Comp. Biochem. Physiol., 1975, Vol. 52B. pp. 187 to 191. Peroamon Press. Printed in Great Britain

SQUID MUSCLE PYRUVATE KINASE: CONTROL PROPERTIES IN A TISSUE WITH AN

ACTIVE ~-GP CYCLE

K. B. STOREY* AND P. W. HOCHACHKA

Department of Zoology, University of British Columbia, Vancouver, B.C., Canada V6T 1W5

(Received 11 October 1974)

Abstract--1. Pyruvate kinase in squid muscle occurs as a single electrophoretic species with an isoelec- tric point at pH 5"9.

2. The enzyme activity is strongly affected by varying temperature and pressure. 3. Under normal assay conditions, saturation curves are Michaelis-Menten rectangular hyperbolas

w i t h K m values of 0"15 mM and 0'4 mM for phosphoenolpyruvate and ADP, respectively. 4. Fructose diphosphate and alanine do not affect the enzyme activity; even ATP is a rather poor

inhibitor. 5. Control of the enzyme activity appears vested in NADH and citrate inhibition. NADH is non-

competitive with P-enolpyruvate while citrate greatly reduces enzyme-substrate affinity and in its pres- ence P-enolpyruvate saturation follows sigmoidal kinetics.

INTRODUCTION

IN MOST mammalian muscles pyruvate kinase (E.C.2.7.1.40) occurs as a tissue specific isozyme of high activity that is often found to be greatly dis- placed from equilibrium (Williamson, 1965). Until recently, it was considered to be a control site closely integrated with phosphofructokinase through adeny- late coupling, a product of the first reaction (ADP) serving as a substrate for the second (Scrutton & Utter, 1968). Although ATP was a known product inhibitor, earlier estimates of the KI for ATP were so high as to rule out potential physiological signifi- cance. Recently, however, Kemp (1973) has found that muscle pyruvate kinase activity is closely coordinated with the energy needs of the cell through synergistic inhibitory actions of ATP and creatine phosphate.

In muscle of bivalve molluscs, control at this point is far more complex. Most of these animals are ex- tremely good anaerobes and here pyruvate kinase functions at a key metabolic branchpoint. Under aer- obic conditions, pyruvate kinase (PK) is fully active, producing pyruvate as a substrate for the Krebs cycle. On transition to anaerobic conditions, pyruvate kinase appears to be fully blocked and its usual sub- strate, P-enolpyruvate (PEP), instead is carboxylated to oxaloacetate (OXA). In obligate anaerobic tissues, such as muscle of adult Ascar i s , achieving this PEP- pyruvate block seems so fundamental that the enzyme is deleted and no PK activity can be demonstrated in these organisms. However, this is the extreme situ- ation. The more usual case is seen in the adductor muscle of bivalves which possess a highly regulated pyruvate kinase, potently inhibited by alanine and H ÷ during aerobic-anaerobic transition and deinhi- bited (or activated) by FDP during the reverse transi- tion to aerobic metabolism. In addition, the enzyme activity is integrated into the overall energy status

* Present address: Department of Zoology, Duke Uni- versity, Durham, NC, U.S.A.

187

of the cell by strong ATP inhibitory control (Mustafa & Hochachka, 1971; Saz, 1971; Hochachka & Mus- tafa, 1972; De Zwann et al., 1973; Livingstone & Bayne, 1974).

When we initiated our study of the squid, S y m p l e c - to t eu th i s oualaniens is , we had no reasons for anticipat- ing any unusual PK properties; but the clearer our understanding became the more sharply did an obli- gatorily aerob ic metabolism in squid muscle contrast with the highly anaerobic metabolism of bivalve muscle. For this reason, and because of novel features at other control sites (Storey & Hochachka, 1975), a detailed description of the regulatory properties of this molluscan enzyme became an imperative. We found that the enzyme occurs in single electrophoretic form of high specific activity. Substrate saturation curves are normal rectangular hyperbolas. Unlike pyruvate kinases from bivalves, squid mantle PK is not at all inhibited by alanine or phenylalanine and it is only poorly inhibited by ATP. The ATP inhibi- tory feature appears to be taken over by NADH and citrate. Being a signal of the overall redox state of the cell, NADH integrates PK with the cytoplasmic arm of the alpha-glycerophosphate (~-GP) cycle and with the general metabolic status of the cell; being a signal of the degree of Krebs cycle activation, citrate control integrates the PK reaction with the pathway for which it is the only major supplier of carbon sub- strate.

MATERIALS AND METHODS

All reagents used in this study were purchased from Sigma Chemical Co., St. Louis, Mo. Ampholines were pur- chased from LKB Products (Stockholm, Br/Smvoa, Sweden). The squid used were captured by jigging off the Kona Coast of Hawaii during November and December and were frozen, packed in dry ice and shipped back to the laboratory for study.

The mantle was cut into small pieces, the skin removed, and homogenized in 50 mM Tris-HC1, pH 7.5 containing

188 K.B. STOREY AND P. W. HOCHACHKA

20 mM fl-mercaptoethanol and 10 mM Mg 2+. The tissue was homogenized in a Sorvall omnimixer for 2 min and then spun at 27,000 × g for 15 min. The supernatant was then heated to 45°C for 2min, spun as above and the pellet discarded. This supernatant was then brought to pH 5"5 for 30 sec, neutralized and spun as above and the pellet again discarded. This supernatant was brought to 47~o ammonium sulphate, stirred at 4°C for 20 min, centrifuged as above and the pellet discarded. The supernatant was then brought to 60~ ammonium sulphate, stirred at 4°C for 20 min, centrifuged as above and the pellet saved. The precipitated protein was dialyzed exhaustively against 1~ glycine, pH 6.5 containing 30mM -mercaptoethanol. Damp precycled phosphocellulose was then added at this pH to remove contaminating enzyme activities; the pI of squid pyruvate kinase is 5.9, and it therefore binds poorly at pH values in this range. The ion exchanger was added until loss of activity was seen. The slurry was centrifuged as above and the supernatant was electrofocused in a pH 5-8 gradient, at 700V for 16hr at 4°C. The purified enzyme (peak tube of the electrofocused protein) had a specific activity of 154moles of product formed per min/ mg of protein at 26°C, compared to 405 units per min/mg of protein at 30°C for rabbit muscle enzyme (Kemp, 1973).

The preparation was free of any enzymes that would either interfere with the basic assay or remove or intercon- vert any of the added metabolites.

Pyruvate kinase was assayed by the method of Biicher & Pfleiderer (1955). Pyruvate formation was coupled to lac- tate dehydrogenase and the rate of pyruvate kinase activity was measured as the decrease in E340 due to NADH. Tris- HC1 buffers were used in all assay reactions. Standard assay mixtures contained the following in a final volume of 2 ml: 50mM Tris-HC1 buffer, pH 7.2, Mg 2÷, K ÷, ADP, P-enolpyruvate, NADH and excess of dialyzed Sigma lac- tate dehydrogenase at concentrations specified in the figure legends. Saturating concentrations for each of the reactants are: PEP, 1.0mM; ADP, 2.0mM; Mg 2+, 2.0mM; K ÷, 30mM; NADH, 0-15 mM. All reactions were started by the addition of pyruvate kinase preparation. All exper- iments were performed at 25°C unless otherwise noted.

Pressure effects on the enzyme were determined in a temperature-controlled high pressure cell installed in a Un- icam SP 1800 recording spectrophotometer (Mustafa et al., 1971).

RESULTS AND DISCUSSION

Electrophoretic properties As pyruvate kinase is known to occur in isozymic

forms in mammalian tissues, it was important at the outset to determine the number of forms of the enzyme in squid mantle muscle. Electrofocusing ex- periments of mantle muscle preparations indicate that all of the recoverable P K activity occurs as a single species with an isoelectric point of pH 5.9 (Fig. 1), a result in close agreement with studies of bivalve muscle (Mustafa & Hochachka, 1971).

Effect of pH The pH profile for squid mantle P K under saturat-

ing concentrations of substrates and cofactors shows an opt imum at pH 7.2-7.4. This is substantially lower than the pH optimum for the enzyme from other mol- luscs (Mustafa & Hochachka, 1971).

Effect of temperature and pressure Pyruvate kinases from other sources are often un-

usually temperature and pressure sensitive (Somero & Hochachka, 1968; Mustafa et al., 1971). Because the squid under study is a vertical migrant, it was

1E

V

14

4=

4=

4t

20 40 60 Tube Number

, * pH ~t

80 100

Fig. 1. Isoelectric focusing of a squid mantle muscle prep- aration on a pH 5-8 gradient at 4°C for 19 hr. Electro- focusing column was drained into a LKB fraction collec- tor; PK activity and pH were assayed in each of the 0.8 ml lractions obtained. PK activity focused into one band with

an isoelectric point of pH 5.9.

important to assess the effects of the physical environ- ment on enzyme reaction velocity. In agreement with previous studies, squid mantle muscle PK is highly temperature sensitive (Qlo values of about 3.0) and is strongly inhibited by pressure. Arrhenius plots of the data are linear between about 5 ° and 20°C, the reaction showing a distinct temperature optimum at about 25°C. Interestingly, this temperature corres- ponds closely with the surface water temperatures where these organisms are normally found. At high pressure the temperature sensitivity of the reaction is further increased; thus, at 2000psi, the apparent Q~o between 10 ° and 20°C is about 3-6 (Fig. 2).

The effect of pressure on the reaction velocity depends upon the temperature, at 25°C, the reaction proceeds with a volume change of activation (AVe) of about 32 cm3/mole under saturating concentrations of substrates and cofactors. At 10°C, the AV:~ is nearly doubled to 53 cma/mole calculated between 200 and 400 atm (Fig'; 3). These values are in the same range as previously reported for the homologous enzymes

100

50

V 2o

10

30 25 20 15 10 5

330 335 341 348 353 360

Fig. 2. Arrhenius plots, log reaction velocity vs l/T, °K for squid mantle muscle pyruvate kinase at 14-7 and 2000 psi under saturating concentrations of substrates and

cofactors.

Squid muscle pyruvate kinase 189

100

20 25°C v , o i

~10°C

2o0 , ~ o ~,,o ~ A T M

pressure

Fig. 3. Log reaction velocity versus pressure (in psi and atm) for squid mantle muscle pyruvate kinase at 25 ° and at 10°C, under saturating conditions of substrate and

cofactors.

from midwater and abyssal fishes (Moon et al., 1971), both sets of experiments being performed under simi- lar conditions. However, the magnitude of AV:~ for pyruvate kinase and possibly for other enzymes as well (Low & Somero, 1975, this series) may be strik- ingly dependent upon the amount and the kind of monovalent cation used to activate the enzyme. Hence, it is important to qualify that in these exper- iments K ÷ was utilized at concentrations that are believed to be in the physiological range, but the in- teracting effects of other ions (such as Na ÷ or NH~-) with K + were not examined. If we assume that these would not significantly alter the pressure sensitivity of the reaction at saturating levels of K ÷, then it is clear that during deep diving of this squid (to waters of about 10°C and perhaps 150atm), PK activity could be reduced to about 1/6 of reaction rates under surface conditions (Fig. 3). This large retardation of PK activity, coupled with similar effects on key con- trol sites such as phosphofructokinase and e-glycero- phosphate dehydrogenase (c~-GPDH) (Storey & Hochachka, 1975a,b, this series), are consistent with the concept of a generalized metabolic depression occurring during deep diving of vertically migrating animals (see Teal & Carey, 1967; Teal, 1971, for detailed discussions of this concept).

Cation requirements

All pyruvate kinases have an absolute requirement for a divalent cation, that can be satisfied with Mg 2÷ or Mn 2÷ and this is likewise the case with squid muscle PK. Mg 2÷ saturation of the enzyme follows Michaelis-Menten kinetics with a linear double-reci- procal plot and a calculated Km value of 0"6 mM. As expected, Ca 2÷ inhibits being competitive with re- spect to Mg 2+ but non-competitive with respect to either substrate. Unlike other pyruvate kinases, which are activated by, but do not necessarily display an absolute requirement for, a monovalent cation, squid mantle PK is catalytically inactive in the absence of K ÷ or NH~-. Saturation of the enzyme with respect to either K ÷ or NH,~ follows Michaelis-Menten

C.B.P.(B} 52/lB M

kinetics with K~ values of 6"5 mM and 4mM for K + and NH2, respectively. Na + cannot substitute even to a slight degree for either K ÷ or NH~-, nor is Na ÷ inhibitory when added at physiological con- centrations (30-40 mM).

Substrate saturation

Under normal circumstances, saturation curves of squid mantle muscle PK with either PEP or ADP also are Michaelis-Menten hyperbolas, with Km values of 0"15 and 0'4 mM for PEP and ADP, respect- ively.

Lack of FDP or alanine effects

Because of the pivotal role of FDP and alanine in the control of other molluscan pyruvate kinases (Mustafa & Hochachka, 1971; De Zwaan et al., 1973; Livingstone & Bayne, 1974), we were particularly sur- prised to find that neither metabolite had any in- fluence on the activity of the enzyme from squid man- tle muscle. The enzyme activity was tested at a variety of substrate concentrations, with alanine levels rang- ing from 1-10 mM, and FDP concentrations ranging from 0" 1 to 0"3 mM and was found to be fully refrac- tory to these two metabolites under all conditions tested. Phenylalanine at concentrations up to 12 mM also has no effect on the enzyme. Other compounds (tested at concentrations of 5 mM) that were found to be without effect on the enzyme include serine, glycine, arginine, proline, valine, aspartate, threonine, tryptophan, isoleucine, cysteine, lysine, histidine, and 3-phosphoglycerate. Three metabolites, however, were discovered to be potent inhibitors. These are NADH, citrate, and ATP.

Regulation by NADH, citrate, and ATP

Unlike the situation in bivalve molluscs, squid mantle muscle PK functions in an unique metabolic "field" in which carbohydrate is the chief and perhaps only substrate and in which the e-GP cycle plays a most prominent role in sparking aerobic metabolism during transition from low activity to high activity states. In this situation, NADH concentrations serve as a most immediate indication of the degree to which the ~-GP cycle is activated. Citrate on the other hand, is a product of the first (and entry) step of the Krebs cycle and the degree of its accumulation is an accu- rate index of the degree of Krebs cycle activation (LaNoue et al., 1973). In this metabolic organization, singly by far the most important source of acetylCoA for the citrate synthase reaction is glucose-derived pyruvate, formed at the pyruvate kinase reaction. Most instructive, then, is the observation that both NADH and citrate are specific and potent inhibitors of the PK reaction in squid mantle muscle (Fig. 4). In metabolic terms, what this probably means is that during low activity states, a relatively high NADH/ NAD ratio holds PK in an inhibited state. During transition from low to high activity, sparked by a strong activation of the ~-GP cycle (Storey & Hochachka, 1975a, b, this series), NADH/NAD ratios drop precipitously because of the extremely active cytoplasmic ~-GPDH, thus leading to a deinhibition of pyruvate kinase. Pyruvate formed by this reaction is then available for conversion to acetylCoA, which condenses with OXA to form citrate. Although all Krebs cycle intermediates are augmented during such

190 K.B. STOREY AND P. W. HOCHACHKA

100

80

60

V

1 2 3 4 5 6 concentration, rnM

Fig. 4. Relative velocity (V) vs concentration of 4 different metabolities for squid mantle muscle pyruvate kinase at pH 7'2, 1 mM PEP, 2 mM ADP, 2 mM Mg 2 +, and 30 mM K ÷, 14.7 psi and 25°C. Chelation effects were avoided by

adding Mg.ATP and Mg2. Citrate.

transitions (LaNoue et al., 1973), citrate is in effect the product of the first step in the cycle and presum- ably for this reason serves as the means for limiting its own further production (thereby limiting further Krebs cycle activation) by pyruvate kinase inhibition as well as by citrate synthase inhibition (Hochachka et al., 1975).

In biochemical terms, NADH and citrate bring about their effects on PK by different mechanisms. With respect to PEP, NADH is a strictly non-com- petitive inhibitor, reducing the calculated Vm~x but having no effect on the enzyme affinity for its sub- strate (Fig. 5). The primary action of citrate, on the other hand, is to greatly reduce enzyme-substrate affinity, converting hyperbolic saturation kinetics to

sigmoidal ones (Fig. 6). In consequence, under physio- logical levels of PEP, citrate inhibitory effects are especially large. At Km concentrations of PEP, for example, 2-5 mM citr~ite reduces the reaction velocity by 15- to 20-fold (Fig. 6).

The regulatory actions of NADH and citrate are probably potentiated in vivo by ATP. The latter is of course the effective end product of energy metabo- lism and it is therefore not surprising that squid man- tie muscle PK, like other molluscan pyruvate kinases (Hochachka & Mustafa, 1972), is product inhibited by ATP. With respect to PEP, ATP is a mixed com- petitive inhibitor (Fig. 6), with large effects on the Km and on the Vmax, while it is strictly non-competi- tive with respect to the cosubstrate, ADP. It is prob- able that NADH and ATP share the same inhibitory binding site as they interact with ADP and PEP in a similar manner (Fig. 7). However, even if that is so, it is evident that NADH binding is greatly favoured over ATP binding (Fig. 4), so much so that ATP control probably comes into play only under

.4

1

S CONTROL

2 6 10 14 18 20 (p'~] "10 3

Fig. 5. Inhibition of squid mantle muscle PK with 0.4 mM NADH but otherwise optimal conditions for the reaction

velocity.

67 ~ ~ ' ~ 2 ~ S r n M CITRATE a

V 5 C O N T R O L . ~ o / r

4 I

3 " 3 mM ATP

2 i1 []

1

0,1 0.2 0.3 0.4 0.5 PEP, mM

Fig. 6. PEP saturation curves for squid mantle pyruvate kinase under control conditions (open circles), in the presence of two concentrations of ATP (squares), and in the presence of 2.5 mM citrate (triangles).

Squid muscle pyruvate kinase 191

"t -leL// /

1 x103 [ADP)

Fig. 7. Lineweaver-Burk plots for NADH and ATP inhibi- tion of squid mantle muscle pyruvate kinase showing that these two metabolites are n6n-competitive with respect to

ADP.

energy saturated circumstances and even under these conditions, its inhibitory action would have to be coupled with that of citrate or NADH to fully block P K activity (Fig. 4).

Finally, attention should also be brought to bear on the problem of reversing P K inhibition by the above three metabolites. In other species, both verte- brate and invertebrate, FDP appears to be of particu- lar utility in this regard Mustafa & Hochachka, 1971 ; Bayne et al., 1974; Storey & Hochachka, 1974), but it is without effect on squid mantle muscle PK. FDP at concentrations between 0.1 and 3.0mM has no positive or negative effect on P K inhibition by NADH, citrate, or ATP. Similarly ineffective are Pi (tested at 0"5 to 25 mM), AMP (tested at 0"5 mM), 3-phosphoglycerate (tested at 1.0 mM), plus a variety of amino acids tested at high (5 mM) levels. The only metabolic reversal that could take on physiological significance relates to citrate inhibition, for it is clear that PEP at high concentrations in effect serves to reverse the action of citrate (Fig. 6). Otherwise, it is clear that t~ae primary strategy of P K regulation ~ln squid mantle muscle involves cycles of inhibition and deinhibition that are closely integrated with the redox state and the energy status of the cell.

Acknowledgements---This work was supported by an NRC (Canada) operating grant to PWH. R/V Alpha Helix operations were supported by the NSF (U.S.). Especial thanks are due Capt. R. Coleman and the crew of the Alpha Helix, without whose full cooperation these studies would not have been possible.

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