Physical and chemical properties of lactate dehydrogenase in Homarus americanus.

Two isoenzymes of lactate dehydrogenase have been purified from Homarus americanus: One is found predominantly in the tail muscles; the other, in the walking leg muscles. This is the first demonstration of multiple forms of l-specific lactate dehydrogenase in an invertebrate organism. These proteins contain four essential sulfhydryl groups titratable by p-hydroxymercuribenzoate and 5,5′-dithiobis(2-nitrobenzoic acid). The molecular weights of these isoenzymes are dependent upon ionic strength. The native tetramer (M_r 145,000) exists in low ionic strength solutions; the active dimer (M_r 75,000), in high ionic strength solutions; this is the only example of lactate dehydrogenase disaggregation without concomitant loss in enzymatic activity. Microcomplement fixation studies suggest that there may be less than 4% difference in the primary structures of these two proteins.     

Diphosphopyridine nucleotide-linked D-lactate dehydrogenases from the horseshoe crab, Limulus polyphemus and the seaworm, Nereis virens. I. Physical and chemical properties

d-lactate dehydrogenase has been purified from horseshoe crab (Limulus polyphemus) skeletal muscle and the seaworm (Nereis virens). The purified Limulus dehydrogenase was shown to be a dimer, with a molecular weight of approximately 70 000. Sephadex gel filtration and equilibrium sedimentation yield molecular weights of about 80 000 and 70 000 respectively. Acid dissociation yields a molecular weight species of about 35 000. The native enzyme has an s^o₂₀^w of 3.95. Extrapolation of para-hydroxymercuribenzoate inhibition curves to 100% inhibition corresponds to two molecules of para-hydroxymercuribenzoate bound per molecule of enzyme. Studies on the stoichiometric binding of reduced coenzyme show two molecules bound per molecule of enzyme. The number of tryptic peptides has been found to be one-half that expected from the amino acid composition. The electrophoretic pattern of isoenzymic forms can be best interpreted as suggesting that the enzyme is dimeric. In vitro high salt, freeze-thaw hybridizations of the isolated Limulus muscle isoenzymes yield the electrophoretic pattern predicted by a dimeric structure.The physical properties ot Nereis lactate dehydrogenase have been found to be similar to those for the Limulus muscle lactate dehydrogenase.     

Some properties of aldehyde dehydrogenase from sheep liver mitochondria.

Aldehyde dehydrogenase from sheep liver mitochondria was purified to homogeneity as judged by electrophoresis on polyacrylamide gels, and by sedimentation-equilibrium experiments in the analytical ultracentrifuge. The enzyme has a molecular weight of 198000 and a subunit size of 48000, indicating that the molecule is a tetramer. Fluorescence and spectrophotometric titrations indicate that each subunit can bind 1 molecule of NADH. Enzymic activity is completely blocked by reaction of 4mol of 5,5'-dithiobis-(2-nitrobenzoate)/mol of enzyme. Excess of disulfiram or iodoacetamide decreases activity to only 50% of the control value, and only two thiol groups per molecule are apparently modified by these reagents.     

Kinetic properties of aldehyde dehydrogenase from sheep liver mitochondria.

The kinetics of the NAD+-dependent oxidation of aldehydes, catalysed by aldehyde dehydrogenase purified from sheep liver mitochondria, were studied in detail. Lag phases were observed in the assays, the length of which were dependent on the enzyme concentration. The measured rates after the lag phase was over were directly proportional to the enzyme concentration. If enzyme was preincubated with NAD+, the lag phase was eliminated. Double-reciprocal plots with aldehyde as the variable substrate were non-linear, showing marked substrate activation. With NAD+ as the variable substrate, double-reciprocal plots were linear, and apparently parallel. Double-reciprocal plots with enzyme modified with disulfiram (tetraethylthiuram disulphide) or iodoacetamide, such that at pH 8.0 the activity was decreased to 50% of the control value, showed no substrate activation, and the plots were linear. At pH 7.0, the kinetic parameters Vmax. and Km NAD+- for the oxidation of acetaldehyde and butyraldehyde by the native enzyme are almost identical. Formaldehyde and propionaldehyde show the same apparent maximum rate. Aldehyde dehydrogenase is able to catalyse the hydrolysis of p-nitrophenyl esters. This esterase activity was stimulated by both NAD+ and NADH, the maximum rate for the NAD+ stimulated esterase reaction being roughly equal to the maximum rate for the oxidation of aldehydes. The mechanistic implications of the above behaviour are discussed.     

The purification and properties of NADP-dependent isocitrate dehydrogenase from ox-heart mitochondria.

The purification of NADP-linked isocitrate dehydrogenase from ox heart mitochondria is described. The molecular weight from gel filtration, sedimentation equilibrium and gel electrophoresis is 90000+/-4000, and there are two subunits in the molecule each of which binds NADPH with enhancement of the coenzyme fluorescence. The amino-acid composition is reported, and the absorption coefficient, A1/280%, estimated from dry weight measurements is 11.8 cm-1.     

Purification and properties of L-3-glycerophosphate dehydrogenase from pig brain mitochondria.

L-3-Glycerophosphate dehydrogenase (EC 1.1.99.5) was purified from pig brain mitochondria by extraction with deoxycholate, ion-exchange chromatography and (NH4)2SO4 fractionation in cholate, and preparative isoelectric focusing in Triton X-100. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single subunit of mol.wt. 75 000. The enzyme contains non-covalently bound FAD and low concentrations of iron and acid labile sulphide. No substrate reducible e.p.r. signals were detected. The conditions of purification, particularly the isoelectric focusing step, lead to considerable loss of FAD and possibly iron-sulphur centres. It is therefore not possible to decide with certainty whether the enzyme is a flavoprotein or a ferroflavoprotein. The enzyme catalyses the oxidation of L-3-glycerophosphate by a variety of electron acceptors, including ubiquinone analogues. A number if compounds known to inhibit ubiquinone oxidoreduction by other enzymes of the respiratory chain failed to inhibit L-3-glycerophosphate dehydrogenase, except at very high concentrations.     

Phosphodiesterase from cultured tobacco cells. Physical and chemical properties.

Phosphodiesterase isolated from suspension cultures of tobacco cells showed high affinity for concanavalin A-Sepharose and gave single superimposed bands of protein and carbohydrates on disc gel electrophoresis, suggesting that it is a glycoprotein. It contains 14% carbohydrate by weight, and has relatively high contents of basic and aromatic amino acids. Its isoelectric point is at pH 8.8, and the molecular weight of its subunits was estimated as 72 000 from a plot of the retardation coefficient on sodium dodecyl sulfate gel electrophoresis versus the molecular weight. The enzyme was catalytically active in an immobilized state on a concanavalin A-Sepharose column.     

Purification and properties of L-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus.

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a 'wall + membrane' fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.     

Physical and enzymatic properties of myosin from porcine brain

Porcine brain myosin is a cytoplasmic protein similar to, but distinct from, its muscle counterpart. It has a high K+-ATPase activity at high ionic strength in EDTA and a low Mg+2-ATPase activity that is activated fivefold by either porcine brain or rabbit skeletal muscle actin. The molecule consists of three classes of subunits, with molecular weights of approximately 195,000 , 19,000, and 16,000. Brain myosin contains less glutamic acid, less lysine, and more threonine, serine, proline, and tyrosine than skeletal muscle myosin. The brain myosin extinction coefficient at 278 nm is 0.810 cm2/mg. Hydrodynamic studies yield an S020,w of 4.95S, a D020,w of 1.07 x 10(-7) cm2/s for brain myosin, and indicate that the molecules aggregate at high ionic strength. The molecular weight of the molecule, as calculated from extrapolation of D020,w/S20,w to zero concentration, is 444,000. The intrinsic viscosity of brain myosin is 0.191 ml/mg. These data are consistent with a highly asymmetric molecular species. Circular dichroism spectroscopy indicates that brain myosin is 58-60% alpha-helical in the presence of Ca+2 ions, and that removal of Ca+2 causes a small change in the spectrum.     

Evidence for the existence of enzymatic variants of beta-hydroxybutyrate dehydrogenase from rat liver and brain mitochondria.

A comparison of rat brain and liver β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) has revealed that significant differences exist between the enzymes with regard to their kinetic and physical properties. In contrast to the liver enzyme, brain β-hydroxybutyrate dehydrogenase is rapidly inactivated at 46° and is unstable when stored at ?20°. The brain dehydrogenase was found to have a larger K_m (apparent) for the 3-acetylpyridine analog of NAD⁺, and a greater energy of activation in the direction of β-hydroxybutyrate oxidation than the liver enzyme. In the reverse direction, the brain and liver dehydrogenase exhibit substrate inhibition by NADH (0.22 mM and 0.36 mM, respectively). The brain and liver β-hydroxybutyrate dehydrogenase did not differ significantly with regard to the Michaelis-Menten constants measured for NAD⁺ and β-hydroxybutyrate. The K_m constants of brain β-hydroxybutyrate dehydrogenase for acetoacetate (0.39 mM) and NADH (0.05 mM) were lower than those determined for the liver enzyme, acetoacetate (0.73 mM) and NADH (0.35 mM) respectively. These results suggest that the β-hydroxybutyrate dehydrogenase from rat brain and liver are isozymic variants.     

Cloning, nucleotide sequences, and enzymatic properties of glucose dehydrogenase isozymes from Bacillus megaterium IAM1030.

Bacillus megaterium is known to have several genes that code for isozymes of glucose dehydrogenase. Two of them, gdhI and gdhII, were cloned from B. megaterium IAM1030 in our previous work (T. Mitamura, R. V. Evora, T. Nakai, Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Ferment. Bioeng. 70:363-369, 1990). In the present study, two new genes, gdhIII and gdhIV, were isolated from the same strain and their nucleotide sequences were identified. Each gene has an open reading frame of 783 bp available to encode a peptide of 261 amino acids. Thus, a total of four glucose dehydrogenase genes have been cloned from B. megaterium IAM1030. In addition, this strain does not seem to have other glucose dehydrogenase genes that can be distinguished from the four cloned genes so far examined by Southern hybridization analysis. The two newly cloned genes were expressed in Escherichia coli cells, and the products, GlcDH-III and GlcDH-IV, were purified and characterized and compared with the other isozymes, GlcDH-I and GlcDH-II, encoded by gdhI and gdhII, respectively. These isozymes showed different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GlcDH-I greater than GlcDH-III = GlcDH-IV greater than GlcDH-II), although they have the same number of amino acid residues. Double-immunodiffusion tests showed that GlcDH-I is immunologically different from the other isozymes and that GlcDH-III and GlcDH-IV are identical to one another but a little different from GlcDH-II. These glucose dehydrogenases were stabilized in the presence of 2 M NaCl. The effect of NaCl was especially large for GlcDH-III, which is most unstable enzyme. Kinetic studies showed that these isozymes are divided into two groups with respect to coenzyme specificity, although they can utilize both NAD and NADP: GlcDH-III and GlcDH-IV prefer NAD, and GlcDH-I and GlcDH-II prefer NADP. The phylogenic relationship of these glucose dehydrogenase genes is also discussed.     

Homoserine dehydrogenase of Rhodospirillum rubrum. Physical and chemical characterization.

A detailed physicochemical characterization of purified homoserine dehydrogenase of Rhodospirillum rubrum is presented. The enzyme has a molecular weight of 110000 and consists of two subunits of identical molecular weight of 55000. Depending on the ionic strength and protein concentration it is possible for the native enzyme to dimerize to produce an enzymatically active species of molecular weight 220000. Titrations of the native and detergent-treated enzyme with a variety of sulfhydryl reagents show 2 mol free--SH groups per 110000 g, one of which is buried in the protein interior. L-Threonine and/or high concentrations of salt can expose the buried--SH group, and this--SH group is essential for the catalytic activity of the enzyme. Two independent lines of evidence show that extensive polymerization of the enzyme caused by L-threonine and/or high concentrations of salt does not involve the formation of intermolecular disulfide bonds.