Abstract
This chapter discusses the assay method and properties of L-lactate dehydrogenase, fructose- 1,6-diphosphate (FDP)-activated, from Streptococcus cremoris . L-lactate dehydrogenase activity is measured spectrophotometrically at 340 nm by following either the oxidation of nicotinamide adenine dinucleotide dehydrogenase (NADH) in the presence of pyruvate and FDP, or the reduction of NAD in the presence of L-lactate and FDP. The steps involved in the purification procedureof L-lactate dehydrogenase are (1) the growth of organism, (2) the preparation of cell-free extracts, (3) fractionation with streptomycin sulfate, (4) first and second fractionation with ammonium sulfate, (5) chromatography on diethylaminoethyl (DEAE)-cellulose, and (6) chromatography on DEAE-Sephadex. The stability of the enzyme is dependent on both the type and pH of the buffer in which it is stored. Cell-free extracts prepared in acidic to neutral buffers and stored at 5°C and retain more lactate dehydrogenase activity than those prepared in alkaline buffers. The activation of lactate dehydrogenase activity by FDP is inhibited by phosphate and by the treatment of the enzyme with pyridoxal phosphate followed by reduction with sodium borohydride.
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