Effects of high dietary methionine on activities of selected enzymes in the liver of kittens (felis domesticus)

• 1.1. Growing male kittens were fed an 18% casein diet supplemented with 2, 3, or 4% l-methionine (MET) for 6 weeks.
• 2.2. Free MET concentration in liver increased 30-fold and cystathionine two- to three-fold; the activity of adenosyl-MET transferase and cystathionase also increased but remained lower than previously found in rats.
• 3.3. Taurine concentration in liver decreased in cats fed excess MET and appeared to depend on taurine intake.
• 4.4. Alanine aminotransferase activity was high in all groups while serine dehydratase activity was very low.
• 5.5. Pyruvate kinase and malic enzyme activities which are normally low in cat liver increased after excess MET. Also, glucose 6-phosphate and 6-phosphogluconate dehydrogenases increased.
• 6.6. Cat liver metabolism showed limited adaptation to an excess dietary intake of methionine compared to that found in rats.

     

Long-term starvation in Xenopus laevis daudin—III. Effects on enzymes in several tissues

• 1.1. Adult, female Xenopus laevis were subjected to 12 months of starvation.
• 2.2. Starvation resulted in a continuous reduction in the activity of both hepatic and renal glucose-6-phosphate dehydroganse.
• 3.3. Fructose-1,6-diphosphatase was significantly reduced at months 10 and 12 in the liver, and at months 4, 10, and 12 in the kidney.
• 4.4. Pyruvate kinase activity of muscle and liver decreased during the experimental period whereas the renal enzyme remained essentially unchanged.
• 5.5. Both hepatic and renal glutamate-pyruvate transaminase (GPT) and hepatic glutamate-oxaloacetate transaminase (GOT) showed a reduction of activity after 2 and 4 months of starvation followed by an increase in GPT but not in GOT.

     

Purification,characterization and kinetic mechanism of glucose-6-phosphate dehydrogenase from mouse liver

• 1.1. Glucose-6-phosphate dehydrogenase (G6PDH EC 1.1.1.49) from mouse liver has been purified 1100-fold by extraction, ion-exchange chromatography on DE-52, absorption chromatography on Bio-Gel HTP and gel filtration through sepharose 6 HR 10/30. The purified enzyme showed a single band in silver stained SDS-PAGE.
• 2.2. The native and subunit molecular weight were 117 and 31 kDa respectively.
• 3.3. The kinetic studies and the patterns obtained from the inhibition by-products suggest that the enzyme follows an ordered sequential kinetic mechanism.
• 4.4. The reduced K_m values for the substrates favour the operativity of the enzyme. The “fine control” of the enzymatic activity was exerted by the NADPH, whose K_i is several fold lower than the in vivo concentration.

     

Effects of soluble corn bran hemicellulose on lipid metabolism

• 1.1. Since soluble corn bran hemicellulose (CBH) was found to reduce serum cholesterol level in the rat fed with a high cholesterol diet, rats were fed with diets containing orotic acid (OA) to investigate the effect of CBH on lipid metabolism.
• 2.2. Hepatic lipid accumulation induced by OA was reduced by feeding with CBH in rats. The reduction was not due to inhibition of intestinal absorption of OA by CBH.
• 3.3. Administration of acetate or propionate, colonie fermentation products of CBH, tended to alleviate the hepatic lipid accumulation by OA in rats.
• 4.4. OA feeding decreased activities of some hepatic enzymes involved in fatty acid synthesis except for acetyl CoA carboxylase. The decreases were reversed by the concurrent feeding of CBH.

     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

A new glutamate dehydrogenase from Halobacterium halobium with different coenzyme specificity

• 1.1. Halobacterium halobium has two chromatographically distinct forms of glutamate dehydrogenase which differ in their thermolability and other properties. One glutamate dehydrogenase utilizes NAD, the other NADP as a coenzyme.
• 2.2. The NADP-specific glutamate dehydrogenase (EC 1.4.1.4) was purified 65-fold from crude extracts of H. halobium.
• 3.3. The Michaelis constants for 2-oxoglutarate (13.3 mM), ammonium (3.1 mM) and NADPH (0.077 mM) indicate that the enzyme catalyzes in vivo the formation of glutamate from ammonium and 2-oxoglutarate.
• 4.4. The amination of 2-oxoglutarate by NADP-specific glutamate dehydrogenase is optimal at the pH value of 8.0–8.5. The optimal NaCl or KCl concentration for the reaction is 1.6 M.
• 5.5. None of the several metabolites tested for a possible role in the regulation of glutamate dehydrogenase activity appeared to exert an appreciable influence on the enzyme.
• 6.6. NAD- and NADP-dependent glutamate dehydrogenases from H. halobium showed apparent molecular weights of 148,000 and 215,000 respectively.

     

Gender-related differences of mouse liver d-aspartate oxidase in the activity and response to administration of d-aspartate and peroxisome proliferators

• 1.1. The hepatic d-aspartate oxidase activity was found to be higher in female ddY and ICR mice than in their male counterparts. On the contrary, the free d-aspartate content in the liver was lower in female mice than in male mice, suggesting that d-aspartate is actually metabolized by d-aspartate oxidase in vivo.
• 2.2. Oral administration of d-aspartate to the animals increased the hepatic d-aspartate oxidase activity 2–3 fold in both genders without any significant difference in the rate of the increase between the genders.
• 3.3. Several peroxisomal enzyme activities other than d-aspartate oxidase examined were not affected by this treatment.
• 4.4. Experiments in vitro suggested that the increase in the d-aspartate activity might be explained in part by stabilization of the enzyme by d-aspartate.
• 5.5. The administration of clofibrate, a peroxisome proliferator, to male mice, increased the hepatic d-aspartate oxidase activity with a significant simultaneous decrease of d-aspartate content in the liver, in agreement with a possible role of the enzyme n vivo.
• 6.6. On the other hand, the administration of clofibrate or dehydroepiandrosterone to female mice decreased the d-aspartate oxidase activity.
• 7.7. The peroxisome proliferators were suggested to act to eliminate the gender difference of hepatic d-aspartate oxidase activity in mice.

     

The regulation of glucose 6-phosphate dehydrogenase from Dicentrarchus labrax (bass) liver

• 1.1. Kinetic constant values of the reaction catalyzed by bass liver glucose 6-phosphate dehydrogenase show to be modified between 10 and 40°C.
• 2.2. The Arrhenius plot between 10 and 50°C shows two slopes with different activation energies.
• 3.3. These results suggest a regulation of this enzyme by environmental temperature.
• 4.4. Kinetics of ATP inhibition were examined between pH 6.2 and 7.8: patterns and K_i values obtained are affected by the pH variation.
• 5.5. NADH is an effective inhibitor of bass glucose 6-phosphate dehydrogenase but this enzyme does not show NAD-linked activity.
• 6.6. Kinetics of pyridoxal 5′-phosphate inhibition have indicated the presence of a lysine in the catalytic site for NADP⁺.

     

Intramitochondrial reductive carboxylation of 2-oxoglutarate in adipose tissue and its contribution to fatty acid synthesis

• 1.1. The reductive carboxylation of 2-oxoglutarate was found to proceed in mitochondria of rat epididymal fat pads and rabbit perirenal adipose tissue at a rate similar to that in liver mitochondria.
• 2.2. In rat fat pads the incorporation of ¹⁴C from [5-¹⁴C]2-oxoglutarate into fatty acids via the carboxylation was suppressed by butylmalonate by 30%.
• 3.3. 2-Oxoglutarate and glutamate stimulated the incorporation into fatty acids of ¹⁴C from [2-¹⁴C]acetate in rat fat pads with the simultaneous reduction of tissue NADP. These effects persisted after inhibition of succinate dehydrogenase by malonate.
• 4.4. It is concluded that in adipose tissue 2-oxoglutarate carboxylation proceeds in both the cytoplasm and mitochondria. Therefore, it can supply carbon atoms as well as NADPH for fatty acid synthesis.

     

The effects of oxalate and glucose on lipogenesis by isolated hepatocytes from normal and biotin-deficient chicks (Gallus domesticus)

• 1.1. Isolated hepatocytes synthesize fatty acids and cholesterol from lactate and acetate with lactate being the more effective substrate.
• 2.2. Biotin deficiency decreased fatty add synthesis from both substrates but stimulated cholesterogenesis.
• 3.3. Exposure of intact hepatocytes to oxalate inhibited fatty acid and cholesterol synthesis from lactate, this effect was enhanced in biotin-deficient chicks. A similar effect was not observed when acetate was the substrate.
• 4.4. Synthesis of fatty acids from lactate and acetate was stimulated by glucose, biotin deficiency increased this response. Cholesterogenesis was reduced in control but not biotin-deficient chicks.

     

Purification and some properties of isocitrate dehydrogenase of a blowfly Aldrichina grahami

• 1.1. NADH-dependent isocitrate dehydrogenase has been purified 110-fold from the crude extract of the flight muscle mitochondria of Aldrichina grahami.
• 2.2. The purification procedure involved Triton X-100 treatment of isolated mitochondria, column chromatography on DEAE-cellulose, Affi-gel blue, and P-cellulose.
• 3.3. The purified enzyme was homogeneous by criteria of the polyacrylamide gel electrophoresis.
• 4.4. The enzyme of the blowfly contains more acidic amino acids and less hydrophobic amino acids than that of pig heart.
• 5.5. The molecular weight was determined to be 330,000 daltons. The subunit construction differs from ghat of mammalian isocitrate dehydrogenase.

     

Human GMP synthetase

• 1.1. The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes. placenta, and liver.
• 2.2. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration.
• 3.3. The K_m values were determined to be 4.9μM for XMP, 270μM for ATP. and 340 μM for glutamine.
• 4.4. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient.
• 5.5. The enzyme was specific for ATP as the energy source.
• 6.6. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound.
• 7.7. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

     

Glycolytic enzymes of fowl and turkey spermatozoa

• 1.1. It was confirmed that, under anaerobic conditions, fowl spermatozoa formed lactate from glucose thirteen times faster than turkey spermatozoa.
• 2.2. The profiles of glycolytic enzyme activities were similar for spermatozoa from both species; however fowl spermatozoal activities were generally 2- to 4-fold higher.
• 3.3. Exceptions were glycerophosphate mutase and lactate dehydrogenase activities which were respectively 9.5 and 41 times greater in fowl spermatozoa.
• 4.4. In both species, spermatozoal glyceraldehyde-3-phosphate dehydrogenase had the lowest activity of the glycolytic enzymes.

     

Adaptive changes in total pyruvate dehydrogenase activity in lipogenic tissues of rats fed high-sucrose or high-fat diets

• 1.1. Pyruvate dehydrogenase complex (PDC) activity was measured in several tissues of rats fed for 7 or 15 days on control, or high-sucrose or high-fat diets.
• 2.2. Total activity in adipose tissue increased in the three groups 3–4-fold as compared with chow-fed animals in the first week. Total activity was 60% lower in rats fed the diet containing 22% corn oil for 2 weeks.
• 3.3. Hepatic total and PDCa activities were 50–80% higher in rats fed the sucrose diet for 7 or 15 days and decreased 30–40% in those fed on the high-fat diet for 2 weeks.

     

Stearoyl-CoA desaturase activity in primary culture of chicken hepatocytes. influence of insulin,glucocorticoid, fatty acids and cordycepin

• 1.1. Stearoyl-CoA desaturase (Δ9-desaturase) activity was measured in chicken primary hepatocytes, as a function of time in culture.
• 2.2. When using fasted donor animals, the desaturase activity was low at the beginning of culture and then increased steadily to a maximum value between 30 and 70 hr of culture. When hepatocyte cultures were prepared from fed animals, enzyme activity was high at the beginning of culture and maintained thereafter at similar values to those obtained in cultured hepatocytes from fasted animals after 30 hr of culture.
• 3.3. Insulin significantly enhanced enzyme activity when added to the culture medium at a 10⁻⁹M concentration, and a small stimulating effect was also observed with 10⁻⁶M dexamethasone.
• 4.4. Linoleic acid (0.5 mM) added to the culture medium as albuminic complex partly inhibited Δ9-desaturase activity.
• 5.5. Cordycepin (3' deoxyadenosine) decreased enzyme activity when present at a 3 μg/ml concentration in the culture medium.
• 6.6. Taken together, the induction of enzyme activity in culture, its impairment by cordycepin and response to insulin and linoleic acid strongly suggest that synthesis and translation of the Δ9-desaturase mRNA occur in chicken hepatocytes in primary culture, and that this cellular model may be a useful tool for further studies on Δ9-desaturase regulatory mechanisms.

     

Enzyme activities and level of SH groups in breast carcinomas

• 1.1. The carcinoma showed higher enzyme activities than the normal mammary tissue.
• 2.2. The ratios of glutamate dehydrogenase, glutathione reductase and catalase to lactate dehydrogenase were lower in carcinomas than in normal tissues. Similarly, the ratios of glutamate dehydrogenase, glutathione reductase and catalase to glucose-6-phosphate dehydrogenase were also significantly lower in carcinomas.
• 3.3. There were no significant differences in enzyme activities between stages I and II of disease, however in the metastatic tissues, there were significant differences between stages I and II.
• 4.4. SH groups were higher in the tissues of cancer patients than in normal tissues. The levels of thiols groups were higher in carcinomas at stage III of disease.

     

Effects of the nonsteroidal anti-inflammatory drug piroxicam on energy metabolism in the perfused rat liver

• 1.1. The actions of piroxicam, a nonsteroidal and noncarboxylic anti-inflammatory drug, on the metabolism of the isolated perfused rat liver were investigated. The main purpose was to verify if piroxicam is also active on glycogenolysis and energy metabolism, as demonstrated for several carboxylic nonsteroidal anti-inflammatories.
• 2.2. Piroxicam increased oxygen consumption in livers from both fed and fasted rats.
• 3.3. Piroxicam increased glucose release and glycolysis from endogenous glycogen (glycogenolysis).
• 4.4. Gluconeogenesis from lactate plus pyruvate was inhibited.
• 5.5. The action of piroxicam on oxygen consumption was blocked by antimycin A, but not by atractyloside.
• 6.6. The action of piroxicam in the perfused rat liver metabolism seems to be a consequence of its action on mitochondria.
• 7.7. It can be concluded that inhibition of energy metabolism and stimulation of glycogenolysis are not specific properties of carboxylic nonsteroidal anti-inflammatory drugs.

     

The purification of kallikrein from human whole saliva

• 1.1. A quick and simple procedure is described for purifying kallikrein from human whole saliva. The enzyme has been purified about 2700-fold with a yield of approx. 30%.
• 2.2. The procedure is based on the immediate fractionation of saliva by ion exchange chromatography. This is followed by a combination of affinity and high performance liquid chromatography.
• 3.3. The results indicate that another protein component binds to the enzyme at pH 8.0.
• 4.4. The homogeneity of the enzyme has been demonstrated by gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate.
• 5.5. A mol. wt of 40,100±1800 has been calculated from gel electrophores is experiments.
• 6.6. Sedimentation equilibrium in an analytical ultracentrifuge gave a mol. wt of 39,700.
• 7.7. The amino acid composition has been determined and it confirms that the enzyme has a low isoelectric point.
• 8.8. The presence of tryptophan has been demonstrated by absorption and fluorescence spectroscopy.

     

Influence of dietary sources of fat on lipid synthesis in mink (Mustela vison) mammary tissue

• 1.1. The fatty acid composition of the triglyceride fraction of mink milk sampled during mid-lactation (day 28 post partum) from two nursing mink was compared to that of plasma samples and to the fatty acid composition of the feed rations used.
• 2.2. Chemical analysis of the triglyceride composition of mink milk demonstrated only minute concentrations of fatty acids with a chain length below C₁₄.
• 3.3. The saturated C_16:0- and C_18:0-unit fatty acids in mink milk made up for 24–40% of the total amount of fatty acids extracted, the remainder being represented by mono and polyunsaturated long-chain (C₁₆-C₂₄) fatty acids.
• 4.4. Preliminary in vitro experiments proved the incorporation of¹⁴C-labelled glucose, acetate or palmitate into triacylglycerols in cultures of mink mammary tissue to be linear for at least 2 hr.
• 5.5. The in vitro capacity for de novo fatty acid synthesis in mink mammary tissue using 14C-labelled glucose or acetate was low, i.e. ranging from 0.096–0.109 nmol/g (fresh tissue)/min, and amounted to only about 5% of that obtained in the case of [¹⁴C]palmitic acid incubation.
• 6.6. Following ¹⁴C-labeIled acetic or palmitic acid incubation of mink mammary tissue neither desaturation nor chain elongation was observed.
• 7.7. In response to long-term feeding on rations with two different sources of animal fat (F = fish oil or L = lard) the influence of compositional changes in dietary neutral lipids on the fatty acid composition of the lipids of mink milk is discussed.

     

Gut microflora can modify fatty acid composition in liver and egg yolk lipids of laying japanese quail (Coturnix coturnix japonica)

• 1.1. The influence of the gut microflora on lipid metabolism was investigated in germ-free (GF) and conventional (CV) laying Japanese quail.
• 2.2. Serum and egg yolk cholesterol concentrations showed comparable values in both GF and CV environments.
• 3.3. The fatty acid compostion of liver lipids was modified by the presence of gut microflora. Notably, in the presence of the gut microflora, proportion of oleic acid was reduced and conversely, stearic and linoleic acids were enhanced.
• 4.4. In egg yolk lipids, the proportion of myristoleic and palmitoleic acids was significantly lowered and that of stearic acid was significantly enhanced by the presence of the gut microflora, though the difference was very small.
• 5.5. It was suggested that oleic acid could be easily either hydrogenated to stearic acid or desaturated to linoleic acid by the action of the gut microflora in Japanese quail.