Samir M. Khoja

Properties of ouabain-resistant Na+K+-transporting ATPase from the excretory system of Poekilocerus bufonius

Abstract The properties of Na + K + - transporting ATPase in microsomal fractions from the excretory system (Malpighian tubules plus hind-gut) of Poekilocerus bufonius were studied. The optimal conditions required for optimal activity of Na + K + - transporting ATPase were as follows: 1. (1) Maximal activation of Na + K + - transporting ATPase occurred at an ATP:Mg 2+ ratio of 1:1.5. The apparent K m value was 0.84 mM and the V max was 166 nmol Pi lib. mg protein −1 min −1 . 2. (2) Maximal activation of Na + K + - transporting ATPase occurred at 80 mM Na + and 20 mM K + . The apparent K m values were 7.4 mM Na + and 3.6 mM K + . 3. (3) The optimal pH of Na + K + - transporting ATPase was 7.5. 4. (4) The enzyme is rather insensitive to the cardiac glycoside ouabain (I 50 = 2 × 10 −4 M). The sensitivity of Na + K + - transporting ATPase of P. bufonius to Na + , K + and ouabain is lower than that of S. gregaria . However, the low sensitivity of the enzyme to ouabain inhibition observed in this insect would possibly indicate that the presence of this enzyme is an important factor in the tolerance of plant cardiac glycosides in P. bufonius .

Regulatory properties of 6-phosphofructo-1-kinase of the mid-gut of the grasshopper, Poekilocerus bufonius

The fine structure of the mid-gut of Poekilocerus bufonius has been examined and three types of epithelial cells were identified; normal epithelial cells with their apical part possessing well developed microvilli, goblet-like cells containing myelin-like figures and the small basal cells with small and round nuclei, nidi. The regulation of 6-phosphofructo-1-kinase (PFK-1) prepared from the mid-gut of the grasshopper, Poekilocerus bufonius, was studied. Mid-gut PFK-1 displayed cooperativity with respect to fructose-6-phosphate at pH 7.0, and the enzyme was inhibited by high concentrations of ATP. The affinity of the enzyme for fructose-6-phosphate was increased by fru-2,6-P2 whereas the inhibition of the enzyme by high concentrations of ATP was relieved by fru-2,6-P2. The activity of mid-gut PFK-1 was highly stimulated in a simultaneous presence of low concentrations of fru-2,6-P2 and AMP. ADP, AMP and c-AMP were all shown to be activators of the mid-gut PFK-1 with AMP being the greatest effector. The enzyme was not inhibited by citrate either in the presence of low or high concentrations of ATP. These results suggest that the PFK-1 of the mid-gut of the grasshopper is highly regulated with positive stimulators, specially fru-2,6-P2, whereas the enzyme is not regulated by citrate or glucose-1,6-bisphosphate.

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.

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.

Utilization of dates in the fermentative formation of citric acid by Yarrowia lipolytica

Summary Yarrowia lipolytica NRRL Y-1095 was found to be the most potent organism for citric acid formation. Utilization of different Barni date-coat sugar extract concentrations in the fermentation medium as carbon source was found to be good carbon in concentration of 25 mg/ml. Date-seed hydrolysate as nitrogen source for citric acid formation was suitable due to the presence of miscellaneous nitrogen sources, especially amino acids. The yield of citric acid was increased with the increase of phosphorus concentration reaching its maximum when the medium was supplemented with about 1.0 mg KH 2 PO 4 /ml. On using different oxaloacetic acid concentrations in the fermentation medium, it was found that citric acid was increased with the increase of oxaloacetic acid concentration reaching its maximum when the medium was supplemented with 0.4 mg/ml.

Alkaline phosphatase from the excretory system of the grasshopper, Poekilocerus bufonius

Abstract Alkaline phosphatase from the excretory system of the grasshopper, Poekilocerus bufonius was purified with ammonium sulphate fractionation and chromatography on Bio-Gel A-0.5 m. The specific activity of the enzyme is 152 units/mg of protein. The enzyme is a tetramer and the M r value of the subunit is 72,000 ± 2500 as shown by gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme has a pH optimum of 9.6 and an apparent K m value of 0.28 × 10 −3 M. The activity of the enzyme reached a maximum at 75°C and the enzyme showed stability at 65°C. The enzyme was inhibited by Ca 2+ , Na + and Fe 3+ and was stimulated by Zn 2+ , Mn 2+ and Mg 2+ .

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.

Phosphofructokinase from the epithelial cells of rat small intestine, comparison of regulatory properties with those of skeletal muscle, liver and brain phosphofructokinase

The regulatory properties of phosphofructokinase from rat mucosa, liver, brain and muscle were investigated. Mucosal phosphofructokinase displayed cooperativity with respect to fructose 6-phosphate at pH 7.0 and so did the muscle, brain and liver isoenzymes. All these four isoenzymes were inhibited by ATP, the mucosal isoenzyme being the least inhibited. They were also inhibited by citrate and creatine phosphate. AMP, ADP, glucose 1,6-diphosphate, fructose 2,6-bisphosphate and inorganic phosphate were all strong activators for the mucosal, brain, liver and muscle phosphofructokinase, but the mucosal isoenzyme was found to be more activated than the others, accounting for the higher rates of glycolysis observed in mucosa. The results suggest that mucosal phosphofructokinase is unique and different from all the other isoenzymes.

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.

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.