We examined the effects of temperature and stabilizing solutes on A 4 -lactate dehydrogenase (A 4 -LDH) from warmand cold-adapted fishes, to determine how extrinsic stabilizers affect orthologs with different intrinsic stabilities. Conformational changes during substrate binding are ratelimiting for A 4 -LDH, thus stabilization due to intrinsic or extrinsic factors leads to decreased activity. A 4 -LDH from a warm-temperate goby (Gillichthys mirabilis ), which has lower values for k cat and the Michaelis constant for pyruvate (K PYR m ), was intrinsically more stable than the orthologs of the cold-adapted Antarctic notothenioids Parachaenichthys charcoti and Chionodraco rastrospinosus, as shown by a higher apparent transition ('melting') temperature (T m(APP) ). We used four solutes, glycerol, sucrose, trimethylamine-N-oxide and poly(ethylene glycol) 8000, which stabilize proteins through different modes of preferential exclusion, to study temperature -solute interactions of the three orthologs. Changes in T m(APP) were similar for all orthologs in each solute tested, but the catalytic rate of G. mirabilis A 4 -LDH was decreased most by solutes and increased most by temperature. In contrast, the K PYR m values of the Antarctic orthologs were more affected than that of the goby by both solutes and temperature. We conclude that (a) preferential exclusion of solutes functions within the native state of A 4 -LDH to favor conformational microstates with minimal surface area; (b) the varied effects of the different solutes on the kinetic properties are due to the interaction between this nonspecific stabilization and the differing intrinsic stabilities of the orthologs; (c) the catalytic rates of A 4 -LDH orthologs are equally affected by stabilizing solutes, if measurements are made at physiologically appropriate temperatures; and (d) global stability and localized flexibility of these A 4 -LDH orthologs may evolve independently.
Unlike birds and mammals, teleost fish express two paralogous isoforms (paralogues) of cytosolic malate dehydrogenase (cMDH; EC 1.1.1.37; NAD+: malate oxidoreductase) whose evolutionary relationships to the single cMDH of tetrapods are unknown. We sequenced complementary DNAs for both cMDHs and the mitochondrial isoform (mMDH) of the fish Sphyraena idiastes (south temperate barracuda) and compared the sequences, kinetic properties, and thermal stabilities of the three isoforms with those of mammalian orthologues. Both fish cMDHs comprise 333 residues and have subunit masses of approximately 36 kDa. One cytosolic isoform, cMDH-S, was significantly more heat-stable than either the other cMDH (cMDH-L) or mMDH. In contradiction to the generally accepted model of vertebrate cMDH evolution, our phylogenetic analysis indicates that the duplication of the fish cytosolic paralogues occurred after the divergence of the lineages leading to teleosts and tetrapods. cMDH-L and cMDH-S differed in optimal concentrations of substrates and cofactors and apparent Michaelis-Menten constants, suggesting that the two paralogues may play distinct physiological roles. Differences in intrinsic thermal stability among MDH paralogues may reflect different degrees of stabilization in vivo by extrinsic stabilizers, notably protein concentration in the case of mMDH. Thermal stabilities of porcine mMDH and cMDH-L, but not cMDH-S, were significantly increased when denaturation was measured at a high protein (bovine serum albumin; BSA) concentration, but the BSA-induced stabilization reduced the catalytic activity.
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