Properties of the V-type ATPase from the excretory system of the usherhopper, Poekilocerus bufonius.

The bafilomycin A(1) and N-ethylmaleimide (NEM)-sensitive (V-type) ATPase was partially purified from the apical membrane-rich fractions of excretory system (Malpighian tubules and hind gut) of P. bufonius. Enzymatic activity was inhibited by bafilomycin A(1) (IC(50) = 1.3 nM) and NEM (IC(50) = 10.1 microM). The V-type ATPase activity is confined to the apical membrane fraction, while the activity of Na(+)/K(+) -ATPase forms the major part of the basal membrane fraction. The optimal pH required for maximal activity of V-type ATPase was pH 7.5. The effect of 30 mM of various salts on ATPase activity was investigated. NaCl and KCl caused increases of 175% and 184%, respectively. Other chloride salts also caused an increase in activity in the following ascending order: RbCl, LiCI, choline Cl, NaCI, KCl and tris-HCl. The activity of V-type ATPase was stimulated by a variety of different anions and cations, and HCO(3)(-) was found to be the most potent cationic activator of ATPase activity. The present results show that the properties of V-type ATPase of P. bufonius are similar to those reported for other insect tissues.

Effect of vanadate on the activity of rat jejunal 6-phosphofructo-1-kinase.

The effect of sodium orthovanadate on the activity of 6-phosphofructo-1-kinase (PFK) in the epithelial cells of rat small intestine was investigated. Injection of vanadate (2.5 mg/rat) into rats at 2-day intervals per week for two consecutive weeks resulted in a significant decrease in the maximal activities and activity ratios (activity at 0.5 mM fructose-6-phosphate at pH 7.0/activity at pH 8.0 [v0.5/V]) of the partially purified PFK in rat jejunum. Also, the sensitivity of jejunal PFK to inhibition by ATP increased in rats treated with vanadate. The addition of 1 microM fructose-2,6-biphosphate and 50 microM AMP in the assays released the enzyme inhibition by ATP, and no significant difference was seen between vanadate-injected and control rats. Moreover, the extent of activation with 1 microM fructose-2,6-bisphosphate was significantly higher (79%) in vanadate-injected rats than in control rats (26%). The present results indicate that rat jejunal PFK is highly inhibited with vanadate in vivo. Therefore, although vanadate has been considered to be an insulin-like agent, because of its insulin-like effects on adipocytes and skeletal muscle, the present results may indicate that this behavior could not be applicable to normal rat tissues, because the effect of vanadate on jejunal PFK is clearly opposite that of insulin.

Effect of vitamins C and E intake on plasma lipid concentrations in rats.

Changes in the plasma lipid levels were investigated among rats fed an atherosclerotic-promoting diet containing 0.5% cholesterol and rats fed the same diet with added vitamin C (ascorbic acid), vitamin E (a-tocopherol) and vitamins C + E from one to seven weeks. Total cholesterol (TC) and triglycerides (TG) were significantly increased in rats fed a hyperlipidemic diet from the third week to the seventh week, whereas high density lipoprotein cholesterol (HDL-C) was not affected. Rats supplemented with 5 mg vitamin C, 5 mg vitamin E or 5 mg vitamin C + 5 mg vitamin E per day for four to seven weeks showed significant decrease in the concentration of TC and TG. HDL-C was only affected at the seventh week with vitamin C alone, whereas it was significantly increased with vitamin E alone and vitamins C + E at five to seven weeks. However, supplementation of vitamins C, E or C + E for less than four weeks has no significant effect on plasma lipid concentrations. The antioxidant effect of vitamins C and E is probably a time-dependent process that significantly lowers plasma lipids between week four and week seven following administration of these vitamins. It is therefore suggested that the incidence of coronary heart disease (CHD) may be reduced in lowering plasma lipid levels by dietary supplementation of vitamins C or E.

Effect of hypothyroidism on glucose transport and metabolism in rat small intestine.

The effect of experimental hypothyroidism on the absorption, transmural transport and metabolism of glucose was studied by perfusion of isolated loops of rat jejunum in vitro. When expressed on a dry weight basis, the rate of absorption was enhanced by 32% (P < 0.01); when expressed on a length basis there was no significant change, since the enhancement per unit weight was almost exactly compensated by a diminution in mass per unit length in the hypothyroid state. When expressed in either units, there was a significant enhancement in transmural transport (+123% and +77%, respectively, both P < 0.001), which reflected in part a diminution in the rate of glucose utilization (-29%, P < 0.01 and -43%, P < 0.001, respectively). The changes in glucose utilization were matched by changes in lactate production. Three factors contributed to the diminution in glucose utilization in the hypothyroid state: a diminution in the concentration of 6-phosphofructo-1-kinase (-35%, P < 0.05), and increase in the S0.5 of 6-phosphofructo-1-kinase for fructose 6-phosphate from 0.4 to 0.6 mM and a fall in the mucosal concentration of fructose 2,6-bisphosphate (-56%, P < 0.05). From the point of view of the whole animal, there is little if any change in the capacity of the intestine to absorb glucose from the lumen, but there is a large enhancement of transmural transport that is metabolically driven.

Effects of hyperthyroidism on glucose, glutamine and ketone-body metabolism in the gut of the rat.

1. The metabolism of glucose, glutamine and ketone-bodies was studied in the small intestine of rats after 5 days of hyperthyroidism. 2. Portal-drained visceral bloodflow increased by 20.1% (P < 0.05) in hyperthyroid rats and was accompanied by a decrease in the arteriovenous concentration difference of glutamine (25.7%, P < 0.05), glutamate (22.0%, P < 0.05), alanine (20.9%, P < 0.05) and ammonia (20.6%, P < 0.05) and an increase in that of glucose (27.2%, P < 0.05), lactate (28.9%, P < 0.05) and ketone-bodies (163.2%, P < 0.001). 3. The gut of hyperthyroid rats showed increased rates of extraction of glucose, lactate and ketone-bodies. 4. Enterocytes isolated from hyperthyroid rats showed increased rates of utilization of glucose and ketone-bodies but that of glutamine were decreased. 5. The maximal activities of hexokinase, 6-phosphofructokinase, pyruvate kinase, citrate synthase and oxoglutarate dehydrogenase were increased (by 13.7-36.2%) in intestinal mucosal scrapings of hyperthyroid rats, whereas the activity of glutaminase was decreased (22.1-31.4%). 6. It is concluded that hyperthyroidism increases the rates of utilization of glucose and ketone-bodies but decreases that of glutamine (both in vivo and in vitro) by the epithelial cells of the small intestine.

Effect of experimental hypothyroidism on the control of 6-phosphofructo-1-kinase activity in rat jejunal mucosa.

Changes in the activity of 6-phosphofructo-1-kinase (PFK, EC 2.7.1.11) from the epithelial cells of rat small intestine during experimental hypothyroidism were studied. Hypothyroidism resulted in significant decreases in the plasma concentrations of total tri-iodothyronine, free tri-iodothyronine, total thyroxine, free thyroxine and insulin. These changes were associated with a significant increase in the plasma concentration of thyrotropin. The total activity and activity ratios (activity at 0.5 mM fructose 6-phosphate at pH 7.0/activity at pH 8.0 (v0.5/V)) of jejunal PFK of hypothyroid rats were significantly diminished as compared to control rats. PFK of hypothyroid rats was more sensitive to inhibition by ATP. The mucosal enzyme of both control and hypothyroid state was sensitive to stimulation by AMP and fructose 2,6-bisphosphate. It is concluded that during hypothyroidism the rate of glycolytic pathway in the small intestine is reduced as a result of a fall in glucose uptake, and the subsequent kinetic changes of PFK are primarily to maintain the concentrations of fructose 6-phosphate (and glucose 6-phosphate) during the reduced glycolytic flux. These changes in PFK activity may be caused by changes in plasma insulin concentrations, glucose utilization and fructose 2,6-bisphosphate concentrations.

Allosteric control of 6-phosphofructo-1-kinase from rat lung.

1. The regulation of 6-phosphofructo-1-kinase (PFK) in the rat lung of normally fed (control), 72 hr-starved and streptozotocin-induced diabetic rats was investigated. 2. No significant changes in the total enzyme activities and the activity ratios [activity at 0.5 mM fructose 6-phosphate at pH 7.0/activity at pH 8.0 (v0.5/V)] of rat lung were observed between the control and 72 hr-starved or streptozotocin-induced diabetic rats. 3. Rat lung PFK was highly stimulated by fructose 2,6-bisphosphate (Fru-2,6-P2) as the affinity of the enzyme for fructose 6-phosphate was highly increased by this metabolite and the enzyme inhibition by ATP was released. 4. Although rat liver and mucosal PFK were found to be highly sensitive to stimulation by Fru-2,6-P2, lung PFK was significantly more sensitive to the stimulation by this metabolite than the other tissues. 5. The enzyme was highly inhibited by citrate and was only slightly inhibited by phosphocreatine. 6. ADP, AMP and c-AMP were shown to be activators of lung PFK with c-AMP being the most effective activator. 7. As a rate limiting enzyme of glycolysis, rat lung PFK is highly controlled by its allosteric effectors, especially Fru-2,6-P2, possibly for surfactant lipid synthesis which usually requires a high rate of glycolysis.

Regulation of 6-phosphofructo-1-kinase in the placenta and small intestine of pregnant streptozotocin-induced diabetic rats.

Changes in the activities of 6-phosphofructo-1-kinase (PFK, EC 2.7.1.11) in the placenta and jejunal mucosa of pregnant rats during the onset of experimental diabetes induced by streptozotocin were investigated. The concentrations of fructose 2,6-bisphosphate were significantly decreased in the placenta and small intestine of the streptozotocin-induced diabetic rats. The total activities and the activity ratios (activity at 0.5 mM fructose 6-phosphate at pH 7.0/activity at pH 8.0 (v0.5/V] of placental and jejunal PFK of diabetic pregnant and virgin rats were markedly diminished as compared to normal control rats. Also the susceptibility of jejunal and placental PFK to inhibition by ATP was increased in the diabetic virgin and pregnant rats. Administration of insulin in vivo completely reversed the effects of diabetes on the regulatory properties and on the total activities of placental and jejunal PFK. It is suggested that the diminished activity of PFK in the placenta and small intestine of streptozotocin-induced diabetic rats could be the result of the decreased concentrations of fructose 2,6-bisphosphate as well as the effect of insulin on the activity of PFK.

Regulation of 6-phosphofructo-1-kinase from the epithelial cells of rat small intestine during pregnancy and lactation.

The regulation of 6-phosphofructo-1-kinase (PFK) in the epithelial cells of rat small intestine was studied during pregnancy and lactation. The total activities and activity ratios (activity at 0.5 mM fructose 6-phosphate at pH 7.0/activity at pH 8.0 (nu 0.5/V] of the partially purified mucosal PFK were found to increase initially in early pregnant rats (11-12 days of gestation) and to fall back to normal in late pregnant rats (19-20 days of gestation). These changes in enzyme activity during pregnancy were associated with similar changes in the circulating levels of progesterone. The maximal activity and activity ratio (nu 0.5/V) were increased in male and female rats injected with progesterone. An increase in the total activity and activity ratio of mucosal PFK was also obtained in lactating rats. However, the enzyme was not strongly activated by inorganic phosphate, fructose 2,6-bisphosphate or glucose 1,6-bisphosphate either in early pregnant or lactating rats. These results indicate that mucosal PFK is already present as an active form during early pregnancy and lactation. Therefore, it is suggested that female sex hormones increase the circulating levels of insulin during early pregnancy which, in turn, positively affect the activity of mucosal PFK which could be also stimulated by the increased levels of fructose 2,6-bisphosphate. The increased activity of PFK in the peak lactating rats could be possible because of an increased demand for lactate production from glucose together with the stimulation of PFK by the increased concentrations of fructose 2,6-bisphosphate which therefore increases the rate of glycolysis.

Effects of jurak smoke condensate on enzyme activity of the mouse.

The effect of jurak smokes condensate on the activities of alkaline phosphatase, gluoce-6-phosphatase, 5'-nucleotidase, and cholinesterase of mouse liver and small intestine was investigated. Jurak smoke condesate was administered orally by stomach tube five times weekly over a three-month period. Fifteen animals were used at 1, 2, and 3 months after the start of the administration, with 5 animals killed on days 1, 5, and 9, and the liver and small intestine removed for enzyme assays. The activities of all four enzymes, which are known to be sensitive to toxic agents, were significantly affected. These results indicate that the low content of tobacco leaves in jurak paste and the filtration of the smoke by water in the sheesha reservoir are not sufficient to make the smoke inhaled by smokers risk free.

6-Phosphofructo-1-kinase of rat placenta.

6-Phosphofructo-1-kinase (PFK) of rat placenta was purified to homogeneity with a recovery of 56% of the enzyme activity in the original extract. The purified enzyme is a tetramer and the Mr value of the subunit is 85,000 +/- 1500 as shown by gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Considering the properties of the native rat placental PFK isoenzyme, it is clear that this tissue is a complex mixture of homotetramer and heterotetramer. Purified placenta PFK displayed little cooperativity at pH 7.0 with respect to fructose 6-phosphate and was markedly inhibited with high concentrations of ATP. The affinity of the enzyme for fructose 6-phosphate was increased by fructose 2,6-biphosphate. The purified enzyme was highly inhibited by citrate, whereas it was only slightly inhibited by phosphoenol pyruvate. ADP, AMP and fructose 2,6-bisphosphate showed little stimulation towards placental PFK. The present study suggests that the placental PFK is a relatively active enzymic form and it is also probably characterized with a high rate of glycolysis possibly because this tissue requires a high energy production for the development and maintenance of the fetus as the placenta tends to be a semipermeable membrane through which substances are exchanged between mother and fetus.

Metabolic regulation of renal gluconeogenesis in response to sepsis in the rat.

1. The regulation of renal gluconeogenesis was studied in rats made septic by a caecal ligation and puncture technique. 2. Blood glucose concentrations were not markedly different in septic rats, but lactate, pyruvate and alanine concentrations were markedly increased, compared with sham-operated rats. Conversely, blood ketone body concentrations were significantly decreased in septic rats. Both plasma insulin and glucagon concentrations were markedly elevated in response to sepsis. 3. The maximal activities of glucose-6-phosphatase (EC 3.1.3.9), fructose-1,6-bisphosphatase (EC 3.1.3.11), pyruvate carboxylase (EC 6.4.1.1) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) were markedly decreased in kidneys obtained from septic rats, suggesting diminished renal gluconeogenesis. 4. Renal concentrations of lactate, pyruvate and other gluconeogenetic intermediates were markedly elevated in septic rats, whereas those of acetyl-CoA and fructose 2,6-bisphosphate were decreased and unchanged, respectively. 5. The rate of gluconeogenesis from added lactate, pyruvate and glycerol was decreased in isolated incubated renal tubules from septic rats. 6. Sepsis decreased the arteriovenous concentration difference for glucose, lactate, and alanine. Septic rats showed decreased net rates of glucose production and net rates of removal of lactate and alanine as compared with sham-operated controls. 7. It is concluded that the diminished capacity for renal gluconeogenesis in septic rats could be the result of changes in the maximal activities or regulation of key non-equilibrium gluconeogenic enzymes or both, but the effect of other factors (e.g. toxins) has not been excluded.

Metabolic control of hepatic gluconeogenesis in response to sepsis.

The regulation of hepatic gluconeogenesis was studied in rats made septic by cecal-ligation and puncture technique. Blood glucose was not significantly different in septic rats, but lactate, pyruvate, and alanine were markedly increased. Conversely, blood ketone body concentrations were markedly decreased in septic rats. Both plasma insulin and glucagon were markedly elevated in septic rats. The maximal activities of glucose 6-phosphatase, fructose 1,6-biphosphatase, pyruvate carboxylase, and phosphenolpyruvate carboxykinase were decreased in livers obtained from septic rats suggesting a diminished hepatic gluconeogenesis. Hepatic concentrations of lactate, pyruvate, and other gluconeogenic intermediates were markedly increased in septic rats, whereas those of fructose 2,6-bisphosphate and acetyl-CoA were decreased. The rate of gluconeogenesis from added lactate, pyruvate, alanine, and glutamine was decreased in isolated incubated hepatocytes from septic rats. It is concluded that the diminished capacity of hepatic gluconeogenesis of septic rats could be the result of changes in the maximal activities or regulation of key nonequilibrium gluconeogenic enzymes or both but do not exclude other factors (e.g., toxins).

6-Phosphofructo-1-kinase isoenzymes of the jejunal mucosa of rabbit, rat and mouse.

1. 6-Phosphofructo-1-kinase (PFK) isoenzymes were studied in the jejunal mucosa of rabbit, rat and mouse. 2. The rat mucosal enzyme was found to be very similar to, although not identical with, the mouse mucosal enzyme, as the physical and regulatory properties of these two enzymes were nearly similar except that the immunological studies were dissimilar. 3. PFK prepared from rabbit mucosa showed different and distinct properties from the rat and mouse mucosal PFK when studied by (NH4)2SO4-precipitation, polyacrylamide gel electrophoresis, immunological cross-reactivity and regulatory properties. 4. The difference between the rabbit enzyme and the rat or mouse enzymes is suggested to be due to the lower rate of glycolysis observed in the rabbit jejunal mucosa as the total enzyme activities of the rabbit were found to be less than half of those activities of the rat and mouse mucosa. 5. The dissimilarities among the species in mucosal isoenzymes obtained in the present study are rather expected since the term isoenzyme is now properly reserved for forms that have been shown to be genetically distinct as shown for different tissues in the same species. Such multigenic control does not appear to have been established for the same tissue in different species.

Effects of starvation and streptozotocin-induced diabetes on the activity of phosphofructokinase in the epithelial cells of rat colon.

The regulation of phosphofructokinase in the colonic mucosa of 48 h-starved and streptozotocin-induced diabetic rats was investigated. The specific activities of phosphofructokinase from colonic mucosa of starved and diabetic rats were found to be diminished compared with normal controls. The enzyme obtained from the colonic mucosa of normal, diabetic and starved rats showed sigmoidal velocity curves with respect to fructose-6-phosphate, with apparent Km values of 0.6, 0.62 and 0.7 mM, respectively. However, the present results indicated that phosphofructokinase from the epithelial cells of rat colon is not regulated in a manner similar to that of the intestinal enzyme, which was shown to be highly regulated.

The distribution of phosphofructokinase isoenzymes in the liver of camel, rat and rabbit.

1. The distribution of phosphofructokinase isoenzymes have been compared among camel, rat and rabbit livers. 2. Only a single phosphofructokinase isoenzyme is present in the camel liver which has shown different physical and regulatory properties from the isoenzymes of rat and rabbit liver. 3. The ammonium sulphate precipitation curves of the camel and rabbit enzymes were monophasic, whereas the rat enzyme was biphasic. 4. Rabbit liver phosphofructokinase was slightly more anodic than the rat enzyme, whereas the camel enzyme was the least anodic as shown by the techniques of DEAE-cellulose chromatography and cellulose acetate electrophoresis. 5. Partially purified camel liver phosphofructokinase showed different regulatory properties from the rabbit and rat isoenzymes as the apparent Km values were 0.58, 0.45 and 0.82 mM respectively.

Effects of female sex hormones on the activity of serum hyaluronidase.

The activities of serum hyaluronidase from female rats were measured during pregnancy. In pregnant female rats, the activity of serum hyaluronidase was found to increase initially and to fall to a minimum by the last day of the gestation period, but the activity of the enzyme increased after delivery and was similar to normals at 21 days post-partum. The activity of hyaluronidase obtained from bovine leucocytes was significantly increased when leucocytes were incubated with various concentrations of progesterone. It is suggested that female sex hormones affect lysosomal membranes making them lyse more readily and hence release hyaluronidase.

Fructose-2,6-bisphosphate in rat mesenteric lymph nodes.

1. The fructose-2,6-bisphosphate (Fru-2,6-P2) content of mesenteric lymph nodes was measured in rats. 2. The effects of Fru-2,6-P2 on the activity of 6-phosphofructo-1-kinase (PFK-1) from rat mesenteric lymph nodes were also studied. 3. The affinity of the enzyme for fructose-6-phosphate was increased by Fru-2,6-P2 whereas the inhibition of the enzyme with high concentrations of ATP was released by Fru-2,6-P2. 4. The activity of lymphocyte PFK-1 was highly stimulated in a simultaneous presence of low concentrations of AMP and Fru-2,6-P2. 5. These results show that rat lymphocyte PFK-1 is highly regulated with Fru-2,6-P2 which means that glycolysis in rat lymphocytes is controlled by Fru-2,6-P2.

The purification, characterization and regulatory properties of liver phosphofructokinase in the Arabian one-humped camel (Camelus dromedarius).

1. Phosphofructokinase from camel liver was purified to homogeneity more than 3600-fold, and the yield of the preparation was 46%. 2. The sodium dodecyl sulphate-treated purified enzyme migrated as a single band in 10% polyacrylamide gel. 3. The enzyme is a tetramer, with a monomer Mr 90,000. 4. The regulatory properties of the purified enzyme from camel liver were studied at pH 7.0. 5. The enzyme displayed cooperativity with respect to fructose 6-phosphate and was inhibited by high concentrations of ATP. 6. The enzyme was also inhibited by citrate, phosphocreatine and 2,3-bisphosphoglycerate. 7. On the other hand, ADP, AMP, glucose 1,6-bisphosphate and fructose 2,6-bisphosphate were all found to be strong activators for camel liver phosphofructokinase.

Allosteric properties of phosphofructokinase from the epithelial cells of thermally injured rat small intestine.

1. The allosteric properties of phosphofructokinase from the epithelial cells of thermally injured rat small intestine were studied and compared with those properties of the normal rats. 2. The fructose 6-phosphate saturation curve of mucosal phosphofructokinase from thermally injured rats (3 days post injury, 33% of body surface area) displayed cooperatively; the ratio of the activity observed at pH 7.0 in the presence of 0.5 mM fructose 6-phosphate and 2.5 mM-ATP to the optimal activity at pH 8.0, v 0.5/V, was 0.42 +/- 0.02 in the normal rats and 0.22 +/- 0.03 in the injured rats. 3. The enzyme from thermally injured rats was very sensitive to inhibition by ATP as compared to that from normal rats. 4. The enzyme from thermally injured rats was inhibited by citrate and phosphocreatine in a synergistic manner with ATP. 5. Activation under nearly cellular conditions was produced by ADP, AMP and glucose-1,6-biphosphate. 6. In general, the mucosal enzyme of thermally injured rats was more susceptible to inhibition or activation by various metabolites than the enzyme of the normal rats. 7. These results may suggest that mucosal phosphofructokinase of thermally injured rats may not be subject to the same control mechanism as the normal rats in vivo due to changes in the concentrations of fructose-2,6-biphosphate.