Cornp. Biochem. Physiol., 1975. Vol. 52B. pp. 175 to 178. Peroamon Press. Printed in Great Britain

THE KINETIC REQUIREMENTS OF CYTOPLASMIC ALPHA-GLYCEROPHOSPHATE (~-GP)

DEHYDROGENASE IN MUSCLES WITH ACTIVE ~-GP CYCLES

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. The specific activities of alpha-glycero-P dehydrogenase (~-GPDH) from squid mantle muscle and bee flight muscle are almost i00 times higher than in oyster adductor muscle.

2. All three enzymes display similar pH optima and appear designed for unidirectional (dihydroxyace- tone-P---*~-glycero-P) function.

3. In both bee and squid muscles, ~-GPDH has a low affinity for DHAP but an extremely high affinity for NADH; it is thus poised to "spark" the ct-glycero-P cycle whenever substrate is available, but can do so only when ATP concentrations fall because ATP is a potent inhibitor of the enzyme in both muscles.

4. The oyster enzyme, in contrast, is insensitive to ATP and displays a high affinity for DHAP but a low affinity for NADH, regulatory properties that would be anticipated if this enzyme functioned primarily during anoxic stresses when NADH levels rise.

I N T R O D U C T I O N

IN MUSCLES that burn carbohydrate, there are two potential functions for ~-glycero-P dehydrogenasc (E.C.I.I.I.8). Firstly, it may function to initiate and sustain the ~-glycero-P (~-GP) cycle, as in insect flight muscle (Sacktor, 1970; Crabtree & Newsholme, 1972) and in squid mantle muscle (Hochachka et al., 1975; Storey & Hochachka, 1975). And secondly, it may contribute to maintaining redox balance during anoxic stresses, as in some vertebrate muscles (White & Kaplan, 1972). In both kinds of cases, of course, it may also serve as a source of ct-GP for other meta- bolic purposes. Although the enzyme has been puri- fied and at least partially characterized from a variety of sources (Storey & Hochachka, 1975; Lee et al., 1973; Marquardt & Brosemer, 1966; Kleitke & Wol- lenberger, 1969); no one has yet identified those cata- lytic and regulatory properties that "fit" ~-GPDH for function in a highly active ~-GP cycle, nor have the kinetic differences between ~-GPDHs playing these different physiological roles been carefully established. To gain some insight into these questions we have compared the catalytic and regulatory properties of ~-GPDHs from flight muscle of the honey bee, from squid mantle muscle, and from the adductor muscle of the oyster. Bee flight muscle and squid mantle muscle are believed to possess a highly active ~-GP cycle and an obligate aerobic, carbohydrate-based metabolism, while the oyster adductor displays a highly anaerobic, glycogen-based metabolism with no evidence for a functional ~-GP cycle (Hochachka et

al., 1973). In both the bee and the squid, we found that ~-GPDH has a low affinity for DHAP but an extremely high NADH affinity, being highest in the bee enzyme. The enzyme is thus poised to spark the c~-GP cycle whenever DHAP becomes available. Since NADH pool size is obviously limited, the problem of NADH levels rising without limit does not arise even if the enzyme should become saturated with reduced coenzyme. By comparison, the DHAP pool size is less limited, and the high Km for DHAP assures that high rates of cycling occur only when glycolytic rates (and hence DHAP levels) are high. Moreover, it prevents the enzyme from saturating with DHAP and thus the more DHAP that is formed, the higher the ~-GP cycling rates that can be achieved, a feature of obvious physiological advantage in extremely active tissues such as squid mantle and bee flight muscles. As ~-GPDH occurs in higher titre than most other enzymes in these muscles (Hochachka et al., 1975), it in effect supplies the tissue with an unlimi- table mechanism for potently sparking the ct-GP cycle. In oyster adductor, on the other hand, ~- GPDH occurs in low specific activity, and displays a high affinity for DHAP but a low affinity for NADH. It therefore appears to be geared to compete well for DHAP but poorly for NADH, as would be anticipated if it functioned primarily during anoxic stress periods when NADH levels are rising. In view of the impressive anaerobic capacities of oyster adductor (Hochachka & Mustafa, 1972), it is reason- able to conclude that ~-GPDH in this tissue repre- sents an emergency pathway for sustaining redox dur- ing unusually prolonged anoxia. Its low specific acti- vity is biologically important since its simultaneous function with glycolysis reduces by one-half the ATP output of an already energetically inefficient system.

* Present address: Zoology Department, Duke Univer- sity, Durham, NC, U.S.A.

175

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

MATERIALS AND METHODS

All substrates, reagents, and coupling enzymes were pur- chased from Sigma Co., St. Louis, Mo. Ampholines were purchased from LKB Products (Stockholm, Br~mvoa, Sweden). The honey bees (Apis mellifera) used in this study were taken from hives and killed by cooling. Flight muscle was dissected out and care was taken to remove any leg muscle contamination. The oysters used were freshly deli- vered from local B.C. suppliers.

In separate preparations, honey bee flight muscle and oyster adductor muscle were homogenized for 2 rain in a Sorvall omnimixer in 50 mM Tri~HC1, pH 7.8, containing 20 mM fl-mercaptoethanol, and 5 mM EDTA. The super- natants were collected after centrifugation at 27,000 x g for 10min, and brought to 40% saturation with ammonium sulfate and stirred at 4°C for 20 rain. The mix- ture was then centrifuged as above and the precipitate dis- carded. The supernatants were then heated to 45°C for 3 min, centrifuged and the precipitates discarded. The oys- ter muscle preparation was then subjected to pH 5.5 briefly, then neutralized, spun, and the precipitate dis- carded. The honey bee enzyme solution was lowered to pH 6.0, left stirring for about 10 min, spun, the precipitate discarded, and then neutralized. This supernatant was then brought to 60% ammonium sulfate in both cases, stirred at 4°C for 20 min as above and the supernatant was then discarded. The precipitated protein was dissolved in 1% glycine at pH 6.4 containing 30 mM fl-mercaptoethanol. Damp, precycled phosphocellulose was then added to each of the enzyme preparations until loss of enzyme activity occurred. The resultant slurries were centrifuged as above and the precipitate discarded.

The final specific activities obtained were l l0#moles product formed/min per mg protein for the honey bee enzyme, and 92 #moles product formed/min per mg pro- tein for the oyster enzyme. Each preparation was free of any enzymes that would either interfere with the basic assay or remove or interconvert any of the added metabo- lites.

Enzyme activity was monitored by measuring the disap- pearance of NADH (decrease in A34o) for the forward di- rection or appearance of NADH (increase in A34o) in the reverse direction. Standard assay mixtures for the forward direction contained the following: 50 mM Tris-HC1, pH 7'0, DHAP and NADH; for the reverse direction: 50 ram Tris-HC1, pH 7'5, ~-glycerophosphate and NAD. All reac- tions were started by the addition of the enzyme prep- aration.

RESULTS AND DISCUSSION

Effect of pH In one general characteristic, pH requirements, all

three ct-GPDHs under study are rather similar. In each case, the forward reaction displays a much lower pH optimum (about pH 7) than the backward reac- tion (about pH 8'5). In the absence of accurate intra- cellular pH data for the different species involved, it is impossible to attach any particular physiological significance to the minor differences noted for the 3 ~t-GPDHs examined (Table 1).

Specific activities

Some general insight into the functional signifi- cance of an enzyme can be obtained simply from knowing how much of it is present in the tissue under study. In the case of oyster ~-GPDH, the enzyme occurs in very low titre, about 3 pM/min/g wet weight of adductor muscle. Squid and bee muscle ~- GPDHs occur at activities nearly 100-fold higher than the oyster. In the case of the squid mantle, ~- G P D H occurs in higher titre than any other enzyme in energy metabolism (Hochachka et al., 1975, this series) and that is the probable case in bee flight muscle as well (Crabtree & Newsholme, 1972). Hence, in comparing the catalytic and regulatory properties of these 3 enzymes, it is important to bear in mind that we are comparing two of extremely high catalytic potential to one of very low catalytic potential.

Substrate saturation kinetics As expected, all three enzymes display hyperbolic

saturation kinetics with respect to substrates and coenzymes. Double reciprocal plots of the data yield estimates of the Km value for each substrate in the forward and the backward reaction. From these data (Table 2), a number of important differences between the enzymes can be noted. Firstly, oyster ~-GPDH, compared to the homologues from the squid and the bee, displays a much higher competitive ability for the carbon substrate, DHAP, the Km values for DHAP being 3- to 4-fold higher for ~-GPDHs that function in active ~-GP cycles (Table 2). At the same time, the squid enzyme shows a 5-fold higher affinity for NADH than does the oyster enzyme while bee ~-GPDH affinity for NADH is 10 times greater than that of the oyster enzyme. In the absence of any further information it is clear that a high activity of ~-GPDH in fast muscles such as the squid mantle or the bee would require a concomitantly high glyco- lytic rate and high DHAP levels. These are functional requirements that are consistent with high aerobic metabolic rates that can be sustained for indefinite time periods. In contrast, the oyster enzyme, a low activity enzyme functioning in a highly anaerobic metabolism, requires highly reducing conditions (i.e. high NADH levels) to achieve its full catalytic poten- tial. This functional requirement also appears to fit the in vivo situation, as it would call for increasing c~-GPDH function with increasing anoxic stress.

Unidirectional catalysis under in vivo conditions As the usual direction of function of ct-GPDH may

depend upon the physiological functions of the tissue (Lee et al., 1973; Sacktor, 1970), it is important to emphasize that all 3 c~-GPDHs under study catalyze essentially one-way reactions under all probable in

Table 1. Optimum pH ranges for c~-GPDHs from the oyster adductor and honey bee flight muscle, compared to the squid enzyme, each enzyme assayed at saturating levels of substrate and coenzyme in Tris-

HC1 buffer

pH Optimum Enzyme source Forward reaction Backward reaction

Oyster adductor 7-2~7.3 7.8-8.5 Honey bee 6.7-6.8 8.3-8.4 Squid 6.5-7.5 8-5-9.0

Oyster, bee and squid ~-GPDHs 177

Table 2. Michaelis constants calculated from double-reciprocal plots of 1/velocity vs l/substrate concentration for the forward reaction at pH 7-0 and for the backward reactions at pH 7.8 for the enzymes from bee and oyster. The squid enzyme was assayed in the forward direction at pH 7.0; in the back reaction,

at pH 7.5 (Storey & Hochachka, 1975)

K m (mM) for ~-GPDHs from Substrate Oyster Squid Bee

For forward reaction DHAP 0.06 0" 15 0.24 NADH 0"05 0.01A).02 0.005

For backward reaction • -GP 0.5 1.0 2.0 NAD 0.14 1.6

vivo situations. Several lines of evidence lead to this conclusion, the first of which derives from the relative affinities for substrates. It will be evident (Table 1) that in all three cases, the affinities for substrates and coenzymes in the forward direction are at least an order of magnitude greater than in the backwards direction. An equally good measure of this impression arises from studies of product inhibition of both for- ward and back reactions. Thus, in the forward direc- tion, ~-GP is a poor inhibitor of all three enzymes (but particularly of the squid and bee c~-GPDHs), a result in fact to be expected since under anoxic stress this metabolite accumulates (Sacktor, 1970; Hochachka et al., 1975, this series). In contrast, when the back reaction is considered, all three enzymes are remarkably sensitive to product inhibition, particu- larly with respect to NADH. In all three cases, NADH is a strictly competitive inhibitor with respect to NAD, and the Kl values are 0"8, 1-0, and 4.0 pM for the oyster, bee, and squid enzymes, respectively. Similarly, DHAP is a competitive inhibitor with re- spect to ~-GP and the K~ values for DHAP are like- wise remarkably low, about an order of magnitude lower than the respective K m values for the forward reaction (Table 3). In functional terms what this means is that in all three species, when the forward reaction is half-saturated with either DHAP or NADH, the back reaction is fully inhibited by either or both metabolites. On these grounds alone, then, one-way in vivo function, DHAP--*~-GP, seems inevi- table and this conclusion is bolstered by thermodyna- mic considerations (the reaction proceeding in this direction with a large, negative free energy change) and by the respective pH optima for the forward and

backward reaction, the back reaction rates being reduced by 2- to 4-fold at physiological pH compared to optimal pH. As all three muscles under study uti- lize carbohydrate as the major carbon and energy source, the direction of net in vivo function that is favoured should in fact be the same; it is in tissues which have an important fat metabolism that the back reaction of ~-GPDH takes on physiological sig- nificance (Lee et al., 1973).

The role of ATP in ct-GPDH regulation

From the above considerations, the picture emerges of two kinds of ~-GPDHs, one exemplified by the enzyme in oyster adductor and primarily concerned with redox regulation under anoxic stresses severe enough to gradually lead to increased NADH levels, the other exemplified by the enzymes in squid mantle and bee flight muscles and primarily concerned with initiating and sustaining the ~-GP cycle. In the adduc- tor tissue, which lacks an ~-GP cycle, activation of ~-GPDH is energetically wasteful because it reduces by about one-half the ATP output of the glycolytic pathway (Hochachka & Somero, 1973); ~-GPDH function is presumably favoured for reasons of redox regulation. In contrast, in squid mantle and bee flight muscles, activation of ~-GPDH sets the stage for an efficient energy-yielding ~-GP cycle and it is this ATP-yielding function that presumably favoured the evolutionary appearance of this metabolic organiza- tion. Most instructive, then, is the observation that only the squid and bee c~-GPDHs are ATP inhibited. The K~ for the squid enzyme is about 1/3 the value for the bee enzyme, but both values are very much within the lower physiological concentration range for ATP

Table 3. K, values obtained from Dixon plots of l/velocity vs inhibitor concentration at least at two different concentrations of the variable substrate

K[ values (mM) for 7-GPDHs from Inhibitor Oyster Squid Bee

For forward reaction ~-GP 2.5 Over 15 mM 9.7 mM ATP No inhibition 0-6 2.0 AMP 2.6 Over 5 mM NH~- 10.0 36.0

For backward reaction NADH 0.0008 0.004 0.001 DHAP 0.013 0-012 0.005 ATP 0.13 1.6 2.0

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

(Williamson, 1965; Sacktor, 1970). What this means is that under energy saturated conditions (for example, under resting conditions), both the bee and the squid ~-GPDHs are held in a highly (probably fully) inhibited state by ATP. Upon transition from resting muscle metabolism to flight work (in the bee) or swimming work (in the squid), a transient drop in ATP levels due to myofibrillar ATPase releases from inhibition the great catalytic potential of ~- GPDH, in this way "sparking" the ~-GP cycle at a metabolically appropriate time. In the oyster adduc- tor, on the other hand, no ATP inhibition of cytoplas- mic ~-GPDH, can be detected. In fact, if there is any ATP effect it is a slight activation of the reaction at low ATP concentrations. The lack of ATP inhibi- tory control is entirely consistent, of course, with the concept of a redox related function for ~-GPDH rather than the energy-yielding function observed in the squid and the bee.

Search for other metabolite modulators

In the course of these studies, a concerted search was made for other potential modulators of this im- portant metabolic locus. All three enzymes were found to be fully refractory to high concentrations of a wide range of glycolytic intermediates, of dicar- boxylic and tricarboxylic acids of the Krebs cycle, of amino acids, and of most other nucleotides. Of the metabolites tested, only AMP, ADP, and GTP are inhibitory; examination of these effects indicates high K~ values for adenylates such as AMP, well beyond the physiological range for these compounds (Table 3). GTP is almost as effective as ATP, but as its concentration in the cytosol is substantially lower than ATP, the latter is considered of greater physiological significance. Otherwise, the only other potentially important inhibitor is NH,~ (Table 3). The KI for the oyster enzyme is well within in vivo con- centration range for this ion and may therefore be of some significance. The honey bee enzyme, in con- trast, is largely refractory to this ion; as in vivo NH~ concentrations in bee flight muscle undoubtedly are far below 36 mM, the observed K 1 (Table 3), we attach no major significance to this inhibitory effect.

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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