origins (i)-(iii) are not responsible. Cloning and sequencing experiments may provide the best approach to examine the remaining alternatives.
ACKNOWLEDGMENT
This research was supported by DHHS NIH Grant GM 27003 from
the National Institute of General Medical Sciences.
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