The activities of fructose diphosphatase in flight muscles from the bumble-bee and role of this enzyme in heat generation

1. The maximum catalytic activities of fructose diphosphatase from flight muscles of bumble-bees (Bombus spp.) are at least 30-fold those reported for the enzyme from other tissues. The maximum activity of fructose diphosphatase in the flight muscle of any particular bee is similar to that of phosphofructokinase in the same muscle, and the activity of hexokinase is similar to or greater than the activity of phosphofructokinase. There is no detectable activity of glucose 6-phosphatase and only a very low activity of glucose 6-phosphate dehydrogenase in these muscles. The activities of both fructose diphosphatase and phosphofructokinase vary inversely with the body weight of the bee, whereas that of hexokinase is relatively constant. 2. There is no significant hydrolysis of fructose 1-phosphate, fructose 6-phosphate, glucose 1,6-diphosphate and glycerol 3-phosphate by extracts of bumble-bee flight muscle. 3. Fructose 1,6-diphosphatase from bumble-bee flight muscle and from other muscles is inhibited by Mn(2+) and univalent cations; the potency of inhibition by the latter varies in the order Li(+)>Na(+)>K(+). However, the fructose diphosphatase from bumble-bee flight muscle is different from the enzyme from other tissues in that it is not inhibited by AMP. 4. The contents of ATP, hexose monophosphates, fructose diphosphate and triose phosphates in bumble-bee flight muscle showed no significant changes between rest and flight. 5. It is proposed that both fructose diphosphatase and phosphofructokinase are simultaneously active and catalyse a cycle between fructose 6-phosphate and fructose diphosphate in resting bumble-bee flight muscle. Such a cycle would produce continuous hydrolysis of ATP, with the release of energy as heat, which would help to maintain the thoracic temperature during rest periods at a level adequate for flight.     

Temperature and the regulation of enzyme activity in poikilotherms. Properties of rainbow-trout fructose diphosphatase

1. The properties of fructose diphosphatase from the liver of rainbow trout (Salmo gairdnerii) were examined over the physiological temperature range of the organism. 2. Saturation curves for substrate (fructose 1,6-diphosphate) and a cofactor (Mg(2+)) are sigmoidal, and Hill plots of the results suggest a minimum of two interacting fructose 1,6-diphosphate sites and two interacting Mg(2+) sites per molecule of enzyme. 3. Mn(2+)-saturation curves are hyperbolic, and the K(a) for Mn(2+), which inhibits the enzyme at high concentrations, is 50-100-fold lower than the K(a) for Mg(2+). 4. Fructose diphosphatase is inhibited by low concentrations of AMP; this inhibition appears to be decreased and reversed by increasing the concentrations of Mg(2+) and Mn(2+). Higher concentrations of AMP are required to inhibit the trout fructose diphosphatase in the presence of Mn(2+). 5. The affinities of fructose diphosphatase for fructose diphosphate and Mn(2+) appear to be temperature-independent, whereas the affinities for Mg(2+) and AMP are highly temperature-dependent. 6. The pH optimum of the enzyme depends on the concentrations of Mg(2+) and Mn(2+). In addition, pH determines the K(a) for Mg(2+); at high pH, K(a) for Mg(2+) is lowered. 7. The enzyme is inhibited by Ca(2+) and Zn(2+), and the inhibition is competitive with respect to both cations. 8. The possible roles of these ions and AMP in the modulation of fructose diphosphatase and gluconeogenic activity are discussed in relation to temperature adaptation.     

Temperature and the regulation of enzyme activity in poikilotherms. Properties of lungfish fructose diphosphatase

1. The properties of fructose diphosphatase from liver of South American lungfish (Lepidosiren paradoxa) were examined. 2. Saturation curves for substrate (fructose diphosphate) and both cofactors (Mn(2+) and Mg(2+)) are sigmoidal and Hill plots of these results suggest about 2 interacting substrate and cofactor sites/molecule of enzyme. 3. Mn(2+) is an efficient positive modulator of the enzyme and K(a) for Mn(2+) is about 20-30-fold lower than the K(a) for Mg(2+). 4. Lungfish fructose diphosphatase is inhibited by low concentrations of AMP, and the affinity of the enzyme for AMP is insensitive to temperature. 5. The affinities of fructose diphosphatase for fructose diphosphate and Mn(2+) appear to be dependent on temperature, whereas affinity for Mg(2+) is temperature-independent. 6. The pH optimum of the enzyme depends on the presence of the particular cofactor. As pH increases, the K(a) values of both cations are lowered, maximum velocities are increased and the saturation curves for cofactor become hyperbolic. 7. The possible roles of these ions, pH and substrate in the modulation of fructose diphosphatase and gluconeogenic activity in the lungfish are discussed in relation to aestivation and temperature adaptation.     

Studies on hepatic injury and antioxidant enzyme activities in rat subcellular organelles following in vivo ischemia and reperfusion

The activities of rat hepatic subcellular antioxidant enzymes were studied during hepatic ischemia/reperfusion. Ischemia was induced for 30 min (reversible ischemia) or 60 min (irreversible ischemia). Ischemia was followed by 2 or 24 h of reperfusion. Hepatocyte peroxisomal catalase enzyme activity decreased during 60 min of ischemia and declined further during reperfusion. Peroxisomes of normal density (d = 1.225 gram/ml) were observed in control tissues. However, 60 min of ischemia also produced a second peak of catalase specific activity in subcellular fractions corresponding to newly formed low density immature peroxisomes (d = 1.12 gram/ml). The second peak was also detectable after 30 min of ischemia followed by reperfusion for 2 or 24 h. Mitochondrial and microsomal fractions responded differently. MnSOD activity in mitochondria and microsomal fractions increased significantly (p < 0.05) after 30 min of ischemia, but decreased below control values following 60 min of ischemia and remained lower during reperfusion at 2 and 24 h in both organelle fractions. Conversely, mitochondrial and microsomal glutathione peroxidase (GPx) activity increased significantly (p < 0.001) after 60 min of ischemia and was sustained during 24 h of reperfusion. In the cytosolic fraction, a significant increase in CuZnSOD activity was noted following reperfusion in animals subjected to 30 min of ischemia, but 60 min of ischemia and 24 h of reperfusion resulted in decreased CuZnSOD activity. These studies suggest that the antioxidant enzymes of various subcellular compartments respond to ischemia/reperfusion in an organelle or compartment specific manner and that the regulation of antioxidant enzyme activity in peroxisomes may differ from that in mitochondria and microsomes. The compartmentalized changes in hepatic antioxidant enzyme activity may be crucial determinant of cell survival and function during ischemia/reperfusion. Finally, a progressive decline in the level of hepatic reduced glutathione (GSH) and concomitant increase in serum glutamate pyruvate transaminase (SGPT) activity also suggest that greater tissue damage and impairment of intracellular antioxidant activity occur with longer ischemia periods, and during reperfusion.     

Age-related quantitative changes in enzyme activities of rat brain

The patterns of brain enzymes linked to energy metabolism have been determined in rats aged between 3 and 21 months and compared to those of the developing brain as an estimate of the senescent energy capacity of this organ. During aging, pyruvate kinase increases, pointing towards an enhancement of the glucose-dependence of this organ. However, NAD-isocitrate dehydrogenase declines, suggesting a reduction of Krebs cycle activity in the aged rat brain. An increase in cytoplasmic NAD-malate dehydrogenase found during aging could provide an alternative mechanism of NAD recovery.     

Induction of rat liver malic enzyme messenger RNA activity by insulin and by fructose

Several human normal and neoplastic cell lines were screened for production of PDGF receptor competing activity. Conditioned medium from two sarcomas and one glioma blocked 125I-PDGF binding to human foreskin fibroblasts in a dose-dependent manner. In each case this effect was abolished when the conditioned medium was pretreated with PDGF-antiserum, indicating that the receptor competing activity was immunologically related to PDGF. Direct evidence for de novo synthesis of a PDGF-like component in the cultures was afforded by 35S-cysteine labeling of the three cell lines, followed by immunoprecipitation with PDGF antiserum. This resulted in the specific precipitation of a 31,000 molecular weight labeled protein, which upon reduction was split into two polypeptides of molecular weights 17,000 and 16,500. The significance of these findings in view of the recently discovered structure homology between PDGF and the transforming gene product of simian sarcoma virus, p28sis, is discussed.     

Postnatal changes in dolichol-pathway enzyme activities in rat liver.

The activity of hepatic protein N-glycosylation was compared in rats of different ages by incubating UDP-[14C]glucose with liver microsomes. Dolichyl-phosphate [14C]glucose, [14C]glucosyl-oligosaccharide-lipid and [14C]glycoproteins formed were increased after birth to maximal levels at 2 weeks; thereafter dolichylphosphate [14C]glucose remained constant, while [14C]glucosyl-oligosaccharide-lipid and [14C]glycoproteins were decreased to constant levels at 4 weeks. The postnatal change in the formation of [14C]glycoproteins was similar to the change in the hexosamine content of N-glycans in liver microsomes and plasma, suggesting that the N-glycosylation of proteins in rat liver increases after birth to a maximum at 2 weeks, and thereafter decreases to a constant level at 4 weeks. The possibility of a regulatory role for dolichyl phosphate in glycoprotein synthesis in rat liver during postnatal development was eliminated by demonstrating the inefficiency of exogenous dolichyl phosphate on the postnatal changes in [14C]glycoprotein formation. The transfer of [14C]glucose from UDP-[14C]glucose to denatured alpha-lactalbumin in liver microsomes increased to a maximum at 2 weeks and then decreased to a constant level, as with transfer to endogenous proteins (i.e. the formation of [14C]glycoproteins). On the other hand, the transfer of oligosaccharide from exogenous [14C]glucosyl-oligosaccharide-lipid to denatured alpha-lactalbumin reached a maximum at 2 weeks and then remained constant. These results strongly suggest that oligosaccharide-lipid available for N-glycosylation is limiting in rat liver after 2 weeks post partum. The activities of dolichyl-phosphate glucose, dolichyl-phosphate mannose and dolichyl-pyrophosphate N-acetylglucosamine synthases increased until 2 weeks post partum. Thereafter, the activity of dolichyl-pyrophosphate N-acetylglucosamine synthase decreased to a constant level at 4 weeks, while the activities of dolichyl-phosphate glucose and dolichyl-phosphate mannose synthases remained constant. These results suggest that N-glycosylation of proteins in rat liver increases until 2 weeks post partum, and that this depends on the activities of dolichol-pathway enzymes as a whole rather than on the activity of specific enzymes. N-Glycosylation then decreases to a constant level at 4 weeks due to decreases in the activities of enzymes responsible for oligosaccharide assembly on lipids, including dolichyl-pyrophosphate N-acetylglucosamine synthase.     

The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase and fructose diphosphatase in muscles from vertebrates and invertebrates

1. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase and fructose diphosphatase in crude homogenates of vertebrate and invertebrate muscles are reported. 2. Pyruvate carboxylase activity was present in all insect flight muscles that were investigated: in homogenates of bumble-bee flight muscle the activity was inhibited by ADP and activated by acetyl-CoA, and it was distributed mainly in the mitochondrial fraction. This is the first demonstration of pyruvate carboxylase activity in muscle. However, the activity appears to be restricted to insect flight muscle, since it was not found in other invertebrate or vertebrate muscles. 3. Since the three enzymes were never found together in the same muscle, it is concluded that these enzymes cannot provide a pathway for the synthesis of glycogen from lactate or pyruvate in muscle. Other roles for these enzymes in muscle are suggested. In particular, pyruvate carboxylase may be present in insect flight muscle for the provision of oxaloacetate to support the large increase in activity of the tricarboxylic acid cycle which occurs when an insect takes flight.     

Density-dependent changes in hexose transport, glycolytic enzyme levels, and glycolytic rates, in uninfected and murine sarcoma virus-transformed rat kidney cells

In normal rat kidney (NRK) cell cultures, increased cell density results in a decrease in the rates of hexose transport, glucose utilization, and lactate production and an increase in the level of hexokinase activity. A murine sarcoma virus (Kirsten)-transformed cell line (KNRK) showed little or no density-dependent variation in sugar uptake, glucose consumption, or lactate production. On the other hand, hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase activities were elevated in dense transformed cultures as compared to sparse or uninfected cultures. In another virus-transformed cell line (ts339/NRK) exhibiting temperature-dependent morphology, growth pattern, and transport of 2-deoxy- -glucose, the levels of glycolytic enzyme activity were related to cell density but not to the culture temperature. The lack of correlation between glycolytic enzyme activity and lactate production by either uninfected or murine sarcoma virus-transformed cultures supports the suggestion that enhanced growth and/or hexose transport capacity rather than elevated glycolytic enzyme activity are responsible for the increased rate of lactate production by virus-transformed NRK cells.