T H E JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 20, Issue of October 25, pp. 10519-10523, 1981 Printed ~n U. S. A.

Platelet AMP Deaminase PURIFICATION AND KINETIC STUDIES*

(Received for publication, March 9, 1981)

Barrie Ashby and Holm Holmsen From the Thrombosis Research Center, Temple University Health Science Center, Philadelphia, Pennsylvania 19140

AMP deaminase has been purified to homogeneity from human platelets by phosphocellulose chromatog- raphy. Kinetic studies showed sigmoidal behavior as a function of AMP concentration with the midpoint of the saturation curve (So,) at 3.5 and 4.0 m~ in NaCl and KCl, respectively, at pH 6.5. Activation by saturating ATP converted the velocity versus substrate plot to hyperbolic with a Michaelis constant of 1.2 mM and the same maximum velocity in either salt. Addition of in- creasing concentrations of GTP in the presence of NaCl led to activation followed by inhibition whereas GTP in the presence of KC1 gave inhibition with no apparent activation.

Platelet secretion requires ATP, some of which may be utilized in an actomyosin contractile apparatus (1). The pro- duction of ATP is stimulated by initiation of secretion and cytoplasmic ATP is converted to IMP and eventually to hypoxanthine (1, 2). A conceivable role for the conversion would be to displace the adenylate kinase equilibrium to generate ATP from ADP which would then be available to the actomyosin ATPase. The rate-controlling step in the process is the irreversible conversion of AMP to IMP and ammonia by AMP deaminase (EC 3.5.4.6). The same reaction sequence is responsible for maintaining the adenylate energy charge above 0.9, which seems to be crucial for platelet re- sponses ( 3 ) .

AMP deaminase has been purified and studied from a number of sources. It has been shown to be regulated by nucleotides such as ATP, GTP, and ADP, and by phosphate and pyrophosphate, and to require monovalent cations such as sodium or potassium ions for optimal activity (4-16). The same ligands have been shown to affect AMP deaminase activity in platelet lysates (17). In the present paper we describe the purification of platelet AMP deaminase and show in detail how its activity is influenced by ATP and GTP and by sodium and potassium ions.

MATERIALS AND METHODS’

RESULTS

Purification of AMP Deaminase-A summary of a purifi- cation is presented in Table I-S. A sodium dodecyl sulfate

* This work was supported by Grant HL 05976 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ Portions of this paper (including “Materials and Methods,” Figs. 1-S through 6-S, and Tables I-S and 11-S) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethe&,

polyacrylamide gel of the phosphocellulose column pools is shown in Fig. 1. The single band in lane 3 corresponds to a molecular weight of 83,000 to 85,000 when compared with protein standards of known molecular weight (not shown).

Kinetic Studies-Results appear in the miniprint supple- ment.

DISCUSSION

Purification-Platelet AMP deaminase has been purified by a method originally described by Smiley et al. (5) for the rabbit skeletal muscle enzyme using modifications described by Yoshino et al. (6). The molecular weight of the subunit was determined to be 83,000 to 85,000, similar to that of the bakers’ yeast enzyme ( 7 ) but higher than that obtained for the enzyme from muscle or erythrocyte by a number of investigators (5, 8-10) and the highest specific activity ob- tained was similar to that of the purified human erythrocyte enzyme ( 10).

Kinetic Properties-Platelet AMP deaminase is strikingly similar in its kinetic properties to the enzyme from calf brain (11-13). In the presence of monovalent cations the two en- zymes show sigmoidal substrate-velocity plots; activation by ATP, that converts substrate-velocity plots to hyperbolic; and inhibition by GTP, that increases the degree of sigmoidicity and reverses ATP activation. In the absence of nucleotide effectors, sodium ions are more effective activators than po- tassium ions of the platelet (Fig. l-S) and calf brain (13) enzyme. In NaC1, GTP both activates (at low concentrations) and inhibits (at high concentrations) the platelet enzyme (Figs. 4-S and 5b-S) and similar behavior has been observed with the calf brain enzyme (13). In KCl, GTP merely inhibits both the platelet enzyme (Figs. 4-S and 5a-S) and the calf brain enzyme (13).

In contrast, platelet AMP deaminase differs markedly in its interaction with ligands from the skeletal muscle enzyme from rabbit, rat and man (4, 5, 8, 14, 15) and from human erythro- cyte AMP deaminase (10). Potassium ions are more effective activators than sodium ions of the erythrocyte enzyme (10) whereas sodium and potassium ions are equally effective activators of the muscle enzyme (4, 5, 14, 15). In KCl, in the absence or presence of ATP both skeletal muscle and eryth- rocyte AMP deaminase shows hyperbolic substrate-velocity plots (5 , 10, 14, 15) that are converted to sigmoidal in the presence of GTP (10,14). At low concentrations ATP can also inhibit the muscle enzyme (16), whereas the platelet enzyme is activated at all concentrations of ATP (Fig. 3-S).

Mechanism of Regulation-Platelet AMP deaminase shows sigmoidal behavior as a function of substrate concen-

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10519

10520 Platelet AMP Deaminase

3 n i

A B FIG. 1. Sodium dodecyl sulfate polyacrylamide gel electro-

phoresis patterns of platelet AMP deaminase. A, partially puri- fied enzyme from the fvst phosphocellulose column; B, homogeneous enzyme from the second phosphocellulose column eluted with ATP.

tration. However, this characteristic apparently plays no part in the regulation of the enzyme since the concentration of AMP in platelet cytoplasm never rises above around 50 PM (17) whereas the So.e for AMP is about 4 mM (Fig. 2-S). The afiinity of the enzyme for ATP, on the other hand, is charac- terized by an activation constant of 2 to 4 C(M (Fig. 3-S) so that ATP is almost an obligatory activator. Activation by high concentrations of AMP in vitro, leading to sigmoidal behavior, results from an overlap in the specificity of an ATP activator site.

Kinetic behavior in the presence of GTP is most simply explained by the assumption that there is a separate GTP activator site that enhances binding of AMP, and presumably ATP, to the ATP/AMP activator site as well as a GTP inhibitor site that weakens binding to the ATP/AMP activa- tor site. Good quantitative fits to initial rate data can be made using this model with respect to AMP activation (Fig. 5-S) and differences in response to GTP in NaCl compared with KC1 can be attributed to changes in the magnitude of certain dissociation constants (Table 11-S). According to this inter- pretation, switching from KC1 to NaCl leads to activation primarily because enzyme with GTP bound to the GTP activator site can bind AMP to the AMP activator site 31 times more tightly in NaCl than in KC1. This is in contrast to the idea that NaCl activates by relief of GTP inhibition by weakening binding to a GTP inhibitor site suggested by Setlow and Lowenstein (13) to explain the behavior of the calf brain enzyme.

Setlow and Lowenstein (13) have suggested that an influx of sodium ions into the cell accompanied by an efflux of potassium ions could lead to activation of AMP deaminase due to a reversal of GTP inhibition. Feinberg et dl . (18) have shown that sodium-potassium exchange can occur upon stim- ulation of platelets with ADP under conditions where secre-

tion does not occur and the ATP-IMP conversion does not take place (1). The magnitude of this change is on the order of 10 mM with the sodium concentration inside the platelet changing from about 42 to 52 mM and potassium from 100 to 90 mM (18, 19). In experiments with GTP and ATP concen- trations chosen to maximize the difference between activity in NaCl compared with activity in KC1 we have shown that over this range of sodium-potassium exchange there is little activation of platelet AMP deaminase (Fig. 643).

These observations do not rule out the possibility of sodium- potassium exchange affecting enzyme activity since it is con- ceivable that agents such as thrombin that do cause secretion and the ATP-IMP conversion could lead to a sodium-potas- sium exchange over a wider range. In addition, the presence of magnesium ions alters the interaction of AMP deaminase with its effectors. Setlow and Lowenstein (13) have shown that ATP in the presence of magnesium but in the absence of monovalent cations leads to a Michaelis constant about 2-fold lower than in the presence of free ATP. Magnesium also increases the effectiveness of GTP as an inhibitor of the enzyme. It is possible that differential changes in kinetic constants between NaCl and KC1 could be enhanced in the presence of magnesium.

Acknowledgments-We wish to thank Dr. James Daniel for help with computer programming, the Penn-Jersey Red Cross for outdated platelets, and the staff of the Temple Hospital Blood Bank for their kind cooperation.

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2. Ireland, D. M. (1967) Biochem. J. 105,857-868 3. Holmsen, H., and Robkin, L. (1977) J. Biol. Chem. 252, 1752-

4. Zielke, C. L., and Suelter, C. H. (1971) in The Enzymes (Boyer, P.

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6. Yoshino, M., Murakami, K., and Tsushima, K. (1979) Biochim.

7. Murakami, K. (1979) J. Biochem (Tokyo) 86,1331-1336 8. Coffee, C. J., and Kofke, W. A. (1975) J. Biol. Chem. 250,6653-

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Biophys. Acta 570, 157-166

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167,626-629 10. Yun, S., and Suelter, C. H. (1978) J. Biol. Chem. 253,404-408 11. Setlow, B., and Lowenstein, J. M. (1967) J. Biol. Chem. 242,607-

12. Setlow, B., and Lowenstein, J. M. (1968) J. Biol. Chem. 243,

13. Setlow, B., and Lowenstein, J. M. (1968) J. Biol. Chem. 243,

14. Ashby, B., and Frieden, C. (1978) J. Biol. Chem. 253,8728-8735 15. Makarewicz, W., and Stankiewicz, A. (1974) Biochem. Med. 10,

180-197 16. Tomozawa, Y., and Wolfenden, R. (1970) Biochemistry 9,3400-

3404 17. Holmsen, H., Ostvold, A.-C., and Pimentel, M. A. (1977) Thromb.

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Grossman, B., and Born, G . V. R. (1977) Biochim. Biophys.

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Acta 470,317-324

Biophys. Acta 600,448-455

Platelet AMP Deaminase 10521

MATERIkLS AND HETHODS

RESULTS

Crude Extract 1497 15,456 0.10 loo

Phasphocelluiase I 1100 6.3 175 73

Phosphacellulose I1 380 0.87 435 25

t 1

10522 Platelet AMP Deaminase

0.5 F i NaCl

0 . 4 -

0 I I I 0 5 10 15 20 I Id

ATP ("MI

FIG.3-5: The ratio Of initlal velocity to maximum velocity as

mM KC1 101. The AMP Concentration was 1 mM in each case a function O t ATP concentration in 100 mM NaCl 1.1 or 100

and the enzyme concenrrarion was 2.5 ug/ml ~n each assay. vmax was determined from Fig.2-s.

fIG.4-s: The ratio of initla1 velocity to maxmum velocity as

mM KC1 1 0 1 . The AMP concentration was 100 LIM I" each case a function Of GTP concentration in 100 mY NaCl (e ) or 100

Vmax was determined from fq.2-s. and the enzyme Concentration was 2.5 ug/ml in each assay.

I I I I I

KC1

I I T I b

I I I I

20

V S

10 t FIG.5-5: a. DlXDn plots of GTP inhiblrlon measured a t Several

AMP Concentrations in the presence of 100 mM KC1. b. Dixon D10f5 Of GTP inhiblfion land actlvationl

measured at &era1 AMP concentrations in the presence of 100 mM NaCl. The solid llnes in all cases Were calculated using Equatlon 111 and constants listed ln Table 11-S. Otner COndltlOnS were de descrlbea in the legend to Fig.3-S.

Cannot be due to GTP binding to the Same activator site as AMP and ATP. This 1s so because 6afUration with GTP would then lead to hyperbolic Substrate- velocity C U I V ~ S , which are not observed IFig.2-51.The same is true for altern- ative descrlptlons of cooperative behavior such as the model of Monod et.. (81. The data ln Fig.5-s Can be most s~mply explained Dy prOpOslng a separate activator slte for GTP that does not compete wlth AMP blndlng but, rather, en- hances bindlng to the AMP activator site. The interactlo" of GTP wlth the en- zyme can then be described by the mechanism

According to the model descrlbed earller the observed activation by GTP

E ~ S E - S E S '> . SE + P

I I I

Platelet AMP Deaminase 10523

TABLE 11-S: Kinetic constants for interaction of AMP deaminase wlth GTP ~n 100 mM NaCl 01 100 m KC1

The rate constants k2,kg and k4 were assumed to be equal since Vmax was the same in the a~sence and presence of GTP. Data could be fit by assuming that GTP had no effect on bindlng of Substrate eo the catalytic site SO that K3,Kg and K L 3 were see equal a i a value Of 1.2 my. K ,K3,Kl* and K13 were determined by fitting data in Fig.2-S using Equation I. ~4.K.l and Kg were determined by computer fltting nf + h s A.+s i n P?o.ci.$ ,nsjnm Emuation 111. The remalninq consrants . . ."

the principle of microscopic reversibillty. I ~.

eraction of the enzyme with its effector ligands. I" normai, resting human platelets the concentratlons Of Nd+ and Ki have been measured to be about 42 and 100 mM of plaCelec vaeer respectively (9). Upon ntimulatlon by ADP (which leads to aggregation but not secretlonl =here zs a net ytake Of Nei and effl- ux Of Kt and che COnCentraflons change to about 52 mM Na and 90 m M K+ (9.101. The effect of a change in the rat10 Of Na+ to Kt On the activity of AMP deami- nase was examined. The toral sa12 COncentratlon was kept constant at 150 nLM and varled in 10 mM steps from 0 NaCl. 150 mM KC1 to 150 mM NaC1, 0 KC1. In rhe absence of GTP the total difference ~n acrlvlfy between 0 and 150 mM NaCl was 4 fold and th15 was enhanced to 22 fold in the Presence Of 10 y M GTP (Fig. 6a-SI. Thls GTP concentration was chosen to maximise the difference between acLIvlty in NaCl and KC1. Over the range expectea for ADP Induced influx of Na+ the actlvation produced w a s 1.2 fold and 1.4 fold in the Rbsence and Dres- ellce Of GTP, respectively. S ~ n c e ADD does not induce secretion and doe5 not

."" I-_ "." "" .. ..~

NaCl 1mM)

KC1 (mM) 150 100 50 0

FIG.6-5: a. Initial velacify measured in the absence 1.1 and Pre- sence ( 0 ) of 10 U M GTP in varying mlxtuees of NaCl and KC1 at 100 )IM AMP. b. Inltial velocity measured in the presence Of 25 UM ATP and I" the absence lo) or presence 1.) of 10 uM GTP at 100 VM AM?. other detalls were an descritied l n Fig.3-S.

- - - - - - . 4 . Bergmeyer. H.U. (ed.1 Methods of Enzymatlc Analysis, 2nd. edition, 1974,

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Biochem. 19, 262-276.

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