Adaptation of Drosophila enzymes to temperature—II. Supernatant and mitochondrial malate dehydrogenase

The observed temperature-dependent Km patterns were similar among species (Drosophila) from the same habitat and different among species from different habitats. The Q10 values in general reflected temperature dependent changes in Km. The observed convergence in terms of Km and Q10 values and assay temperature strongly suggests that natural selection is operating at the malate dehydrogenase locus in the genus Drosophila with the temperature being one of the mediating agents. Our data, based on biochemical analysis, show that mitochondrial malate dehydrogenase seems to be a ‘conservative’ enzyme from the evolutionary point of view. This enzyme isolated either from temperate or tropical species exhibits ‘symptoms’ of tropical enzyme in terms of Km vs. temperature. The fact that mitochondrial malate dehydrogenase and soluble malate dehydrogenase do not react coordinatly, bears on the significance of the existence of two forms of the enzyme.

INTRODUCTION SINCE temperature is known to affect the higher structures of proteins (3 ° and 4 ° ) and the interactions of proteins with low molecular weight ligands, many studies have focused on the effects of temperature on the formation of enzyme-substrate complexes and on the conversion of this complex into an 'activated complex' (Low et al., 1973;Low and SOMERO, 1976). From these studies significance was placed on the mechanism of positive thermal modulation, for metabolic rate compensation (HOCHACHKA and SOMERO, 1973). According to this mechanism decreases in temperature within the physiological temperature range of an organism, which would normally reduce enzyme reaction rates, are accompanied by an immediate increase of the enzyme substrate affinity. This leads to reaction rate constancy possibly buffering the individual against changes which could take place during daily or seasonal temperature fluctuations.
The present work deals with the temperature dependent kinetic parameters of the double enzymic system of the mitochondrial-soluble malate dehydrogenase (m-Mdh, s-Mdh) by comparing several temperate and tropical species of Drosophila. Investigation, utilizing such enzymic systems, is of considerable interest for studies in biochemical evolution. The aim of this study was to determine the extent to which the temperature-compensatory system mentioned above, is taking place in terrestrial poikilotherms (in the genus of Drosophila). The specific questions whether this is a common scheme and if so, whether there exists a convergence in enzymatic function among terrestrial species inhabiting similar habitats (tropical versus temperate species) were examined.

Stocks and culture conditions
We examined three tropical (D. willistoni, D. arizonensis, D. equinoxialis) and two temperate species (D. virilis, D. americana). Stocks were provided by the Stock Center, University of Texas, Austin (PATTERSON and STONE, 1952) and were maintained at 25°C in a dead-yeast-sugar-agar food medium (ALAHIOTIS and PELECANOS, 1978).

Enzyme separation and assay techniques
For the separation of the cytoplasmic from the mitochondrial malate dehydrogenase a simple method was applied based on the observation that the soluble enzyme migrates to the anode while the mitochondrial enzyme migrates to the cathode in starch electrophoresis (McREYNOLDS and KXTTO, 1970). This technique and the gel electrophoresis are described in detail elsewhere (ALAmOTIS, in press). In short, crude homogenates [300 mg flies/ml 0.1 M potassium phosphate buffer pH 7.5 with 1 mM EDTA (Na2), 1 mM DTF] of 4-10-day-old adults were made in a glass homogenizer. Following two centrifugations (at 20,000 g for 20 min at 4°C) both the soluble and mitochondrial Mdh were present in the supernatant. To collect cytoplasmic or mitochondrial enzyme, electrophoresis was carried out (tris--eitrate buffer at pH 7.0), (ALAmOT1S, in press), for 2 hr on starch plates and a small longitundinal strip of gel removed and stained for malate dehydrogenase (the staining solution was 25 mg NAD, 15 mg NBT, 1 mg PMS, 50 mg L-malate 10 ml 0.1 M glycine-NaOH, pH 10.3, 40 ml H20; activity appeared within 5-10 rain). Just anodal to the position in 189 which staining took place a section of starch was removed from across the entire gel and substituted with a buffer saturated strip of sponge. Current was turned on again and the enzyme was electrophorized into the sponge during the next 50 min. A strip of dialysis membrane was inserted anodal to the sponge to prevent enzyme from migrating out of the sponge. The sponge was then removed and squeezed to recover the enzyme. To remove starch and sponge particles the solution was centrifuged at 5000 g for 10 min. We tested the purity of the soluble or mitochondrial enzyme by a second electrophoresis (Fig. 1). The partially purified enzyme was used immediately. Malate dehydrogenase activity was assayed by monitoring the oxidation of NADH at 340 nm in a Gilford recording spectrophotometer. Assay temperatures were maintained in the cuvette chamber by a Haake circulating water bath. The reaction was initiated by the addition of 20/tl partially purified enzyme preparation in a total 1.0 ml reaction mixture, and activity was monitored for 2 min. The 1.0 ml reaction mixture contained 0.35 mM NADH, 0.94 ml 0.05 M glycine-NaOH at pH 9.45, varying levels of OAA (or 3 mM OAA in the standard assays) and enzyme.

K,, determination
For estimating kinetic parameters, six-substrate concentrations were assayed with three to four replicates (in at least two separate experiments) at each of four temperatures. V~ and K m values were determined from 1/S vs. 1/v plots.

Thermal stability studies
Partially purified enzyme preparations were incubated at 5T:C in a water bath. Samples were removed from the water bath, cooled on ice, and assayed for activity. The results were plotted as percent of original activity vs time of incubation.

Starch-gel electrophoresis; heat inactivation test
Crude extract from each of the five species was subjected to starch-gel electrophoresis at pH 7.0. The mobility of the s-Mdh shows very high interspecific variation (Fig. 1). The same is true when the heat inactivation profiles ofs-Mdh are compared (Fig. 2). It seems that enzymes extracted from tropical species are not more stable than those from temperate. The same is true for the acetylcholinesterase (Ache) and NADPdependent isocitrate dehydrogenase (Idh-NADP) (ALAHIOT1S and BERGER, 1978) although in the case of e-glycerophosphate dehydrogenase (~t-Gpdh) (ALAHIOTIS et al., 1977) a correlation was observed between inactivation rate and natural habitat temperature. The heat inactivation patterns of s-Mdh were found to be species specific which indicate that the primary sequence of s-Mdh for each of the five species was different. Figure 3 shows that the enzyme-substrate affinity of s-Mdh is temperature-dependent, indicating that it is sensitive to environmental temperature. All species had a pattern of positive thermal modulation the extent of which differed between tropical and temperate species. In comparison with those ot ~ tropical species, K,., values of the temperate species, although indistinguishable at low temperatures are higher at high temperatures. The observation suggests that the K,~-temperature plots, again are habitat temperature specific. It must be noted that the K,, values obtained here for partially purified enzyme are higher than those reported earlier (McREYNOLDS and NITro, 1970;HAY and ARMSTRONG, 1976) for D. virilis and D. melanogaster. Moreover, enzymes other than Mdh which are present in crude extracts, or partially purified enzymepreparation mayalso use oxalo acetate as substrate. Thus the discrepancy in K,, values may be due in fact to variation in the degree of purification (and assay conditions); alternatively, it could be due to the interference of low molecular weight compounds that are removed during purification. A parallel case has been reported for the alcohol dehydrogenase of D. melanogaster (McDoNALD et al., 1977).

Qlo and E,
From the kinetic data temperature coefficients (Qlo) at three 10°C temperature intervals were determined at two substrate concentrations. As one can see from Table I the Qlo values are lower at below substrate saturation conditions (0.2 mM OAA) than those at saturating concentrations of substrate (3 mM OAA), where Km is unimportant in determining reaction velocity (SoMERO, 1969). Furthermore, on the basis of positive thermal modulation model, and below saturation concentrations of the substrate (0.2 mM; Table 1) the Qlo values of tropical species are higher than those of temperate as predicted (HOCHACHKA and SO~RO, 1973). Activation energies (E,) were calculated (ROBERT and GRAY, 1972) from Arrhenius plots of log V~,~vs lIT. All species had similar values (10.24_.+0.23 kcal/mole).

pH optima
As in the case of D. melanogaster (ALAmOTIS, in press) all Drosophila species examined here exhibited activity peaks at two different pH values (the main peak at pH 9.45 and a shoulder at pH 7.95) although it has not been noticed before (McREYNOLDS and KITTO, 1970). It is quite likely that the observed bimodality in pH optima indicates the presence of two isozymic types (ALAHIOTm, in press) of s-Mdh (it has been suggested by O'Brien (1973), that s-Mdh is a dimeric enzyme with epigenetic modification).  (1)  s-Mdh isozymes s-Mdh is one of the D. melanogaster enzymes which shows no developmental isozymes when single flies, pupa~, larvae and embryonic cells are subjected to starch electrophoresis (ALAHIOTIS and BERGER, 1977). Moreover, homogenates of fly parts (head, thorax, abdomen from either females or males) exhibited Mdh activity without showing isozymic differences on starch gel electrophoresis. Hence, the present study was based on enzyme isolated from the whole organism and not from a particular organ.

Mitochondrial Mdh (m-Mdh )
Particularly interesting in this study is the observed K. vs. temperature patterns of the m-Mdh. Figure 4 shows dearly that m-Mdh, isolated either from tropical or temperate species, has 'symptoms' of tropical enzyme, in terms of K,.-temperature dependence. This finding, in combination with the absence of interspecific variation in electromorphs ( Fig. 1)   melanogaster was more thermostable than the s-Mdh (McREYNOLDS and KITTO, 1970) increase, considerably, the possibility that m-Mdh is cons6i'vative in evolutionary changes m the genus of Drosophila. Such a 'conservative' enzyme like Drosophila ~t-glycerophosphate dehydrogenase (LAKOVARA et al., 1977) has much taxonomic value and is apparently useful as a key in classification. Our results are strengthened by those of MCREYNOLDS and KITTO (1970), where mitochondrial malate dehydrogenase from two different species of Drosophila (D. melanogaster and D. virilis) is catalytically more similar to each other than to the supernatant enzyme from the same species and vice versa. Conservation, but in the opposite direction in electrophoretic mobility of s-Mdh and m-Mdh, was also observed in birds (KITTO and WILSON, 1966). The fact that s-Mdh and m-Mdh do not react coordinately in terms of K m vs temperature also supports the existence of two forms of the enzyme. While both s-Mdh and m-Mdh are under nuclear control (O'BRIEN, 1973) it appears that they have different evolutionary capabilities.

CONCLUSION
It is widely accepted that the K m and Q]o for any given enzyme is not a change characteristic, but one of major importance in metabolic control and regulation of reaction rates. Hence, it must be under selective pressure (HocHACHKA and LEWIS, 1971). In conclusion, the observed temperature-dependent Km profiles described in the present paper, as predicted, seem to be based on the species distribution rather than phylogeny. The data strongly suggest that natural selection is operating on malate dehydrogenase in the genus Drosophila, temperature being one of the controlling agents; although it cannot be said with any certainty how these kinetic differences are translated into physiological differences in vivo. This is also true for Ache, Idh-NADP (ALAHIOTXS and BERGER, 1978), tt-Gpdh (ALAH1OTIS et'al., 1978) of Drosophila as well as for s-Mdh of reptiles (HosKINS and ALEKS]UK, 1973).