Role of pyridoxal phosphate in mammalian polyamine biosynthesis. Lack of requirement for mammalian S-adenosylmethionine decarboxylase activity.

1. Polyamine concentrations were decreased in rats fed on a diet deficient in vitamin B-6. 2. Ornithine decarboxylase activity was decreased by vitamin B-6 deficiency when assayed in tissue extracts without addition of pyridoxal phosphate, but was greater than in control extracts when pyridoxal phosphate was present in saturating amounts. 3. In contrast, the activity of S-adenosylmethionine decarboxylase was not enhanced by pyridoxal phosphate addition even when dialysed extracts were prepared from tissues of young rats suckled by mothers fed on the vitamin B-6-deficient diet. 4. S-Adenosylmethionine decarboxylase activities were increased by administration of methylglyoxal bis(guanylhydrazone) (1,1'-[(methylethanediylidine)dinitrilo]diguanidine) to similar extents in both control and vitamin B-6-deficient animals. 5. The spectrum of highly purified liver S-adenosylmethionine decarboxylase did not indicate the presence of pyridoxal phosphate. After inactivation of the enzyme by reaction with NaB3H4, radioactivity was incorporated into the enzyme, but was not present as a reduced derivative of pyridoxal phosphate. 6. It is concluded that the decreased concentrations of polyamines in rats fed on a diet containing vitamin B-6 may be due to decreased activity or ornithine decarboxylase or may be caused by an unknown mechanism responding to growth retardation produced by the vitamin deficiency. In either case, measurements of S-adenosylmethionine decarboxylase and ornithine decarboxylase activity under optimum conditions in vitro do not correlate with the polyamine concentrations in vivo.     

Activation of branched-chain alpha-keto acid dehydrogenase complex by exercise: effect of high-fat diet intake

The effect of exercise on the activity of branched-chain alpha-keto acid dehydrogenase complex in liver and muscle was studied in rats fed a high-fat (FAT) or a high-carbohydrate (CHO) diet. Both diet groups of rats were offered isoenergetic diets by a meal-feeding method and were trained by treadmill running. On the final day of the experiment, half of the rats in each diet group were exercised by 2 h of running just before they were killed. The activity state of the enzyme complex was elevated maximally by exercise in liver of rats fed the FAT diet but not in liver of rats fed the CHO diet, suggesting that catabolism of branched-chain amino acids in rat liver during exercise was enhanced by the FAT diet. The activity state of the enzyme complex in muscle was enhanced by exercise in both groups of rats, but a significant difference was not observed between the groups. The concentration of branched-chain amino acids was elevated in liver and muscle by exercise in both groups of rats, but the elevated levels in liver were lower in rats fed the FAT diet than in those fed the CHO diet. Serum branched-chain amino acid concentrations were significantly lower in rested rats fed the FAT diet than in those fed the CHO diet, and the leucine and isoleucine concentrations in the former were elevated by exercise, but the serum concentrations in the latter were not significantly affected by exercise. ATP and ADP concentrations in muscle were not significantly affected by either diet or exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of clofibrate feeding on some lipogenic enzyme activities induced by high carbohydrate diet

Clofibrate administration by stomach tube or intraperitoneally for 3 successive days to rats fed standard diet or starved for 72 hr caused about 2-fold increase of malic enzyme activity in the liver and adipose tissue. The drug administered by stomach tube (but not intraperitoneally) to the rats fed fat free-high carbohydrate diet significantly blocked the inducing effect of the diet on malic enzyme activity in both tissues. Clofibrate blocked the induction by fat free-high carbohydrate diet of hexose monophosphate shunt dehydrogenases and ATP-citrate lyase in the liver. The amount of fat free-high carbohydrate diet consumed by rats received clofibrate by stomach tube was much less than by untreated animals. It is concluded therefore that the significant decrease of food consumption by rats receiving clofibrate by stomach tube is responsible for the inhibitory effect of the drug on some lipogenic enzymes activity induced by fat free-high carbohydrate diet.     

Differential effects of fat deficiency on hepatic and pulmonary drug metabolizing enzymes in rat and mouse

In the present study, we have evaluated the effect of dietary fat deprivation on the carcinogen/drug metabolizing enzymes in rats and mice. In rats, hepatic AHH, Cyt.P-450 Cyt.b5 and Cyt.c-reductase were significantly decreased due to fat deficiency, whereas in lungs, AHH and Cyt.c-reductase were decreased without any change in Cyt.P-450 level. In mice, feeding of fat-free diet did not cause any alteration in hepatic AHH and Cyt.P-450, but the levels of Cyt.b5 and Cyt.c-reductase were significantly reduced. In contrast to liver, Cyt.P-450 and Cyt.b5 were increased in lungs. Activity of UDPGT was lowered both in liver and lungs of rats whereas GST and GSH were increased in liver only. In mice, a decrease in UDPGT and appreciable increases in GST and GSH in liver were observed. However, in lungs, UDPGT activity was enhanced by feeding fat-free diet. These observations suggest that mice and rats respond differentially to the depletion of fat in the diet.     

Effect of estrogens on β-hydroxy-β-methylglutaryl coenzyme a reductase activity and cholesterol levels

The effect of estrogens on hepatic β-hydroxy-β-methylglutaryl coenzyme A reductase activity and cholesterol in serum and liver of ovarietcomized rats on normal diet, 2% cholestyramine diet or 2% cholesterol diet was investigated. Estrogen administration to ovariectomized rats on normal diet resulted in increased reductase activity and was correlated with decreased serum cholesterol and increased liver cholesterol levels wlth mestranol (ME), ethinyl estradiol (EE) and estradiol benzoate (EB, 250 μg) but increased serum and liver cholesterol levels with 25 μg and 100 μg EB administration. The increased stimulation of reductase activity by estrogen administration was absolished when rats were fed a 2% cholesterol diet. Cholestyramine feeding markedly increased reductase activity in livers of ovariectomized rats. These studies show that estrogens are not absolutely required for the stimulation of reductase activity and therefore is consistent with the model in which cholesterol functions as a feedback repressor of reductase activity.     

The influence of actinomycin D on the activity of delta-aminolaevulinic acid synthetase and dehydratase in the liver of mice and rats treated by griseofulvin.

The changes of delta-aminolaevulinic acid dehydratase--the second enzyme of porphyrin synthesis--were studied and compared in the liver of mice and rats treated with griseofulvin. The results showed that griseofulvin increased the activity of delta-aminolaevulinic acid dehydratase in the liver of mice and rats treated by griseofulvin. No differences were found between mice and rats as to the effect of griseofulvin on the activity of delta-aminolaevulinic acid dehydratase. The influence of the administration of actinomycin D on the activity of delta-aminolaevulinic acid synthetase and dehydratase in the liver of mice and rats was studied at an early period of experimental porphyria induced by griseofulvin. It was found that the administration of actinomycin D blocked the observed effect of griseofulvin on the activity of both enzymes in the liver of mice and rats.     

Role of pectin from cucumber (Cucumis sativus) in modulation of protein kinase C activity and regulation of glycogen metabolism in rats

The regulatory role of protein kinase C (PKC) in glycogen metabolism in pectin fed rats was investigated. Administration of pectin (5 g/kg body wt/day) from cucumber (Cucumis sativius L.) led to inhibitory effects on PKC activity in the liver of rats. In the brain and pancreas, PKC activity was significantly higher in pectin-treated rats as compared to the control group. Level of blood glucose was significantly lowered and the level of glycogen in the liver was significantly increased in pectin-administered rats. Glycogen synthase activity was enhanced, while glycogen phosphorylase enzyme showed inhibition in pectin-treated rats. Results indicated that pectin administration might have caused an increase in the secretion of the insulin, which, in turn, had a stimulatory effect on the PKC activity in the pancreas. The decreased PKC activity in the liver and increased PKC activity in the brain and pancreas on pectin administration indicated enhanced glycogenesis and reduced glycogenolysis.     

The isoelectric fractionation of hen''s-egg ovotransferrin

1. ATP sulphurylase was assayed in various organs from vitamin A-deficient and pair-fed control rats at different stages of deficiency. Activity decreased slightly in the liver and markedly in the adrenal gland. Striking differences in liver activity were observed between pair-fed control and ad libitum-fed animals. This observation suggested that diet (apart from vitamin A) strongly influenced the activity of ATP sulphurylase. 2. Total starvation caused a severe decrease in activity in liver within 48hr. This was due to a lack of protein intake. 3. By feeding groups of vitamin A-deficient and pair-fed control rats on a diet containing 80% protein, the specific activity of the liver ATP sulphurylase was maintained in the pair-fed control group at the normal level of an ad libitum-fed rat, whereas it decreased by 25% (statistically significant at P<0.01) in the deficient rat. On a 20%-protein diet, there were no significant differences between vitamin A-deficient and pair-fed control rats. These relationships held also for enzyme activity expressed per g. of liver, per total liver and per g. of DNA. There were no differences in liver protein or DNA concentration between vitamin A-deficient and control rats on either protein intake. 4. Control rats on a 20%-protein diet had liver specific enzyme activities about one-half of those in control rats on an 80%-protein diet, as well as lower liver protein concentrations. 5. It is concluded that, when the effect of protein deprivation on ATP sulphurylase is separated from the effect of vitamin A deficiency, a lowering of the enzyme activity caused by the vitamin deficiency is demonstrable.     

Decrease of rat liver cysteine dioxygenase (cysteine oxidase) activity mediated by glucagon.

The hepatic cysteine dioxygenase activity of rats was markedly decreased by the intraperitoneal administration of glucagon. The enzyme activity was also decreased by either dibutyryl cyclic AMP or theophylline. The prior administration of actinomycin D completely blocked the glucagon-mediated decrease of enzyme activity, while administrations of this inhibitor of protein synthesis after glucagon injection did not block the decrease of enzyme activity. A single administration of actinomycin D resulted in a slight increase of cysteine dioxygenase activity in the rat liver. On the other hand, the injection of cycloheximide resulted in a rapid decrease of the hepatic cysteine dioxygenase with a half-life of 2.5 h. The half-life of the enzyme in rat liver after glucagon administration was one hour. The administration of hydrocortisone or insulin had no effect on the glucagon-mediated decrease of cysteine dioxygenase of rat liver. The enzyme activity of alloxan diabetic rat liver was almost the same as that of the intact rat liver. The evidence obtained here suggests that enhancement of degradation or inactivation of cysteine dioxygenase is responsible for the glucagon-mediated decrease of the enzyme activity in rat liver.     

Adaptive changes in enzyme activity and metabolic pathways in adipose tissue from meal-fed rats

A number of metabolic factors and the activity of a number of enzymes were determined in meal-fed (animals fed a single daily 2 hr meal) and nibbling (ad libitum-fed) rats. The dependency of the observed adaptive changes on the ingestion of carbohydrate was studied by feeding diets high in carbohydrate or fat. Glucose-6-phosphate dehydrogenase and NADP-malic dehydrogenase were more active in adipose tissue from high carbohydrate meal-fed rats than in tissue from ad libitum-fed rats. The activity in adipose tissue of isocitric dehydrogenase, 6-phosphogluconate dehydrogenase, and NAD-malic dehydrogenase did not increase significantly in response to meal-feeding the high carbohydrate diet. No increase in lipogenesis or enzyme activity could be demonstrated in adipose tissue from rats meal-fed a high fat diet. Lipase activity of adipose tissue was increased by high carbohydrate meal-feeding and decreased by feeding a high fat diet. The in vitro uptake of palmitate-1-(14)C by adipose tissue was depressed by a high fat diet and enhanced in rats meal-fed a high carbohydrate diet. Diaphragm or slices of liver from high fat-fed rats oxidized palmitate-1-(14)C more rapidly than did tissue from ad libitum-fed animals. Evidence is presented for the quantitative importance of citrate as a source of extramitochondrial acetyl CoA in adipose tissue of meal-eating and ad libitum-fed rats. The relationship of extramitochondrially formed citrate to the NAD-malic dehydrogenase-malic enzyme system in adipose tissue is discussed.     

Induction of cytosolic aspartate aminotransferase by a high-protein diet

Induction of cytosolic aspartate aminotransferase (glutamic oxaloacetic transaminase) was observed in rat liver on administration of a high-protein diet. The enzyme activity in the liver of rats given 60% and 80% protein diet increased to 1.8- and 1.9-fold that in the liver of rats maintained on 20% protein diet, with about 2-fold increases in the levels of functional sGOT mRNA, measured by in vitro translation. Whereas the activity of mitochondrial aspartate aminotransferase did not increase. Induction of cytosolic aspartate aminotransferase was also detected immunocytochemically.     

Differential effect of sunflower seed oil on hepatic and renal D-amino acid oxidase in the rat.

1. The specific activity of hepatic and renal peroxisomal D-amino acid oxidase (D-AAOX) was measured in rats fed diets containing various quantities of vegetable oil. 2. Increasing the amount of dietary sunflower seed oil (SSO) from 10 to 25% (w/w) reduced the specific activity of hepatic D-AAOX by up to 30% after 10 days. 3. In both tissues, the enzyme activity was moderately decreased during the first two-day period after administration of the 25% SSO diet was begun. Unlike hepatic D-AAOX, renal D-AAOX returned to its baseline level in the kidney after the third day. 4. In contrast to SSO, hydrogenated coconut oil (HCO) did not evoke alterations of D-AAOX activity. 5. The activity levels of another peroxisomal enzyme, L-2-hydroxy acid oxidase (L-HAOX), in the liver of rats fed the high-SSO diet vs those fed the control diet were similar. 6. The subcellular distribution of D-AAOX and L-HAOX was not altered in the liver of rats fed the 25% SSO diet during the 10-day period.     

Thiamine diphosphate pool and its biosynthesis in the liver in galactosamine hepatitis

The hepatitis-like changes were induced in the liver of albino female rats weighing 120-150 g and fed on the appropriate vivarium diet by single parenteral administration of hydrochloride galactosamine in a dose of 0.9 or 1.8 mmol per 1 kg of body weight. The thiamine diphosphate level in the cytosol fraction of the liver decreased 24 h after the preparation administration, the same in blood but with the higher dose used. The activity of pyruvate dehydrogenase, a thiamine diphosphate dependent enzyme, decreased similarly. The cytosol transketolase activity lowered by 38-39%. The coenzyme biosynthesis disturbance due to a fall by 49-58% in the thiamine pyrophosphatase activity is considered to be responsible for hydrochloride galactosamine-induced decrease in the thiamine diphosphate pool. Specificity of the thiamine diphosphate pool disturbance and discoordination of thiamine diphosphate dependent enzymes in the liver are observed under administration of hydrochloride galactosamine.     

Effect of dietary protein content on the activity of rat liver asparagine synthetase.

The activity of rat liver asparagine synthetase [EC 6.3.1.1]increased when animals maintained on 25% protein diet were placed on 15% or 6% protein diet. The enzyme activity level rose within one day, reached a maximum in 7 or 10 days after switching the diet and thereafter dropped gradually. During the purification of the enzyme from rats on 25% or 6% protein diet, the yield and increase of the specific activity were similar in the two groups. Combination of the liver extracts from two such groups demonstrated that the amount of endogeneous inhibitors of the enzyme did not change on replacing the diet. The elevation of the enzyme activity in rats fed 6% casein diet was suppressed by an injection of cycloheximide or actinomycin D. It is suggested that the change in the enzyme activity was due to alteration of the amount of the enzyme.     

Rat cytosolic aspartate aminotransferase: regulation of its mRNA and contribution to gluconeogenesis

Induction of cytosolic aspartate aminotransferase (cAspAT) was observed in rat liver on administration of a high-protein diet, or glucagon and during fasting. The enzyme activity in the liver of rats given 80% protein diet or glucagon injection during starvation increased to 2- to 2.4-fold that in the liver of rats maintained on 20% protein diet, with about 2-fold increases in the levels of hybridizable cAspAT mRNA, measured by blot analysis using the cloned rat cAspAT cDNA as a probe. No increase in the enzyme was detected in kidney, heart, brain, or skeletal muscle. The activity of mitochondrial aspartate aminotransferase (mAspAT) did not increase. Induction of cAspAT was observed when glucose metabolism tended toward gluconeogenesis. The physiological function of the induction of cAspAT is considered to be to increase the supply of oxaloacetate as a substrate for cytosolic phosphoenolpyruvate carboxykinase (PEPCK) [EC 4.1.1.32] for gluconeogenesis.     

D-galactosamine induced changes in rat liver plasma membranes lipid composition and some enzyme activities

The influence of D-galactosamine administration on rat liver plasma membranes lipid composition, fluidity and some enzyme activities was investigated. D-Galactosamine was found to induce an increase of the total phospholipids, the cholesterol level and membrane rigidity. In liver plasma membranes of D-galactosamine-treated rats the exogenous phospholipase A2 activity was enhanced about 2 fold, whereas the endogenous activity was slightly decreased. No alteration of the neutral sphingomyelinase activity was observed.     

Effect of thyroid hormone on peroxisomal flavin enzymes in rat liver and kidney

The effect of thyroid hormone on peroxisomal enzyme activity was studied in thyroidectomized- and T4-administered-thyroidectomized rats. In liver, the activities of isozyme A of L-alpha-hydroxyacid oxidase, D-amino acid oxidase, urate oxidase and catalase were decreased by thyroidectomy, and the diminished enzyme activities were restored by T4 administration to rats. These modifications induced by thyroidectomy or by T4 administration, however, were prominent only in immature animals (20-day-old rats). Although the changes in-alpha-hydroxyacid oxidase and D-amino acid oxidase activities, induced by thyroidectomy or by T4 administration, were also observed in 40-day-old rats, those in urate oxidase and catalase activities were not significant in 40-day-old rats. Acyl CoA oxidase activity was not affected by thyroidectomy or by T4 administration in either 20- or 40-day-old rats. In the kidney, isozyme B of L-alpha-hydroxyacid oxidase activity was reduced by thyroidectomy and the diminished enzyme activity was restored by T4 administration in both 20- and 40-day-old rats. D-Amino acid oxidase and catalase activities in kidney, however, were not significantly modified by thyroidectomy or by T4 administration in either 20- or 40-day-old rats. The results suggest that thyroid hormone can modify the peroxisomal enzyme activity, which is prominent in immature animals.     

Early changes in mitochondrial monoamine oxidase activity of rat liver during 2-acetylaminofluorene feeding

The activities of mitochondrial type A and B monoamine oxidase were determined in the liver of rats fed a diet containing 2-acetylaminofluorene (AAF). Three days after the initiation of AAF-feeding, there was a significant decrease of type B monoamine oxidase activity without affect on type A enzyme. The decreased activity of type B monoamine oxidase, which reached a minimum after three weeks, was sustained for as long as AAF-feeding was continued. Sex-related difference in response to AAF was seen in the rat with respect to the onset and the intensity of the decreased type B monoamine oxidase activity, male rats being more sensitive to the carcinogen than female rats. In contrast to the in vivo effect, AAF showed a potent inhibitory effect on type A monoamine oxidase, rather than on type B enzyme, when added in vitro. The pI₅₀ values were estimated to be 7.5 against type A monoamine oxidase and 4.1 against type B enzyme, respectively. The in vitro inhibition of both types of monoamine oxidase by AAF was competitive. The K_i values for AAF were calculated to be 9.51 · 10^?9 M for type A monoamine oxidase and 1.30 · 10^?5 M for type B enzyme, respectively. In accordance with the potent inhibitory effect of AAF on type A monoamine oxidase in vitro, a single administration of the carcinogen, at a dose of 50 mg/kg, resulted in a marked and temporal decrease of the enzyme activity in the mitochondria of male rat liver. Recovery of the decreased type B monoamine oxidase activity was slow, and the enzyme activity did not return to control levels, even if rats were fed the basal diet for 2 or 4 weeks after the cessation of AAF-feeding.     

Activity of nicotinamide–adenine dinucleotide pyrophosphorylase in liver nuclei. Effects of partial hepatectomy, hepatotoxins and dietary changes

1. The activity of NAD pyrophosphorylase is lower in nuclei isolated from regenerating rat liver than in normal nuclei, and this is due to leakage of the enzyme from the nuclei during the isolation. 2. The NAD pyrophosphorylase activity is lower in liver nuclei from newborn rats, and from rats on a protein-free diet, but no leakage occurs in these cases. 3. Poisoning with alpha-amanitin brings about a transient enhancement of NAD pyrophosphorylase activity in mouse liver nuclei. 4. No changes of enzyme activity were observed after 72hr. starvation, administration of actinomycin D or infection with MHV3 virus.     

Studies on the mechanism of 5-fluoroorotic acid inhibition of serine dehydratase induction

Administration of glucagon to rats fed a protein-free diet caused a significant induction of the liver enzyme, serine dehydratase. This effect of glucagon is inhibited by the concomitant administration of fluoroorotic acid. This inhibition was enhanced by pretreatment with glucosamine or galactosamine, probably through depletion of the intracellular uridine pools. Although less than a doubling of enzyme activity was observed after glucagon plus fluoroorotic acid administration, the amount of protein precipitable by antisera specifically reactive against serine dehydratase increased 4.5 times. Ouchterlony double-diffusion analysis showed a completely cross-reacting single precipitin band from liver extracts of untreated animals and rats treated with the analog. Analysis of the antigen-antibody complex by Na dodecyl sulfate-gel electrophoresis indicated that a single protein was being immunochemically precipitated from both the glucagon- and glucagon plus fluoroorotic acid-treated rats. In the latter, the precipitated protein had a molecular weight similar to purified serine dehydratase. These results are consistent with the concept that the incorporation of fluoroorotic acid into mRNA results in the synthesis of a protein with characteristics similar to authentic serine dehydratase but without normal enzymatic activity. Other possible mechanisms to explain the production of this abnormal protein are discussed.