Autophosphorylation of lactate dehydrogenase

Incubation of rabbit muscle lactate dehydrogenase in the presence of Mg[alpha-32p]ATP results in the incorporation of the label into the protein. The autophosphorylation reaction is strongly pH-dependent. The maximal phosphorylation is observed at pH 6.8 with 3-4 moles of phosphate bound per mole of tetrameric enzyme. The enzyme-phosphate complex is readily hydrolyzed by hydroxylamine.     

Protein fluorescence of lactate dehydrogenase

1. There is a non-linear decrease in the protein fluorescence (F) of lactate dehydrogenase with the increase in the fraction (alpha) of the coenzyme-binding sites occupied with NADH. 2. By a curve-fitting procedure it is shown that the fluorescence intensity can be represented by the equation F=[1-alpha(1-x)](n) where n is the number of identical and indistinguishable coenzyme-binding sites per protein molecule and x=F(s) (1/n) (F(s) is the protein fluorescence at alpha=1). This equation implies that the relative protein fluorescence of molecules bearing j ligands form the geometric series x(j). 3. Non-linear quenching of protein fluorescence for this enzyme is probably due to radiationless transfer of energy from the protein molecule to the bound NADH and should also be observed when other potential acceptors of protein fluorescence are bound at unique sites. 4. The intercept with F(s) of an initial tangent to a curve of protein fluorescence against alpha will be at a value of alpha equal to (K(d)+[E(0)]). (1-x(n))/n.(1-x) and not at a value equal to the sum of the dissociation constant (K(d)) and the concentration of identical ligand-binding sites ([E(0)]). 5. A use of non-linear protein fluorescence quenching to investigate the state of aggregation of a protein is discussed.     

Pulse radiolysis of lactate dehydrogenase.

Pulse radiolysis has been used to investigate the rates and transient spectra for the reactions of free radicals with beef heart lactate dehydrogenase at pH 7. Analysis of the results leads to second-order rate-constants for eaq-, .OH, .I, .Br2-, .I2- and .(CNS)2- which are, respectively, 24, 21, 10, 0.55, 0.43 and 0.15 in units of 10(10) M-1 s-1 with uncertainties of +/- 20 per cent. Those for .I and .I2- are similar to the corresponding rate-constants for the related enzyme alcohol dehydrogenase. The spectra of the transient species produced by .OH, .Br2- and .(CNS)2- all showed evidence for reactions with tyrosine and tryptophan residues, and in general terms the magnitudes of the rate-constants appeared to increase with the oxidizing abilities of the radicals. The implication of the results for understanding the mechanism of deactivation by free radicals is discussed.     

Phosphorylation of lactate dehydrogenase by ATP

Evidence is presented indicating that phosphorylation of porcine muscle lactate dehydrogenase by [gamma-32P] ATP occurs at carboxyl residues of the protein. The phosphoenzyme complex was moderately stable at pH 6.8 and 25 degrees C, with a half-life of 3.5 h. In the presence of NADH rapid dephosphorylation occurred. Formation of an abortive complex with NAD-pyruvate also caused hydrolysis of the phosphoenzyme. The phosphorylated lactate dehydrogenase was shown to serve as a phosphate donor for phosphorylation of ADP.     

Malate dehydrogenase and lactate dehydrogenase in trematodes and turbellarians

Studies have been made on the activity and properties of malate and lactate dehydrogenases from the cattle rumen trematodes Eurytrema pancreaticum, Calicophoron ijimai and the turbellarian Phagocata sibirica which has a common free-living ancestor with the trematodes. All the species studied have a highly active malate dehydrogenase, its activity in the reaction of reducing oxaloacetate being 6-14 times higher than in the reaction of malate oxidation. The affinity of malate dehydrogenase to oxaloacetate was found to be higher than that to malate. The activity of lactate dehydrogenase (reducing the pyruvate) was lower than the activity of malate dehydrogenase, the difference being 50 times for C. ijimai, 4 times for E. pancreaticum and 10 times for P. sibirica.     

Drosophila lactate dehydrogenase: Developmental aspects

Partially purified lactate dehydrogenase (LDH) from third-instar larvae displays two bands (one major and one minor) on polyacrylamide gels. Analogous preparations from pupae and adults exhibit three LDH-staining bands (one major and two minor) in a similar pattern. The migration of the major band is similar for larvae, pupae, and adults, while the two minor LDH bands of pupae and adults migrate more slowly than the minor larval band. It has been shown that larval LDH incubated with beta-nicotinamide adenine dinucleotide exhibits two additional minor bands with an electrophoretic mobility similar to that of the minor bands of both pupae and adults. The intensity of the minor larval LDH band (exhibited also by untreated preparations) is drastically reduced. This fact indicates that the life-cycle stage-dependent LDH isozymic distribution is possibly due to a posttranslational effect(s). Highly purified LDH from larvae, pupae, or adults, obtained by an affinity chromatography procedure, displays just one dispersed band, located in the area between the band 5 and the band 6 exhibited by crude extract preparations. These data, in combination with the lack of difference in catalytic properties among enzymes from larvae, pupae, and adults, suggest that LDH synthesis is controlled by the same single structural gene at all developmental stages.     

Refolding of lactate dehydrogenase by zeolite beta

We used zeolite beta as an adsorbing matrix to refold recombinant lactate dehydrogenase (LDH) protein collected as an insoluble aggregate from a bacterial expression system. The adsorption isotherm revealed that 1 g of zeolite adsorbed 200 mg of denatured LDH solubilized with a buffer containing 6 M of guanidine hydrochloride. The pH of the buffer had little effect on the adsorption, but this property was abolished by preincubation of the zeolite with polyethylene glycol (PEG) in a weight ratio of 1:10. These data suggest that the adsorption of LDH depends on the hydrophobicity of the zeolite surface, and that the adsorption of PEG to zeolite is sufficient to release LDH from its surface. LDH was thus released by refolding buffer containing PEG and arginine, and soluble LDH was obtained in its active enzymatic form. The addition of arginine dramatically increased the yield of LDH in a dose‐dependent manner. The overall refolding efficiency was optimized to 35%. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009