Purification and properties of a fructose-1,6-diphosphate activated L-lactate dehydrogenase from Staphylococcus epidermidis.
L-(+)-lactate dehydrogenase (LDH) from Staphylococcus epidermidis ATCC 14990 was purified by affinity chromatography. The purified enzyme was specifically activated by fructose-1,6-diphosphate (FDP). The concentration of FDP required for 50% maximal activity was about 0.15 mM. The enzyme activity was inhibited by adenosine diphosphate (ADP) and oxamate. The inhibition by ADP appeared to be competitive with respect to reduced nicotinamide adenine dinucleotide (NADH). The catalytic activity of the LDH for pyruvate reduction exhibited an optimum at pH 5.6. The enzyme is composed of four, probably identical, subunits. Sephadex gel filtration and sedimentation velocity at pH 5.6 Yielded molecular weights of about 130 000 and 126 000, respectively. The molecular weight at pH 6.5 and 7.0 was found to be only about 68 000. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and sedimentation velocity at pH 2.0 or 8.5 revealed monomeric subunits with an approximate molecular weight of 36000. The thermostability of the heat labile enzyme was increased in the presence of FDP, NADH and pyruvate. The purified LDH exhibited an anomalous type of kinetic behavior. Plots of initial velocity vs. different concentrations of pyruvate, NADH or FDP led to saturation curves with intermediary plateau regions. As a consequence of these plateau regions the Hill coefficient alternated between lower and higher n-values. Some distinguishing properties of the S. epidermidis LDH and other LDHs activated by FDP are discussed.
D000227:Adenine Nucleotides / Q000494:pharmacology; D004355:Drug Stability; D004789:Enzyme Activation / Q000187:drug effects; D005636:Fructosephosphates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D007773:Lactates; D008970:Molecular Weight; D009243:NAD; D010072:Oxamic Acid / Q000494:pharmacology; D011773:Pyruvates / Q000378:metabolism; D013210:Staphylococcus / Q000201:enzymology*; D013237:Stereoisomerism; D013696:Temperature
D000227:Adenine Nucleotides; D005636:Fructosephosphates; D007773:Lactates; D011773:Pyruvates; D009243:NAD; D007770:L-Lactate Dehydrogenase; D010072:Oxamic Acid