Cloning and sequence analysis of a cdna encoding bovine cytosolic aspartate aminotransferase

• 1.1. Complementary DNA encoding cytosolic aspartate aminotransferase was isolated from an adult bovine heart library.
• 2.2. The amino add sequence deduced for the protein (412 amino acids) is extremely similar (> 94% identity) to that of porcine cytosolic aspartate aminotransferase but interesting differences were noticed comparing the position of cysteine residues.

     

The effect of pyridoxine deficiency on aminotransferase activity in liver and white muscle of rainbow trout (Salmo gairdneri Richardson)

• 1.1. Aspartate aminotransferase activity in the white muscle of rainbow trout dropped significantly after the animals had received a pyridoxine-deficient diet for 7 days.
• 2.2. Alanine aminotransferase activity in the white muscle of rainbow trout dropped significantly after the animals had been fed on a pyridoxine-deficient diet for 21 days.
• 3.3. Alanine and aspartate aminotransferase activity in the liver of rainbow trout did not drop significantly until the animals had received a pyridoxine-deficient diet for 28 days.
• 4.4. After rainbow trout have received a pyridoxine-deficient diet for 35 days, feeding with complete diet for 7 days is sufficient to restore the aminotransferase activities to the levels observed in control animals.

     

The effects of cholesterol feeding on the membranes of liver cells and on the cholesterol metabolism in the rat

• 1.1. Feeding of rats with a 2% cholesterol diet for 6 weeks increased the serum cholesterol concentration. The activity of lecithin cholesterol acyltransferase was also increased during the feeding time.
• 2.2. The activities of aspartate aminotransferase and alanine aminotransferase remained on a constant level during the experiment on rats having cholesterol in their diet. Omitting cholesterol from the diet enhanced the activities of both enzymes and the increase in alanine aminotransferase activity was more pronounced.
• 3.3. The activity of alkaline phosphatase was on higher level during the whole experiment in the rats having cholesterol in the diet than in those fed a cholesterol-free diet.
• 4.4. Present data suggest that excluding cholesterol from the diet labilizes the membranes of hepatocytes and facilitates the release of aspartate and alanine aminotransferases in the blood.

     

Regulation of gluconeogenesis from propionate and propanol in the perfused guinea pig liver

• 1.1. Gluconeogenesis from propanol in perfused guinea pig liver is stimulated by aspartate, glutamate and phenazine-methosulfate, and inhibited by octanoate and by aminooxyacetate.
• 2.2. Gluconeogenesis from propionate is inhibited by octanoate and by NH₄⁺, and stimulated by ethanol.
• 3.3. Phosphoenolpyruvate formation from propionate in isolated guinea pig liver mitochondria is inhibited by octanoate and by NH₄⁺ or by both.
• 4.4. The data are discussed in terms of regulation of gluconeogenesis from both substrates in guinea pig liver.

     

Pyrimidine degradation in tomato cell suspension cultures and in Euglena gracilis—localization of enzymes

• 1.1. Subcellular location of dihydropyrimidinase and NCβA-amidohydrolase2 was studied in a cell suspension culture of tomato (Lycopersicon esculentum cv. Lukullus) and in Euglena gracilis.
• 2.2. By differential centrifugation, crude extracts were separated into ten fractions. Activities of both enzymes were found mainly in cytosolic fractions marked by EDH (tomato) and glu-6-P-DH (E. gracilis).
• 3.3. A cytosolic location was also found by a 20–60% and a 17.5–30% sucrose density gradients.
• 4.4. Using mitochondrial marker enzymes such as fumarase, SDH, CS and MDH, a mitochondrial occurrence of both enzymes or their release from mitochondria can be excluded by sucrose gradient centrifugations. This can also be achieved using purified mitochondria prepared from tomato cells by two subsequent sucrose gradients.
• 5.5. A possible vacuolar location of dihydropyrimidinase and NCβA-amidohydrolase was excluded by comparing their activities in isolated protoplasts and purified vacuoles which were characterized by their marker enzyme α-mannosidase.
• 6.6. A nuclear location of both enzymes and/or their release from the nucleus during procedures used cannot be excluded.
• 7.7. The results are discussed in relation to subcellular location to other pyrimidine-metabolizing enzymes in plant cells.

     

Regulation of glutamine catabolism in the perfused guinea-pig liver in relation to ureogenesis and gluconeogenesis

• 1.1. L-Glutamine conversion into ammonia, urea and glucose by the perfused liver of 48 hr starved guinea-pigs was concentration dependent attaining the maximal rate at 4 mM.
• 2.2. The activity of glutaminase I (EC 3.5.12), measured in isolated liver mitochondria was high enough to account for the observed rate of ammonia, urea and glucose formation by the perfused liver. Neither NH4C1 (5 mM) nor aminooxyacetate (0.5 mM) affected the rate of glutamine conversion into glutamate by isolated liver mitochondria.
• 3.3. Gluconeogenesis and ureogenesis from glutamine was inhibited by octanoate, Dt-3-hydroxybutyrate, aminooxyacetate, ethanol and p-hydroxyphenylpyruvate while ammonia formation was stimulated by aminooxyacetate. 2,4-Dinitrophenol stimulated the rate of the formation of all three metabolites from glutamine.
• 4.4. The major changes induced by aminooxyacetate, as determined in livers perfused with glutamine and stopped by freeze-clamping technique, consisted in a decrease in the content of ATP, aspartate and malate and in a slight increase in the content of glutamate.
• 5.5. Glutamine is an effective precursor of phosphoenolpyruvate in isolated liver mitochondria. Its formation was inhibited by octanoate and by DL-3-hydroxybutyrate.
• 6.6. The data are discussed in terms of regulation of glutamine catabolism in liver with emphasis on ureogenesis and gluconeogenesis.

     

In vitro-studies on the anaerobic formation of ethanol by the larvae of Chironomus thummi thummi (Diptera)

• 1.1. The anaerobic formation of ethanol by the larvae of Chironomus thummi thummi was investigated in homogenates and isolated mitochondria.
• 2.2. It was found that homogenates transform fructose-1,6-bisphosphate into ethanol and acetate. The accumulation of ethanol decreases substantially and the formation of acetate almost ceases when arsenite, an inhibitor of pyruvate dehydrogenase, is present in the incubation medium.
• 3.3. The cytosolic fraction of the homogenate was shown to degrade fructose-1,6-bisphosphate to pyruvate only. No ethanol could be detected, although there is high activity of alcohol dehydrogenase in the cytosol.
• 4.4. Isolated mitochondria produce large quantities of ethanol from pyruvate during anoxia. This ethanol production was shown to depend on the presence of NADH. It is deduced that this cosubstrate originates from intramitochondrial formation of acetate from pyruvate, which was always found to accumulate alongside ethanol.

     

Flight effects on certain blood parameters in homing pigeons Columba livia

• 1.1. Various blood parameters were monitored in resting and flown homing pigeons. A homing flight of 48 km lasting 60–80 min did not significantly alter plasma levels of total protein, electrolytes and plasma osmolality, which indicated maintenance of the homeostatic stability of the internal milieu during moderate exercise.
• 2.2. Plasma concentrations of marker enzymes such as alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), laetate dehydrogenase (LDH) and creatine phosphokinase (CPK) that tend to denote muscle damage and metabolic flux in prolonged exercise, were also not altered, thereby indicating the steady state of tissue structure and function during a flight of this magnitude.
• 3.3. Significant increases in plasma levels of uric acid and creatinine and decreases in plasma albumin were observed in the flown pigeons.
• 4.4. The flight-induced increase in blood uric acid could be attributed to increased purine catabolism and the increase in creatinine to increased nucleotide turnover.
• 5.5. It is suggested that the higher uric acid levels should not only enhance water conservation, but may also reduce flight-induced hyperthermia besides acting as an antioxidant defence against oxidative tissue injury.
• 6.6. The rise in creatinine is indicative of the breakdown of phosphocreatine for energy during the initial period of flight prior to the utilization of carbohydrate and lipid as fuels.
• 7.7. The decrease in plasma albumin should account for the albumin as lipid carrier lost in transport to the muscles during flight.

     

Properties of enzymes hydrating epoxides in human epidermis and liver

• 1.1. Cytosolic and microsomal epoxide hydrolyzing enzymes of human skin and liver were compared and found to be different.
• 2.2. Epidermal and hepatic cytosolic epoxide hydrolases were different in terms of substrate selectivity, pI, inhibitor sensitivity and affinity Chromatographic properties.
• 3.3. Microsomal epoxide hydrolases had the same pIs but different substrate selectivities.
• 4.4. Cytosolic epoxide hydrolase from adults had higher specific activity than that from neonates or cultured epidermis, but lower activity than adult hepatic enzymes.
• 5.5. The sizes of cytosolic epoxide hydrolase from epidermis and liver were similar and lower than that from cultured fibroblasts.
• 6.6. Cytosolic epoxide hydrolase from all sources shared similar antigenic determinants.

     

Toxicity of cadmium administration to the toad and the treatment of its poisoning with EDTA

• 1.1. The effect of cadmium administration on female Bufo regularis was studied. The median lethal doses were 22, 18, 15 and 6.2 Cd²⁺/kg after 24, 48, 72 and 96 hr respectively.
• 2.2. After a single intramuscular injection of 6.2 Cd²⁺/kg (representing 96-hr ld₅₀), the results indicated that Cd²⁺ causes severe physiological abnormalities to this experimental animal.
• 3.3. The serums alanine aminotransferase (AlAt), aspartate aminotransferase (AAt), alkaline phosphatase (A1P) and lactic dehydrogenase (LDH) were elevated while the calcium serum was not influenced by Cd²⁺ throughout the experimental period
• 4.4. On the other hand, phosphorus, total protein and total bilirubin were increased.
• 5.5. EDTA treatment (0.2 mmole/kg protected female toads from mortality up to 20 mg Cd²⁺/kg. It overcame the physiological alterations that were caused by the Cd²⁺ injection.
• 6.6. This may be due to the fact that Cd²⁺ is bound to EDTA in a strong complex which is readily excreted via the kidneys.

     

A comparison of aspartate transcarbamylase activity in juvenile pacific oysters (Crassostrea gigas) when fed various diets

• 1.1. The optimum pH for measurement of aspartate transcarbamylase activity in oyster tissue was determined to be 9.35 while the optimum temperature was 39.5°C.
• 2.2. Aspartate transcarbamylase activity varied significantly over short periods of time (hr) possibly due to fluctuations in the amount of food digested.
• 3.3. The composition of the oyster's diet also affected the levels of aspartate transcarbamylase activity in oyster tissues.
• 4.4. Those oysters fed an egg yolk-starch diet contained significantly lower aspartate transcarbamylase activity than oysters fed an egg yolk-starch-salmon oil diet or a casein-starch-salmon oil diet.
• 5.5. The aspartate transcarbamylase activities in oysters fed Phacedactylum tricornutum or a starch diet were not significantly different from the activities in oysters fed the egg yolk-starch diet.

     

Malate dehydrogenase isoforms from ribbed mussel (Modiolus demissus) gill tissue

• 1.1. The malate dehydrogenase (MHD) activity from the ribbed mussel gill is polymorphic with two distinct mitochondrial forms (M1 and M2) and five forms that could be resolved from cytosolic extracts (C1 to C5) by DEAE-cellulose chromatography and starch gel electrophoresis.
• 2.2. Two of the cytosolic forms (C3 and C4) may represent interchangeable conformational states.
• 3.3. With kinetic analysis there appear to be three distinct cytosolic forms (C1, C2 and C3–C4), with C2 possibly behaving as a heterodimer.
• 4.4. The identity of C5 is uncertain.
• 5.5. The forms isolated from the mitochondria (M1 and M2) exhibited lower apparent K_ms for oxaloacetate (OAA) than the cytosolic forms.
• 6.6. For all isozymic forms, the apparent K_ms for OAA increased as the pH increased between pH 6 and 9
• 7.7. Increasing the salt concentration raised the K_m for OAA for all forms.
• 8.8. The mMDHs were more sensitive to inhibition by NaCl than the cMDHs.
• 9.9. Representative cMDH (C1) and mMDH (M2) isozymes exhibited substrate inhibition by high concentrations of OAA with the mMDH possessing lower K_is for substrate inhibition than the cMDH at each pH tested.
• 10.10. Differences and similarities in K_m app. for OAA at the different pHs and salt concentrations indicated that C1, C2 and C3–C4 and C5 were distinct forms, that M1 and M2 were distinct but very similar to each other, and that C1, C2, C3–C4 and C5 were distinct from M1 and M2.

     

Prenatal induction of tyrosine aminotransferase in rat liver

• 1.1. Hepatic tyrosine aminotransferase activity from adult rat can be resolved into four components on hydroxylapatite column.
• 2.2. A similar profile of enzyme distribution can be obtained from late foetal liver.
• 3.3. Insulin administration to pregnant rats result in induction of two isoenzymes of tyrosine aminotransferase in foetal rat liver. Similarly Cyclic AMP injection to foetal rats in utero results in the induction of the same two forms of the enzyme.
• 4.4. Triaminolone injection to foetal rats in utero leads to the induction of three of the isoenzymes of tyrosine aminotransferase.

     

Effect of methyl methacrylate on mitochondrial function and structure

• 1.1. Treatment of isolated rat liver mitochondria with methyl methacrylate (MM) produced membrane disruption as evidenced by the release of citrate synthase, and changes in the ultrastructure of mitochondria.
• 2.2. At concentration 0.1%, MM uncoupled oxidative phosphorylation as evidenced by stimulation of state 4 respiration supported either by pyruvate plus malate or succinate (+rotenone) and ATP-ase activity in intact mitochondria.
• 3.3. At concentration 1% MM stimulated ATP-ase activity in intact mitochondria and succinate (+rotenone) oxidation at state 4 and was without effect on this substrate oxidation at state 3.
• 4.4. MM inhibited pyruvate plus malate oxidation either at state 3 or in the presence of uncoupling agents.
• 5.5. MM inhibited the NADH oxidase of electron transport particles at a concentration which failed to inhibit either succinic oxidase or the NADH-ferricyanide reductase activity.
• 6.6. The data presented suggest that in the isolated mitochondria MM inhibits NADH oxidation in the vicinity of the rotenone sensitive site of complex I.
• 7.7. The general conclusion is that MM may block an electron transport and to uncouple oxidative phosphorylation in rat liver mitochondria. The overall in vitro effect would be to prevent ATP synthesis which could result in cell death under in vivo conditions.

     

The influence of seasonal changes on energy metabolism in Mytilus edulise (L.).—III. Anaerobic energy metabolism

• 1.1. Seasonal changes in the accumulation of end products after 48 hr of exposure to air and in the composition of the free amino acid pool were studied in Mytilus edulis.
• 2.2. The accumulation levels of succinate and acetate showed only weak seasonal changes.
• 3.3. Conversion of succinate to propionate was high in summer and virtually zero in winter
• 4.4. Alanine and most other free amino acids were present in relatively high concentrations in summer and early autumn and reached minimal values in winter and early spring.
• 5.5. Exceptions were glutamate, aspartate and taurine, which showed hardly an season related changes and glycine, which changed inversely to the majority of the free amino acids.
• 6.6. The anaerobic formation of alanine was inversely proportional to the endogenous concentration.
• 7.7. The only other free amino acids affected by anaerobiosis were glutamate and aspartate, which respectively increased and decreased under these conditions.

     

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.

     

Tyrosine aminotransferase activity of frog (Rana esculenta) liver—III. A circannual study

• 1.1. A circannual study of tyrosine aminotransferase and other metabolic enzymes in frog liver is reported. The subcellular distribution of all enzymatic activities under investigation was also studied.
• 2.2. Results show significant oscillations of all enzymatic activities throughout the year; in particular tyrosine aminotransferase has a marked summer maximum.
• 3.3. The subcellular distribution of tyrosine aminotransferase shows significant variations: the soluble activity of the enzyme presents a bimodal circannual distribution, which has its counterpart in an increased activity of heavier fractions.

     

Purification and characterization of a manganese containing superoxide dismutase from bovine heart mitochondria

• 1.1. A manganese containing superoxide dismutase from bovine heart mitochondria was isolated and characterized.
• 2.2. It has a molecular weight of about 86,000 and is composed of 4 noncovalently bound subunits of equal size.
• 3.It appears to contain 2 mole manganese per mole enzyme.
• 4.The carbohydrate content is very low.
• 5.The specific activity and amino acid composition are similar to those of other mitoehondrial superoxide dismutases.
• 6.The enzyme forms complexes with ampholytes and can therefore not be analysed by isoelectric focusing.

     

The intracellular compartmentation of metabolites in isolated kidney cortex tubules

• 1.1. A method is described to measure cytosolic and mitochondrial metabolites in isolated kidney tubule suspensions.
• 2.2. With the use of digitonin, 95% of cytosolic metabolites are released into the supernatant fraction whereas mitochondrial matrix enzymes are retained in the pellet fraction.
• 3.3. In a study of adaptation to acidosis, different α-oxoglutarate gradients between the tubular cell compartments are obtained, when medium pH is changed from 7.0 to 7.8.

     

Serum and urine biochemical indicators of nutritional status in adult female collared peccaries,Tayassu tajacu (tayassuidae)

• 1.1. Physiological responses of 13 adult female collared peccaries (Tayassu tajacu) to high quality and low quality diets, fed for 15 weeks, were examined. The low quality diet simulated energy and protein intake of peccaries during poor range conditions resulting from drought. Blood samples were collected after 10 and 15 weeks of dietary treatment; urine samples were collected after 15 weeks of treatment.
• 2.2. Females receiving the low quality diet for 15 weeks lost 27.4% of their original body weight, compared to no weight change among high quality-fed females.
• 3.3. Red blood cell counts, hematocrits, and hemoglobin concentrations were significantly greater among females fed a high quality diet compared to those receiving a low quality diet. High quality-fed females also had a higher mean corpuscular hemoglobin concentration. Plasma fibrinogen concentration was nearly twice as great among females receiving the low quality diet compared to the high quality group.
• 4.4. Consumption of the low quality diet resulted in significantly elevated serum levels of nonesterified fatty acids, alkaline phosphatase, phosphorus, alpha-2 globulin and alpha globulin: beta globulin ratio.
• 5.5. Consumption of the low quality diet resulted in significantly lowered serum levels of urea nitrogen, calcium, zinc, calcium: phosphorus, urea index, beta-1 flobulin, beta globulin: albumin ratio, thyroxine and triiodothyronine.
• 6.6. Serum levels ofcreatinine, total bilirubin, glucose, cholesterol, gamma glutamyltransferase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, potassium, copper, magnesium, sodium chloride, total protein and gamma globulin were unaffected by diet quality.
• 7.7. Urine chemistry results suggested pH, osmolarity, albumin, creatinine phosphokinase, calcium and phosphorus concentrations might be useful indices for assessing nutritional status in female peccaries.