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.

     

Purification and characterization of carbonyl reductases from bovine liver cytosol and microsome. the cytosolic enzyme has a novel 3α/17β-hydroxysteroid dehydrogenase activity

• 1.1. Carbonyl reductase, which is distributed in both cytosolic and microsomal fractions in bovine liver, were purified to homogeneity on 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis and shown to have molecular weights of 32 kDa and 68 kDa, respectively.
• 2.2. Both carbonyl reductases can catalyze the reduction of many carbonyl compounds including ketone, quinones and aldehyde with relatively low K_m values.
• 3.3. From the absorption spectrum result, microsomal carbonyl reductase closely resembles cytochrome P-450 reductase.
• 4.4. Cytosolic carbonyl reductase is a novel enzyme which can act on both testosterone and androsterone at low concentration.

     

31P NMR studies on kidney intracellular pH in acute renal acidosis

• 1.1. Phosphorus nuclear magnetic resonance (³¹P NMR) provides a non-destructive method for the quantitative observation of the most abundant phosphate-containing metabolites in isolated perfused kidney and in kidneys inside an anaesthetised animal.
• 2.2. In this paper, the NMR method is introduced and the intracellular pH changes following acute renal acidosis are measured (from the resonance position of P_i) for the isolated perfused rat kidney.
• 3.3. The results are discussed in relation to the biochemical data obtained by freeze extraction.

     

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.

     

Purification and characterization of monkey albumin and proalbumin

• 1.1. Albumin purified from rhesus monkey (MSA) shows immunological cross-reactivity with human serum albumin (HSA) by RIA.
• 2.2. The amino-terminal sequence of MSA shows a high degree of homology to HSA.
• 3.3. Thirty minutes after injection of radioactive leucine directly into the portal vein, albumin was purified chemically from the liver, kidneys and serum.
• 4.4. At this time, 15% of the label was incorporated into liver homogenate protein.
• 5.5. A highly labelled immunoreactive albumin form was purified from liver to constant specific radioactivity and separated from tissue and serum albumin.
• 6.6. The specific radioactivity of this proalbumin was 36-times higher than the specific radioactivity of albumin in liver tissue.
• 7.7. These similarities to HSA suggest that this non human primate species can serve as a useful model of human albumin synthesis in vivo.

     

Cloning and characterization of rabbit pancreatic colipase

• 1.1. Among the digestive enzymes synthesized by pancreas, lipase is the principle lipolytic enzyme which hydrolyses dietary glycerides.
• 2.2. For its action it requires a coenzyme, colipase.
• 3.3. The molecular mechanisms of the interaction of these two are not fully understood.
• 4.4. Further, molecular events that regulate and influence lipid absorption are ill denned.
• 5.5. The rabbit is the conventional animal model for the study of lipid absorption. We have undertaken the molecular cloning, and characterization of rabbit pancreatic colipase, the coenzyme for pancreatic lipase.
• 6.6. Colipase has been cloned from a gt 11 library of an adult rabbit pancreatic cDNA by probing with an oligonucleotide derived from human colipase sequence.
• 7.7. The total reading frame consists of 321 nucleotides coding for 90 amino acids of the functional protein and 17 nucleotides of the leader peptide.
• 8.8. Northern blot analysis revealed a distinct band around 0.5kb. Comparison with other species revealed an over all homology of 75% at the nucleotide level.
• 9.9. At the amino acid level highest conservation is observed at the lipase-binding region (AA 53–73).
• 10.10. Rabbit enzyme also retained the N-terminal pentapeptide of it preform.
• 11.11. The regions of homology and conservation may aid to define the sites of interaction of colipase with lipase.

     

Metabolic and contractile differentiation of rabbit muscles during growth

• 1.1. A study was carried out of post-natal evolution of the oxidative, glycolytic and contractile capacities in various types of rabbit muscle.
• 2.2. At birth, muscles are non-differentiated and present very limited metabolic and contractile activity, metabolism is mainly oxidative in all muscles.
• 3.3. Although muscular discrimination is manifest from the sixth week after birth, the glycolytic metabolism reaches its maximum capacity only after six to eight weeks.
• 4.4. Subsequently, oxidative metabolic capacity steadily decreases until adulthood.

     

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.

     

Analysis of the circadian rhythm in energy metabolism of rat liver

• 1.1. To study the temporal organization of energy metabolism in rat liver the steady state concentrations of key intermediates of carbohydrate and phosphorus metabolism were determined during 24 hr.
• 2.2. The circadian rhythm in energy metabolism of rat liver has been analysed by four different approaches. It was shown that neither apparent PEP synthesis nor crossover theorem were acceptable for the elucidation of the temporal organization of multi-enzyme systems.
• 3.3. Correlations analysis explained the temporal organization of energy metabolism most satisfactorily.
• 4.4. Based on the results of this analysis it was suggested that circadian regulation of energy metabolism in liver was realized at the level of the citric acid cycle.

     

Interference of benzo[a]pyrene with cytochrome P-450-mediated steroid metabolism in pyloric caeca microsomes of the sea star Asterias rubens L.

• 1.1. The effects of benzo[a]pyrene (BaP) on the metabolism of progesterone and pregnenolone, and the effects of steroids on BaP metabolism were examined in pyloric caeca microsomes of female Asterias rubens.
• 2.2. The patterns of metabolism of progesterone and pregnenolone in microsomes were similar to those found in previous studies for homogenates and tissue incubations of pyloric caeca.
• 3.3. BaP reduced the rate of hydroxylation of progesterone and pregnenolone, but had no effect on metabolite formation by non-cytochrome P-450-catalysed reactions.
• 4.4. Microsomal BaP hydroxylase activity was reduced by the presence of progesterone, but pregnenolone and testosterone had no such effect.
• 5.5. The reductions in steroid or BaP metabolism were progressive with increasing ratios of the concentration of the interfering compound to that of the assay substrate and were maximally 50% or less at ratios of × 100.
• 6.6. It is concluded that isoenzymic forms of cytochrome P-450 are present, with preferences towards either steroid or BaP metabolism. The implications of the results for the in vivo situation are discussed.

     

Immunochemical and immunohistochemical determination of ubiquitin in molluscan (Mytilus edulis) smooth muscle

• 1.1. Immunochemical and immunohistochemical distribution of ubiquitin in the anterior byssus retractor muscle (ABRM) of Mytilus edulis was investigated.
• 2.2. In immunostaining, specific ubiquitin immunoreactivity was observed in the cross-sectioned ABRM, and was uniformly localized in this section.
• 3.3. The amount of free ubiquitin in the ABRM homogenate was 130 ± 4.6 ng/mg protein by western blot analysis, and ubiquitin conjugates were found at about 25, 29 and 200–230 kDa.
• 4.4. These findings were similar to those obtained in the skeletal muscle of rat.

     

Characterization of NaK ATPase from the gills of the freshwater prawn Macrobrachium rosenbergii (de Man)

• 1.1. The specific activity of Na-K ATPase was determined from the microsomal preparation of gills dissected from adult Macrobrachium rosenbergii.
• 2.2. Maximal ATPase activity was achieved at a substrate concentration of 0.5 mM ATP.
• 3.3. Optimal enzyme activity was obtained at pH of 7.5.
• 4.4. The Arrhenius plot of Na-K ATPase activity revealed a marked discontinuity at 30°C. “Mg” ATPase activity did not exhibit a marked discontinuity.
• 5.5. The E_a for Na-K ATPase and “Mg” ATPase was 14.6 kCal/mole and 9.31 kCal/mole respectively. Q₁₀ values for Na-K ATPase was 2.34 and for “Mg” ATPase 1.65.
• 6.6. ATPase activity and gill homogenate protein concentration exhibited a linear relationship up to 130 μg protein/ml.
• 7.7. Na-K ATPase activity was inhibited by 10⁻³ M ouabain. It was equally inhibited by the removal of K⁺ from the reaction medium.

     

Spingophosphonolipids in microsomes of Diplodon delodontus. Distribution and effect on the membrane microviscosity

• 1.1. The distribution of ceramide aminoethylphosphonate (CAEP) in microsomal membranes obtained from different tissues of the bivalve mollusc Diplodon delodontus was determined.
• 2.2. The concentration of CAEP reached from 9 to 19% of the total microsomal polar lipids, depending on the kind of tissue.
• 3.3. Palmitic acid was the main fatty acid in the ceramide moiety, followed by stearic and eicosamonoenoic acids.
• 4.4. Artificial membranes were prepared with microsomal phospholipids or phospholipids plus sterols, with and without the addition of CAEP.
• 5.5. It was shown that the phosphonate confers minor mobility to the membranes. This effect is more effective when the membrane contains the natural sterols and the phospholipids are unsaturated.

     

Evidence for extracellular deamination of adenosine in the rat heart

• 1.1. In rat heart perfused with adenosine (10⁻⁶M), dilazep (10⁻⁴M) inhibited incorporation of adenosine into nucleotides (an index of nucleoside transport and phosphorylation) to a greater extent (70%) than metabolism to inosine and uric acid (40%) and actually increased the recovery of inosine to 30% of the adenosine infused.
• 2.2. Extrapolating for complete inhibition of transport suggested that 60% of adenosine metabolism was intracellular and 40% extracellular.
• 3.3. Static incubations of atria also gave an estimate for extracellular metabolism of 40%.
• 4.4. Adenosine deaminase was localised by immunocytochemistry to the extracellular surface of endothelial cells of small coronary arteries.
• 5.5. Extracellular deamination may explain the lack of effect of nucleoside transport inhibitors on responses to adenosine in rat heart.

     

Phospholipid requirement for fatty acid desaturase activity in microsomes of Ceratitis capitata

• 1.1. Lipids from purified microsomal preparations of Ceratitis capitata have 72.4% of phospholipids and their participation in the fatty acid desaturase activity has been examined.
• 2.2. Treatment of microsomal preparations with phospholipase C decreased notably the enzyme activity than can be restored by adding phosphatidylcholine.
• 3.3. These data suggest that phosphatidylcholine derives from the immediate lipid environment of the microsomal desaturase.

     

Effect of photoperiod on steroid metabolism in the sea star Asterias Rubens L

• 1.1. The effect of photoperiod on steroid metabolism in Asterias rubens was studied.
• 2.2. Daylength was artificially shortened in 3 weeks from long-day (LD 18/6) to short-day (LD 6/18) conditions and its effect on the metabolism of pregnenolone and dehydroepiandrosterone was studied in homogenates of gonad and pyloric caeca tissue from male and female seastar.
• 3.3. Pregnenolone metabolism did not change during the experiment when the animals were kept continuously under the same (long-day) conditions. Pregnenolone metabolism was intensified by decreasing daylength. The production of progesterone reached its maximum at a daylength comparable to that in autumn (LD 12/12), and that of an unidentified steroid at an even shorter daylength.
• 4.4. Metabolism of dehydroepiandrosterone was influenced by photoperiod. There were indications that androstenedione production is maximal at fall conditions. This was evident for an as yet unidentified steroid.
• 5.5. Metabolism of DHEA strongly increased during the experiment in animals which were kept continuously under long-day conditions. It is discussed that this may be a reaction to crowding.

     

1H NMR study of metabolic alterations in the small intestine of rats infected with Hymenolepis diminuta

• 1.1. 1H NMR spectra of the duodenum, jejunum and ileum tissues of the small intestine of a rat showed metabolic gradients.
• 2.2. The concentrations of metabolites in these gut regions were altered by the presence of the tapeworm Hymenolepis diminuta.
• 3.3. In the infected duodenum there was significantly less glycogen, glucose and phosphocreatine/creatine, but significantly more lactate than in the corresponding controls.
• 4.4. Infected jejunum contained significantly less betaine but significantly more succinate, alanine and lactate.
• 5.5. Infected ileum had significantly less glycogen and taurine but significantly more alanine and lactate.

     

Transamination and transsulphuration of l-cysteine in ehrlich ascites tumor cells and mouse liver: The nonenzymatic reaction of l-cysteine with pyruvate

• 1.1. The activity of cysteine aminotransferase (CAT), 3-mercaptopyruvate sulfurtransferase (MPST) and rhodanese is much lower in Ehrlich ascites tumor cells (EATC) than in mouse liver.
• 2.2. Contrary to mouse liver homogenate, no synthesis of sulphane sulphur-containing compounds from L-cysteine is observed in EATC homogenate.
• 3.3. 2-Methyl-thiazolidine-2,4-dicarboxylic acid (CP), 2-methyl-thiazolidine-4-carboxylic acid (CA) and thiazolidine-4-carboxylic acid (CF) can be used as sources of low molecular-weight thiol compounds both in EATC and mouse liver homogenate.
• 4.4. Pyruvate formed from phosphoenolopyruvate (PEP) in EATC homogenates reacts with L-cysteine (l-CYS) to CP.

     

31P-NMR study on effects of electric shock on swimming leeches,Hirudo medicinalis

• 1.1. We evaluated the effect of electric shock on swimming leeches by measuring changes in high-energy phosphate metabolism using in vivo³¹P-NMR.
• 2.2. Leeches electrically stimulated during swimming showed anodal galvanotaxis, and stopped swimming with stimulation at strong current.
• 3.3. Comparison of the concentrations of high-energy phosphate metabolites before and after electric shock using ³¹P-NMR revealed a marked decrease in β-ATP and an increase in that of Pi.
• 4.4. Electric shock apparently induces excessive muscle fatigue in leeches, resulting in transient paralysis.

     

Comparative studies of glucose metabolism on mammals' red blood cells

• 1.1. Glucose utilization was measured in human, pig, cow, rabbit, mouse and rat red blood cells. Mean values are variable from species to species and range from 0.27 μmol/hr/ml RBC for pig erythrocytes to 2.85 μmol/hr/ml RBC in mouse red cells.
• 2.2. The amount of glucose metabolized through the pentose phosphate pathway ranges from 2.1 to 7.0% of the total glucose utilized.
• 3.3. Variable recycling values have been obtained for the red blood cells of the species studied but with the exception of mouse (14 nmol/hr/ml RBC) all the other values do not show great differences.
• 4.4. The hexokinase levels of the erythrocytes studied when correlated with the glucose utilization and the pentose phosphate pathway show that this enzyme could play a central role in the regulation of glucose metabolism.