Anne Faulkner

Pyruvate kinase isoenzymes in tissues of the developing guinea pig

Abstract Pyruvate kinase activities and isoenzymes have been followed during the development of the fetal and neonatal guinea pig. The kinetic properties of the adult isoenzymes were not substantially different from those previously reported for the rat except pyruvate kinase 1 isolated from liver was far less sensitive to l -alanine inhibition. The kinetic properties of the isoenzymes isolated from the fetal tissues were the same as those of the adult. Fetal liver contained pyruvate kinase 1 and 4 in comparable activity throughout the period of gestation studied. Up to five bands of activity in the region of pyruvate kinase 3 and 4 were detected in the brain and muscles of developing guinea pig after electrophoresis. Early in gestation the bands with mobility close to pyruvate kinase 4 were predominant; during development these disappeared and bands of activity with mobility close to pyruvate kinase 3 were seen. The properties and distribution of the pyruvate kinase isoenzymes are discussed in relation to the control of glycolysis in developing tissue. The possible molecular significance of multiple forms between pyruvate kinase 3 and 4 is discussed.

Hexokinase isoenzymes in tissues of the adult and developing guinea pig

Abstract Changes in the activities and isoenzyme distribution of hexokinase were determined in a number of tissues during the development of the guinea pig. The total activity in the fetal liver showed a large fall during the second half of gestation to reach adult values by term. With normal diet the fetal, neonatal, and adult livers had isoenzymes I and III but little or no detectable IV (glucokinase). The fetal liver had predominantly type I, but the proportion of type III increased during development. The kinetics of the guinea pig isoenzymes were similar to those reported for the rat. Two additional isoenzymes with mobility between I and II were detected in the fetal liver and blood. They appear to have kinetic properties similar to type I. Detectable liver glucokinase activity was induced by glucose administration to adult guinea pigs. The total activity in kidney, brain and skeletal muscle showed a postnatal rise while in the fetal heart it was high and declined after birth. These tissues contained predominantly type I with varying proportions of type III hexokinase. The ratio of particulate-bound to soluble hexokinase varied from tissue to tissue. All except the liver showed a significant increase in binding after birth. The changes are discussed in relation to the control of glucose utilization in the fetal and neonatal periods.

Pyruvate kinase isoenzymes in tissues of the human fetus

Four major isoenzymes of pyruvate kinase (EC 2.7.1.40) have been identified in various animal tissues which, based on increasing mobility, have been labelled PK4* (alternatively M,), PK3 (alternatively M,), PK2 and PKl (alternatively liver L) [l-7]. The presence of pyruvate kinase isoenzymes in tissues of the fetal rat [8-l 31 and fetal guinea pig [ 141 has been described. The fetal rat liver possesses little PKl activity particularly early in gestation while we found that PKl represented a major portion of the total pyruvate kinase activity of the fetal guinea pig liver. Moreover Balinsky et al. [ 151 recently demonstrated that PKl may represent a major portion of total activity in the 20 week human fetal liver. In addition to the 4 major isoenzymes fetal guinea pig tissues were found to possess additional pyruvate kinase isoenzymes of mobilities intermediate between PK3 and PK4 [14]. The present work describes further studies on the pyruvate kinase isoenzymes in fetal tissues. Pyruvate kinase activity has been measured in tissues from human fetuses and the distribution of isoenzymes determined by electrophoresis and chromatography. PKI and a previously unidentified isoenzyme have been found in substantial amounts in the fetal liver. The pattern in the other tissues is discussed in relation to the changes observed during development.

Metabolite concentrations in the liver of the adult and developing guinea pig and the control of glycolysis in vivo.

Abstract A range of metabolite concentrations have been determined in the liver of the adult and fetal guinea pig during the latter half of gestation. Adenine nucleotides showed little change during development of the fetal liver and the only major difference from the adult was a low ADP concentration. The hexose phosphates, particularly fructose 1,6-diphosphate, were higher and the triose phosphates in the glycolytic pathway after glyceraldehyde 3-phosphate were lower in the fetal liver. Cytosolic NAD + NADH ratios were comparable in both adult and fetal livers as were cytosolic NADP + NADPH ratios for the last 15–20 days of gestation. The metabolite concentrations have been used to indicate that glycolysis in the fetal guinea pig liver is controlled largely by hexokinase, glyceraldehyde 3-phosphate dehydrogenase, and pyruvate kinase.

Pyruvate dehydrogenase and the regulation of glucose metabolism in ruminant tissues

Ruminants absorb little glucose from their diet, >90% being synthesized by gluconeogenesis [l-3]. Glucose ‘sparing’ mechanisms are thought to operate in the ruminant to conserve glucose for essential purposes, since glucose oxidation contributes only 5-10% of COz production [4] and the rate of fatty acid synthesis from glucose is very low in ruminant tissues [2]. Pyruvate dehydrogenase (PDH) is critically placed to regulate the irreversible loss of carbon from the pool of glucose and glucogenic precursors and its key role in this respect is well established in the rat [S]. The role of PDH in the control of glucose utilization in the ruminant has not been explored; the activity of the enzyme has not even been reported for most ruminant tissues. In this study we report the activity of PDH and the proportion in the active state (PDHa) for sheep adipose tissue, liver and placenta. The results suggest that PDH has an important role in the control of glucose utilization in sheep.

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig.

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase. The calculated cytosolic NAD+/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.

Changes in the activities of some glycolytic enzymes during the development of the guinea pig

Abstract 1. 1. The activities of some enzymes of glycolysis have been determined in the livers of the fetal, neonatal and adult guinea pig. 2. 2. The activities of phosphoglucose isomerase, phosphoglyceromutase and lactate dehydrogenase showed no change, but all the other enzymes increased in activity by 2–7-fold in the fetal liver. 3. 3. The activity of aldolase was substantially lower than that of any of the other equilibrium enzymes and was similar to that of phosphofructokinase. 4. 4. The ratio of activity of the equilibrium and regulatory enzymes increased substantially in the fetal liver. Thus major differences in the regulation of glycolysis may occur during development.

Renal gluconeogenesis in pregnant and non-pregnant sheep

Abstract 1. 1. Glucose production was determined in ovine kidney tubules using a variety of precursors. 2. 2. Similar rates of glucose production were observed with tubules isolated from kidneys of fed or 3-day starved sheep. 3. 3. No significant differences in the rates of gluconeogenesis were observed in kidney tubules prepared from late-pregnant or non-pregnant animals. 4. 4. In some instances the rates of gluconeogenesis observed in the presence of two precursors was in excess of the sum of the rates observed when these precursors were supplied individually.