Lack of temperature-sensitivity of rat liver pyruvate kinase

Abstract

Rat liver pyruvate kinase, prepared by Sephadex treatment of a 10(5)g supernatant in phosphate buffer, is quite stable and gives reproducible results when a variety of parameters are altered in the enzyme assay. Incubation of this preparation at 25 degrees C or 0-2 degrees C has no effect on the activation by fructose 1,6-diphosphate or inhibition by ATP or alanine.

Kinetic properties of rat liver pyruvate kinase at cellular concentrations of enzyme, substrates and modifiers

“…The present paper reports results of studies to assess the simultaneous effects of H+, fructose 1,6-diphosphate, phosphoenolpyruvate, L-alanine, Mg2+, adenine nucleotides and enzyme concentrations at physiological concentrations on enzymic activity in phosphate buffer. The enzyme preparations used exhibited reproducible fructose 1,6-diphosphate stimulation unaffected by temperature, as reported by Flory & Koeppe (1973).…”

“…Therefore for results with low concentrations of enzyme the more extensive data with Sephadex-treated crude enzyme are cited. These results were completely reproducible and unaffected by incubation at 00 or 25°C iffructose 1,6-diphosphate was adequately removed from the enzyme preparation (Flory & Koeppe, 1973). Addition of 0.02mM or more fructose 1,6-diphosphate to low concentrations of enzyme (less than lpg/mI) completely reversed inhibitions by MgATP and L-alanine giving approx.…”

“…This assay at 25°C gave the same reaction rates within experimental error as that described by Tanaka et al (1967), but avoided the inconvenience of using 37°C. The essentially twofold greater enzymic activity at 25°C in our assay solution compared with Tanaka's at 37°C is due principally to the presence of 1.0mM-fructose 1,6-diphosphate in our assay, which is also recommended in order to avoid possible errors from residual fructose 1,6-diphosphate which frequently occurs in crude pyruvate kinase preparations (Flory & Koeppe, 1973). For all stopped-flow measurements, the lactate dehydrogenase was dialysed exhaustively against the assay buffer before use and its concentration (units of enzyme/ml) determined by assay at 340nm with 0.4mM-pyruvate and 0.15mM-NADH in the same buffer.…”

“…Preparation of enzyme. Data in Table 1 were obtained on Sephadex-treated crude enzyme, prepared as described by Flory & Koeppe (1973). All other data were obtained with pyruvate kinase I, purified by the method of Tanaka et al (1967) up to the DEAE-Sephadex A-50 chromatography.…”

“…significant regulatory role in glycolysis (Rozengurt et al, 1969; for other pertinent references see introductory paragraph of Flory & Koeppe, 1973). It is difficult, however, to evaluate the probable physiological activity of the enzyme, or factors most important in its regulation from previously available information.…”