Enzymatic properties of platelet actomyosin

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

Enzymatic MAZHAR

161, 268-274

Properties

N. MALIK,2

(1974)

of Platelet

Actomyosin’

JOEL ABRAMOWITZ, THOMAS ALFRED STRACHER

C. DETWILER,

AND

Department

of Biochemistry,

State University of New York Downstate Brooklyn, New York 1ldOd Received

October

Medical

Center,

19, 1973

The effects of various modifiers on the ATPase activity of bovine platelet actomyosin has been studied. The order of activation by monovalent cations was NHq“>> K+ > Li* > Na+. The order of activation by divalent cations was Ca2+ > Mn2+ = Srzi > Baa+> Co2’ > Mg 2+ > Zn2+. Ethylenediaminetetraacetate inhibits. Activity increased with increasing concentrations of monovalent cations, except for inhibition by increasing concentrations of NHd+ in the presence of Ca’J’ . Adenosine triphosphatase activity was increased by low concentrations of urea and trypsin, but was unaffected by low concentrations of N-ethylmaleimide. For all enzymatic properties where direct comparisons are possible, actomyosin from platelets is unlike that from skeletal muscle, but is similar to that from smooth muscle and nonmuscle sources. An about

actomyosin-like 15% of the total

protein protein

erties, platelet actomyosin differed from that from skeletal muscle and was similar to that from smooth muscle and certain nonmuscle sources. In addition, we have recently observed cooperative substrate binding by platelet actomyosin, a property not previously reported for any actomyosin system (13). We have now systematically investigated the enzymatic properties of platelet In nearly every actomyosin. propert’y studied, platelet actomyosin is unlike actomyosin from skeletal muscle, but resembles closely that from smooth muscle.

constitutes content of

human platelets. Suggestions have been made that it functions in clot retraction (1, 2), morphological changes (3), secretion (4), or aggregation (4, 5), but there is as yet no definitive

evidence

of the

exact

function

of

this protein. This contractile system is important not only for a better understanding of platelets, but also for comparative studies of contractility in muscle and nonmuscle cells. Although there have been many detailed studies of the interaction

of ATP

with

contractile

proteins

of

skeletal muscle (6-ll), little is known of this interaction with nonmuscle contractile systems. Do all these systems share a common basic mechanism, and, if so, can the current concept of the contractile mechanism

in striated

muscle

be applied

EXPERIMENTAL

to these

other contractile systems? Preliminary observations (12) suggested that in certain important enzymatic prop’ This research was supported in part by grant HL 14020 from USPHS and by a grant from the New York Heart Association. 2 Fellow of the New York Heart Association. 268 Copyright All rights

@ 1974 by Academic Press, of reproduction in any form

Inc. reserved.

PROCEDURES

Preparation of platelets. Platelets from 1 liter of freshly drawn bovine blood collected into 0.1 vol of 120 rnM NaCl and 54 mM EDTA as anticoagulant were prepared by differential centrifugation at 4°C in a swinging bucket centrifuge. Red and white cells were removed by centrifugation at 5008 for 30 min, and platelets were collected and washed twice in 150 mM NaCl, 2.5 mM EDTA, pH 7.0, by centrifuging at 12oOg for 30 min. Approximately 2 ml of a platelet pellet was obtained. Preparation oj platelet actomyosin. Actomyosin was extracted as described (14) with the modifica-

PROPJ’RTIII:S

OF PLATELET

tion that 5 mM dithiothreitol was included t,hroughout the preparation procedure. Proleitl determif&io~c. Protein concentration was determined by the modified Folin method of Lowry et al. (15). 12eactior~ oj 1~~-ethylnlnleil?lide with platelet actomyosi,l. Actomyosin (protein concentration 8.0 mg/ml) was reacted wit,h increasing amounts of ,Vethylmaleimide (in ice) in 0.6 M KCl, 0.05 M Tris-HCl buffer, pH 7.2. Aliquots of the protein were taken after intervals to measure the ATPase activity. It was observed that the reaction of Nl5M3 with the enzyme is complete in 3 hr in ice, since a comparison of the sample after 3 hr or after 24 hr reaction with NEM showed no difference in the activity of the enzyme. Elczyme assay. ATPase activity was determined in 0.085 M KC1 or 0.6 Y KC1 containing 50 mM Tris-HCl, pH 7.2, and 1 mM ATP. For the experiment.s described in Figs. 1 and 2, the cation composit)ion of the assay mixture was changed by dialyzing the enzyme against the respective salt solutions. Other conditions are described in legends. Protein concentration ranged from 0.15 to 0.5 mg/ml. After 10 or 15 min incubation at 37”C, the reaction was stopped with cold 1O7o trichloroacetic acid. The tubes were centrifuged and the inorganic phosphate in the supernatant was determined by the method of Marsh (16). Re‘actions are linear for at least 20 min. MATER

I AL8

l\T-Ethylmaleimide and trypsin were obtained from Sigma Chemical Co., St. Louis, Missouri. ATP, CTP, GTP, UTP and ITP were purchased from P-L Biochemicals, Inc. RESULTS

Idffect of cations on activity. Since different cations are known to have characteristic effects on the enzymatic activity of muscle actomyosin, we investigated the effect of various monovalent and divalent cations on the ATPase activity of platelet actomyosin. The effects of monovalent cations are shown in Fig. 1. In the absence of CaClz (Fig. IA), there is little activity with any concentration of Na+, a very slight activation by Li+, a five- to eightfold activation by K+, and a very great activation by NH,+. Thus, the sequence of activation is in the order of NHd+ >> K+ > Li+ > Na+. In t’he presence of 2.5 mu CaClz (Fig. lB), all four monovalent cations a Abbreviation

used: NEM,

Xethylmaleimide.

ACTOMYOSIN

269

activate, with maximum activity at 0.20.3 M. Comparison of Fig. 1A and B reveals that activity with Ca2+ is greater than without for each monovalent cation except XH,+., where Ca2t- actually inhibits. These contrasting effects of Ca2+ are clear in P’ig. 2, where Ca2+ is varied in the prtsclnce of either NH4+- or K+. There is lit~tlc effect of Ca2+ at low ionic strength, but at high ionic strength Ca2t- activates in the presence of KC1 and inhibits in t#he presence of NH&l. The Ca2+ inhibition of KHJ+.-activated ATPase suggests t#hat there may be a simple competit’ion between these ions. Such inhibition by Ca2f in the presence of XH4+ has also been observed for skeletal muscle myofibrillar proteins (9). The data prcscxnted in Fig. 1 and 2 differ from t,hat for skcblet8al muscle actomyosin in the following respects. (i) The Ca2+-activated ATPase activity of skeletal muscle actomyosin is inhibited by increasing ionic strength (17), whcrcxas the opposite is true in the case of platclct actomyosin. (ii) The activity of skeletal muscle actomyosin increases with an inrrcase in the concentration of CaC12 to a greater extent in low KC1 than in high KCl. In contrast, the pronounced act’ivation of platelet actomyosin by Ca2+ is observed only at high KCl, with only slight (>ffccts in low KCl. We previously reported (14) that the ATPase activity of platelet actomyosin is not activated by Mg2+, but is actually inhibited, even by very low concentrations. This is in striking contrast to skeletal which is activated muscle actomyosin, equally by Ca2+ or Mg2+. We have now investigated the effect’ of other divalent cations on the ATPase act’ivity, and the results are shown in Table I. Only Ca2+ activates at both low and high KC1 concentrations. Zn2+, Mg2+, and Co2+ inhibit, whereas Mn2+, Sr2+, and Ba2+ have lit’tle effect. EDTA also inhibits at either low or high KCl; this is also in contrast to skeletal muscle actomyosin, which is slightly activated by EDTA at low KC1 and activated about tenfold at high KC1 (18). E$ect of NEM on the ATPase activity. It has been shown that N-ethylmaleimide and other sulfhydryl reagents affect the ATPase

270

MALIK

0

0

0.2

0.4

0.6

ET AL.

0

0.2

,

,

04

0.6

[M’CI] , M

FIG. 1. Effect of monovalent cations on the ATPase activity of platelet actomyosin. The assay was as described under Experimental Procedures. (A) in the absence of added Caz+, (B) in the presence of 2.5 mM CaC12. (O,+) NH&l, (0 , 0) KCl, (A, A) LiCl, (0, q ) NaCl.

2401

FIG. 2. Effect of CaClz on the ATPase activity of platelet actomyosin in the presence of NH&l and KCl. The ATPase activities were measured as described under Experimental Procedures. (0) 0.085 M NH&I, (0) 0.085 M KCl, (0) 0.6 M NH&l, (m) 0.6 M KCl.

activity of actomyosin and myosin from other sources (6, 19, 20). It was therefore of interest to study the effect of NEM on the ATPase activity of platelet actomyosin (Fig. 3). Low concentrations of NEM do not affect’ the Ca2+-activated ATPase, but above 0.1 pmole NEM/mg protein, the activity is completely inhibited. Similar results were obtained when Ca2+ was replaced by Mg”+. These results suggest the absence of the “rate retarding” SH groups present on skeletal muscle actomyosin, which is activated by increasing concentrations of NEM. Smooth muscle actomyosin also fails to show the characteristic NEM activation (21, 32).

E$ect of urea. Urea has also been shown to have different effects on actomyosins from skeletal muscle or smooth muscle (17, 22). Figure 4 demonstrates that platelet actomyosin is first activated and then inhibited by increasing concentrations of urea, with complete inhibition at 3.0 M. This pronounced activation and inhibition has also been observed with smooth muscle actomyosin (21). Under similar conditions the Ca2+activated ATPase activity of skeletal muscle actomyosin is not affected by urea concentrations to 1.0 M and is partially inhibited by 2.0 M urea (17). Effect of trypsin. Needham and Williams

PROPERTIES

OF PLATELET

271

ACTOMYOHIX

(‘3) have shown that the Ca2+-ATPaa: activity of uterus actomyosin is increased by twatment with trypsin, while trypsin has very little effect on the activity of skeletal muscle actomyosin. Figure 5 shows that t’rypsin also activates tht Ca2+-ATPase actlvity of platelet actomyosin. Hydrolysis of various rwxleoside triphosphates. A comparison of initial rat’es of hyTABLE

I

I~JFFECTOF DIVALENT CATIONSAND EDTA ON ATP.lsls ACTIVITY OF PL.ITELET ACTOMYOSIN' Additions

Specific activity (nmole Pi/mg protein/min) 0.085 M KC1

None CaCls MgClz MnCl? ZnCls SrCle BaCle COCl? El1T.4

0.6

10 30 3 7 2 10 5 4 5

M KC1 38 100 4 40 2 35 29 17 7

(1The ATPase activities were measured as described under Experimental Procedures. Divalent cations were 2.5 IIIM, and EDTA was 1 mM.

lZO-

[Urea],

M

FIG. 4. Effect of urea on the Ca2+-ATPase act,ivity of platelet actomyosin. The reaction mixture contained 0.05 M KCl, 25 mM Tris-HCl buffer (pH 7.2), 2.5 mM ATP, 2.5 mM CaC12, and urea. ATPase activities were determined as described under Experimental Procedures.

drolysis of various nucleoside triphosphates in the presence of Ca2+ or Mg2+ is presented in Table II. In the presence of Ca2+, none of the nucleotides was hydrolyzed to an appreciable extent compared to ATP except for UTP at high KCl. In the presence of Mg2+, there was little difference in the rate of hydrolysis of the different nucleotidcs, except for UTP, which was hydrolyzed more slowly. The extremely slow rate of ITP hydrolysis by platelet actomyosin is noteworthy, since skeletal muscle actomyosin (19) hydrolyzrs ITP faster than ATP or other nucleosidc triphosphatcs. DISCUSSION

(moles

NEM/

mg protein)

x IO’

FIG. 3. The effect of S-ethylmaleimide on the Ca*+-moderated ATPase activity of platelet a(%omyosin. Treatment of platelet actomyosin with iv-ethylmaleimide and the activity measurements were as described under Experimental Procedures except that 2.5 mM CaClz was added in the assay mixture. (0) 0.085 M KCI, (0) 0.6 M KCl.

The results reported in this paper have been compared v,ith those of actomyosins from different sources (Table III). It is clear that the propcrtics of platelet actomyosin differ from those of skeletal muscle but are similar to those of smooth muscle and nonmusclc actomyosins. Because of the very low ATPase activities of platelet actomyosin in the presence of Mg2+, which is actually a strong inhibitor (Table I and Ref. 14), it seems unreasonable to consider platelet actomyosin a Mg-activated ATPase, as is skeletal muscle actomyosin. This is important to several lines of investigation. First, studies of the actin activation of myosin ATPase are done with MgATP, and t’he slight’ activations reported (24) are simply a reflection of the very low actfivit.y of platelet actomyosin with Mgz+. In-

272

MALIK

0

” 0 HO0 130

ET AL.

-I

1:10 TRY PSIN: ACTOMYOSIN

1:: RATIO

FIG. 5. Effect of digestion with trypsin on the Ca* -activated ATPase activity of platelet actomyosin. The actomyosin was digested with trypsin in a medium containing 0.6 M KC1 and 20 rnM Tris-HCl (pH 7.0), for 20 min at room temperature. The reaction was then stopped by adding trypsin inhibitor. An aliquot of the trypsin digested enzyme was used to measure ATPase activity. The conditions under which the activity was determined were as described in the legend to Fig. 4 except the KC1 concentration was 0.1 M. TABLE II SUBSTRATE SPECIFICITY OF PLATELET ACTOMYOSIN Substrate

Specific activity (nmole Pi/mg protein/min) 2.5 mM CaC12

ATP CTP GTP ITP UTP

0.085 M

0.6 M

KC1

KC1

25 8 8 9 10

100 9 6 3 70

2.5 mM MgClz 0.085 M KC1

0.6 Y KC1

4

6 7 10 7 2

5 7 4 2

deed, comparisons of activities at low ionic strength with those at high ionic strength (where actin and myosin are dissociated) raise some doubts as to whether there is any actin activation of myosin ATPase. Second, attempts to demonstrate regulation of platelet actomyosin by a Ca2+-dependent troponin-tropomyosin system similar to that in skeletal muscle involve determination of the effect of very low concentrations of Ca2+ on Mg-ATPase activity, but it is not clear that there is any significance in the observed small changes (25, 26) in the

Mg2+-inhibited activity. Thus there is no convincing evidence for a troponin-tropomyosin type of regulation of plat’clet actomyosin, and the similarities between platelet actomyosin and smooth muscle actomyosin (for which there is no evidence for troponin-tropomyosin) suggest that t’his mechanism is not operative in plat’elets. In evaluating the role of divalent cations in the function and regulation of platelet actomyosin, it is perhaps significant that on stimulation of platelet’s there is a massive flux of Ca2+ from intracellular, nonexchangcable storage sites to the extracellular phase (27, 28). The amount of Ca2+ involved, if uniformly distributed throughout the platelet, would represent an intracellular concentration of about 40 rnNi; thus, almost any local concentration of Ca2+ after stimulation is possible. Interestingly, the changes in ATPase activity between no Ca2+ and 2.5 mM Ca2+ are much more pronounced than those reported with addition of very low concentrations of Ca2+ in the presence of 2.5 mM Mg2+. The pronounced activation of the ATPase activity with r\;H4+ ions (Fig. 1) has also been observed by Kaldor et al. (9) with

PROPERTIES

OF PLATELET TABLE

I~FFECT

OF

VARIOUS MODIFIERS

273

ACTOMYOSIN

III ON ACTOMYOSIN ATPasx

ACTIVITY

Source of actomyosin Modifiers

Smooth muscle and nonmuscle

Skeletal muscle Ionic strength (I) IXvalent, cations (Low I) Nb;M (low concentration) Urea (0.5-1.5 M) Part.ial trypsin digestion Srtbst,rate analogs 15I)TA

(high I)

a The numbers

Platelet

high Z < low Z (17)” Ca2+ = Mg2+ (19)

high Z > low Z high Z > low Z (21,32) Ca2+ > Mg2+ (22, 33, 35) Ca*+ >> Mg2+

Activation

No effect

(6, 20)

Partial inhibition No effect (17)

(17)

ITP > UTP > (:TP ATP > CTP (19) Activation (18) in parentheses

indicate

Activation Activation >

REFERENCES 1. BETTICX-(;11~~~4~~, M., BND LUSCHER, E. F. (1965) Advan. Protein Chem. 20, 1. 2. PZ~I~~~~~~2~~~, 8. (1961) Thromb. Diati2. Haemorrh. 6, 517. 3. HOVIG, T. (1962) Thromb. Diath. Haemorrh. 8, 455. 4. LuscnEti, E. F., AND BETTEX-GALL~ND, M. (1971) i?z The Circulating Platelet (Johnson, S. 9., ed.), p. 225, Academic Press, New York.

(al, 22) (22, 23)

ATP > ITP > (:TP (32) Inhibition (32)

the appropriat,e

sk(>lctal muscle myosin. He suggested that NH4+ may combine with the enzyme to suppress an inhibitory group that may interact with the G-NH2 group of the ATP t’o prevent the actomyosin from exhibit,ing its full activity. It is also possible that iYH4+ ions may cause some conformational change in the protein so that the hydrolysis of ATP now occurs faster at the catalytic site. Such COW formational changes of skelet’al muscle myosin have, for example, been observed with addition of alkali metal ions (29). The data presented in this paper strongly suggest that although skeletal and platelet actomgosin have the same molecular weight, the same molecular shape (30, 31), and the same substrate, there are definite diff ercnces in their cxnzymatic properties. Whether these diffcrcnccs arc related to the structure of the artiw site or other structural aspect,s remain opc11 to question.

(34)

No effect Activation ilctivation ATP > UTP > ITP (:TP = CTP Inhibition

=

references.

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274

MALIK

W. W., ;~ND BUDLEY, L. B. (1956) Chem. 218, 653. WACHSBERGER, P., .IND KALDOR, (+. (1971) Arch. Biochem. Biophys. 143, 127. BBRANY, M., B.~R.INY, K., ~~~~~~~~~~~ E., END B.ULIN, G. (1966) Arch. Biochem. Biophys. 113, 205. NEEDH.IM, D. M., .\ND WILLIAMS, J. M. (1959) Biochem. J. 73, 171. BOOYSE, F. M., HOVEKE, T. P., .IND RAFELSON, M. E., JR. (1973) J. Bid. Chem. 248, 4083. COHEX, I. (1973) Fed. Eur. Biochem. Sot. Letters 34, 315. ALEDORT, M. L., PUSZKIN, S., PUSZIIIN, E., HANSON, J., AND K.ITZ, A. M. (1973) Biochim. Biophys. Acta 6, 411.

20. KIELLEY,

J. Biol.

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23. 24.

25. 26.

ET AL. 27. MURER, E. H., END HOLME, R. (1970) Biochim. Biophys. Acta 222, 197. 28. DETXVILER, T. C., AND FEINMSN, It. D. (1973) Biochemistry 12, 282. 29. CHEUNG, H., AND COOKE:, R. (1971) Biopolymers 10, 523. R. S., POLLARD, T. L>., .IND 30. ADELSTEIN, KUEHL, W. M. (1971) Proc. Nat. Acad. Sci. USA 68, 2703. 31. PXOBST, E:., AND LUSCHER, F. (1972) Biochim. Biophys. Ada 278, 577. 32. ADELM.IN, M. R. .\ND TIYLOR, E. W. (1969) Biochemistry 8, 4976. W. E. (1973) Eur. J. Biochem. 33,459. 33. RUSSELL, 34. Y.~M.IGUCHI, M., M~az,~w.%, Y., .\ND SEKINE, T. (1970) Biochim. Biophys. Acta 216, 411. 35. HAT~NO, S., .&ND Tj~z~~>v~~,M. (1968) Biochim. Biophys. Acta 164, 507.