Some kinetic properties of lactate dehydrogenase activity in cell extracts from a mammalian (ovine) corneal epithelium.
The purpose of the following study was to examine the kinetic properties of lactate dehydrogenase (LDH) in cell extracts of corneal epithelium. The epithelium, from quality- and size-selected ewe corneas, was dispersed in 10 mM Trizma maleate with or without sucrose at pH 7.0 and cell fractions obtained by homogenisation and differential centrifugation for evaluation of LDH at 37 degrees C. Pyruvate-dependent oxidation of NADH was optimal at pH 6.5, while lactate-dependent reduction of NAD was optimal at pH 9.5; the former activity averaged 3 to 3.5 fold higher than the latter. NADPH and NADP were poor cofactors. The apparent Km values for pyruvate and lactate were 99 micromoles and 3.9 millimoles respectively. At pH 6. 5, substrate inhibition was observed with pyruvate in excess of 0.25 mm with 50% activity measured at 1.2 mm. Excess substrate effects were not seen with lactate at pH 9.5, but pyruvate inhibited lactate-dependent reduction of NAD with 50% activity measured at 1.1 mm. Differential centrifugation confirmed that the activity was predominantly localised in the soluble (post mitochondria-lysosome) fraction of the cells.
Doughty
M J
MJ
Department of Vision Science, Glasgow-Caledonian University, Cowcaddens Road, Glasgow, G4 0BA, Scotland.
D000818:Animals; D002457:Cell Extracts; D004795:Enzyme Stability; D019573:Epithelium, Corneal / Q000201:enzymology*; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D019289:Pyruvic Acid / Q000378:metabolism; D012756:Sheep
D002457:Cell Extracts; D009243:NAD; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase