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The kinetic quantitation of ATP: D‐glucose 6‐phosphotransferases
Author(s) -
Gumaa K.A.,
McLean Patricia
Publication year - 1972
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(72)80644-6
Subject(s) - citation , medical school , medicine , library science , computer science , medical education
It is currently held that the cytosolic fraction of rat liver contains four isozymes of ATP : D-glucose 6_phosphotransferase, three of which exhibit high affinities for D-glucose and are designated hexokinases [EC 2.7.1. l] and the fourth, namely glucokinase [EC 2.7.1.21, has a high Km for glucose. Sols et al. [l] postulated that glucokinase is unique to the liver parenchymal cell and that the hexokinase activity of liver extracts represents a contamination with non-parenchymal cells. Evidence for the existence of more than one form of ATP : D-glucose 6-phosphotransferase in rat liver extracts was furnished by Walker [2] who in a later report [3] found that the isozyme with the high Km for glucose was highly adaptive, and decreased significantly in liver extracts from fasted and diabetic rats. Sols et al. [l] could not confirm the existence of residual glucokinase activity in livers from alloxan diabetic rats and attributed this to the interference by the unmodified hexokinase activity. Fractionation of tissue extracts by chromatography on DEAE-cellulose columns [4-71 and by starch-gel electrophoresis [6-91 confirmed the kinetic evidence for the existence of high and low Km-glucose ATP : Dglucose 6-phosphotransferases [2] and, in addition, resolved the dilemma around the Michaelis constants of the hexokinases obtained from various sources, which was due to the presence of various proportions of the isozymes in the different tissues. The isozymes were designated Types I-IV in accordance with their rates of mobility towards the anode upon electrophoresis, type IV being the fastest [7]. In non-hepatic tissues isozyme II is highly adaptive [lo-l 51 and appears to parallel the sensitivity of the tissue to insulin [7] as does glucokinase [ 161. The study of adaptive changes of the ATP : Dglucose 6-phosphotransferases in various tissues has, to date, been qualitative through the electrophoretic separation on starch-gels or on cellulose acetate membranes [ 171, or at most, semiquantitative where electropherograms were optically scanned. The quantitative approach of Hansen et al. [8] to the study of the adaptive changes in isozymes I and II of rat epididjrmal fat pads is the first kinetic approach to the problem, and the present study extends the procedure of Hansen et al. [8] to cover a range of tissues with diverse isozymic patterns. The differing kinetic properties of the four isozymes of ATP : D-glucose 6-phosphotransferase, in addition to their varying stabilities to heating [6] makes it possible to quantitate each of the four isozymes separately from their mixture in crude tissues extracts.