The preparation of crystalline human L-lactate--nicotinamide--adenine dinucleotide oxidoreductase isoenzyme 1 involvoing preparative polyacrylamide-gel electrophoresis.
A method is presented for the preparation of human heart lactate dehydrogenase (l-lactate-NAD(+) oxidoreductase; EC 1.1.1.27) isoenzyme 1; this involves the use of polyacrylamide-gel electrophoresis as a preparative step. The yield was about 10% with a final specific activity of 220 units/mg of protein, one unit being defined as the amount of enzyme catalysing the oxidation of 1mumol of NADH/min at 25 degrees C, in the presence of 0.33mm-pyruvate. The crystalline preparation contained less than 2% of the other isoenzymes, was homogeneous in the ultracentrifuge and showed only a trace of protein contamination on polyacrylamide-gel electrophoresis. Some properties of the crystalline isoenzyme are reported; E(1%) (1cm)=13.2 at 280nm, s(0) (20,w)=7.43S, pI=4.6, and the apparent K(m) for pyruvate=1.02x10(-4)m. The human isoenzyme and the isoenzyme from pig heart differ with respect to amino acid composition, electrophoretic mobility and solubility. It is possible that these differences do not involve the active site, or sites, but are due to changes in amino acid residues elsewhere in the molecule. The importance of purified human LDH-1 isoenzyme with regard to enzyme radioimmunoassay is emphasized.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002848:Chromatography, DEAE-Cellulose; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification*; D009206:Myocardium / Q000201:enzymology; D009243:NAD; D010084:Oxidation-Reduction; D011863:Radioimmunoassay
D000596:Amino Acids; D007527:Isoenzymes; D009243:NAD; D007770:L-Lactate Dehydrogenase