cgmp-stimulated protein kinase phosphorylates pyruvate kinase in an anoxia-tolerant marine mollusc

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1 J Cmp Physil B (1990) 160: Jurnal f Cmparative B~..,~,. Systemic, and Envirn- Physilgy B melltal Physilgy 9 Springer-Verlag 1990 cgmp-stimulated prtein kinase phsphrylates pyruvate kinase in an anxia-tlerant marine mllusc Stephen P.J. Brks and Kenneth B. Strey* Institute f Bichemistry and Department f Bilgy, Carletn University, Ottawa, Ontari K1S 5B6, Canada Accepted April 3, 1990 Summary. A cystslic prtein kinase that phsphrylates pyruvate kinase (PK) in vitr has been identified in crude hmgenates f heart, radular retractr, and ft muscle frm the anxia-tlerant marine whelk Busycn eanaliculatum. Prtein kinase actin was measured by fllwing changes in PK kinetic parameters: phsphrylated PK has a higher K,, value fr phsphenlpyruvate and a lwer 15 value fr L-alanine. The crude prtein kinase readily phsphrylated PK in a Mg 2+and ATP-dependent manner in the absence f any added effectr. This activity was nt affected by the additin f either camp (a stimulatr f prtein kinase A) r Ca 2 + plus phrbl 12-myristate 13-acetate (stimulatrs f prtein kinase C) t the incubatin medium. Additin f cgmp t the hmgenate, hwever, increased the rate f PK phsphrylatin giving a 3-4-fld increase in the rate f change in PK kinetic parameters that was readily apparent after 5 h. Cmplete time-curses f changes in PK kinetic parameters in the presence and absence f cgmp shwed that cgmp increased the rate, but nt the final extent, f PK phsphrylatin. These results indicate that PK inactivatin by enzyme phsphrylatin in respnse t anxia in whelk tissues may be mediated by a cyclic GMP stimulated prtein kinase in respnse t changing levels f cgmp. This cnclusin was further supprted by data indicating that the ttal activity f prtein kinase was the same in bth anxic and aerbic animals, and that the ttal PK phsphatase activity was als cnstant. Changes in PK phsphrylatin during anxia are nt, therefre, the result f changes in the ttal amunt f prtein kinase r phsphatase. * T whm ffprint requests shuld be sent Abbreviatins: camp adensine 3':5'-mnphsphate; egmp guansine 3' : 5'-mnphsphate; PK pyruvate kinase; PMA phrbl 12-myristate 13-acetate; PEP phsphenlpyruvate; Km Michaelis cnstant; Is0 inhibitr cncentratin that reduces enzyme activity by 50% Key wrds: Anaerbisis - Pyruvate kinase - cgmpdependent prtein kinase - Marine mlluscs - Reversible phsphrylatin cntrl f enzymes Intrductin Many marine mlluscs have the ability t survive extended perids f time in the absence f envirnmental xygen (Hchachka 1980). In the anxic state, the survival f these facultative anaerbes depends n bichemical adaptatins that rerganize metablic prcesses s that energy prductin balances energy demands ver the lng term with limited accumulatin f txic end prducts (de Zwaan 1983; Livingstne and de Zwaan 1983 ; Strey 1985). A central part f this re-rganizatin is the ability t depress metablic rates t values that are nly 5-10% f the nrmxic rates (de Zwaan and Wijsman 1976; Shick et al. 1983; Strey 1988a). The verall prcess by which facultative anaerbes crdinate metablic depressin is nt well understd, althugh the end results f this prcess are easily identified in marine invertebrates. Three cntrl mechanisms have been identified in the whelk Busycn canaliculatum that act t regulate glyclytic flux in the anxic state: (1) cvalent mdificatin f regulatry enzymes via reversible prtein phsphrylatin (affecting glycgen phsphrylase, 6-phsphfruct-l-kinase, 6-phsphfruct-2-kinase, and pyruvate kinase; Strey 1984, 1988 b; Plaxtn and Strey 1984, 1985; Bsca and Strey 1990); (2) changes in the subcellular lcatin f enzymes via reversible binding t macrmlecular structures (Plaxtn and Strey 1986); and (3) fructse 2,6-bisphsphate regulatin f 6-phsphfruct-l-kinase t cntrl the bisynthetic use f carbhydrate reserves (Strey 1988b). Cntrl ver glyclytic rate is particularly imprtant in facultative anaerbes since carbhydrate fermentatin is the primary means f ATP synthesis during anxia.

2 310 S.P.J. Brks and K.B. Strey: Prteinkinase phsphrylates pyruvate kinase Studies n whelk tissues have clearly demnstrated that fr pyruvate kinase (PK) the changes in kinetic parameters bserved during anxia result frm changes in the degree f enzyme phsphrylatin; bth the anxia-stimulated incrpratin f 32p int PK, and the actin f exgenus phsphatases in recnverting the anxic enzyme frm t the aerbic frm, cnfirm this (Plaxtn and Strey 1984). Changes in glycgen phsphrylase activity and in 6-phsphfruct-l-kinase and 6-phsphfruct-2-kinase kinetic parameters are als knwn t be caused by changes in the phsphrylatin state f the enzyme in mammalian systems (Chen 1980; Sale and Dentn 1983; Kemp and Fe 1983). In mammalian rgans, the phsphrylatin f phsphfructkinase, PK, and glycgen phsphrylase can be initiated and crdinated thrugh the additin f specific hrmnes that act by changing the cncentratin f secnd messengers (mst ntably camp and Ca 2 + ; see Chen 1980; Sale and Dentn 1985; Kagimt and Uyeda 1979; Claus et al. 1979; Feliu et al. 1976). Available evidence suggests, therefre, that the crdinated cntrl f regulatry enzymes f glyclysis in marine facultative anaerbes is prbably achieved via changes in the activity f either prtein kinases r phsphprtein phsphatases during the transitin t the anxic state. Preliminary tests f this hypthesis used cmmn prtein kinase activatrs such as dibutyryl camp (Krebs and Beav 1979; Pallen et al. 1987) r Ca 2 (with the calcium inphre A23187) plus phrbl 12-myristate 13-acetate (Cnnelly et al. 1987; Mieskes et al. 1987) in an attempt t mimic anxia-induced changes in viv, but failed t detect changes in either PK activity, 6-phsphfruct-1- kinase activity, r fructse 2,6-bisphsphate cncentratins in islated whelk tissues (Brks and Strey 1989). The study did, hwever, demnstrate the existence f a stable endgenus prtein kinase activity which culd phsphrylate PK. The present study analyzed the nature f the prcesses regulating PK phsphrylatin in whelk tissues. The activities f prtein kinases and phsphatases were analyzed in anxic and aerbic tissues and the secndmessenger-dependence f prtein kinase activity was identified. Materials and methds Animals and chemicals. Whelks (Busycn canaliculatum) were btained frm the Marine Bilgical Labratry, Wds Hle, Mass., and were held in recirculating (aerated) sea water (1000 mosm) at ~ withut feeding until use. Cntrl animals were sampled directly frm the tank. Anxia was impsed accrding t Plaxtn and Strey (1986). Briefly, animals were placed in a large pail cntaining sea water (previusly bubbled with N2 gas fr 12 h) fr 16 h prir t sampling. Animals were held at ~ during the expsure t anxic cnditins. Alt bichemicals were purchased frm Sigma Chemical C. (St. Luis, MO) and were f the highest purity available. Pyruvate kinase assays. The kinetic parameters f PK were determined by cupling the reactin t excess lactate dehydrgenase and mnitring the xidatin f NADH at 340 nm using a Gilfrd mdel 240 spectrphtmeter. The assay was perfrmed by varying phsphenlpyruvate (PEP) r L-alanine cncentratins in 50 mm imidazle HCl-buffer (ph 7.0) cntaining 50 mm KCI, 5 mm MgC12, 1 mm ADP, and 0.15 mm NADH at 22 ~ The I5 values fr L-alanine were determined in the presence f 0.2 mm PEP. Phsphrylatin fpyruvate kinase. Whelk tissues (radular retractr, ft, r ventricle) frm either aerbic r anxic animats were hmgenized 1:2 (w/v) in ice-cld 50 mm imidazle, ph 7.0, 100 mm NaF, 25% (v/v) glycerl, 5 mm EDTA, 5 mm EGTA, 10 mm fl-mercaptethanl (Buffer A) cntaining 0.1 mm phenylmethylsulfnyl fluride using a Plytrn PT 10 hmgenizer. Hmgenates were then centrifuged at x g fr 10 min, and the supernatant was desalted by centrifugatin thrugh a Sephadex G25 "spun" clumn (Helmerhrst and Stkes 1980) pre-equilibrated in 50 mm imidazle, ph 7.0, 25% (v/v) glycerl, 10 mm /~mercaptethanl, 0.1 mm EDTA, 10 mm ptassium phsphate (Buffer B). Fr the analysis f prtein kinase activity in extracts frm anxic whelks, enzyme hmgenates were first treated t dephsphrylate the endgenus PK; 300 gl supernatant was incubated vernight at 20 ~ in the presence f 15 mm MgC12, and then desalted prir t use. A 40-gl aliqut f desalted hmgenate (either incubated vernight (anxic) r freshly prepared (aerbic)) was added t 80 ~1 Buffer B, and the apprpriate effectrs where then added t give a final ttal vlume f 150 gl. All tubes fr phsphrylatin time-curse determinatins cntained 30 mm NaF t inhibit prtein phsphatases (ptimal cncentratin under ur cnditins) and 10 mm ATP_ Experimental tubes als cntained ther effectrs as nted in individual figure and table legends. Reactins were incubated fr specified times at 30 ~ and stpped by immersin in liquid N2. Samples were stred at - 80 ~ (fr nt lnger than 4 d) until assayed fr K,, r Is values. Strage at -80 ~ did nt affect the maximal PK activity, the K,, r I5 values. Samples were desalted by centrifugatin thrugh "spun clumns" immediately prir t assay. Dephsphrylatin f pyruvate kinase. Dephsphrylatin time curses fr PK were measured in desalted extracts frm either cntrl r anxic animals. This prcedure relied n endgenus phsphatases t alter the PK kinetic cnstants. In rder t measure dephsphrylatin in aerbic animal hmgenates, a phsphrylatin experimental tube was set up cntaining 40 gl desalted extract, 80 gl Buffer B, 15 mm MgC12, and 10 mm ATP (ttal vlume= 150 gl). This mixture was incubated fr 16 h at 30 ~ t phsphrylate PK, and then desalted t remve Mg 2+ and ATP. Dephsphrylatin f PK was then initiated by adding 4.5 gl 0.5 M MgC12 (15 mm MgC12 final cncentratin) t 150 gl f this eluate. Samples were remved at specified time pints and the reactin stpped by freezing at -80 ~ The PK kinetic cnstants were determined frm desalted samples thawed immediately prir t assay. Dephsphrylatin time-curses f PK frm anxic animals were measured by preparing PK-entaining hmgenates frm anxic animals essentially as described fr cntrl animal extracts. Tissues frm freshly killed animals were hmgenized 1 : 2 in Buffer A and centrifuged fr 10 min at x g. The supernatant was then desalted thrugh a Sephadex G-25 "spun clumn" previusly equilibrated in Buffer B. A 40 gl aliqut f eluant was incubated with 80 gl Buffer B cntaining 15 mm MgC12 in a ttal vlume f 150 gl fr defined perids as described abve. Dephsphrylatin time-curses were fitted t a mdified Hill equatin which served as a general relatinship between the initial Km values (K,, p, the K~ value fr the phsphrylated enzyme), the final Km values (K,, dep, the K,, value fr the dephsphrylated enzyme), the half time fr enzyme phsphrylatin (t.5), the time f incubatin (t), the Hill cefficient (h) and the K,, value bserved at any time t (Km~ K,, ~ = K,~p - ( K, V -- K,,aeP) * th / ( t. 5 ~ + t h) (1) Equatin 1 is a generalized equatin fr changes in the kinetic patterns f multi-subunit enzymes and as such is nt intended as a specific slutin t a detailed phsphrylatin mechanism.

3 S.P.J. Brks and K.B. Strey: Prteinkinase phsphrylates pyruvate kinase 311 The slutin t Eq. I gives the midpints f the dephsphrylatin time curses (t.s) as well as the Kz values fr phsphrylated and nn-phsphrylated PK. Results Phsphrylatin f pyruvate kinase by endgenus prtein kinases The data f Figs. 1 and 2 demnstrate that whelk hmgenates cntain an endgenus prtein kinase activity that can alter PK kinetic cnstants under apprpriate cnditins. This is shwn by the dependence f changes in the Km fr PEP (Fig. 1) and the I5 fr L-alanine (Fig. 2) n the presence f bth ATP and Mg 2 ins in the incubating medium. Thus, extracts f whelk radu i i i l i RADULA A lar retractr, ft, and ventricle shwed n changes in kinetic parameters when incubated in the presence f ATP plus EDTA r in the absence f ATP. In the presence f bth ATP and Mg 2+, hwever, the Km values fr PEP increased and the I5 values fr L-alanine decreased with increasing length f incubatin until a new, cnstant, value was btained. These new final values (bserved after 24 h f incubatin) agree with previus values frm whelk muscle tissues btained by hlding the whle animal under anxic cnditins fr 21 h (Plaxtn and Strey 1985; Whitwam and Strey 1990) and with changes bserved in radular retractr muscle PK during in vitr incubatins with exgenus E. cli phsphatase (Plaxtn and Strey 1984). This suggests that phsphrylatin by endgenus prtein kinases in vitr is a gd mdel system fr studying changes bserved during anxia in viv. Althugh the curves f Figs. I and 2 were btained using tissue hmgenates frm aerbic animals, identical curves were als btained when the enzyme system in tissue hmgenates f anxic animals were tested. PK in these preparatins was first dephsphrylated by vernight incubatin f the hmgenate t allw the actin f endgenus phsphatases. When phsphrylatin was then stimulated by the additin f ATP and / 1.0 I I I I." I FOOT B 1 t /! I l I I ~ I r - - A // e~ /,, I i Jl i VENTRICLE C I! FOOT B 1.0 ///~/-'~ / / 1 _ ~ ' ~ ~ ~.r =~ t!.-. /0 i i i m tl m time (hurs) Fig. IA-C. PK phsphrylatin time-curse: effect f cgmp n the PEP K,, value fr PK frm radular retractr muscle A, ft muscle B, and ventricle C. K,. values fr PEP (in ram) were measured after 2, 4, 8, 16, and 24 h f incubatin under ne f three cnditins: /,, plus 20raM EDTA; O, plus 15mM MgC12; 9 plus 0.4 mm cgmp and 15 mm MgC12. All tubes cntained 10 mm ATP. All curves were determined in tissue hmgenates frm aerbic animals. I i i 1 # i time (hurs) Fig. 2A, B. PK phsphrylatin time-curse: effect f cgmp n the I5 value fr L-alanine fr PK frm radular retractr muscle A r ft B. Is values (in mm) fr L-alanine were measured after 2, 4, 8, 16, and 24 h f incubatin under ne f three cnditins: zx, plus 20 mm EDTA;, plus 15 mm MgC12; 9 plus 0.4 mm egmp and 15 mm MgCI2. All tubes cntained 10 mm ATP. All curves were determined in tissue hmgenates frm aerbic animals

4 312 S.P.J. Brks and K.B. Strey: Prteinkinase phsphrylates pyruvate kinase Table 1. Effect f camp, cgmp, and Ca 2+ plus PMA n PK phsphrylatin in vitr. Values are mean +- SEM (n = 3). Samples were incubated at 30 ~ fr 5 h befre assay. All tubes cntained 10 mm ATP and 15 mm MgC12. Other additins were either: 0.4 mm camp, 0.4 mm cgmp, r 2 mm CaCI2 plus 40 gg.m1-1 phrbl 12-myristate 13-acetate (Ca PMA) Tissue Experimental cnditin Km PEP I5 L-alanine (ram) (ram) Radular N secnd messenger retractr + camp _ cgmp " 2.5_+1.5" +CaZ+ +PMA 0.052_ _+8.9 +camp+ca2+ + PMA 0.071_ _+6.9 +cgmp+ca 2+ +PMA _+4.& Ft N secnd messenger _ camp cgmp 0.325_ a " +Ca 2+ +PMA _2.9 +camp+_ca 2+ +PMA _2.3 +cgmp+ca2+ +PMA _ _+3.2 Ventricle N secnd messenger 2.65 N.D. +camp _0.14 N.D. +cgmp 3.78 _+1.02 ~ N.D. +Ca2+ +PMA 0.83 _+0.12" N.D. +camp+ca 2+ +PMA 0.92 _+0.26 a N.D. +cgmp+ca 2++PMA N.D. a Significantly different frm cnditin withut added secnd messenger as determined by the Paired Student's t-test at the P<0.05 level. N.D. nt determined Mg 2+ (with r withut effectrs), the same respnse f PK was fund (data nt shwn). The effect f varius prtein kinase secnd messengers n the extent f PK phsphrylatin is presented in Table 1. These measurements were made after 5 h incubatin at 30 ~ because the data f Figs. 1 and 2 indicated this time wuld shw a maximal difference between cntrl and experimental values; the kinetic cnstants were just beginning t change after 5 h f incubatin. Three different types f secnd messengers were used: camp (activates prtein kinase A; Krebs and Beav 1979; Pallen et al. 1987); cgmp (activates cgmp activated prtein kinase; Lincln 1983); and Ca 2 + plus phrbl 12-myristate 13-acetate (activates prtein kinase C and calmdulin dependent prtein kinase; Cnnelly et al. 1987; Mieskes et al. 1987). The effect f adding each cmpund separately, r in cmbinatin, t the desalted PK-cntaining supernatants is shwn in Table 1. It is apparent frm Table 1 that neither camp alne, nr Ca 2+ plus phrbl 12-myristate 13-acetate (PMA), nr camp plus Ca 2 + plus PMA, had any effect n the extent f PK phsphrylatin in radular retractr r ft muscle after 5 h f incubatin. In ventricle, hwever, supernatants incubated with either Ca 2+ +PMA alne, r camp plus Ca PMA, shwed significantly lwer Km values fr PEP. This latter result is ppsite frm that expected fr an activatin f prtein kinase. The effect f cgmp n PK kinetic parameters is als presented in Table 1. In cntrast t the actins f the ther secnd messengers, the additin f cgmp t PK-cntaining supernatants resulted in a significant increase in the Km value fr PEP, and a decrease in the I5 value fr L-alanine, in all three muscle tissues assayed. This effect was ablished when Ca z + + PMA was added t the cgmp-cntaining incubatins; the cgmp+caz++pma samples were nt significantly different frm the cntrl tubes except fr the L-alanine Is value fr radular retractr muscle PK. The data f Table 1 shw that the additin f cgmp significantly altered the PK kinetic parameters measured after 5 h f incubatin. The data f Figs. I and 2 shw that this effect was due t an enhanced rate f PK phsphrylatin and nt t an increase in the verall extent f PK phsphrylatin. This was demnstrated by the fact that cgmp affected nly PK kinetic cnstants measured befre 12 h f incubatin. After this time, the presence f cgmp had n effect n the measured PK kinetic cnstants. The cgmp effect was bserved in all three muscle tissues examined; cgmp increased the rate f change f bth the Km fr PEP and the I5 fr L-alanine (pen circles, Figs. 1 and 2). A cmparisn f the time-curses fr K,, (Fig. 1) and I5 (Fig. 2) values shws that the changes did nt ccur ver the same time perids. Figures I and 2 demnstrate that the I5 values changed first, fllwed changes in the K,, value. This pattern is identical t that bserved in viv (Whitman and Strey 1990) suggesting that this time-curse differential is a prperty f the PK phsphrylatin prcess and nt f the in vitr system used. Measurement f PK activity als shwed n significant changes in Vmax ver the time-curse f Fig. 1 (data nt shwn). This shws that increased enzyme phsphrylatin affected nly the PK, Kin, and I5 values and did nt effect enzyme turnver rate. The prtein kinase activity bserved in these hmgenates was cmpletely sluble as demnstrated by measurements with crude hmgenates which cntained membrane fragments. In these experiments, the centrifugatin step was mitted during the purificatin prcedure (thus retaining large membrane vesicles and insluble cellular material) and samples were dialysed fr 4 h versus 2 x 500 ml Buffer B. Bth secnd-messenger-insensitive and cgmp-sensitive endgenus prtein kinase activity was identical t that measured in samples centrifuged at x g fr 10 rain. Dephsphrylatin f pyruvate kinase by endgenus phsphatases Initial experiments int PK phsphrylatin by endgenus prtein kinases shwed that the extent f PK phsphrylatin was a functin f the amunt f NaF added t the incubatin mixture. In ur system, 30 mm NaF gave ptimal phsphrylatin after a 5-h incubatin. Since NaF is a well-knwn phsphatase inhibitr, this suggested that an endgenus phsphatase activity was present in the PK-cntaining supernatant. The existence f this phsphatase activity is demnstrated in Fig. 3; adding nly Mg 2+ ins t phsphrylated PK led t

5 S.P.J. Brks and K.B. Strey: Prteinkinase phsphrylates pyruvate kinase 313 I/I 1.2 :Eq 0.8 E U E I RADULA c- u I FOOT 0 i I a w! A I B IN Q 0.4 x b.2 Fig. 3A, B. Time-curses fr PK dephsphrylatin: cmparisn f endgenus PK phsphatase activity in tissue extracts frm aerbic versus anxic animals. Figure shws PEP Km values (in mm) measured after 0, 1, 2, 4, 8, 12, 23, and 30 h f incubatin. Reactins were initiated by the additin f 15 mm MgC12 t desalted tissue extracts and represent endgenus phsphatase activity. Values represented by slid circles were btained by phsphrylating PK in vitr vernight in hmgenates frm aerbic animals (PK frm aerbic animals is dephsphrylated) prir t the start f the experiment (see Materials and methds). Phsphatase activity in anxic hmgenates (pen circles) was btained by measuring the Km value fr PK btained directly frm tissue hmgenates (PK frm anxic tissues is phsphrylated). The slid lines were btained by a nn-linear regressin (Gauss-Newtn algrithm) fit f the data t Eq. 1. The half-time values fr the reactin are presented in Table 2 time (hurs) a decrease in Km fr PEP in bth radular retractr and ft muscle. This prcess was time-dependent; the Km values decreased with increasing length f incubatin until a final value was btained. Figure 3 als shws the PK dephsphrylatin time-curses fr hmgenates frm bth anxic animals and frm aerbic animals where the hmgenate was previusly treated t phsphrylate PK in vitr. These dephsphrylatin time-curses were btained using hmgenates diluted by the same factr in rder t cmpare the rates f dephsphrylatin in the tw different hmgenates. Table 2. Actin f endgenus phsphatases in tissue extracts frm aerbic versus anxic animals n a phsphrylated PK substrate. t.s values are best-fit values (+1 standard deviatin) btained by a nn-linear least squares regressin (Gauss-Newtn algrithm) fit f the data f Fig, 3 t Eq. 1. t,5 values represent the time required fr ne half the maximal respnse and are thus prprtinal t the ttal phsphatase activity in the samples. The phsphrylated PK required t measure phsphatase activity in hmgenates f aerbic tissues (PK frm aerbic tissues is dephsphrylated) was btained by phsphrylating PK in vitr vernight prir t the start f the experiment (see Materials and methds). Phsphatase activity in anxic hmgenates was btained by measuring its effect n phsphrylated PK (btained directly frm anxic tissues), Phsphrylatin was carried ut by endgenus phsphatases; the reactin was initiated simply by additin f 15 mm MgC12 (see Materials and methds) Tissue Experimental cnditin t.s (h) Radula aerbic 3.4 _+ 1.1 Radula anxic Ft aerbic 1.7 +_ 0.7 Ft anxic 1.5 _+ 0.2 Because it is difficult t cmpare the curves using nly graphical prcedures, the data f Fig. 3 was fitted t Equatin 1. The t.s values (half-time fr cmplete reactin) frm the data f Fig. 3 are presented in Table 2. Since the rate f PK dephsphrylatin is prprtinal t the time required t btain the half maximal change in the Km value (t.~), a cmparisn f this value fr hmgenates frm bth aerbic and anxic animals allwed a relative measure f the phsphatase activity present in anxic and aerbic tissues. Table 2 shws a 2.4-fld lwer t.5 fr phsphatase actin in hmgenates frm anxic as cmpared t aerbic radular retractr muscle, whereas ft muscle shwed n difference between phsphatase activity in aerbic and anxic extracts. These data reveal that the phsphatase activity in anxic animals is equal t r greater than that f aerbic animals. Nte that the curves in Fig. 3 are sigmidal (the cperativity factr fr these prcesses was greater than 1.0) suggesting that the bserved decrease in the Km value fr PEP required the remval f mre than ne phsphate per active subunit (data nt shwn). Discussin The data presented in this paper shw that pyruvate kinase in whelk muscle tissues can be reversibly phsphrylated in vitr by endgenus prtein kinases t give an enzyme with altered kinetic prperties. This phsphrylated enzyme was similar t that islated directly frm tissues f anxic animals in bth the directin and the magnitude f the effect n the Km value fr PEP and the Is value fr L-alanine. Table 3 cmpares data fr the in vitr phsphrylated and in vitr

6 314 S.P.J. Brks and K.B. Strey: Prteinkinase phsphrylates pyruvate kinase Table 3. Cmparisn f in vitr phsphrytated and in vitr dephsphrylated PK kinetic parameters t values fr PK islated frm aerbic and anxic whelks Enzyme surce K,, PEP I5 L-alanine (mm) (mm) Radular retractr purified anxic PK b in vitr phsphrylated a purified aerbic PK b in vitr dephsphrylated" 0.18 N.D. Ft muscie in viv anxic PK ~ in vitr phsphrylated" in vitr aerbic PK c in vitr dephsphrylated a 0.11 N.D. Ventricle in viv anxic PK r in vitr phsphrylated" 1.2 N.D. in viv aerbic PK r in vitr dephsphrylated" N.D. N.D. a Values determined frm this study. Phsphrylated values are frm Figs. I and 2, dephsphrylated values are frm Fig. 3 b Values are fr PK purified frm aerbic and anxic individuals (Plaxtn and Strey 1984) Values are frm Whitwam and Strey (1990) and represent measurements perfrmed in crude hmgenates frm aerbic and anxic individuals dephsphrylated PK kinetic parameters btained frm the present study with values measured directly in tissues frm aerbic and anxic whelks. In all cases, the kinetic parameters f the in vitr phsphrylated PK are similar t thse f the enzyme islated frm anxic animals, and the kinetic parameters f in vitr dephsphrylated PK are similar t thse f the aerbic enzyme frm. This indicates that the endgenus prtein kinase activity identified in these tissue hmgenates is respnsible fr generating the bserved changes in PK during the aerbic t anxic transitin in viv. It als shws that the phsphatase activity measured in ft and radular retractr muscles catalyses the changes in kinetic parameters during the anxic t aerbic recvery in whelks. In whle animals, a significant decrease in PK maximal activity is assciated with expsure t anxic cnditins (Plaxtn and Strey 1985). The present data suggest that this decrease results frm changes in PK prtein turnver rates, and nt frm changes in the degree f enzyme phsphrylatin since n changes in Vmax were bserved during the curse f ur experiments. It is apparent, then, that phsphrylatin alters nly enzymesubstrate and enzyme-inhibitr affinities, and des nt affect maximal enzyme activity. The results f Figs. 1 and 2 demnstrate that a substantial prtein kinase activity exists in the absence f any added secnd messenger. Thus, in the presence f Mg 2+ and ATP alne, ft and radular retractr PK were phsphrylated with a half-time f apprximately 11 h, whereas the half maximal respnse fr ventricle PK ccurred at apprximately 6 h. These results suggest the existence f a secnd messenger-insensitive prtein kinase activity present in all three muscles. This activity may result either frm prtease actin n a prtein kinase C (t give a PK-M prtein kinase) r frm the dissciatin f an active subunit frm a larger '" regulatry prtein-catalytic subunit" prtein kinase cmplex. Secnd-messenger-independent prtein kinase activities have als been identified frm systems such as Ascaris suum muscle (Thalhfer et al. 1988) and vertebrate muscle (see Edelman et al. 1987). These ften represent the active subunit f a camp dependent prtein kinase. Althugh this explanatin may accunt fr the secnd messenger-insensitive activity bserved in whelk muscle hmgenates, it seems mre likely that the secnd messenger-insensitive prtein kinase activity cmes frm dissciatin f a nn-camp dependent prtein kinase. This is supprted by the cmplete insensitivity f endgenus prtein kinase activity t the additin f camp in the whelk hmgenates; it appears that camp is nt invlved in the phsphrylatin f PK in the three muscle tissues studied. The secnd-messenger insensitive prtein kinase is als nt, apparently, regulated by a membrane-bund cfactr, r by binding t membranes (such as bserved fr prtein kinase C; Mruzzi et al. 1987) since mitting the centrifugatin step during the sample preparatin did nt alter the expressin f either the secnd-messenger-insensitive r cgmp-sensitive prtein kinase activities. Hwever, if the secnd-messenger-insensitive activity represents the dissciatin f a larger prtein cmplex, the affinity between catalytic subunit and regulatry prtein must nt be very high because the tw putative subunits were dissciated under the very mild cnditins f ur tissue hmgenizatin. It is pssible, then, that this activity represents a genuine secnd-messengerinsensitive prtein kinase, distinct frm the cgmp catalytic subunit. Such prtein kinases have been fund in several animals including Ascaris suum muscle tissue (Thalhfer et al. 1988) and usually frm part f a larger phsphrylating cascade such as that described fr insulin stimulatin f serine kinases (Czech et al. /988). Thus, the secnd-messenger-insensitive prtein kinase activity may represent the final enzyme in a cascade respnsible fr mediating the respnse f PK t anxia. This cascade may be similar t thers (such as the serine kinase pathway) with several '~ layers" f phsphrylated kinases. The results frm Pigs. 1 and 2 als shw that the endgenus prtein kinase activity respnded t increased cncentratins f cgmp, and nt t camp r Ca 2 + plus PMA. This effect was maximal after apprximately 5 h f incubatin and was the result f an increase in the rate, but nt the verall extent f PK phsphrylatin as measured by the changes in enzyme kinetic parameters. Althugh the existence f cgmp-dependent prtein kinases has been well established in invertebrates (Bdnaryk 1983; Ku et al. /971; Higgins and Greenberg 1974) and mammalian tissues (Lincln et al. /988), endgenus prtein substrates have nt yet been identified fr this enzyme (Takahashi 1985) even thugh

7 S.P.J. Brks and K.B. Strey: Prteinkinase phsphrylates pyruvate kinase 315 artificial substrates have included PK, fructse 1,6- bisphsphatase, glycgen synthase, and phsphrylase b kinase (Lincln and Crbin 1977). The present paper, hwever, demnstrates a physilgically relevant substrate, and functin, fr a cgmp activated prtein kinase. Althugh cgmp dependent prtein kinases are nt ubiquitus (see Krebs and Beav 1979), the level f cgmp stimulated activity can be higher than the camp stimulated activity in sme arthrpd tissues (Ku et al. 1971) and in bivalve mycardium (Higgins and Greenberg 1974). Changes in cgmp cncentratin~ have als been linked t develpmental changes in insects (Bdnaryk 1983; Takahashi et al. 1975), t anxia in marine bivalves (Hlwerda et al. 1981) and t increased acetylchline cncentratins in bivalves (Khler and Lindl 1980), shwing that cgmp prtein kinases regulate imprtant changes in invertebrate cellular prcesses. The present study suggests that cgmp may play a similar rle in the rerganizatin f metablism assciated with the transitin frm aerbic t anxic cnditins in the channelled whelk, Changes in prtein kinase secndmessenger cncentratins in respnse t differing metablic cnditins are well dcumented in hrmne-regulated mammalian systems (Krebs and Beav 1979; Lincln 1983) as well as in invertebrate tissues (Bdnaryk 1983; Takahashi 1985; Hlwerda et al. 1981). This hypthesis, therefre, prpses that an increase in cellular cgmp levels, stimulating cgmp prtein kinase activity, prbably mediates the anxia-induced phsphrylatin f PK, and pssibly als the crdinated cvalent mdificatin f glycgen phsphrylase, 6-phsphfruct-1- kinase, and 6-phsphfruct-2-kinase that is seen in whelk tissues (Strey 1988b; Bsca and Strey 1990). A decrease in the rate f PK dephsphrylatin during anxia, which wuld als result in increased PK phsphrylatin, is ruled ut by the data f Fig. 3 and Table 2 which shw identical rates f phsphatase activity in fd tissue hmgenates f aerbic and anxic animals, and an apparent higher phsphatase activity in anxic radula. An increase in the cncentratin f prtein kinase activity is als ruled ut by measurements f identical ttal and cgmp-stimulated activity in extracts frm bth anxic and aerbic ft and radular retractr muscles. Finally, the data in Table 1 als suggest that either a Ca 2 + activated PK phsphatase activity is present in B. canaliculatum muscle tissue, r that lw cncentratins f Ca 2+ ins inhibit the endgenus prtein kinase. This is mst clearly illustrated when the K,, values fr PEP f ventricle PK are cmpared fr 5-h cntrl versus additin f Ca 2+ (in the presence r absence f ther effectrs); ventricle hmgenates incubated in the presence f Ca 2 + ins and cgmp had a PEP K,, value that was identical t the 0-h cntrl. This shws that PK was dephsphrylated with respect t 5-h cntrl PK and suggests either Ca 2 + activatin f an endgenus phsphatase activity and/r Ca 2 + inhibitin f the cgmp-stimulated prtein kinase. Althugh this effect was mst evident in ventricle hmgenates (because f the greater degree f ventricle PK phsphrylatin after 5 h incubatin) the effect is als seen in ft and radular retractr tissue (Table 1). Interactins between Ca 2 and cgmp secnd messenger systems are well dcumented: Ca 2+ ins indirectly mediate an increase in the rate f cgmp synthesis (increase guanylate cyclase activity) and directly stimulate its degradatin (increase Ca 2 + dependent phsphdiesterase; Tremblay et al. 1988). These results suggest that the cntrl f PK phsphrylatin during anxia in whelks is the result f a cmplex balance between several cellular signals including cgmp and Ca 2 + cncentratins. Acknwledgements. Supprted by an N.S.E.R.C. Canada perating grant t KBS and a pst-dctral fellwship t SPJB. Thanks t J. Strey fr prfreading. References Bdnaryk RP (1983) Cyclic nucletides. In: Dwner RGH, Laufer H (eds) Invertebrate endcrinlgy, vl 1. Alan R. Liss Inc., New Yrk, pp Bsca L, Strey KB (1990) 6-Phsphfruct-2-kinase and glyclytic cntrl in a facultative anaerbe. Bichim Biphys Acta in press Brks SPJ, Strey KB (1989) Influence f hrmnes, secnd messengers, and ph n the expressin f metablic respnses t anxia in a marine whelk. J Exp Bil 145:31-43 Claus TH, E1-Maghrabi R, Pilkis SJ (1979) Mdulatin f the phsphrylatin state f rate liver pyruvate kinase by allsteric effectrs and insulin. J Bil Chem 254: Chen P (1980) Recently discvered systems f enzyme regulatin by reversible phsphrylatin. 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