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The activation of human platelet adenylate cyclase by vanadate
THROMBOSIS RESEARCH 29; 371-376, 1983 0049-3848/83/030371-06$03.00/0 Printed Copyright (c) 1983 Pergamon Press Ltd-
BRIEF
Latalk
_kjtai,
in the C;SA. All rights reserved.
COMMUNICATION
I
‘l&a
a&.
2.Z.i:.
Sir6
.>Gepatment of Ziochemistry, Ztvijs Lor5nd University Puskin u. j, ii-lC88 Budapest, Hungary (Received 4.7.1982; in revised form 18.10.1982. Accepted by Editor R. Machovich)
Over the past few years a great deal of interest has been paid to the effects of orthovanadate ions on different ATPases (1). The E2VO4 (Vi) ion, a powerful inhibitor of these enzymes is in many respects analogous to orthoghosphate, one of the products of the ATP-ase reaction. As the involvement of phosphate is not a parent in the reaction catalysed by adenylate cyclase (ACP it seems rather nuzzling that V. ions h s Seen affect this enzyme, too: marked activatzon by Vi -a detected in the AC of fat cell (2), heart tissue (3,4) membrane preparations, of pancreatic enzyme (5) rat brain (6) and of turkey erythrocyte (7). In this communication some preliminary results on the activation by ViOf human platelet AC are described. ,Thte effect of vanadate, second of its combination with prostaglandin hormones, third wit'? re&ative guanine nucleotides then combined erithboth were studied on Ihumanplatelet AC, It was found that the activation of human platelet AC by Vi is related to the presence of guanine nucleotides, thus it seems that the G?F binding protein (G-protein, activator subunit) is somehow involved in the effect of vanadate. The key words: adenylate cyclase, human Rlatelet, vanadate, GTE', GDT, prostaglandins.
371
372
HUXAN PLATELET AC AJiDVAXADATE
vo1.29, so.3
to (9,1*2). 'i‘reassay L-C activi*i ijas dete-mined accoAinG aj_>+ce contained ir; a total vol;lr;le of 1 fl I.5 ti; l~2?,C:.i)S I-1 Tris-Xl 2;; 7.6, 6 di TigGC4, C-1 EGIp.~z_~eri_ne, U-4 5I XL!, C,6 & ZT;;, 1 ndnl bovine serum albmim, 0.6-1,c' q/o1 nezkmaze srotem. '7he assay nixture vas melincubated for j xinutes &.th or without activators, and the reactios -.:asstarted ‘oy t’ne addition of 1’_‘2l?.incubations wer;? gade h dqlicates, lzhe refereace ni::ture contained heat izz.cti.vatedzeareact;ion was ltie3.r Yith t'ne (u.2 braze >roto:jl?,Yae enz oroteF11 coacentration. 3uAas to at least 15 ni;mtes incubation 20 -ore tha;l 5 ;i o? the substrate rras s_olit37 the contarni_2atj_ng X!Z?-ases.After I(;Sm.lteo of i2cu~2ati.o~at vas stopFed by boiling for j k.zuPcs. ?:Lf37co, t;le reaction determined (in duniiter coo1i.q a;ld.centrifuzation c-Z2 :-;a.~ cates) according to (11) from'the diluted su~ematazts. pz;;r,TB 2he results me sumxxrised in labie I, ;?hen increasing concentrations of Vi were tested (not SHOVED;!st.?.tistxcallg
fi,-st:-6th 160 ~2; Vi. si:AiG_ficmt activation peas o.asei-Jed nLher elLTeri_ Tllj_sco centratio of Vi was adopted in all fu-b--
rot; safe, because 'Ji nents. 4 5!0 use hi:;hel concentrat~ons,is be&ls to polperize at aromd 1 tii (A). ) zct;i-~ati,ol?iz all conGZ added alone causes a si,yaifj_caE_t ce:_tlations used, but 'ii Save a further [email protected] activation only vhen GYF concentrations >rere 1ol.q($ fl). l:le 3ost ate_restj_ng fbding imu the first zroup_,of eqer~kellts is the coab;?ed effect of GDl' and Vi: while GLJL*alone has X0 effect, \:ith‘ii it kcreases the acti-C,Q by a factor of t o+&er
about 3. FTti an as73ecific actj.vato,of all X-S ?Tn:redQ sctivated 4-1 t:lis edE%qced xtivity is mactical8 -latelet enzpe, but their ;G i;lseF_si
G1.29, No.3
H'JkW PLATELET AC AND VA?UDATE
373
(?Gsl i (?l? + Vi)kcreaSeS the H2.ree activators together of 2 relative to the roughly br a factor enzymic activity _. activity with hormone aLone. PGI2 was found to be a somewhat more potent activator, then PGEq. d nore conspicuous difference between the two prostaglandin hormones is that PGI stimulation of human platelet AC like other well is strongly de?endent on the ?GZq, G!?Phas &J_y a modest alone does not affect the PGi2 stimulated AC activity. neither is it of influence on the ?GI2+GTP stimulated system. however, t'ne sgstem containing Vi besides PG12 + (Gap shorrsa nearly twofold activation relative to the PGI2 + GDP case.
3elative activities in $ of human platelet membrane preparations in tine absence or presence of vazadate with the addition of varied agonists of the AC system.
&eat
100 #I vanadate present .absent
added
none
effect of vanadate
(100) 151 + 17 26GT 34 '1047 14 1703 2 w 957 2 5'00
1839 _t 358 1507 t 373
n-s. n-s.
10 pi-1 PGXq 10 fi Pc'iq + 10 zlI:i GTP 10 $1 PGXl + IO #I GDP
1464 + ?I9 1790 'T 261 1458 z 203
1466 2 149 2284 2 376 1758 + 250
n-s.
lci III?
1679 + 232 2333 7 2OC _-' 15027347
1553 2 210 ;!19? L 375 2!202 2 355
n.s. n-s, s.
GTP IO nI.1GTP 10 nI1 GDP IC mH Hal? I
Fti;r
IO
ti
BaIi’+lO
~$1
GW
?GIq
&I PGSS + 10 $1 GTP
10
IO UN PC17 + 10 fl GDP S
f
SO
S.
n. se = not s?&alxlc~t significant at the 5 >J’ level; Activities expressed in percentage of the activie of the same preparation without addition of any of the agents referred to the table. This basal activity varied between 20-90 pmole ck?Z/mg protein in 10 min. The values in the table are the means of results with 6-10 independent preparations. The results were evaluat;ed statistically, by the t-test, for paired comparisons (12). Because of the approximately lognormal distribution of the measured activities their 1oi;arithmic transforms were used t>rouzhout the computations. Since ue have tested the effect of tiown activators, the one tailed t-test was used.
374
E rM;LUPLATELET AC iWD VANADATE
Vo1.29, No.3
The results presented in thrs note (besides show~h,-that platelet AC can also be activated by Vij give some clue to tne problem of the mechanism of this activation. As phosphate is not a product of the AC reaction itself, Vi ion, an analogue of phosphate, cannot act directly on the catalytic unit of the AC complex. Like all well coupled AC-s (13,14,15), human platelet AC comists of three subunits (16): catalytic unit, prostaglandin hormone receptor and GTP-binding protein. The latter is a regulatory unit, which is responsible for the connection of the hormone receptor to the catalytic unit. The hydrolysis of GTP is involved in t&e regulation of the AC system. All the observations presented above suggest a close link between activation by Vi plus guanine nucleotides and the GTP hydrolysis of platelet G-protein. This idea is supported by the following details of the observations presented here: The most interesting finding is that GDP ?Ihich does not activate any AC systen in the absence of a nucleotide regenerati 'ing system causes in combination with Vi a high activation kcnether or not a hormone is added. GDP + Vi seems to play the role of GTP. In the hormone (PGZq or PGI2)-activated state linenGTP hydrolysis is highly stimulated (l7), activation by Vi is observable only upon the addition of GTP or GDP. In the absence of activating hormones, activation by V- is observed only when no or relatively low concentrations o? GTP or GDP are added. (Tie action of Vi without the addition of any exogenous GT? is not in contradiction with the assumption of a concerted action of this anion and GE?/GDP, 5'he concentration of CJPP + GDP in the medium used for enzymic activity measurement can be calculated from the 'knownnucleotide content of intact platelets (18) as roughly IO-7 wihh seerasto be high enough to maintain the G-protein GTP-ase(Xm = IO-7 -10-8j(l ) in a state charged with substrate.) Pluori 1 e causes an activation insensitive to the addition of Vi or any of the above combinations as expected since its binding site is known to be different from that of GTP, and its action does not involve the G-protein Gi!P-ase. The conclusion that Vi activation of XC is mediated somehow by G-protein is supported by some reported data. in the presence of the strongly activating nonhydrolysable GTP analog GptiXp n further ac$ig:tj,,p. could be elicited by Vi in fat A more direct supsortzng evicells (27 or 1~1. hearb a dence is that AC of S 49 lymphoma cells, deficient in Gprotein, are insensitive to the addition of Vi. Moreover vi inhibits the G-protein GTP-ase in turkey erythrocyte AC f7). Similar inhibition by Vi was observed with several ATPases (I). This inhibitions are due to the formation of an BD?. Vi. enzyme comolex (19). There are indications at least in the case of myos'=m ATP-ase, that the conformation of this "freezed-in" complex is similar to that of one of the intermediates of the ATP-ase reaction (20). it seems possible that a similar "freezed in" complex forms in the G-protein GTP-ase in the presence of Vi + GDP. It is assumed that its conformation is mimicking the activating state of G-protein.
Vo1.29, No.3
HUMAN PLATELET AC AND VASADATE
'ihemechanism of the interaction of Vi with G-protein requires further investigations which are nOPi in progress.
'.leare indebted to Dr. F. Dartha for the statistical computations (Biological Research Center of the &ngarian Academy of Sciences, Institute of ~zymolo~gy) and to Rozsa SzSke and T?&ria Katona-for excellent technicai-assistance; p~P~~;C~S 1.
*
Vanadate- a new tool for biologists.
2.: 2. SC:~!ABE u. , ?UC;_SyEIN, C., H~~i~i.X:, a., S(jC;i"JIC;, Activation of adenylate cyclase by vanadate. 'liature, a, 143-145, 1979.
K., ERDIWT, N.U.: Stimulation of 3. KRA'.:IEFZ,X., i:!X??~W, &Se human cardiac adenylate cyclase by vane-date.?aSiC. Cardiol. E, 433-437, 1980. SCEOLZ, E., and VTZ-EL, 2.: 4. SCIIKCTZ ;,.,r H&lx3ARTfrI I Effects'of.&adate on'th~'cNP system of the heart. Basic.Res.Cardiol. 2, 438-443, 1980.
5. DALUZDU@, A., DIXR‘ICK, P., IMBXR'I',PI., SVODODA, PI., CE?IS"OPHE, & J.: Vanadate stimulates the activity of pancreatic adenylate cyclase. Arch.Int,Physiol-biochim. 88, B'I27, 1980. 6. 1xtVAHX, J,: Effect of vanadate on adenylate cyclase of the rat brain. Phvs.Biochem. 2, 437-439, 19Sl. R,\!_ , SFI2GXL, A&. , AUX3MXi, G.3. : \I., DOl;fliS, 7. KR=1!jIiE'ifZ, Vanadate stimulates adenylate cyclase via the regulatory protein by a mechanism differeing from that of fluoride. Biochem.Pharm. z, 843-848, 1982.
8. EASlXki, 3.J. , Lm1iLQI, J .A.: Activation and inhibition of blood platelet adenylate cgclase by adenosine or by 2-chloroadenosine. Life Sciences, 2, 1143-1154, 1972. GFEXXX?? P.: Dopamine9. lX3BBIA?$ , J . il. , PE2ZOLD, G L -sensitive adenylate c-rclale.!n caudate nucleus of rat brain, and its similarity to the dopamine receptor. ~roc.rcatl.Acad.Sci.USA. a, 2145-2149, 1972. CO_~;Iy~_, k__C,, mDADl-_Qr, J.::., pzTZOL3, G.L,, P . : Dopamine-sensitive adenylate cyClase in mammalian brain: a possible site of action of anti,g;hotio drugs. Proc,Batl.Acad.Sci.USA. 21, 1113-1117, .
IO. Cz;D:T
GRXXGlS3l,
375
376
HUMAN PLATELET AC AND VAXADATE
b-01.29,so.3
LY%_J, “0"‘ T ?? 12. ._l --.-i. , ZCZZ, P.:;.:, Yreenzn, zd Co. San Francisco, 13.
BODXLL , 21. : T& role of homone receptors and CXJBregulatory proteins in nembranc transductions, Xature, 17-22, 1980. s,
14. 2033, XJI., GIIJLW, X-G,: Biochemical properties of hormone sensitive adenylate cyclase. .kn,Eev.i3iochera. 3, 533-564,
1980.
15. LIMDIRD, L-2.: Activation and attenuation of adenylate
role of GTP-binding proteins as macronocyclase. 'TIhe lecular messengers in receptor-cyclase coupling. 3iochem.J. B, I-13, 1981, 1r3.
:sTzLy,
fi,L. , rlOOD, A. : Regulation of human platelet adenylate cyclase by epinephrin, PG3q and guanine nucleotides. 3vidence for separate gwnine nucleotide sites mediating stimulation and inhibition. J.Biol.Chem. e, 10791-10797, 1979.
17. LXX'EE,
EA., S!XZiZ, ti.L,, LZVITZXI, A.: Prostaglandin stinulated GTP hydrolysis associated with activation of adenylate cyclase in human platelet nembranes, Proc.Xat. .kad,Sci.USA. 22, 719-723, 1982.
18. PRO%, S-X., KLEIN, T.\!. and FISIIEL, C,';i,: Ribonucleoside and iiucleotide Components of Normal Human 3ood Lymphocytes and Platelets. Proc. of the Societg for Experimental Diologv and I'ledicine, z, $8-512, 1977. 19. GOODBO, C.C.: Inhibition of myosin ATP-ase by vanadate ion.Proc.iIQ" u~l.Acad.Sci,USA. & 2620-2624, 1979.
IC SZILAGYI, L. and BIRO, X.X.A.: Study of the 20. XTAI struckrl'of IIkiIk vanadate complex, FEDS Lett. 1y1, 74-77,
1982.
BRIEF
Latalk
_kjtai,
in the C;SA. All rights reserved.
COMMUNICATION
I
‘l&a
a&.
2.Z.i:.
Sir6
.>Gepatment of Ziochemistry, Ztvijs Lor5nd University Puskin u. j, ii-lC88 Budapest, Hungary (Received 4.7.1982; in revised form 18.10.1982. Accepted by Editor R. Machovich)
Over the past few years a great deal of interest has been paid to the effects of orthovanadate ions on different ATPases (1). The E2VO4 (Vi) ion, a powerful inhibitor of these enzymes is in many respects analogous to orthoghosphate, one of the products of the ATP-ase reaction. As the involvement of phosphate is not a parent in the reaction catalysed by adenylate cyclase (ACP it seems rather nuzzling that V. ions h s Seen affect this enzyme, too: marked activatzon by Vi -a detected in the AC of fat cell (2), heart tissue (3,4) membrane preparations, of pancreatic enzyme (5) rat brain (6) and of turkey erythrocyte (7). In this communication some preliminary results on the activation by ViOf human platelet AC are described. ,Thte effect of vanadate, second of its combination with prostaglandin hormones, third wit'? re&ative guanine nucleotides then combined erithboth were studied on Ihumanplatelet AC, It was found that the activation of human platelet AC by Vi is related to the presence of guanine nucleotides, thus it seems that the G?F binding protein (G-protein, activator subunit) is somehow involved in the effect of vanadate. The key words: adenylate cyclase, human Rlatelet, vanadate, GTE', GDT, prostaglandins.
371
372
HUXAN PLATELET AC AJiDVAXADATE
vo1.29, so.3
to (9,1*2). 'i‘reassay L-C activi*i ijas dete-mined accoAinG aj_>+ce contained ir; a total vol;lr;le of 1 fl I.5 ti; l~2?,C:.i)S I-1 Tris-Xl 2;; 7.6, 6 di TigGC4, C-1 EGIp.~z_~eri_ne, U-4 5I XL!, C,6 & ZT;;, 1 ndnl bovine serum albmim, 0.6-1,c' q/o1 nezkmaze srotem. '7he assay nixture vas melincubated for j xinutes &.th or without activators, and the reactios -.:asstarted ‘oy t’ne addition of 1’_‘2l?.incubations wer;? gade h dqlicates, lzhe refereace ni::ture contained heat izz.cti.vatedzeareact;ion was ltie3.r Yith t'ne (u.2 braze >roto:jl?,Yae enz oroteF11 coacentration. 3uAas to at least 15 ni;mtes incubation 20 -ore tha;l 5 ;i o? the substrate rras s_olit37 the contarni_2atj_ng X!Z?-ases.After I(;Sm.lteo of i2cu~2ati.o~at vas stopFed by boiling for j k.zuPcs. ?:Lf37co, t;le reaction determined (in duniiter coo1i.q a;ld.centrifuzation c-Z2 :-;a.~ cates) according to (11) from'the diluted su~ematazts. pz;;r,TB 2he results me sumxxrised in labie I, ;?hen increasing concentrations of Vi were tested (not SHOVED;!st.?.tistxcallg
fi,-st:-6th 160 ~2; Vi. si:AiG_ficmt activation peas o.asei-Jed nLher elLTeri_ Tllj_sco centratio of Vi was adopted in all fu-b--
rot; safe, because 'Ji nents. 4 5!0 use hi:;hel concentrat~ons,is be&ls to polperize at aromd 1 tii (A). ) zct;i-~ati,ol?iz all conGZ added alone causes a si,yaifj_caE_t ce:_tlations used, but 'ii Save a further [email protected] activation only vhen GYF concentrations >rere 1ol.q($ fl). l:le 3ost ate_restj_ng fbding imu the first zroup_,of eqer~kellts is the coab;?ed effect of GDl' and Vi: while GLJL*alone has X0 effect, \:ith‘ii it kcreases the acti-C,Q by a factor of t o+&er
about 3. FTti an as73ecific actj.vato,of all X-S ?Tn:redQ sctivated 4-1 t:lis edE%qced xtivity is mactical8 -latelet enzpe, but their ;G i;lseF_si
G1.29, No.3
H'JkW PLATELET AC AND VA?UDATE
373
(?Gsl i (?l? + Vi)kcreaSeS the H2.ree activators together of 2 relative to the roughly br a factor enzymic activity _. activity with hormone aLone. PGI2 was found to be a somewhat more potent activator, then PGEq. d nore conspicuous difference between the two prostaglandin hormones is that PGI stimulation of human platelet AC like other well is strongly de?endent on the ?GZq, G!?Phas &J_y a modest alone does not affect the PGi2 stimulated AC activity. neither is it of influence on the ?GI2+GTP stimulated system. however, t'ne sgstem containing Vi besides PG12 + (Gap shorrsa nearly twofold activation relative to the PGI2 + GDP case.
3elative activities in $ of human platelet membrane preparations in tine absence or presence of vazadate with the addition of varied agonists of the AC system.
&eat
100 #I vanadate present .absent
added
none
effect of vanadate
(100) 151 + 17 26GT 34 '1047 14 1703 2 w 957 2 5'00
1839 _t 358 1507 t 373
n-s. n-s.
10 pi-1 PGXq 10 fi Pc'iq + 10 zlI:i GTP 10 $1 PGXl + IO #I GDP
1464 + ?I9 1790 'T 261 1458 z 203
1466 2 149 2284 2 376 1758 + 250
n-s.
lci III?
1679 + 232 2333 7 2OC _-' 15027347
1553 2 210 ;!19? L 375 2!202 2 355
n.s. n-s, s.
GTP IO nI.1GTP 10 nI1 GDP IC mH Hal? I
Fti;r
IO
ti
BaIi’+lO
~$1
GW
?GIq
&I PGSS + 10 $1 GTP
10
IO UN PC17 + 10 fl GDP S
f
SO
S.
n. se = not s?&alxlc~t significant at the 5 >J’ level; Activities expressed in percentage of the activie of the same preparation without addition of any of the agents referred to the table. This basal activity varied between 20-90 pmole ck?Z/mg protein in 10 min. The values in the table are the means of results with 6-10 independent preparations. The results were evaluat;ed statistically, by the t-test, for paired comparisons (12). Because of the approximately lognormal distribution of the measured activities their 1oi;arithmic transforms were used t>rouzhout the computations. Since ue have tested the effect of tiown activators, the one tailed t-test was used.
374
E rM;LUPLATELET AC iWD VANADATE
Vo1.29, No.3
The results presented in thrs note (besides show~h,-that platelet AC can also be activated by Vij give some clue to tne problem of the mechanism of this activation. As phosphate is not a product of the AC reaction itself, Vi ion, an analogue of phosphate, cannot act directly on the catalytic unit of the AC complex. Like all well coupled AC-s (13,14,15), human platelet AC comists of three subunits (16): catalytic unit, prostaglandin hormone receptor and GTP-binding protein. The latter is a regulatory unit, which is responsible for the connection of the hormone receptor to the catalytic unit. The hydrolysis of GTP is involved in t&e regulation of the AC system. All the observations presented above suggest a close link between activation by Vi plus guanine nucleotides and the GTP hydrolysis of platelet G-protein. This idea is supported by the following details of the observations presented here: The most interesting finding is that GDP ?Ihich does not activate any AC systen in the absence of a nucleotide regenerati 'ing system causes in combination with Vi a high activation kcnether or not a hormone is added. GDP + Vi seems to play the role of GTP. In the hormone (PGZq or PGI2)-activated state linenGTP hydrolysis is highly stimulated (l7), activation by Vi is observable only upon the addition of GTP or GDP. In the absence of activating hormones, activation by V- is observed only when no or relatively low concentrations o? GTP or GDP are added. (Tie action of Vi without the addition of any exogenous GT? is not in contradiction with the assumption of a concerted action of this anion and GE?/GDP, 5'he concentration of CJPP + GDP in the medium used for enzymic activity measurement can be calculated from the 'knownnucleotide content of intact platelets (18) as roughly IO-7 wihh seerasto be high enough to maintain the G-protein GTP-ase(Xm = IO-7 -10-8j(l ) in a state charged with substrate.) Pluori 1 e causes an activation insensitive to the addition of Vi or any of the above combinations as expected since its binding site is known to be different from that of GTP, and its action does not involve the G-protein Gi!P-ase. The conclusion that Vi activation of XC is mediated somehow by G-protein is supported by some reported data. in the presence of the strongly activating nonhydrolysable GTP analog GptiXp n further ac$ig:tj,,p. could be elicited by Vi in fat A more direct supsortzng evicells (27 or 1~1. hearb a dence is that AC of S 49 lymphoma cells, deficient in Gprotein, are insensitive to the addition of Vi. Moreover vi inhibits the G-protein GTP-ase in turkey erythrocyte AC f7). Similar inhibition by Vi was observed with several ATPases (I). This inhibitions are due to the formation of an BD?. Vi. enzyme comolex (19). There are indications at least in the case of myos'=m ATP-ase, that the conformation of this "freezed-in" complex is similar to that of one of the intermediates of the ATP-ase reaction (20). it seems possible that a similar "freezed in" complex forms in the G-protein GTP-ase in the presence of Vi + GDP. It is assumed that its conformation is mimicking the activating state of G-protein.
Vo1.29, No.3
HUMAN PLATELET AC AND VASADATE
'ihemechanism of the interaction of Vi with G-protein requires further investigations which are nOPi in progress.
'.leare indebted to Dr. F. Dartha for the statistical computations (Biological Research Center of the &ngarian Academy of Sciences, Institute of ~zymolo~gy) and to Rozsa SzSke and T?&ria Katona-for excellent technicai-assistance; p~P~~;C~S 1.
*
Vanadate- a new tool for biologists.
2.: 2. SC:~!ABE u. , ?UC;_SyEIN, C., H~~i~i.X:, a., S(jC;i"JIC;, Activation of adenylate cyclase by vanadate. 'liature, a, 143-145, 1979.
K., ERDIWT, N.U.: Stimulation of 3. KRA'.:IEFZ,X., i:!X??~W, &Se human cardiac adenylate cyclase by vane-date.?aSiC. Cardiol. E, 433-437, 1980. SCEOLZ, E., and VTZ-EL, 2.: 4. SCIIKCTZ ;,.,r H&lx3ARTfrI I Effects'of.&adate on'th~'cNP system of the heart. Basic.Res.Cardiol. 2, 438-443, 1980.
5. DALUZDU@, A., DIXR‘ICK, P., IMBXR'I',PI., SVODODA, PI., CE?IS"OPHE, & J.: Vanadate stimulates the activity of pancreatic adenylate cyclase. Arch.Int,Physiol-biochim. 88, B'I27, 1980. 6. 1xtVAHX, J,: Effect of vanadate on adenylate cyclase of the rat brain. Phvs.Biochem. 2, 437-439, 19Sl. R,\!_ , SFI2GXL, A&. , AUX3MXi, G.3. : \I., DOl;fliS, 7. KR=1!jIiE'ifZ, Vanadate stimulates adenylate cyclase via the regulatory protein by a mechanism differeing from that of fluoride. Biochem.Pharm. z, 843-848, 1982.
8. EASlXki, 3.J. , Lm1iLQI, J .A.: Activation and inhibition of blood platelet adenylate cgclase by adenosine or by 2-chloroadenosine. Life Sciences, 2, 1143-1154, 1972. GFEXXX?? P.: Dopamine9. lX3BBIA?$ , J . il. , PE2ZOLD, G L -sensitive adenylate c-rclale.!n caudate nucleus of rat brain, and its similarity to the dopamine receptor. ~roc.rcatl.Acad.Sci.USA. a, 2145-2149, 1972. CO_~;Iy~_, k__C,, mDADl-_Qr, J.::., pzTZOL3, G.L,, P . : Dopamine-sensitive adenylate cyClase in mammalian brain: a possible site of action of anti,g;hotio drugs. Proc,Batl.Acad.Sci.USA. 21, 1113-1117, .
IO. Cz;D:T
GRXXGlS3l,
375
376
HUMAN PLATELET AC AND VAXADATE
b-01.29,so.3
LY%_J, “0"‘ T ?? 12. ._l --.-i. , ZCZZ, P.:;.:, Yreenzn, zd Co. San Francisco, 13.
BODXLL , 21. : T& role of homone receptors and CXJBregulatory proteins in nembranc transductions, Xature, 17-22, 1980. s,
14. 2033, XJI., GIIJLW, X-G,: Biochemical properties of hormone sensitive adenylate cyclase. .kn,Eev.i3iochera. 3, 533-564,
1980.
15. LIMDIRD, L-2.: Activation and attenuation of adenylate
role of GTP-binding proteins as macronocyclase. 'TIhe lecular messengers in receptor-cyclase coupling. 3iochem.J. B, I-13, 1981, 1r3.
:sTzLy,
fi,L. , rlOOD, A. : Regulation of human platelet adenylate cyclase by epinephrin, PG3q and guanine nucleotides. 3vidence for separate gwnine nucleotide sites mediating stimulation and inhibition. J.Biol.Chem. e, 10791-10797, 1979.
17. LXX'EE,
EA., S!XZiZ, ti.L,, LZVITZXI, A.: Prostaglandin stinulated GTP hydrolysis associated with activation of adenylate cyclase in human platelet nembranes, Proc.Xat. .kad,Sci.USA. 22, 719-723, 1982.
18. PRO%, S-X., KLEIN, T.\!. and FISIIEL, C,';i,: Ribonucleoside and iiucleotide Components of Normal Human 3ood Lymphocytes and Platelets. Proc. of the Societg for Experimental Diologv and I'ledicine, z, $8-512, 1977. 19. GOODBO, C.C.: Inhibition of myosin ATP-ase by vanadate ion.Proc.iIQ" u~l.Acad.Sci,USA. & 2620-2624, 1979.
IC SZILAGYI, L. and BIRO, X.X.A.: Study of the 20. XTAI struckrl'of IIkiIk vanadate complex, FEDS Lett. 1y1, 74-77,
1982.