K.A. Gumaa

The kinetic quantitation of ATP: D-glucose 6-phosphotransferases

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.

Effect of clofibrate on the CoA thioester profile in rat liver

Abstract Acute and chronic treatment with clofibrate increased the total CoA content of rat liver and altered the profile of the various CoA thioesters. There resulted a 2–3 fold increase in the contents of long chain acyl CoA, acetyl CoA and free CoA, contrasting with significant decreases found in succinyl CoA, malonyl CoA and acetoacetyl CoA contents. It is postulated that the known increase in fatty acid binding protein and/or the increased extramitochondrial β-oxidation of fat by an increased peroxisomal population may direct the compartmentation and metabolic fate of fatty acids and their CoA derivatives following clofibrate treatment.

Sequential control of hexokinase in ascites cells

Studies of transient changes in the total rate of glucose phosphorylation compared with the changes in glucose-6-phosphate (G6P), ATP, ADP and fructose diphosphate (FDP) have suggested a sequence of operation of control mechanisms following the addition of glucose to ascites cells. These are: (a) a rapid rate of glucose phosphorylation (50% Vmax) for 15 sec. which appears to be independent of feedback inhibition by G6P which accumulates to inhibitory concentrations within 3 sec.; (b) a release of bound hexokinase by G6P; (c) a slow rate of glucose phosphorylation under feedback control by G6P; (d) the possible involvement of FDP inhibition; this control may depend on Mg++ availability.

A possible interrelationship between binding of hexokinase and the site of ATP formation in Krebs ascites cells

Abstract The overall utilisation of ATP and the partitioning of ATP regeneration between the mitochondrial and cytoplasmic compartments of ascites cells were calculated from the metabolite profiles over a period of 3 – 30 sec. after the addition of glucose. Both compartments contribute about equally to the regeneration of ATP, the glycolytic lagging by 5 sec, pending the “filling up” of glycolytic intermediates between 3-phosphoglycerate and lactate. Regeneration of ATP by adenylate kinase appears to be linear over these time intervals. ATP regeneration slows down appreciably between 15 and 20 sec., coinciding with the release and partial inhibition of mitochondrial hexokinase by glucose-6-phosphate with consequent limitation of the ADP supply. Other possible modulators of mitochondrial phosphorylation are discussed.

Effect of insulin and diet on the steady state concentrations of intermediates of the pentose phosphate pathway of glucose metabolism in liver.

Measurement of the steady state concentrations of intermediates of the pentose phosphate pathway have been made in conditions where marked changes are known to occur in the pattern of glucose metabolism and where it has been established that alterations occur in both the oxidative and non-oxidative reactions of this pathway. The conditions chosen were alloxandiabetes with and without insulin treatment, starvation and starvation followed by refeeding a high carbohydrate diet or a high fat diet. In alloxan-diabetes, the most striking change was a marked increase in sedoheptulose-7-phosphate. Changes in 6-phosphogluconate and pentose phosphate suggested an inhibition of 6-phosphogluconate dehydrogenase. Insulin treatment for 3 days partially reversed this latter effect but the sedoheptulose-7-phosphate concentration remained high. Starvation and starvation followed by refeeding a high fat diet which decreases the flux of glucose through the pentose phosphate pathway, were found to cause a lowering of metabolite levels. Refeeding high carbohydrate diet restored the pentose phosphate level to control value, the sedoheptulose-7-phosphate remained low in this condition. These results suggest control points at both the oxidative reactions of the pentose phosphate cycle and at transketolase.

The changes of metabolite content and the control of phosphofructokinase in rat liver in different dietary and hormonal conditions

It is generally believed that, in most tissues, the control of the glycolytic flux is regulated at the phosphofructokinase (PFK) step and that the main effecters are ATP and citrate (inhibitors) and CAMP, AMP, Pi, F6P and FDP (activators) [l-5, see also 61. Although little doubt exists that these effecters act on the PFK of all tissues, lipogenic and non-lipogenic alike, it seems probable that some differences exist in their relative effectiveness between tissues of different function. Thus, it is logical that in muscle, where glucose metabolism is largely directed to energy production, the main control is vested in ATP and citrate as inhibitors and AMP as an activator. In a lipogenic tissue, such as liver, other considerations apply, namely the requirement for a high glycolytic flux to provide the acetyl-CoA for lipogenesis at a time when there is no necessity for an appreciable change in the activity of the tricarboxylic acid cycle. In this situation a logical case could be made for a control system which is linked to the blood glucose. Such a system would act to permit a high flux rate in glycolysis when the blood glucose is high and the distribution of the acetyl-CoA so produced between oxidation and lipogenesis would then be regulated by the redox state of the nicotinamide nucleotides and the phosphorylation state of the adenine nucleotides in the mitochondria. The present communication examines changes in the content of effector molecules in intact livers from rats in different dietary and hormonal conditions and,

Transient and steady state concentrations of the intermediates of the pentose phosphate pathway in Krebs ascites tumour cells metabolising glucose

The rate of glucose consumption by ascites tumour cells following the addition of glucose to cells incubated in vitro is subject to rapidly acting control mechanisms. There is evidence for an important regulatory site at hexokinase. Three phases have been distinguished: (1) an initially rapid rate of glucose uptake and phosphorylation with apparently maximal hexokinase activity; (2) a marked inhibition of hexokinase; (3) a slow steady rate which is only lo-20% of the initial rate [ 141. While the concentrations of certain glycolytic intermediates during these different phases are known [l-4], relatively little is known about the concentration changes of intermediates of the pentose phosphate pathway under these varied rates of glucose&phosphate formation. Measurements have therefore been made of the concentrations of glucose&phosphate (G6-P), 6-phosphogluconate, the pentose phosphates, sedoheptulose 7-phosphate, erythrose 4-phosphate, glyceraldehyde 3-phosphate and ATP during the transient initial stage and the later steady state conditions of glucose metabolism. It was found that while 6-phosphogluconate, the pentose phosphates and triose phosphate changed in parallel with alterations in G6P, the concentration of both sedoheptulose 7-phosphate and erythrose 4phosphate behaved differently. Sedoheptulose 7phosphate, in particular, was sustained at a high and relatively constant concentration and showed a tendency to be inversely related to the G6P concentration. These results are discussed in relation to the con-