Plasmalemma- and tonoplast-ATPase activity in mesophyll protoplasts,vacuoles and microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana

Adenosine-triphosphatase activity on the plasmalemma and tonoplast of isolated mesophyll protoplasts, isolated vacuoles and tonoplast-derived microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana Hamet et Perr., was localized by a cytochemical procedure using lead citrate. Enzyme activity was detected on the cytoplasmic surfaces of the plasmalemma and tonoplast. The identity of the enzymes was confirmed by various treatments differentiating the enzymes by their sensitivity to inhibitors of plasmalemma and tonoplast H⁺-ATPase. Isolated vacuoles and microsomes prepared from isolated vacuoles clearly exhibited single-sided deposition on membrane surfaces.Abbveviations CAM Crassulacean acid metabolism - H⁺-ATPase proton-translocating ATPase     

Human hepatocyte and kidney cell metabolism of 2-acetylaminofluorene and comparison to the respective rat cells

The metabolism and mutagenic activation of 2-acetylaminofluorene by human and rat hepatocytes and kidney cells were measured. High performance liquid chromatography was used to separate the 2-acetylaminofluorene metabolites, and a cell-mediated Salmonella typhimurium mutagenesis assay was used to detect mutagenic intermediates. Rat and human differences were observed with cells from both organs and levels of metabolism and mutagenesis were higher in human cells. Within a species, liver and kidney cell differences were also evident, with levels of hepatocyte-mediated metabolism and mutagenesis being greater than kidney cells. Human inter-individual variation was apparent with cells from both organs, but the variation observed was significantly greater in hepatocytes than kidney cells. A knowledge of such differences, including an understanding that they may vary with the chemical being studied, should be useful in the extrapolation of rodent carcinogenesis data to humans.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - DMSO dimethylsulfoxide - HPLC high performance liquid chromatography - N-OH-AAF N-hydroxy-2-acetylaminofluorene - 1-OH-AAF 1-hydroxy-2-acetylaminofluorene - 3-OH-AAF 3-hydroxy-2-acetylaminofluorene - 5/9-OH-AAF a combination of 5 and 9-hydroxy-2-acetylaminofluorene - 7-OH-AAF 7-hydroxy-2-acetylaminofluorene - 8-OH-AAF 8-hydroxy-2-acetylaminofluorene     

马齿苋叶片PEP羧化酶的分子特性

马齿苋叶片PEPCase由四个相同的亚基组成,亚基分子量为83kD。远紫外CD光谱分析表明,此酶含有36.6％α—螺旋结构。马齿苋叶片PEPCase可被G6P激活,但不能被Gly、Ser激活。G6P可防止酶的尿素变性和枯草杆菌蛋白酶的作用。这种保护效应与G6P诱导的酶构象变化有关。 从酶对低温、高温及尿素的反应来看,马齿苋叶片PEPCase的稳定性高于高粱叶片PEP—Case,两者的免疫特性和电泳特性亦不同。     

Synthesis and secretion of lipids by long-term cultures of female rat hepatocytes.

The objective of this work was to characterize lipid metabolism in long-term cultures of adult rat hepatocytes from female rats and explore the potential use of this culture system to study the effect of hormones, drugs and toxic chemicals on it. Hepatocytes, seeded on a feeder layer of 3T3 cells, maintained for 2 weeks their typical morphology. The cultures were able to take up [14C]acetic and [14C]oleic acid from the culture medium and incorporate them into lipids. The synthesis and secretion of lipids by [14C]acetic acid-labeled cultures had a maximum value after 11 and 13 days in culture. Triacylglycerols were the main lipidic species synthesized and secreted by hepatocytes (up to 67% of the total lipids); they also synthesized and secreted phospholipids, cholesterol and cholesterol esters from [14C]acetic acid. Similarly, [14C]oleic acid-labeled cultures synthesized and secreted mostly triacylglycerols (up to 60-70% of the total lipids), but they were also able to incorporate the labeled precursor into both cellular and secreted phospholipids and cholesterol esters. The activity of glycerol-phosphate-dehydrogenase, marker enzyme of glycerolipid synthesis, decreased slightly during the culture time whereas the activity of malic enzyme, marker of fatty acid synthesis, increased. Our results show that long-term cultures of female rat hepatocytes are able to synthesize and secrete several lipids, specially triacylglycerols, from both [14C]acetic and [14C]oleic acid for at least 2 weeks and that they maintain enzyme activities related with the synthetic pathways of glycerolipids and fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of diabetes and insulin on ketone bodies metabolism in heart

This work investigates the effect of alloxan-induced short-term diabetes (24 h) on D-3-hydroxybutyrate metabolism at physiological and non-physiological concentrations of the ketone body in the isolated non-working perfused rat heart. Also the effect of insulin (2 mU.ml⁻¹) on D-3-hydroxybutyrate metabolism was investigated in hearts from normal and diabetic rats. The rates of D-3-hydroxybutyrate utilization and oxidation and of acetoacetate production were proportional to D-3-hydroxybutyrate concentration. The utilization of D-3-hydroxybutyrate showed saturation kinetics in hearts from normal and diabetic rats, in the presence and absence of insulin. Acute short-term diabetes augmented D-3-hydroxybutyrate utilization and oxidation at 1.25 and 2.5 mM DL-3-HB, with no significant effect at higher concentrations, but increased acetoacetate production at all investigated concentrations. In hearts from normal rats, insulin enhanced D-3-hydroxybutyrate utilization and oxidation at 2.5, 5, and 10 mM DL-3-HB, but no effect was observed at the lowest (1.25 mM) and highest (16 mM) DL-3-HB concentrations. Insulin had no effect on D-3-hydroxybutyrate metabolism in hearts from diabetic rats. No significant effect of insulin on the rate of acetoacetate production in normal and diabetic states was observed.     

Amino acid metabolism in several tissues of the obese Zucker rat as indicated by the tissue accumulation of α-amino[1-14C]isobutyrate

Measurements of the tissue accumulation of α-amino[1-¹⁴C]isobutyrate [1-¹⁴C]AIB) in lean (+/?) and obese (fa/fa) Zucker rats showed an augmented tissue/plasma ratio in the liver of the obese animals. In contrast, brown adipose tissue AIB accumulation was lower in the fa/fa animals. In response to a 24h starvation period AIB accumulation was significantly elevated in the liver and plasma of the lean animals and was unchanged in the liver of the fa/fa animals. The circulating concentration of alanine and branched-chain amino acids was elevated in the fa/fa animals as compared to their lean counterparts. These observations suggest that amino acid uptake is not involved in the impaired muscle development observed in the obese Zucker rat and that the ability of brown adipose tissue for amino acid utilization is decreased in the obese animals suggesting that this may partially explain the impaired thermoregulatory capacity observed in brown adipose tissue of obese Zucker rats.     

Phosphorus-31 magnetic resonance spectroscopy of forearm flexor muscles in student rowers using an exercise protocol adjusted for differences in cross-sectional muscle area

To assess exercise energy metabolism of forearm flexor muscles in rowers, six male student rowers and six control subjects matched for age and sex were studied using phosphorus-31 magnetic resonance spectroscopy (31P-MRS). Firstly, to adjust for the effect of differences in cross-sectional muscle area, the maximal cross-sectional area (CSAmax) of the forearm flexor muscles was estimated in each individual using magnetic resonance imaging. Multistage exercise was then carried out with an initial energy production of 1 J.cm-2 CSAmax for 1 min and an increment of 1 J.cm-2 CSAmax every minute to the point of muscle exhaustion. A series of measurements of 31P-MRS were performed every minute. The CSAmax was significantly greater in the student rowers than in the control subjects [19.8 (SD 2.2) vs 17.1 (SD 1.2) cm2, P less than 0.05]. The absolute maximal exercise intensity (J.min-1) was greater in the rowers than in the control subjects. However, the maximal exercise intensity per unit of muscle cross sectional area (J.min-1.cm-2) was not significantly different between the two groups. During mild to moderate exercise intensities, a decrease in phosphocreatine and an increase in inorganic phosphate before the onset of acidosis were significantly less in the rowers, indicating a requirement of less adenosine 5'-diphosphate to drive adenosine 5'-triphosphate production. The onset of acidosis was also significantly delayed in the rowers. No difference was observed in forearm blood flow between the two groups at the same exercise intensity (J.min-1.cm-2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of formaldehyde on growth of obligate methylotroph Methylobacillus flagellatum in a substrate non-limited continuous culture

The ribulose monophosphate cycle methylotroph Methylobacillus flagellatum was grown under oxyturbidostat conditions on mixtures of methanol and formaldehyde. Formaldehyde when added at low concentration (50 mg/l) increased the methanol consumption and the yield of biomass. The presence of 150–300 mg/l of formaldehyde resulted in an increase of the growth rate from 0.74 to about 0.79–0.82 h^-1. The presence of 500 mg/l of formaldehyde in the inflow decreased culture growth characteristics. Activities of methanol dehydrogenase and enzymes participating in formaldehyde oxidation and assimilation were measured. The enzymological profiles obtained are discussed.Abbreviations MDH methanol dehydrogenase - NAD-linked FDDH NAD-linked formaldehyde dehydrogenase - DLFDDH dye-linked formaldehyde dehydrogenase - DLFDH dye-linked formate dehydrogenase - GPDH glucose-6-phosphate dehydrogenase - PGDH 6-phosphogluconate dehydrogenase - RuMP cycle ribulose monophosphate cycle     

Anaerobic metabolism of Nitrosomonas europaea

Nitrosomonas europaea is capable of maintaining an anaerobic metabolism, using pyruvate as an electron donor and nitrite as an electron acceptor; utilization of nitrite depends upon supply of both pyruvate and ammonia. The role of ammonia in this reaction was not determined. N europaea also assimilates CO₂ anaerobically into cell material in the presence of nitrite (0.5–1.0 mM), pyruvate and ammonia. This reaction was partially inhibited by nitrite which apparently competed with CO₂ for reducing power. Anaerobic nitrite respiration is sensitive to ionophores, FCCP being the most effective.Non-standard-abbreviations TCA trichloroacetic acid - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazon     

Genetic,biochemical and immunological evidence for the involvement of two alcohol dehydrogenases in the metabolism of propane by Rhodococcus rhodochrous PNKb1

NAD⁺-dependent propan-1-ol and propan-2-ol dehydrogenase activities were detected in cell-free extracts of Rhodococcus rhodochrous PNKb1 grown on propane and potential intermediates of propane oxidation. However, it was unclear whether this activity was mediated by one or more enzymes. The isolation of mutants unable to utilize propan-1-ol (alcA^-) or propan-2-ol (alcB^-) as sole carbon and energy sources demonstrated that these substrates are metabolized by different alcohol dehydrogenases. These mutants were also unable to utilize propane as a growth substrate indicating that both alcohols are intermediates of propane metabolism. Therefore, propane is metabolized by terminal and sub-terminal oxidation pathways. Westernblot analysis demonstrated that a previously purified NAD⁺-dependent propan-2-ol dehydrogenase (Ashraf and Murrell 1990) was only synthesized after growth on propane and sub-terminal oxidation intermediates (but not acetone), and not propan-1-ol or terminal oxidation intermediates. Therefore, our evidence suggest that another dehydrogenase is involved in the metabolism of propan-1-ol and this agrees with the isolation of the alcA^- and alcB^- phenotypes. The previously characterized NAD⁺-dependent propan-2-ol dehydrogenase from R. rhodochrous PNKb1 is highly conserved amongst members of the propane-utilizing Rhodococcus-Nocardia complex.