Phosphatases of ragweed pollen

Phosphatases of Ragweed Pollen l George R. Nakamura 2 and Elmer L. Becker Prom the Department

of Biological Chemistry, College of Medicine, Chicago, Illinois Received

February

University

of Illinois,

21, 1951

The presence of phosphatase activity in raglveed pollen was observed by E. L. Becker in this laboratory; the present study was undertaken t.o investigate the charact,eristics of ragweed phosphatase activity.

EXPERIMENTAL Material The ragweed pollen selected for this study was from the short ragweed variety, Ambrosia

elatior.

The three principal substrates employed for the assay procedures were Bodium a-glycerophosphate, sodium @-glycerophosphate, and disodium p-nitrophenyl phosphate. The a-glycerophosphate, prepared by the procedure described by King and l’yman (I), was found to be 94yo pure according to the method adapted from Leva :tnd Rapaport (2). Thtj P-isomer of sodium glycerophosphate was obtained from Boots Pure Drugs Ltd., Nottingham, England. The periodic acid oxidation method of T,eva and Rapaport showed that it contained less than 3% a-isomer. The preparat.ion of p-nitrophenyl phosphate contained 5oJ, free nitrophenol which was removed by purification with butanol and ether by a procedure described by Bessey, Lowry, and Rrock (3).

Assay Methods The determination of ragweed phosphatase activity was based on the quantity of phosphate liberated (4) from an excess of sodium &glycerophosphate in 0.1 M acetate buffer, pH 5.5, after 30 min. of incubation at 37.5”. It was found t,hat the rate of hydrolysis, in an excess of the substrate, ww linear up to 30 min., and the rate of inorganic phosphate cleavage was observed to be dirent,l,v proportional to the quantity of enzyme used (Fig. 1). 1 Taken from a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biological Chemistry in the Graduate College of the Kniversity of Illinois, 1949. 2 l’rrsent, address: Virus Laboratory, University of California, Berkeley, California. 78

RAGWEED

POLLEN

79

PHOSPHATASES

Another sukrate, disodium p-nitrophenyl phosphate, was employed for the phosphatase activity measurement,; the t,echnique employed was essentjially that of Bessey el al. (3). The activity unit was defined as the amount of enzyme which liberated 1 mg. phosphorus from the substrate at pH 5.5 and 3i.5"C. The specific activity of enzyme preparations was expressed as GP or PSP activity/mg. protein depending on thus I

MI

OF

ENZYME

I

,

SOLUTION

FIQ. 1. Proportionality of the rate oi phosphorus liberation from +glycerophosphate to the varying concentrations of pollen phosphatase solution (Prepn. C) buffered at pH 5.6. respective use of glywrophosphate (GP) or p-nitrophenyl phosphate (PNP) as :t substrate. Protein nitrogen was determined by a trichloroacetic acid precipitation method of Sorthrup (.5).

Before extraction with alkali the pollen wax defatted \vith ether and then washed with cold 957o ethanol. Two grams of dried defatted pollen was extracted with 20 ml. of 0.25% sodium carbonate for 1 hr. at 10’ in a large test tube with frequent shaking. One hour was found sufficient to obtain maximum activity. The pollen residue was treated again with 5 ml. of dilute sodium carbonate and t,he result’ing extract, was combined with the previous one. The final pF1 of’ the Pxt,rac+

80

GEORGE

R.

N-ZKAMURA

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ELMER

L.

BECKER

was usually about 7. The specific activity of the crude ext,ract was determined. The pa-activity curves of the crude extract are shown in Fig. 2. The pH optima for the crude extract were manifested-at pH 3.9, 4.7, and 5.7 with each of the three different substrates. ,

I

,

I

,

I

I

!

,

IJO -

c

90 -

1 5 I;”

80 -

F 3 E 70

-

GO -

’ B- GLYCEROPHoSPHATE o a- GLYCEROPHOSWATE A p-

NlT”OP”ENYL

FWXPH,,TE

FIG. 2. pH-Activity curves of the hydrolysis of a-glycerophosphate, fi-glycerophosphate, and disodium p-nitrophenyl phosphate by a crude extract (Prepn. A) containing 17.6 GP units or 1100 PNP units/mg. protein nitrogen. Standard assay conditions were observed. The optimum activity at pH 5.6 was arbitrarily taken as 100.

The crude extract (Prepn. A) was made 0.7 saturated with ammonium sulfate. The pH was usually about 5-5.5 after the salt was added; if not, it was adjusted to that pH range. The material was centrifuged and the precipitate was taken up with about 15 ml. of cold water. The solution was dialyzed in a cellophane bag against cold wa,ter. Any precipitate formed was removed by centrifugation. The supernatant liquid contained the active fraction which was designated as Prepn. B. To this dialyzate was added 500 mg. of cohoidal kaolin and the suspension was shaken for a few minutes and filtered. The filtrate, or Prepn. C, was used to plot pH-activity curves with three different

RAGWEED

POLLEN

81

PHOSPHATASES

substrates. Two discrete pH optimum peaks were demonstrated (Fig. 3), one being more pronounced than the other: pH 5.5-5.7 > pH 4.2-4.3. Selective Destruction Preparation C was adjusted to pH 8 with dilute NaOH, and kept for 20 hr. in the refrigerator, and then neutralized with dilute HCl.

2

4

5

6

7

9

PH

FIG. 3. Prepn. C conditions maximum

pH-Activity curves for the hydrolysis of three different substrates by containing 50.6 GP units or 3960 PNP units/mg. nitrogen. Standard assay were observed in each case. The curves were plotted by arbitrarily taking activity as 100.

There was a disappearance of the less pronounced peak at pH 4.2-4.3, at the expense, however, of about a 50yo decrease in the activity of the other peak (Fig. 4). Here again, three different substrates were employed. There was an unexplained shift in the pH optimum from pH 5.5 to pH 5.3. This preparation was called D. The activity values for the different preparations are shown in Table I.

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Bauman and Saleer (6) similarly employed this method of selective pH inact’ivation to demonstrate two distinct phosphomonoesterases in the mold Aspergillus oryzae.

FIG. 4. The comparison of the pH-activity curves for the hydrolysis of p-nitrophenyl phosphate by Prepns. C and D, the latter containing 1807 PNP units/mg. protein nitrogen.

pH Stability Samples of Prepn. C were mixed with a series of acetate and Verona1 buffers, the pH of the solutions ranging from pH 1 to 8 (solutions of pH l-3 were made so by adding dilute HCl or dilute acetic acid), and allowed to stand in the refrigerator at 5’. After 0, 1, and 24 hr., the mixtures were assayed using glycerophosphate as substrate. The results presented in Fig. 5 showed that the phosphatases are partially destroyed in an acid milieu of pH 2 within 1 hr. and totally inactivated immediately at pH 1. At pH 8, a 55% decrease in the activity was noted at the end of 24 hr., and a total loss at pH 9.

HAGWEED

Pm$catiwn

POLLEI\:

of Phosphntnses ,from Pollen Extract Protein nitrogen/g. pollen Illrl.

Preparation

83

PHOSPHATASES

G.P. unit/c. pollrn

G.P. unit/ mg. protein nit,rogen

PNP unit/ mg. protein nitrogen

PNP unit/g. pollen

A

Crude extract,: dialyzate

1.1

19.4

17.6

1210

1100

H

Dialyzate made 0.7 saturated with (XH4)2S04; precipitate dissolved in HZ0 and dialyzed; supernatant liquid from centrifuged dial,yzate.

0.5

16.6

33.2

1260

2520

Prepn. B after kaolin adsorption; filtrat,e

0.27

13.7

50.6

1071

3960

Prepn. C adjusted to pH 8 for 20 hr. and then neutralized

0.27

7.1

26.2

488

1807

c

D

I

e

3

4

5

6

*

0

9

PH

Fro. 5. Stability

of pollen phosphat,ase

(Prepn. 6) as :I fun&on

of pH

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ELMER

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BECKER

l’hermolability A tube containing 0.5 ml. of acetate buffer, pH 5.5, plus 0.1 ml. of Prepn. C was immersed in a water bath maintained at a desired temperature and the samples were assayed at various time intervals. The enzyme retained 10yo of its activity despite being subjected to a temperature of 70” for 1 hr. Seventy-five per cent of its activity remained after 3 hr. of treatment at 55”. A heating period of 2 hr. at 60” produced a 55yo decrease in activity. Booth (7) reported a critical inactivation temperature (the temperature at which 50yo of the enzyme activity is destroyed irreversibly in 1 hr.) of 55” for wheat extract phosphatases, whereas the inactivation temperature for the ragweed phosphatases was found to be between 60” and 70”. E#ect of Xubstrate Concentration Michaelis-Menten constants (KJ and maximum velocities (V) for p-nitrophenyl phosphate and a- and fl-glycerophosphates were calculated from the experimental data. The results are summarized in Table II. TABLE

II

h’ffect of Substrate Concentration pH 5.5

pH 4.5

Substrate -__--

Ka

pH 5.6 _____--

Va

Kf

pH 6.0

Vu

Ksa

va

a-Glycerophosphate

2.2 X 1O-3 29.6

2.5 X 1O-3 45.5

2.6 X 1O-3 42.3

B-Glycerophosphate

3.3 x10-3

3.0 x 10-3 32.0

4.3 x 10-3 29.8

p-Nitrophenyl phosphate

17.7 _~---~~

__-

7.5 x10-4 83.0

n The Michaelis-hlenten constants (K,) and maximum velocities (V) for the three substrates were determined from linear curves which were obtained when l/v was plotted against I/S using the equation: T/s K, is expressed as moles/l. and V represents under standard assay conditions.

the micrograms

of phosphorus

liberated

RAGWEED

POLLEN

PHOSPHATlSES

85

Energy of Activation The time activity studies for Prepn. C in buffered solutions, yielded curves representative of a zero-order reaction when substrates were used in excess. The velocity constants were determined from the experimental data under the standard assay conditions.

SLOPE . 1667 .E

* 7640

CAl.ORIES

Y

4 3.0 -

SLOPE m 1620 2.0 -

-E * 1400

CAumES PER

MOLE

,6 GLYCERCWOSP”AlE

x1032

003s I/T

(ABS)

FIG. 6. Effect of reaction temperature on phosphatase activity (Prepn. C). The energy of activation for the hydrolysis of the substrates, &glycerophosphate and p-nitrophenyl phosphate, was determined.

These constants were employed to derive the energies of activation which are expressed as calories per mole. Straight lines were plotted using Arrhenius’ equation (8). The curves in Fig. 6 obey the Arrhenius’ equation where E is a constant value in both instances.

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NAKAMUR.4

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ELMER

L.

BECKER

The effect of various compounds on phosphatase activity was observed under standard conditions using fl-glycerophosphate as substrate and Prepn. C. Table III summarizes the effects of various substances upon phosphatase action. While no activator was noted among the compounds tried, copper sulfate, sodium arsenate, sodium cyanide, and sodium fluoride exerted inhibitory effects on the rate of phosphorus liberation. TABLE

III

I$ffet:tsqf Cotnpomd on Phosphatase Activity Molar concn.in Relative

colllpoundr.

wnction

mixture

(no addition

activity -100)

M

Sodium cyanide Sodium cyanide Sodium arsenak Copper sulfate Sodium fluoride Sodium fluoride Ammonium sulfate Calcium chloride Glycine Magnesium chloride Magnesium chloride Potassium oxalate Sodium iodoacet,ate Sodium citrate Strychnine sulfate Thiourea Urea

Specilficity

0.05 0.01 0.01 0.001 0.02 0.01 0.40 0.02 0.10 0.40 0.02 0.10 0.10 0.10 0.001 0.02 0.02

0 52 25 25 63 100 100 100 100 100 loo 100 100 100 100 100 100

of Action

The action of pollen phosphatases, Prepn. C, on 11 different substrates was observed. All reactions were carried out under identical conditions: pH 5.5; time of incubation, 30 min.; temperature, 38”; and the concentration of each substrate was made so that 1.0 ml. of a substrate contained about 250 pg. of organic phosphorus. The amount of phosphorus split from each substrate under the above conditions was observed. The results are given in Table IV.

RAGWEED

POLLEX

TABLE Specijkity

of Pollen

87

PHOSPH.%l’ASES

I\’ Phosphattrse

Activit~y

Total organic P in reaction mixture

),-Sitrophenyl phosphate a-Glycerophosphate p-Glycerophosphate Fructose g-phosphate Fructose diphosphate ntiPhosphoglyceric acid Sodium pyrophosphate :I-Adenylic acid Adenosine triphosphate Ribonucleic acid Diphenyl phosphoric acid

Pg.

I’ liberaterl Pg.

220 250 250 250 242 276 250 218 285 220 240

82.7 46.5 31.3 21.0 17.6 17.0 16.5 14.8 14.0 0 0

DISCUSSIOK

Three pH optima were demonstrated for the crude extract of ragweed pollen when a pH-activity curve (Fig. 2) was plotted, and therefore it was possible tJo postulate the presence of three phosphomonoesterases in the preparation. On preliminary purification, the pH curve (Fig. 3) of the material indicated the possible presence of at least two phosphomonoesterases, one of which exhibited, in addition to the optimum pH 5.6 with P-glycerophosphate as substrate, a second and less prominent peak in the vicinity of pH 4.2. A similar PI-I-activity curve was observed when p-nitrophenyl phosphate substrate was employed. It would be hasty to postulate the occurrence of two phosphomonoesterases in this purified extract, yet the fact that the two activities could be separated by selective pH inactivation makes this assumption plausible. It is not assumed, however, that there is a specific enzyme for each substrate used. The pollen phosphatases were inhibited by fluoride, copper, arsenate, and cyanide ions, thus providing information with respect to the characteristics of the enzymes. That copper and cyanide retarded the action of phosphatases suggests that sulfhydryl groups are necessary. The absence of iodoacetate action on the pollen phosphatase need not contradict the general rule that iodoacetate inhibits sulfhydryl groupcontaining enzymes; for example, iodoacetate does not inhibit’ adenosinetriphosphatase which is a, slllfhydryl-containing enzyme (9). Study

88

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L. BECKER

of the phosphatases of the higher plants by other investigators demonstrated a general absence of the influence of Mg. The lack of activation by bhe addition of Mg salt did not preclude the possibility that the purified pollen phosphatases may exist as a Mg-enzyme complex, as the fluoride action may have indicated. However, it seemed unlikely that Mg or Ca are cofactors in the enzymes because of the lack of inhibition by citrate and oxalate. In specificity studies, Prepn. C showed a variable reactivity with several monoesters and pyrophosphates; however, it had no apparent diesterase or ribonuclease activities. Since Becker and Berman (10) described the presence of a ribonucleic acid-splitting enzyme in crude pollen extracts, this indicates that Prepn. C was purified to the extent of removing the diesterase activity, which is probably due to separate enzymes. ACKNOWLEDGMENTS The authors are greatly indebted to Dr. S. I’. Colowick of Johns Hopkins University and Dr. C. A. Knight and Dr. A. B. Pardee of the University of California for reading the manuscript and suggesting improvements. SUMMARY

1. Phosphoric ester-splitting enzymes have been obtained from defatted ragweed pollen by extraction with 0.25% sodium carbonate. The crude extract showed the presence of three optima in the pHactivity curve. -After fractionation, a preparation showing two optima, one at pH 5.6 and another smaller one at 4.2 was obstained. 2. The more alkaline optimum was 5.6 for sodium P-glycerophosphate, 5.7 for sodium a-glycerophosphate, and 5.5 for disodium nitrophenyl phosphate. 3. On standing at pH 8.0 for 20 hr., t,he optimum at pH 4.2 was eliminated giving a preparation containing only one optimum. -l. The Michaelis-Menten constants for the enzymatic cleavage of fi-glycerophosphate and p-nitrophenyl phosphate were 3.0 X 10e3 M and 7.5 X 10e4 il;l, respectively, at the optimum pH. 5. The critical inactivation temperature of the purified preparation when tested on /&glycerophosphate was observed to be 60-70” C. 6. The purified phosphatase remained stable in a milieu of pH 3--7 for a minimum period of 24 hr. at Ei”C.

RAGWEED

POLLEN

PHOSPHATASES

89

7. The energies of activation for the hydrolysis of P-glycerophosphate and p-nitrophenyl phosphate by the same enzyme preparation were found to be 7400 calories and 7600 calories, respect’ively. 8. No compound was found to activate the ragweed pollen phosphatases. Copper, arsenate, cyanide, and fluoride were inhibitory to the enzyme system. 9. The pollen phosphatase failed to hydrolyze a diester of ribonucleic acid, indicating the absence of diesterase and ribonuclease activkies. The enzyme preparation possessed, in addition to a phosphomonoest,erase activity, pyrophosphatSasr and wlenosinetriphosphatxsr act’ion. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

KING, H., AND P~MAN, F. L., J. Chem. Sot. 1914, 1238. LEVA, E., AND RAPAPORT, S., .I. Biol. ChewA. 149, 47 (1943). BESSEY, 0. A., LOWRY, 0. H., AND BROCK, ;\I. .J.. J. Riob. Chrrn. 164, 321 (1946). HOLMAN, IV. I. M., Biochem. J. 37, (1943). NORTHRUP, J. H., J. Gen. Physiol. 16, 33 (1932). BAUMAN, E., AND SALZER, W., Biochem. Z. 287, 299 (1936). BOOTH, R. G., Biochem. J. 38, 355 (1944). ARRHENIUS, S., Z. physik. Chem. 4, 226 (1889). XEEDHAM, D. M., Biochem. J. 36, 113 (1942). BECKER, E., AND BERMAN, H., unpublished work, 1949.