Lactate dehydrogenase isozymes in type I,IIA and IIB fibres of rabbit skeletal muscles

Summary Lactate dehydrogenase (LDH) isozyme patterns were analysed by polyacrylamide (PAA) slab gel electrophoresis in extracts prepared from various rabbit skeletal muscles of defined fibre composition and by PAA microelectrophoresis of microdissected, histochemically typed single muscle fibres. The results obtained by electrophoresis of whole muscle extracts generally agreed with the data obtained from single fibre electrophoresis, i.e. the LDH isozyme pattern corresponded to that of the predominant fibre type. Type I Fibres from soleus and semitendinosus muscles were characterized by a unique pattern of all 5 LDH isozymes with a predominance of LDH-1, 2 and 3. The major fraction (80%) of the type II fibres from extensor digitorum longus and tibialis anterior muscles contained only LDH-5 (M₄). About 20% of the type II fibres contained in addition to LDH-5 small amounts of LDH-4 and LDH-3. The fraction of fibres containing LDH-5, LDH-4, and LDH-3 was similar (ca. 20%) in the histochemically defined IIA and IIB subpopulations In view of the fact that the major fractions of rabbit IIB fibres display low and of IIA fibres high aerobic oxidative capacities (Reichmann and Pette 1982), these data indicate that the expression of the H-subunit of LDH is not correlated with the aerobic-oxidative capacity of the fibre. It also appears not to be correlated with the presence of different myosin isoforms in IIA and IIB fibres.     

Interaction between mammalian glyceraldehyde-3-phosphate dehydrogenase and L-lactate dehydrogenase from heart and muscle

The exceptionally high protein concentration in living cells can favor functional protein-protein interactions that can be difficult to detect with purified proteins. In this study we describe specific interactions between mammalian D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenase (LDH) isozymes from heart and muscle. We use poly(ethylene-glycol) (PEG)-induced coprecipitation and native agarose electrophoresis as two independent methods uniquely suited to mimic some of the conditions that can favor protein-protein interaction in living cells. We found that GAPDH interacts with heart or muscle isozymes of LDH with approximately one-to-one stoichiometry. The interaction is specific; GAPDH shows interaction with two LDH isozymes that have very different net charge and solubility in PEG solution, while no interaction is observed with GAPDH from other species, other NAD(H) dehydrogenases, or other proteins that have very similar net charge and molecular mass. Analytical ultracentrifugation showed that the LDH and GAPDH complex is insoluble in PEG solution. The interaction is abolished by saturation with NADH, but not by saturation with NAD(+) in correlation with GAPDH solubility in PEG solution. The crystal structures show that GAPDH and LDH isozymes share complementary size, shape, and electric potential surrounding the active sites. The presented results suggest that GAPDH and LDH have a functional interaction that can affect NAD(+)/NADH metabolism and glycolysis in living cells.     

Purification and structural properties of isozymes of isocitrate dehydrogenase from the mouse

Summary Cytoplasmic and mitochondrial isozymes of NADP⁺-dependent isocitrate dehydrogenase were purified from kidney and heart tissue of an inbred strain of mice. The cytoplasmic isozyme was purified from kidney of DBA/2J mice by means of a four-step procedure which included affinity chromatography with an 8-(6-aminohexyl)-amino-NADP⁺-Sepharose column. The heart mitochondrial isozyme of DBA/2J mice was purified by a two-step procedure involving the use of 8-(6-aminohexyl)-amino-AMP-Sepharose and 8-(6-aminohexyl)-amino-NADP⁺-Sepharose columns. The specific activity of the homogeneous cytoplasmic and mitochondrial isozymes was 40 units/mg and 45 units/mg, respectively. Native and subunit molecular weights of these two isozymes were determined by chromatography on Sephadex G-100, G-150 and G-200 Superfine and polyacrylamide gel electrophoresis. Both isozymes were found to be dimers with the subunit molecular weight of approximatively 35,000. The sedimentation coefficients were determined to be 5.9 and 6.1 for the mitochondrial and cytoplasmic isozyme, respectively. The amino acid compositions of these two isozymes revealed distinct differences in arginine and proline contents. A modified procedure regarding the use of affinity columns for the purification of the weakly bound enzymes is also discussed.National Institute of Health Visiting Fellow.     

Purification and some properties of isozymes 1 and 5 of lactate dehydrogenase from fox heart and skeletal muscles]

An improved method is described for the isolation of isozymes 1 and 5 of lactate dehydrogenase (LDH) from heart and skeletal muscles of foxes. The method includes salt fractionation with ammonium sulphate, chromatography on DEAE- and CM-celluloses and affinity chromatography on AMP-Sepharose. The preparations of LDH isozymes 1 and 5 turned out to be homogeneous both in 7,5% polyacrylamide gel electrophoresis and under immunodiffusion analysis. It is shown that the pH optimum for LDH-1 is 10.2-10.4 for LDH-5 it is 9.5-9.6 in the case of the direct reaction, and the pH optimum is 7.6-7.8 for LDH-1 and 7.3-7.4 for LDH-5 in the case of reverse reaction. The values of Mikhaelis constants were determined for substrates and coenzymes in direct and reverse reactions. It is found that the excess of lactate and pyruvate causes substrate inhibition of both LDH-1 and LDH-5. The activities of LDH-1 and LDH-5 showed an unexpected similar sensitivity to the inhibitory effect of high pyruvate concentrations.     

Lactate dehydrogenase isozymes of the leopard danio,Brachydanio nigrofasciatus: their characterization and ontogeny

• 1.1. The tissue specific patterns and ontogeny of lactate dehydrogenase (LDH) are reported.
• 2.2. While all tissues (eye, brain, heart, intestine, liver, ovary and skeletal muscle) show isozymes of A and B subunit composition, only liver extracts possess isozymes resulting from C subunit synthesis.
• 3.3. The A₄ homopolymer appears simultaneously with initial muscle contractility and is correlated with the physiological function of muscular contraction.
• 4.4. The activation of the Ldh-C locus is correlated with the first functioning of liver. It is suggested that the state of differentiation of liver cells may be the stimulus required for C locus expression.

     

Enzymatic properties of isozymes and variants of glucose dehydrogenase from Bacillus megaterium

Three glucose dehydrogenases (GlcDH) from Bacillus megaterium, GlcDH-I, GlcDH-II and GlcDH-IWG3, were purified from Escherichia coli cells harboring one of the hybrid plasmids, pGDK1, pGDK2 and pGDA3, respectively, pGDK1 and pGDK2 contain two isozyme genes, gdhI and gdhII, respectively, from B. megaterium IAM 1030 and pGDA3 contains an isozyme gene from B. megaterium IWG3; GlcDH-IWG3 is a variant of GlcDH-I. GlcDH-I and GlcDH-II have similar pH/activity profiles and the profile for GlcDH-IWG3 is identical to that of GlcDH-I. The pH/stability profiles of these enzymes show that GlcDH-IWG3 is the most stable enzyme in the acidic region, while GlcDH-II is the most stable in the alkaline region, and GlcDH-I is the most unstable throughout the entire pH range examined. As for thermostability, GlcDH-II is the most resistant against heat inactivation at pH 6.5. The values of the first-order rate constant for heat inactivation at 50 degrees C are 0.27 min-1, 0.05 min-1 and 0.11 min-1 for GlcDH-I, GlcDH-II and GlcDH-IWG3, respectively. Kinetic studies show that these enzymes have similar kinetic constant values except that there are some differences in Kia for NAD(P) and Ka (the limiting Michaelis constant) for NAD; the values of the ratio of Kia for NAD and NADP are 11,340 and 8.7 for GlcDH-I, GlcDH-II and GlcDH-IWG3, respectively. GlcDH-I and GlcDH-IWG3 have very similar substrate specificities and GlcDH-II has a slightly higher specificity for D-glucose and 2-deoxy-D-glucose than the others. The results are discussed on the basis of the amino acid substitutions between the enzymes.