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.

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 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.

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.

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.

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.