Comp. Biochem. Physiol., 1975, Vol. 52B, pp. 179 to 182. Pergamon Press. Printed in Great Britain

SQUID MUSCLE GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE: CONTROL OF THE ENZYME IN A

TISSUE WITH AN ACTIVE ~-GLYCERO-P 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. Squid muscle glyceraldehyde-3-phosphate dehydrogenase occurs as a single electrophoretic form that can be readily purified to a specific activity of over 130#moles product/min/mg protein at 25°C.

2. The enzyme displays optimum activity at pH 7.0-7.5 and 35-40 mM K ÷ or NH~. 3. The Michaelis constants for glyceraldehyde-3-phosphate, NAD, and inorganic phosphate are about

0.2 mM, 0'1 mM, and 10 mM, respectively, when determined at optimal pH. 4. The enzyme is strongly inhibited by the adenylates but this inhibition is reversed by inorganic

phosphate. 5. NADH is also a potent inhibitor and is thought to play an important role in vivo in integrating

the activity of glyceraldehyde-3-phosphate dehydrogenase with other key enzymes in squid muscle energy metabolism.

INTRODUCTION

GAPDH* (E.C.1.2.1.12), catalyzing the following reaction,

GAP + NAD + Pi---' 1,3,DPG + NADH

serves an important function in redox regulation in all cells that utilize carbohydrate. In vertebrate muscle which converts glucose to lactate under anoxic con- ditions, GAPDH functions at an identifiable control site (Williamson, 1965), the reaction being held from equilibrium at periodic time intervals after glycolytic activation. In vertebrate systems, the regulatory properties of the enzyme are well described for muscle, a highly glycolytic tissue, and for liver, a highly gluconeogenic one. In muscle, the enzyme is well fit- ted for glycolytic function chiefly by maintaining an inhibitory control over its activity. Thus, ATP, Cr-P, 1,3,DPG, and NADH are all known inhibitors; of these, the latter two apparently never reach con- centrations high enough to be effective inhibitors in vivo. ATP and Cr-P on the other hand occur at con- centrations easily adequate to hold the enzyme in an almost fully inhibited state under resting conditions; with initiation of muscle contraction, dropping Cr-P and ATP levels lead to a potent deinhibition of the enzyme (Oguchi et al., 1973a,b), that may establish a steady maximum for flux through the glycolytic pathway.

In the liver, the control requirements at this point are reversed during periods ofgluconeogenesis. In this direction, the substrate saturation curves are highly

sigmoidal, indicating substrate and activator roles for 1,3,DPG. NAD, the product of the reaction in this direction, serves also as a potent activator particularly at low concentrations; at high concentrations it inhi- bits in a strictly competitive manner with respect to NADH (Smith & Velick, 1972). As a result, the liver is able to sensitively regulate flux in either direction at this locus in metabolism.

In squid mantle muscle, lactate never accumulates as an anaerobic end product, since muscle work seems to be linked absolutely to an aerobic metabo- lism, based on an active ~-glycero-P cycle (Hochachka et al., 1975a). In muscles with an active ~-glycero-P cycle, the properties of GAPDH have never been carefully examined (Sacktor, 1970) although they may be expected to be different from mammalian muscle since the control requirements at this point during periods of glycolytic activation are different. We therefore isolated and purified the enzyme from squid muscle. We found that although the usual ATP inhibitory control is expressed, it is markedly reversed by Pi, which in vivo occurs at un- usually high levels. Hence, we conclude that ATP control is of lesser significance than is a potent NADH inhibition of the enzyme reaction. Further- more, a control system based on NADH con- centration changes effectively integrates GAPDH function with phosphofructokinase, the chief glycoly- tic control site, and with the cytoplasmic arm of the ~-glycero-P cycle.

* Present address: Department of Zoology, Duke Uni- MATERIALS AND METHODS versity, Durham, NC, U.S.A. All reagents used in this study were purchased from

t Abbreviations used: GAPDH, glyceraldehyde-3-P Sigma Chemical Co., St. Louis, Mo., U.S.A. Ampholines dehydrogenase; LDH, lactate dehydrogenase; PFK, phos- were purchased from LKB Products (Stockholm, phofructokinase; GAP, glyceraldehyde-3-P; 1,3,DPG, 1,3 Br6mvoa, Sweden). The squid used in this study were cap- diphosphoglycerate, tured by jigging off the Kona Coast of Hawaii during

179

180 K.B. STOREY AND P. W. HOCHAClJr,.a

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 50mM EDTA, 20mM fl-mercap- toethanol, 0.1 mM NAD at pH 8.4. The tissue was homo- genized in a Sorvall omnixer for 2 rain and then spun at 27,000 × O for 15 min. The supernatant was collected and brought to 75~o saturation with (NH4)2SO4 and stirred at 4°C for 20 min. The mixture was centrifuged for 10 rain at 27,000 × g and the supernatant dialyzed exhaus- tively against 20 mM fl-mercaptoethanol, 5 mM EDTA, at pH 7.5. Damp, pre-cycled phosphocellulose was then added to the solution in order to remove other proteins (the pI of squid glyceraldehyde-3-phosphate dehydrogenase is 6.7-7.0), and to concentrate the solution. The pH was then briefly lowered to 6.3 and some precycled DEAE Sephadex was added, again to remove contaminating enzyme activities. The ion exchangers were added at their respective pH's until loss of GAPDH activity was noted and then the slurry was spun at 27,000 x g for 10min and the exchanger discarded. The supernatant was then eleetrofocused at pH 3-10 at 300V for 24hr at 4°C, using previously described methods (Hochachka, 1972). The purified enzyme obtained from the electrofocus column had a specific activity of 130 moles of product formed per min/mg of protein at 25°C, compared to a specific activity of 154 moles of product formed/rain per mg of protein at 25°C for the rabbit liver enzyme (Smith & Velick, 1972). The preparation was free of any enzymes that would either interfere with the basic assay or remove or interconvert any of the added metabolites.

Enzyme activity was 'assayed by measuring the appear- ance of NADH by monitering increase in A340 on a Un- icam SP 1800 recording spectrophotometer. Standard assay mixtures contained the following: 20 mM Tris-HC1 buffer, pH 7.3, glyceraldehyde-3-phosphate (DL, free acid; prepared from the diethylacetal, monobarium salt), NAD +, P~, KC1. Saturating concentrations for each of the reactants were: 2raM DL G-3-P, 4mM NAD +, 35mM Pg, and 35 mM K +. All reactions were performed at pH 7.3, and started by the addition of GAPDH preparation, Effects of pressure on reaction velocity were assessed using a high pressure cell previously described (Mustafa et al., 1971).

RESULTS AND DISCUSSION

Effect of pH

G A P D H from rabbit muscle shows a sharp pH profile with a strongly alkaline (8.5-9.0) pH optimum (Oguchi et al., 1973a). In contrast, squid mantle

enzyme shows a distinct pH optimum at pH 7-7.5 (Fig. 1).

Salt effects

It is well known that monovalent cations influence the activity of G A P D H in mammalian systems (Smith & Velick, 1972). We found that both K + and NH~ activate the squid enzyme. Opt imum concentrations are about 35-40mM for both cations. At higher con- centrations, particularly of NH2, the enzyme activity is somewhat inhibited (down to 70~o of control rates at 100mM NH,~). All of our assays were performed at 100mM K +, a value representing a compromise between optimal K + levels for the enzyme and in vivo concentrations.

Effect of temperature and pressure on the Vm, x

AS the squid under study is a vertical migrant, it was important at the outset to determine the effects of temperature and pressure on the reaction velocity. For the data at low pressure Arrhenius plots show a distinct break at about I I°C that is not evident for the data at high pressure (9000 psi). The Q10 for the reaction is 1"5 between 12 and 30°C at 14-7 psi. The pressure sensitivity of the reaction is quite sharply reduced by increasing levels of Pi (Fig. 2), an effect which may be of physiological importance because of the "protection" Pi offers the enzyme against inhibition by regulatory metabolites and because of the high in vivo Pi concentrations found in Cephalopods (Schoffeniels & Gilles, 1972).

Saturation kinetics

Substrate saturation curves for all three substrates of the reaction are rectangular hyperbolas, as is the case for the muscle enzyme in mammals. Lineweaver~ Burk plots of l/velocity vs 1/substrate concentration are simple linear functions in all cases. Values of the Km for each substrate obtained from such plots are summarized in Table 1, and are comparable to the values reported for the mammalian enzyme. As the K m for Pi (about 7-11 mM, depending upon the pH) is considered far lower than in vivo concentrations of Pi, it is probable that the enzyme under normal circumstances is always saturated with respect to this substrate. In contrast, the K,,1 values for NAD and

30

20

/ / pH

Fig. 1. The pH dependence of squid muscle GAPDH under saturating substrate concentrations, 14-7 psi, and 25°C. Phosphate buffer is shown in triangles; data using TribHCl buffer shown in circles.

Squid muscle GAPDH 181

Fig. 2. A plot of pressure (psi x 1000) vs maximum velocity of the reaction catalyzed by squid GAPDH. DL-GAP, 2'0 mM; NAD, 2-0 mM, and phosphate concentrations as shown. At each phosphate concentration, the reaction rate

at 14.7 psi is set at 100.

GAP are well within the expected physiological range (Table 1), and it is not surprising therefore that con- trol is vested in the enzyme's affinity for one or both of these substrates.

Adenylate inhibition

Squid mantle muscle GAPDH is strongly inhibited by the adenylates, AMP and ADP being as effective as ATP (Table 2). ATP inhibition with respect to NAD is strictly competitive. Dixon plots of the data at two different concentrations of Pi (Fig. 3), indicate that Pi strongly protects the enzyme against ATP in- hibition, the KI for ATP at 10mM P~ being about ½ the value at 20 mM Pi. With respect to GAP, ATP inhibition is non-competitive with K~ values of about 6-7 mM depending again on P~ levels (Table 2).

AMP inhibition is almost identical to that obtained with ATP. In agreement with studies on the mam- malian enzyme (Oguchi et al., 1973b), AMP inhibi- tion is competitive with respect to NAD and non-com- petitive with respect to GAP.

Summary of K m values for GAP, NAD, and Pi for squid

muscle glyceraldehyde-3-phosphate dehydrogenase. Unless

otherwise specified, assay conditions for these data are

given in Methods and Materials, K I values for ATP, AMP,

and NADH are also given, in each case calculated from

Dixon plots.

Conditions ~ or K I (raM)

GAP i0 mM Pi 0.2

20 ram Pi 0.2

NAD 5 mM Pi 0.5

I0 mM Pi 0.i

20 mM Pi 0.i

Pi pH 7.1 ii.i

pH 8.3 6.7

ATP 10 mM Pi 1.2 (with respect to NAP)

20 mM Pi 2.3

ATP i0 mM Pi 6.2 (with respect to GAP)

20 mM Pi 7.7

AMP i0 mM Pi 1.2 (with respect to NAD)

AMP I0 mM Pi 0.8 (with respect to GAP

NADH i0 mM Pi 0.027 (with respect to NAP)

ATP, mM

Fig. 3. Dixon plots of ATP inhibition of squid muscle GAPDH at three concentrations of NAD and at two con- centrations of phosphate: 10mM phosphate runs are shown in dark symbols; 20 mM phosphate shown in open

symbols.

Although it is possible that these effects are of im- portance in vivo, as appears to be the case in mam- malian muscle (Oguchi et al., 1973a), we consider this unlikely in the squid for two reasons. Firstly, the effects of ATP are quite strongly reversed by 20 mM Pi, and since in vivo concentrations in cephalopods may approach 100 mM (Schoffeniels & Gilles, 1972), the inhibitory action of ATP may become insignifi- cant. Secondly, for ATP inhibition to be maximally advantageous, it should be coupled to deinhibition by AMP or ADP. This clearly is not the case and since concentration changes of AMP and ADP counter those of ATP, it is difficult to understand how the three compounds could regulate GAPDH activity in a manner consistent with glycolytic require- ments of the cell. A final reason for minimizing the significance of the adenylate effects on the enzyme arises from studies with NADH. As this metabolite appears to play a particularly pivotal control func- tion in squid muscle metabolism (Hochachka et al., 1975a,b), we were particularly interested in examining its effects on GAPDH.

N A D H inhibition As anticipated from previous studies with the mam-

malian enzyme, NADH is a potent inhibitor of squid GAPDH. The inhibition is competitive with respect to NAD with a Kt of about 25-30 pM (Fig. 4). At concentrations of NADH approaching 0.1mM, a value well within the physiological range (Edington, 1970), the enzyme affinity for NAD is decreased by a factor of 4-5.

Metabolic significance

In mammalian muscle, GAPDH occurs in approxi- mately a 1 : 1 activity ratio with LDH and such a ratio is required functionally to sustain redox balance dur- ing anaerobic glycolysis. Although GAPDH functions in maintaining redox balance, it is part of an energy- yielding reaction pathway; hence, it is not unreason- able that its activity be brought under the control of ATP and Cr-P. The concentrations of the latter

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

iO5mM NAD

.02 .04 .06 NADH, mM

Fig. 4. Dixon plot of NADH inhibition of squid mantle GAPDH at two NAD concentrations. Assay conditions: GAP, 1 mM; phosphate, 10mM; K ÷, 100 raM, and NAD

as shown, 14.7 psi, 25°C.

are a good measure of the energy status of the cell and any drop in their level is a sensitive signal for glycolytic activation (Oguchi et al., !973a). Although a similar mechanism may be operative in squid man- tle muscle, it appears that because of the presence of an active ~-glycero-P cycle, N A D H control of G A P D H is of greater potential significance. In this regard, especial attention is attached to N A D H inhi- bition of this enzyme locus because it supplies a means for integrating G A P D H activity with the acti- vity of P F K (perhaps the chief control site in aerobic glycolysis in this tissue) and with ~-GPDH, the cyto- plasmic arm of the c~-glycero-P cycle. Under resting conditions, it appears that energy metabolism is held at reduced levels by N A D H inhibition of P F K (Storey et al., 1975a), of citrate synthase (Hochachka et al., 1975b), and of pyruvate kinase (Storey & Hochachka, 1975b). The same metabolite signal as a means for "turning off" G A P D H is thus most appro- priate. During transition to more active muscle work, activation of the cytoplasmic arm of the ~-glycero-P cycle would dramatically diminish N A D H con- centrations, because of the extremely high catalytic potential of cytoplasmic ~-GPDH, and would lead to a deinhibition of all of the above enzyme loci in a readily coordinated manner.

Acknowledoemenis--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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