THE JOURNAL OF BIOLOQICAL CHEMISTRY

Vol. 235, No. 8, August 1960

Printed in U.S.A.

Purification and Properties of a Nonspecific Acid Phosphatase

from Wheat Germ*
BARBARA K. JOYCE AND SANTIAGO GRISOLIA

From the Mcllvain Laboratories, Department of Medicine, University of Kansas Medical Center, Kansas City 12, Kansas

(Received for publication January 25, 1960)

While studying the phosphoglyceric acid mutase of wheat Protein was estimated by the method of Lowry et al. (8).
germ, we noted marked phosphatase activity when extensively However, since this method can give misleadingly high values
purified mutase preparations were tested with n-3-phospho- in crude plant extracts, the proteins of the crude preparations
glyceric acid and D-Z, 3diphosphoglyceric acid as substrates (1). were precipitated with 30 volumes of acetone, dissolved in water,
Since we had found low phosphatase activity for these reagents and then measured by the method of Lowry et al. and by the
in many biological materials (2), there was the possibility, pre- biuret method of Mokrasch et al. (9). There was excellent agree-
viously discussed (l), that the enzymatic hydrolysis of these ment between the two methods. Phosphodiesterase activity
substrates was a dual function of the purified wheat germ mutase. was assayed by the method of Sinsheimer and Koerner (10).
Further examination, however, showed there was no correlation 3-P-glycerate was estimated by determining phosphoenolpyruvic
between phosphatase and mutase activities at several stages of acid formed after enzymatic equilibration of this substrate with
purification, indicating phosphatase contamination of the wheat P-glycerate mutase and enolase (6). P-glycerate mutase was
germ mutase preparations. Additional evidence for the non- assayed as previously described (1). Pi was measured by the
identity of the two enzymatic activities has now been obtained method of Gomori (11).
by extensive purification of the phosphatase from wheat germ, Assay-For the routine assay of phosphatase activity the
by the method presented in this paper. The purified prepara- following components were mixed in a final volume of 1.5 ml:
tions of nonspecific acid phosphatase described in this paper have 75 pmoles of sodium acetate buffer, pH 5.7, 20 pmoles of MgC12,
activities of the same order of magnitude as those of the exten- 6 pmoles of 3-P-glycerate, and approximately 0.5 unit of en-
sively purified intestinal monoesterase (3) and of the crystalline zyme. After 15 minutes’ incubation at 37”, the reaction was
pyrophosphatase of yeast (4). The best phosphatase prepara- stopped by the addition of 1 ml of 1 nf HC104, and any insoluble
tions are essentially free of P-glycerate mutase activity. material was removed by centrifugation. Aliquots of the super-
natant fluids were tested for Pi.
EXPERIMENTAL PROCEDURE Dejinition of Enzyme Unit and SpeciJicActivity-A unit of en-
zyme is the amount of enzyme which liberates 1 pmole of Pi
Materials and Methods under the conditions of the assay indicated above. Specific
activity is defined as the enzyme units per milligram of protein.
2,3-dip-glycerate’ and 3-P-glycerate free from 2,3 P-glycerate
Fractionation-All operations during fractionation were carried
were prepared as previously described (5, 6). 2-P-glycerate,
out at O’, and all centrifugations were for 10 minutes, unless
phosphoenolpyruvic acid, p-nitrophenyl phosphate and bis
indicated otherwise. A speed of 5500 X g in the Lourdes cen-
p-nitrophenyl phosphate were obtained from the Sigma Chemical
trifuge, with rotor number 3RA, was used through Fraction II.
Company. DEAE-cellulose, lot number 1038, reagent grade,
In the succeeding steps, centrifugations were at 4000 x g in the
was purchased from the Brown Company, Berlin, New Hamp-
International centrifuge. The (NH)$OI solutions were satu-
shire. Anheuser-Busch dried brewers’ yeast was a gift of Dr.
rated at pH 5.5.
Henry Buehler and it was the source of enolase (containing P-
Purification-Wheat germ was extracted with 4 volumes of
glycerate mutase) which was partially purified by the method
water for 30 minutes, and the preparation was centrifuged. To
of Warburg and Christian (7) through the first ethanol fractiona-
each liter of the supernatant fluid (crude extract) were added
tion. Wheat germ, General Mills (S-50), was a gift of Dr. J. S.
20 ml of 1 M MnCIZ, and the mixture was centrifuged (12). To
Andrews. Samples of corn and rice germ were kindly provided
each liter of the supernatant fluid (Mn++ fraction), 538 ml of
by Dr. Pekka Lmko of Kansas State University and Professor
saturated (NH&S04 (35% saturation) were added. The insolu-
M. C. Kik of the University of Arkansas, respectively. Ben-
ble material was removed by centrifugation. To the superna-
tonite (Volclay SPV), a gift from Dr. Paul Bechtner of the
tant fluid, 788 ml of saturated (NH&S04 were added (57%
American Colloid Company, was washed2 as previously described
saturation). While swirling in a 65-70” water bath, the mixture
(1). Other materials were commercial products.
was heated to 60’ and held at this temperature for 2 minutes.
* Supported by grants from the American Heart Association and The preparation was rapidly chilled to 8” and centrifuged. The
the Kansas Heart Association.
1 The abbreviations used are: 2,3-dip-glycerate, D-2,3-diphos- 2 For the suspension of large quantities of bentonite, it is con-
phoglyceric acid; 3-P-glycerate, n-3-phosphoglyceric acid; 2-P- venient to use a paint shaker. Unwashed bentonite suspensions
glycerate, o-2-phosphoglyceric acid; and EDTA, ethylenediamine- (centrifuged at 300X g for 1 minute) can be used. However, the
tetraacetic acid, disodium salt. purification appeared not to be as successful.

2278
August 1960 B. K. Joyce and X. Grisolia 2279

precipitate was suspended in water, the volume of which was TABLE I

one-third that of the Mn++ fraction, and was then centrifuged Purification of nonspecific phosphatase from wheat gerrr
for 30 minutes. The supernatant fluid, Fraction II, may be The conditions of assay were as described in the text.
stored frozen. Total Specific
Fraction Total units Total protein activity Yield
For each new bentonite and enzyme preparation a preliminary VOlUDI~

titration for adsorption is required. Routinely, l-ml samples of ml mg %

Fraction II containing 7.5 & 0.5 mg of protein per ml were
Crude extract. 2,630 106,000* 126,000 0.84 100
adsorbed for 7 minutes with 0.2, 0.25, and 0.3 ml of washed Fraction II. 1,020 75,000 7,850 9.6 71
bentonite (60 to 70 mg per ml) and water to a final volume of 1.5 Fraction III. 1,450 53,500 2,180 24.5 42
ml. The samples were centrifuged at 2000 X g and the super- Fraction IV.. 95.5 34,400 898 34.4 26.5
natant fluids were assayed. The best conditions obtained in Fraction V.. 28.4 15,650 22.4 700 14.8
the preliminary bentonite treatment were followed in purifying Fraction VI.. 1.0 4,150* 1.43,ooot 3.9t
the remainder of the preparation. The supernatant fluid is Fraction Via.. 0.6 1,170 0.6 1,950f 1.1t
Fraction III. To each liter of this fraction, 272 g of solid (NH&-
* The crude extract and Fraction VI showed P-glycerate-mu-
SO4 were added (47y0 saturation). The insoluble material was
tase-phosphatase ratios of 1500 and 0.003, respectively.
discarded after centrifugation and 97 g of (NH&SOe were added
t The specific activity and recovery rates for Fractions VI and
to the supernatant fluid (61 y0 saturation). The mixture was Via were probably higher than indicated in the table. When
centrifuged and the precipitate was dissolved in water (& vol- these fractions were diluted in 0.5yo serum albumin and tested
ume of Fraction III) to give Fraction IV. This fraction may be (final concentration of serum albumin in the assay, 25 pg) they
stored frozen. showed 50yo higher activity. This effect most likely was due to
Fraction IV was adjusted to 4.5 f 0.5 mg of protein per ml. enzyme inactivation during dilution to low protein concentration
To each ml of Fraction IV 0.11 ml of 0.2 M EDTA, 0.05 ml of The serum albumin had no phosphatase activity.
saturated (NH&SO+ and 1.75 volumes of methanol (measured
and added at -20’) were added. After centrifugation the pre- TABLE II
cipitate was suspended in the minimal volume of water and the Effect of (NHd)dO., upon thermal stability
bulky, insoluble material was discarded after centrifugation. of wheat germ phosphatase
The supernatant fluid was dialyzed for approximately 11 hours Samples (6-mg) of Fraction II (specific activity, 2.4) fraction-
against 100 volumes of 0.01 M EDTA (with five changes). The ated with (NH*) zSO~ without heating ((NH,) zSO~ concentration
dialyzed material, Fraction V, may be frozen overnight. For approximately 0.1 M) were held at the indicated temperatures for
best results in this step, it is best to do a preliminary titration 5 minutes in a total volume of 2.5 ml. After cooling, appropriate
(2-ml samples and 3 to 4 ml of methanol). Fraction V was aliquots were tested under standard conditions of assay.
percolated through a DEAE-cellulose column (approximately (NHa)601 molarity
1 X 7 cm) about one-fourth the volume of the enzyme solution. Temperature
The elution was carried out with successive one-column volumes 0.1 I 1 / 1.8 j 3.4
of Tris, pH 7.4, at increasing molarities (0.01, 0.02, 0.04, and
% activity renz&ing
0.08 M). The highest specific activity usually was eluted with
50
approximately 0.02 of solutions. The best fractions were dia- 60
lyzed with three successive changes for 3 hours against 100 70
volumes of 0.01 M EDTA. Samples were lyophilized to dryness
and can be stored as such in the cold or dissolved in the minimal
volume of water and kept frozen. The best two fractions3 were pH values 5, 4.5, and 4, retained 96, 95, and SO%, respectively,
Fractions VI and Via. A summary of the purification procedure of its original activity. This preparation, when heated for 5
is shown in Table I. minutes at 50, 60, and 70”, retained 24, 4, and 0.6% activity. A
crude extract, when heated for 5 minutes at 50, 55, and 60” lost
Distribution of the Enzyme 67, 76, and 85% activity, respectively. (NH&SOI protected
Water extracts of rice germ, corn germ, ground lentils, and the phosphatase when heated, as is shown in Table II.
split peas were assayed for phosphatase activity under standard E$ect of pH-A crude enzyme preparation shows an optimal
conditions, but with no Mg++. It has been calculated that 32, pH of 5.1 with 3-P-glycerate and 2,3-dip-glycerate as substrates.
21, 8.8, and 7 pmoles of Pi were split per gram of dry material. The optimal pH of Fraction V is 5.8, as shown in Fig. 1.
InJluence of Substrate Concentration on Enzyme Activity-The
Properties of Enzyme effect of 3-P-glycerate concentration on velocity with Fraction
Stability with pH and Heat-A partially purified enzyme prep- Via is shown in Fig. 2. The Lineweaver and Burk plot (13)
aration (specific activity, 15) when held for 10 minutes at O”, at shown in the figure indicates a Michaelis-Menten constant (K,)
of 2.7 x 1O-4 M. This value is also obtained with crude prepara-
3 As indicated in the legend of Table I, Fraction VI and Via, tions; the K, value when 2,3-dip-glycerate as the substrate is
when diluted in 0.5% albumin, showed higher activity than when
diluted in water. With 3-P-glycerate, phosphoenolpyruvate, and about the same.
sodium pyrophosphate, Fraction VI in albumin had specific ac- Effect of Time of Incubation and Enzyme Concentration-Under
tivities of 3400, 4000, and 6270, respectively, whereas the specific the standard conditions of assay and with Fraction Via there is
activity was 2330 for the same enzyme diluted in water and assayed proportionality for the dephosphorylation of 3-P-glycerate with
with 3-P-glycerate as substrate. This preparation had lost 20%
of its original activity upon storage at a concentration of 0.3 mg time up to 20 minutes. There is some decrease in activity rate
of protein per ml. beyond this period. The dephosphorylation of 3-P-glycerate is
2280 Wheat Germ Phosphatase Vol. 235, No. 8

glycerate in 15 minutes). Further, at pH 5.7 (optimum for the

phosphatase) there would be negligible diesterase activity since
the activity for this enzyme is practically nil at pH 6 and below
(14).
E$ect of Ions--A partially purified enzyme fraction with
specific activity of 15 was completely inhibited with 0.3 pmoles
of Hg++, when 3-P-glycerate, 2-P-glycerate or 2,3-dip-glycerate
were used as substrate, under standard conditions of assay.
This is in contrast to the stimulation of muscle glycerate 2,3-
diphosphatase by Hg++ (2, 15). Under the standard conditions

TABLE III
Specijicity of acid phosphatase during purification
The conditions of assay were as described in the text. When
testing 2,3-dip-glycerate, 2.8 pmoles were used; 6 pmoles each
of the other substrates indicated in the table were used. Frac-
tion V and Fraction Via had specific activities of 330 and 1800,
respectively.

PH Fractions
FIG. 1. The effect of pH. The standard conditions of assay Substrate
were used, and Fraction V. For pH 4 to 5.8,75 rmoles of the cor- Crude
extract Fraction V Fr%?
responding acetate buffer were used. For pH 6.2 to 7.6,75 pmoles I I
of the corresponding Tris maleate buffer were used. des of P
3-P-glycerate. ............... 0.381 0.578 0.406
2,3-P-glycerate .............. 0.498 0.554
2-P-glycerate. ............... 0.380 0.547
ATP ........................ 0.500 0.616
ADP ........................ 0.375 0.477
AMP ....................... 0.067 0.166
Glucose B-phosphate. ....... 0.308
Fructose 1,6-diphosphate .... 0.291 0.450
B-Glycerophosphate......... 0.300 0.581
Phosphoenolpyruvate. ....... 0.870 0.905 0.533
p-Nitrophenyl phosphate. ... 0.336
Sodium pyrophosphate...... 0.677

0.8

0.7

1 1 I I
0.6
I 2 3 4
S go.5
FIG. 2. Influence of substrate concentration upon velocity.
Fraction V and the standard conditions of assay were used except Eo.4
that the substrate (8) was varied as indicated in the abscissa of
the figure, expressed in micromoles per ml. Velocity (V) is ex- v)
pressed as micromoles of Pi liberated during 15 minutes. O---O, g 0.3
data expressed as reciprocals of velocity and substrate.
0.2
linear between 0.2- and 0.5~pmole utilization of substrate under
the standard conditions of assay. There is lack of linearity at
0.1
very high turnovers, probably as a result of product inhibition
(see below). J I I
E#ect of Temperature upon Rate-The rate with Fraction Via 0 .5 I .o
increased 1.4 times between 30 and 37” under the standard con- )J Moles Pi Added
ditions of assay. FIG. 3. Effect of phosphate and Mg++ on phosphatase. The
Specificity-As is shown in Table III, the enzyme is nonspecific. standard conditions of assay were used except for the quantities
However, it appears to have very little diesterase activity. of Mg++ and Pi added to the tests, as indicated in the figure. In
Under the conditions (3 hours’ incubation, 37”, pH 8.8) of Sins- all cases Fraction Via was diluted 150-fold. O-O, 0.05 ml of
enzyme; O-0, 0.1 ml of enzyme; q -----0, 0.05 ml of enzyme
heimer and Koerner (lo), 0.13 pmoles of p-nitrophenol were and 20 pmoles of Mg++; m----m, 0.1 ml of enzyme and 20 rmoles
liberated from calcium bis(p-nitrophenyl) phosphate by 89 of Mg++; A-----& 0.05 ml of enzyme and 40 pmoles of Mg++;
units of Fraction VI (which would hydrolyze 89 pmoles of 3-P- A----A, 0.1 ml of enzyme and 40 pmoles of Mg++.
August 1960 B. K. Joyce and S. Grisolia 2281

of assay, 0.67, 1.3, and 2.6 x 1O-2 M Mg++ stimulated the activity interfere with Pi estimation, particularly after hydrolysis of
of Fraction Via 21, 32, and 22%, whereas Mn++ at 1.3 x lo-2 M small samples.
stimulated slightly. Pb++ at this concentration inhibited the
enzyme 75 %. Ag++, 8 X 1O-3 M, zn++ and Cu++, 1.2 X 1O-2 M, SUMMARY

and U6+, 3.3 X 1O-4 M completely inhibited the activity.4 A method for the extensive purification of an acid phosphatase
As is shown in Fig. 3, Pi in the presence or absence of Mg++ from wheat germ is presented. Some properties of the non-
inhibited the hydrolysis of 3-P-glycerate. This probably ex- specific phosphatase are described. A wide range of reagents
plains the decrease in activity when large aliquots of enzyme are substrates for this phosphatase, including pyrophosphate.
and long incubations were used. However, the purified enzyme preparations have little phospho-
Extent of Hydrolysis-Under otherwise standard conditions, diesterase activity, as tested with bis(p-nitrophenyl) phosphate.
4.4 units of Fraction Via were incubated with 1.39 pmoles of Complete hydrolysis has been demonstrated with such substrates
3-P-glycerate (estimated enzymatically). Analysis showed 1.28 as n-3-phosphoglycerate and glucose 6-phosphate, which are
pmoles of Pi and no residual 3-P-glycerate. Essentially under difficult to hydrolyze by chemical methods.
the same conditions, but during 25 minutes of incubation, 0.41
pmoles of glucose 6-phosphate were hydrolyzed completely. REFERENCES
However, with 0.375 pmoles of ATP, ADP, and AMP, 0.79, 1. ITO, N., AND GRISOLIA, S., J. Biol. Chem., 234, 242 (1959).
0.43, and 0.29 pmoles of Pi were liberated, respectively. 2. JOYCE, B. K., AND GRISOLIA, S., J. Biol. Chem., 233,350 (1958).
3. MORTON, R. K., Biochem. J., 67, 595 (1954).
DISCUSSION 4. KUNITZ, M., J. Gen. Physiol., 36, 423 (1952).
5. GRISOLIA, S., AND JOYCE, B. K., J. Biol. Chem., 233, 18 (1958).
Although the presence of phosphatases in plants and portions 6. TOWNE, J. C., RODWELL, V. W., AND GRISOLIA, S., J. Biol.
thereof has been known for many years and acid phosphatase Chem., 226, 777 (1957).
of low activity from wheat germ is available commercially,5 few 7. WARBURG, O., AND CHRISTIAN, W., Biochem. Z., 310,384 (1941).
studies have been made with 3-P-glycerate as substrate except 8. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL,
with crude preparations (17). The procedure described here R. J., J. Biol. Chem., 193, 265 (1951).
9. MOKRASCH, L. C., DAVISON, W. D., AND MCGILVERY, R. W.,
permits, with little effort, the preparation from wheat germ of J. Biol. Chem., 222, 179 (1956).
nonspecific acid phosphatase preparations of very high specific 10. SINSHEIMER, R. L., AND KOERNER, J. F., J. Biol. Chem., 198,
activity. Although physical studies have not been conducted 293 (1952).
as yet with the purified preparations, the extensive purification 11. GOMORI, G., J. Lab. Clin. Med., 27,955 (1942).
and the high turnover of the purified preparation suggest that 12. TCHEN, T. T., AND VENNESLAND, B., J. Biol. Chem., 213, 553
(1955).
they may be of high purity. 13. LINEWEAVER, II., AND BURK, D.. J. Am. Chem. Sot.. I 66. 658
Since the specificity varies little during a 3000-fold purifica- (1934).” ”
tion, it seems reasonable that the phosphatase activity for 3-P- 14. PRIVAT DE GARILHE, M., AND LASKOWSKI, M., Biochim. et
glycerate estimated in other germs or seeds (see above) reflects Biophys. Acta., 18, 370 (1955).
15. RAPOPORT, S., AND LUEBERING, J., J. Biol. Chem., 189, 683
the presence of this nonspecific phosphatase.6 It seems para- (1951).
doxical for the germ of plants to have such a large nonspecific 16. SINGER, T. P., J. Biol. Chem., 174, 11 (1948).
phosphatase activity, since apparently it would be desirable, 17. ROBERTS, D. W. A., J. Biol. Chem., 226, 751 (1957).
particularly during initiation of germination, to conserve organic
phosphate esters. The phosphatase activity (per mg of extract- Note Added in Proof-Dr. L. A. Heppel has made some ob-
able protein), with 2,3-dip-glycerate as substrate, of crude servations on the hydrolysis of polynucleotides by the purified
wheat germ extracts is about 10 times larger than in bakers’ Fraction V. In all cases the incubations were at pH 6.0 and
yeast, the best known source for specific 2,3-dip-glycerate contained 1.5 pmoles of MgClz in a final volume of 0.1 ml. Oli-
phosphatase (2). goribonucleotides, as pApA, ApAp, pUpU, and ApApApAp are
Whereas physical evidence for purity of the preparations is converted to the nucleosides. About 14 fig of enzyme are re-
also desirable, the present method results in enzymatic prepara- quired to cleave 0.2 pmole of pApA in 1 hour. Intermediates
tions useful, for example, for estimation of phosphate monoesters accumulate during the hydrolysis, but these have not been in-
such as the phosphoglycerates, which are difficult to hydrolyze vestigated. The deoxypolynucleotide, pTpTpT (obtained from
by chemical means? Enzymatic hydrolysis may also be helpful Dr. H. G. Khorana) is also attacked. Polymers formed with
in avoiding high concentration of acid or salt, or both, which may bacterial polynucleotide phosphorylase (poly A, poly C, poly U)
4 Uranium acetate per se hydrolyzes P-glycerate. Appreciable are completely hydrolyzed to nucleosides, but more slowly than
hydrolysis occurs at 37” from pH 4 to 7. Uranium acetate, 6.6 X oligoribonucleotides. The nondialyzable residue obtained from
10-a M, completely hydrolyzed 1.28 and 0.73 rmoles of 3-P-glycer- a digest of RNA with pancreatic ribonuclease is easily hy-
ate and 2,3-dip-glycerate, respectively, when heated in a 100”
drolyzed. By contrast, commercial yeast RNA is quite slowly
water bath for 35 minutes. This hydrolysis will occur in the pres-
ence of 75 rmoles of acetate buffer but is inhibited almost com- and incompletely cleared. Yeast RNA prepared by the pro-
pletely by 75 pmoles of citrate buffer. cedure of Crestfield and Allen shows no hydrolysis after incu-
6 For example, Worthington Biochemical Corporation markets bating 0.3 mg of substrate with 0.75 pg of enzyme for 6 hours.
a product which “is essentially the 0.35-0.55 saturated ammonium Soluble RNA from yeast prepared by the procedure of Davis
sulfate fraction from wheat germ described by Singer.” This is
taken from the procedure of Singer (16) for preparation of lipase. (I .6 pmoles as base) shows only 5 % hydrolysis after incubation
6 The pH curve with 3-P-glycerate as substrate of crude rice with 300 /Ig at 37” for 3 hours. The resistance shown by RNA
germ extract is identical to that of wheat germ extract (M. Fer- preparations is puzzling and interesting.
nandez and S. Grisolia, unpublished experiments). It is suggested that the purified wheat germ phosphatase is a
7 Dr. E. Racker (private communication) has found our prepa-
useful reagent for gently converting small polynucleotides to
rations useful for hydrolysis of glucose 6-phosphate and sedohep-
tulose 7-phosphate. nucleosides, as part of their structural determination.