The effect of amino acids,monoamines and polyamines on pyruvate dehydrogenase activity in mitochondria from rat adipocytes

Summary The ability of polyamines and other cationic compounds including monoamines, amino acids, poly-L-arginine, poly-D-lysine and poly-L-lysine, to alter pyruvate dehydrogenase (PDH) activity in mitochondria from rat epididymal adipocytes was determined. PDH was assayed with the substrate [1-¹⁴C] pyruvate in the presence of 0.05 mM Ca²⁺ and Mg²⁺. Nine of the fourteen compounds tested at 0.1 mM caused a significant increase (procaine, 3-(-morpholinopropionyl) benzo[b]thiophene [VII], spermine, spermidine, putrescine, lysine and tryptophan) or decrease (poly-L-arginine, 3-(-piperidinopropionyl) benzo[b]thiophene) in PDH activity. None of these compounds nonenzymatically decarboxylated [1-¹⁴C] pyruvate to release ¹⁴CO₂. NaF, a PDH phosphatase inhibitor, suppressed the stimulatory effects of those compounds tested: procaine, tryptophan, VII, spermine and spermidine. These results imply that these five compounds activate PDH activity through stimulation of the PDH phosphatase. When the Mg²⁺ concentration was increased from 0.05 to 4.5 mM, the stimulatory effect of spermine was increased, consistent with the finding by others that spermine lowers the Km of the enzyme for Mg²⁺. However, at Mg²⁺ concentrations greater than 0.3 mM, the stimulatory effect of VII was unaltered, procaine failed to alter PDH activity, lysine inhibited PDH activity, and poly-L-lysine stimulated PDH activity. Therefore, polyamines and other positively charged small molecules may be physiologic regulators of PDH activity.     

Melatonin prevents δ-aminolevulinic acid-induced oxidative DNA damage in the presence of Fe2+

-aminolevulinic acid (ALA), a heme precursor which accumulates during lead poisoning and acute intermittent porphyria, is reported to cause liver cancer. The carcinogenic mechanisms of ALA may relate to its ability to generate free radicals through metal-catalyzed oxidation which cause oxidative DNA damage. The aim of this study was to compare the efficacy of melatonin, trolox (vitamin E) and mannitol in altering DNA damage induced by ALA. Herein, we found, in the presence of Fe²⁺, that ALA-induced formation of 8-hydroxydeoxyguanosine in calf thymus DNA was dose and time-dependent. Melatonin, mannitol and trolox, all of which are free radical scavengers, inhibited the formation of 8-hydroxydeoxyguanosine in a concentration-dependent manner. The concentration of each (melatonin, mannitol and trolox) required to reduce DNA damage by 50%, i.e., the IC₅₀, was 0.52, 0.84 and 0.90 mM, respectively.     

Effect of α-Ketoisocaproate and Leucine on the in Vivo Oxidation of Glutamate and Glutamine in the Rat Brain

Leucine and -ketoisocaproate (-KIC) were perfused at increasing concentrations into rat brain hippocampus by microdialysis to mimic the conditions of maple syrup urine disease. The effects of elevated leucine or -KIC on the oxidation of L-[U-¹⁴C]glutamate and L-[U-¹⁴C]glutamine in the brain were determined in the non-anesthetized rat. ¹⁴CO₂ generated by the metabolic oxidation of [^l4C]glutamate and [¹⁴C]glutamine in brain was measured following its diffusion into the eluant during the microdialysis. Leucine and -KIC exhibited differential effects on ¹⁴CO₂ generation from radioactive glutamate or glutamine. Infusion of 0.5 mM -KIC increased [^l4C]glutamate oxidation approximately 2-fold; higher concentrations of -KIC did not further stimulate [¹⁴C]glutamate oxidation. The enhanced oxidation of [¹⁴C]glutamate may be attributed to the function of -KIC as a nitrogen acceptor from [¹⁴C]glutamate yielding [¹⁴C]-ketoglutarate, an intermediate of the tricarboxylic acid cycle. [¹⁴C-]glutamine oxidation was not stimulated as much as [¹⁴C-]glutamate oxidation and only increased at 10 mM -KIC reflecting the extra metabolic step required for its oxidative metabolism. In contrast, leucine had no effect on the oxidation of either [¹⁴C]glutamate or [¹⁴C]glutamine. In maple syrup urine disease elevated -KIC may play a significant role in altered energy metabolism in brain while leucine may contribute to clinical manifestations of this disease in other ways.     

Interaction between perchlorate and nifedipine on insulin secretion from mouse pancreastic islets

In order to elucidate the mechanisms responsible for the stimulatory effect of perchlorate (ClO ₄ ^– ) on insulin secretion, we have investigated the interaction between this chaotropic anion and the organic calcium antagonist nifedipine. This drug, known as a blocker of L-type calcium channels, was chosen as a tool to test the idea that ClO ₄ ^– acts on insulin secretion by stimulating the gating of voltage-controlled Ca²⁺ channels. ClO ₄ ^– amplified the stimulatory effect of D-glucose on insulin release from perfused pancreas (first and second phases) as well as from isolated islets incubated in static incubations for 60 min. This indicates that ClO ₄ ^– amplifies physiologically regulated insulin secretion. Nifedipine reduced D-glucose-induced (20 mM) insulin release in a dose-dependent manner with half-maximum effect at about 0.8 M and apparent maximum effect at 5 M nifedipine. In the presence of 20 mM D-glucose, the inhibitory effects of 0.5, 1 or 5 M nifedipine were only slightly, if at all, counteracted by perchlorate. When 12 mM ClO ₄ ^– and 20 mM D-glucose were combined, calculation of the specific effect of ClO ₄ ^– revealed that nifedipine produced almost maximum inhibition already at 0.05 M. Thus, the perchlorate-induced amplification of D-glucose-stimulated insulin release shows higher sensitivity to nifedipine than the D-glucose-effect as such. This supports the hypothesis that perchlorate primarily affects the voltage-sensitive L-type calcium channel in the -cell.     

Reversal of the growth inhibitory effect of α-difluoromethylornithine by putrescine but not by other divalent cations

Summary Treatment with -difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC), depletes the putrescine and spermidine content, and reduces the growth rate of Ehrlich ascites tumor cells.The addition of putrescine, which is the immediate precursor of spermidine, promptly replenished the intracellular putrescine and spermidine pools and completely reversed the antiproliferative effect of DFMO. A sequential accumulation of spermine, spermidine and putrescine was observed.1,3-diaminopropane, a lower homolog of putrescine, did not reverse the antiproliferative effect of DFMO, despite its structural similarity and identical positive charge. By inhibiting remaining ODC activity, resistant to 5 mM DFMO, and possibly by inhibiting spermine synthase activity, 1,3-diaminopropane produced a further decrease in total polyamine content by reducing the spermine content.Mg²⁺, which can replace putrescine in many in vitro reactions, completely lacked the capacity to reverse the antiproliferative effect of putrescine and spermidine deficiency.Abbreviations DFMO -difluoromethylornithine - ODC ornithine decarbxylase     

Identification of DNA Polymerase γ in Eggs of a Teleost Fish (Loach)

DNA polymerase found in an extract from eggs of the teleost fish Misgurnus fossilis (loach) has been identified as an enzyme of the type. The enzyme was purified 4000- to 5000-fold from the extract by liquid chromatography. The DNA polymerase activity was sensitive to the inhibiting action of aphidicolin but resistant to N2-(p-n-butylphenyl)-2´- deoxyguanosine 5´-triphosphate (BuPdGTP). The enzyme activity correlates with the presence of a polypeptide with molecular mass of 120-130 kD that interacts specifically with polyclonal antibodies against calf thymus DNA polymerase as revealed by Western blotting and is presumably the catalytic subunit of the enzyme. The loach DNA polymerase possesses the 3´5´-exonuclease activity specific to single-stranded DNA and catalyzes distributive elongation of primers in primer–template complexes.     

Spinach ferredoxin is a calcium-binding protein

Spinach-leaf ferredoxin was identified as a calcium-binding protein by ⁴⁵Ca autoradiography on nitrocellulose membranes and with the cationic carbocyanine dye 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl] naphtho[1,2-d]thiazolium bromide (stains-all). Binding of ⁴⁵Ca was observed at pH 6.8 and pH 7.8 and in the presence of 5 mM and 20 mM MgCl₂. At the higher MgCl₂ concentration the Ca²⁺-binding capacity is reduced. Only micromolar concentrations of LaCl₃, however, are required to achieve a similar effect. Both the oxidized and reduced forms of ferredoxin bind calcium.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - stains-all 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl] naptho[1,2-d]thiazolium bromide     

Regulation of Phospholipase C Activity by Calcium Ions and Guanine Nucleotide in the Normoxic Cat Carotid Body

The carotid bodies (CB) are a paired chemoreceptor organ located at the bifurcation of the common carotid arteries. High O₂ tension suppresses while low tension activates afferent carotid chemoreceptor activity and the chemoreflex ventilatory response in the cat. The intracellular mechanism of chemotransduction is till now unknown. Previously we have shown different activities of phospholipase C (PLC) in normoxic, hypoxic and hyperoxic cat carotid body. Now we have addressed the question whether calcium ions and G-protein could be regulators of the formation of lipid derived messenger molecules in the cat carotid body. To answer this question, the PLC acting against [³H] inositol-phosphatidylinositol (PtdIns) and [³H] inositol-phosphatidylinositol-4, 5-bisphosphate [PtdIns(4,5)P₂] in the cat CB were investigated using labelled phospholipids as a source of the substrate. CB homogenate was used as a source of the enzyme. The results indicate that PLC acting on PtdIns is Ca²⁺-dependent, in contrary to that acting on PtdIns(4,5)P₂ which remains active in the presence of 10 mM EGTA. PtdIns(4,5)P₂-PLC is stimulated by GTPS. In the presence of Ca²⁺, GTPS has a synergistic stimulatory effect. PLC acting on PtdIns is not activated by GTPS. In the presence of calcium ions dopamine and a nonhydrozylable analogue of acetylocholine, carbachol, have a small stimulatory effect of about 30 % on PLC acting on PtdIns(4,5)P₂. GTPS enhances this effect. These results allow us to suggest that there are two pathways of phosphoinositides degradation in the CB, one of them is regulated by calcium ions/PtdIns-PLC/, the other one by G-protein/PtdIns(4,5)P₂-PLC/.     

Clearance and metabolism of arachidonic acid by C6 glioma cells and astrocytes

Effects of increased levels of arachidonic acid (AA) were analyzed in vitro by employment of C6 glioma cells and astrocytes from primary culture. The cells were suspended in a physiological medium added with arachidonic acid (AA) in a concentration range from 0.01 to 0.5 mM. The concentration profiles of the fatty acid and AA-metabolited were subsequently followed for 90 min. AA was measured by gas chromatography, whereas the AA-metabolites PGF₂ and LTB₄ by radioimmunoassay (RIA). Following administration of AA at 0.05 or 0.1 mM the medium was completely cleared from the fatty acid within 10 to 15 min. However, when 0.5 mM were added, AA concentrations of 0.36±0.055 mM were found at 20 min, while 0.275±0.045 mM at 90 min. Addition of AA (0.1 mM) to cell-free medium was also associated with a steady decline of its concentration, although the decrease was markedly delayed as compared to the clearance in the presence of glial cells. AA was subjected to dose-dependent metabolisation in the cell suspension as demonstrated by the production of PGF₂ and LTB₄. Following addition of 0.01 or 0.5 mM, concentrations of PGF₂ increased to a 1.9- or 4.9-fold level within 10 min, whereas those of LTB₄ rose to a 1.3- or 33.7-fold level. This was attenuated or completely blocked, respectively, by the cyclo- and lipoxygenase inhibitor BW 755C. Formation of both metabolites from AA was also observed when studying astrocytes from primary culture. The current findings demonstrate an impressive efficacy of C6 glioma cells and astrocytes to clear arachidonic acid from the suspension medium and to convert the lipid compound into prostaglandins and leukotrienes. Uptake and metabolisation of AA by the glial elements may play an important role in vivo, for example in cerebral ischemia.     

α-Lipoic acid dependent regeneration of ascorbic acid from dehydroascorbic acid in rat liver mitochondria

Rat liver mitochondria were examined for their ability to reduce dehydroascorbic acid to ascorbic acid in an -lipoic acid dependent or independent manner. The a-lipoic acid dependent reduction was stimulated by factors that increased the NADH dependent reduction of -lipoic acid to dihydrolipoic acid in coupled reactions. Optimal conditions for dehydroascorbic acid reduction to ascorbic acid were achieved in the presence of pyruvate, -lipoic acid, and ATP. Electron transport inhibitors, rotenone and antimycin A, further enhanced the dehydroascorbic acid reduction. The reactions were strongly inhibited by 1 mM iodoacetamide or sodium arsenite. Mitoplasts were qualitatively similar to intact mitochondria in dehydroascorbate reduction activity. Pyruvate dehydrogenase and -ketoglutarate dehydrogenase reduced dehydroascorbic acid to ascorbic acid in an -lipoic acid, coenzyme A, and pyruvate or -ketoglutarate dependent fashion. Dehydroascorbic acid was also catalytically reduced to ascorbic acid by purified lipoamide dehydrogenase in an -lipoic acid (K _0.5=1.4±0.8 mM) and lipoamide (K _0.5=0.9±0.3 mM) dependent manner.     

Partial purification and biochemical characterization of alkaline 5'-phosphodiesterase from barley malt sprouts

An alkaline 5-phosphodiesterase (5-PDE) from barley (Hordeum distichum) malt sprouts was partially purified by thermal treatment and acetone precipitation to diminish phosphomonoesterase (PME) activity. 5-PDE was purified 40-fold to a specific activity of 30 U mg^–1 protein with a final yield of about 32%. With synthetic substrate, the enzyme had an optimum pH of 8.9, maximum activity at 70 °C over 10 min, and a Km of 0.26 mM. The partially purified enzyme was activated by 10 mM Mg²⁺ up to 168% of the original activity, while Zn²⁺, Mn²⁺ and Cu²⁺ ions, chelating agent (EDTA) and NaN₃ (1–10 mM), and 5-ribonucleotides (1–5 mM) were inhibitory. Final enzyme preparation was stable over 8 d at 4 °C), at 70 °C for up to 120 min and without loss of activity over 90 d at –18 °C.     

An endogenous factor from soybean (Glycine max L.) cell cultures activates phosphorylation of a protein which is dephosphorylated in vivo in elicitor-challenged cells

The existence of specific binding sites for a -glucan elicitor of phytoalexin synthesis derived from the fungus Phytophthora megasperma f.sp. glycinea at the plasma membrane of soybean (Glycine max L.) tissues (W.E. Schmidt, J. Ebel (1987) Proc. Natl. Acad. Sci. USA 84, 4117–4121) might imply that stimulation of phytoalexin formation by the elicitor is a membrane-mediated process. Addition of the -glucan elicitor to soybean cellsuspension cultures, which has previously been shown to induce phytoalexin accumulation, also results in rapid changes in the phosphate turnover of several phosphoproteins. The effect of the elicitor on protein phosphorylation was tested after labeling of the cells with [³²P]orthophosphate. As shown by analysis using one-and two-dimensional gel electrophoresis, decreases as well as increases in the labeling of several phosphoroteins occurred rapidly, being detectable within 5 min after elicitor application, and persisted for at least 15 min. As judged by their relative molecular masses (M_r) and isoelectric points (pI), a number of proteins which were radioactively labeled in vivo were also phosphorylated in vitro by endogenous protein-kinase activity in the presence of Ca²⁺. The most pronounced effect was observed with a protein substrate with M_r=69000 and pI=5.7 (pp69) whose phosphate labeling markedly decreased in response to elicitor treatment in vivo. Phosphorylation of pp69 in vitro in the presence of -[³²P]ATP was strongly enhanced by a phosphorylation-stimulating factor (effector) derived from soybean cell cultures and occurred predominantly at serine residues. The effector possessed a low apparent M_r (1000), was negatively charged at pH 7.3, and was relatively heat stable. The effector was inactivated by treatment with alkaline phosphatase from calf intestine. Phosphorylation of pp69 was only slightly stimulated by Ca²⁺, and was insensitive to cAMP, cGMP, calmodulin, a lipid mixture, a ganglioside mixture, or spermine under the assay conditions used. A 10 mM concentration of 3-phosphoglycerate increased pp69 phosphorylation to the extent of about 50% of that induced by the soybean effector. There was no evidence, however, that such concentrations of 3-phosphoglycerate occurred in effector preparations. The results are discussed in relation to hypothetical signal transduction during elicitor action on soybean cells.Abbreviations M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TPCK L-1-tosylamide-2-phenylethyl chloromethyl ketone     

The uptake and metabolism of fructose‐1,6‐diphosphate in rat cardiomyocytes

Fructose1,6diphosphate (FDP) is a glycolytic intermediate which has been theorized to increase the metabolic activity of ischemic tissues. Here we examine the effects of externally applied FDP on cardiomyocyte uptake and metabolism. Adult rat cardiomyocytes were isolated and exposed to varying concentrations (0, 5, 25 and 50 mM) of FDP for either 1, 16 or 24 h of hypoxia (95% N₂/5% CO₂), each time period followed by a 1 h reoxygenation (95% air/5% CO₂). The uptake of FDP by rat cardiomyocytes was more concentrationdependent than timedependent. Furthermore, the uptake of FDP by the cardiomyocytes was similar in the hypoxia and normoxia treated cells. Alamar Blue, a redox indicator that is sensitive to metabolic activity, was used to monitor the effects of the FDP on cardiomyocyte metabolism. In the 1 h hypoxia or normoxia group, the 5, 10 and 25 mM FDP showed a significant increase in metabolism compared to the control cells. When the length of hypoxia was extended to 16 h, all doses of FDP were greater than control. And at the 24 h hypoxia or normoxia time period, only the 10, 25 and 50 mM FDP groups were greater than control. The results indicate a non-linear trend between the external concentration of FDP and the changes noted in metabolism. The findings from this study indicate that a narrow concentration range between 5–10 mM augments cardiomyocyte metabolism, but higher or lower doses may have little additional affect.     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

Effects of the γ-aminobutyrate transaminase inhibitors gabaculine and γ-vinyl GABA on γ-aminobutyric acid release from slices of rat cerebral cortex

The release of [³H]-aminobutyric acid (GABA) from pre-loaded slices of rat cerebral cortex was investigated in the presence and absence of the GABA-transaminase inhibitors gabaculine and -vinyl GABA. In the experiments carried out without an inhibitor, an ion-exchange column chromatographic technique was used to separate [³H]GABA from tritiated metabolites released with it into the superfusate. The presence of gabaculine (5 M) substantially reduced the Ca²⁺-dependence of the release of [³H]GABA evoked by a 4 min 30 mM K⁺ pulse, whereas this was not appreciably reduced by the presence of -vinyl GABA (2 mM or 10 mM). Nevertheless, the characteristics of [³H]GABA release were not identical in the presence and absence of either inhibitor.     

Competition between β-ketothiolase and citrate synthase during poly(β-hydroxybutyrate) synthesis inMethylobacterium rhodesianum

The enzymes -ketothiolase and citrate synthase from the facultatively methylotrophicMethylobacterium rhodesianum MB 126, which uses the serine pathway, were purified and characterized. The -ketothiolase had a relatively highK _m for acetyl-CoA (0.5 mM) and was strongly inhibited by CoA (K _i 0.02 mM). The citrate synthase had a much higher affinity for acetyl-CoA (K _m 0.07 mM) and was significantly inhibited by NADH (K _i 0.15 mM). The intracellular concentration of CoA metabolites and nucleotides was determined inM. rhodesianum MB 126 during growth on methanol. The level of CoA decreased from about 0.6 nmol (mg dry mass)^-1 during growth to the detection limit when poly(-hydroxybutyrate) (PHB) accumulated. Nearly unchanged intracellular concentrations of NADH, NADPH, and acetyl-CoA of about 0.5, 0.6–0.7, and 1.0 nmol (mg dry mass)^-1, respectively, were determined during growth and PHB synthesis. During growth, the -ketothiolase was almost completely inhibited by CoA, and acetyl-CoA was principally consumed by the citrate synthase. During PHB accumulation, the -ketothiolase had about 75% of its maximum activity and showed much higher activity than citrate synthase, which at the actual NADH concentration was about 75% inhibited. NADPH concentration was sufficiently high to allow the unlimited activity of acetoacetyl-CoA reductase (K _mNADPH 18 M). PHB synthesis is probably mainly controlled by the CoA concentration inM. rhodesianum MB 126.Abbreviation PHB Poly(-hydroxybutyrate)     

Adenosine 5′-phosphosulfate sulfotransferase from the marine macroalgaPorphyra yezoensis Ueda (Rhodophyta): stabilization,purification, and properties

Adenosine 5-phosphosulfate sulfotransferase (APSSTase) was purified over 2700-fold to homogeneity from the thalli of the marine macroalgaPorphyra yezoensis Ueda (Rhodophyta), using a combination of ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography and gel-filtration. The native M_r measured by gel-filtration was 350 000. The subunit M_r was estimated to be 43 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In addition, APSSTase had a relatively broad pH optimum of pH 9.0–9.8 with a peak at pH 9.5. The apparentK _m value for adenosine 5-phosphosulfate (APS) was 2.1 M, when dithiothreitol was acceptor substrate. 3-Phosphoadenosine 5-phosphosulfate and inosine 5-phosphosulfate could not substitute for APS as a sulfate donor. The enzyme utilized several organic thiols as acceptor substrates (artificial substrates): dithiothreitol (apparentK _m = 1.5 mM) and dithioerythritol (apparentK _m = 1.5 mM) gave the highest activity, and appreciable activity was also obtained usingl-glutathione (reduced form) which exhibited slight substrate inhibition (apparentK _m = 0.6 mM; the initial velocity was maximal at 3.0–4.0 mM). While APSSTase was markedly unstable in vitro: the half-life for activity loss at 25°C and pH 9.5 was about 8 min, the instability was decreased in the presence of a relatively high concentration of Na₂SO₄ or (NH₄)₂SO₄, and in the presence of APS or its analogs (AMP and -methylene-APS). Most of the thiols, with the sole exception of glutathione, were found to inactivate APSSTase irreversibly. The thiol-mediated inactivation was completely inhibited by the high concentration of Na₂SO₄, and by the analogs of APS.Abbreviations APS adenosine 5-phosphosulfate - APSSTase adenosine 5-phosphosulfate sulfotransferase - -m-APS -methylene-adenosine 5-phosphosulfate - DTT dithiothreitol - IPS inosine 5-phosphosulfate - PAPS 3-phosphoadenosine 5-phosphosulfate We wish to thank Mr. I. Kashiwase, Mr. Y. Endo and Mr. Y. Mimura, School of Fisheries Sciences, Kitasato University, for their technical assistance in this study. The research described in this paper was partly supported by the Kitasato Research Grant (H5-9 and H6-13 to N.K.).     

Nitrite reduction in Bacteroids of Rhizobium “hedysari” strain HCNT 1

Ex planta, bacteroids of the sulla-symbiont Rhizobium hedysari strain HCNT 1 terminated reduction of nitrite at nitrous oxide irrespective of the presence or absence of acetylene. Nitrate was not reduced during the experimental period, but slight nitrate reductase activity occurred if incubation with nitrate was prolonged (up to 15 h). As was observed in free-living cells, exposure of the bacteroids to the metal chelator, diethyldithiocarbamate, prevented reduction of nitrite, indicating the presence of a copper-containing nitrite reductase. Pulses of 10–75 M nitrite transiently impeded O₂ uptake in bacteroids, which resumed consumption of O₂ when the nitrite had been reduced. Exposure to >1.0 mM nitrite for 24h greatly inhibited nitrogenase activity (assayed as acetylene reduction activity) of bacteroids in planta. Exposure to the same concentrations of nitrite after 1h of incubation in the presence of acetylene almost completely stopped ongoing ethylene production in bacteroids of strain HCNT 1 extracted from nodules. Free cells of the non-nitrite-reducing R. hedysari strain CC 1335 were lacking in nitrogenase (acetylene-reduction) activity, whereas identically cultured (low-oxygen) strain HCNT 1 cells reduced both nitrite and acetylene.Abbreviations PMS phenazine methosulfate - DDC diethyldithiocarbamate     

Alpha2-adrenoreceptor stimulation does not inhibit L-type calcium channels in mouse pancreatic β-cells

The effects of ₂-adrenergic stimulation on the Ca²⁺-current in mouse pancreatic -cells were investigated using the patch-clamp technique. When using the conventional whole-cell recording configuration (dialysis of cell interior with pipette solution), addition of adrenaline (1 M) or the ₂-adrenergic agonist clonidine (5 M) failed to reduce the Ca²⁺-current, irrespective of whether intracellular GTP (or GTP S) was present or not and at both physiological (1.3 mM) and elevated (10.2 mM) Ca²⁺-concentrations. In fact, in the absence of added guanine nucleotides, the agonists tended toincrease the Ca²⁺-current amplitude in the presence of the higher Ca²⁺-concentration. Ca²⁺-channel activation measured at 1.3 mM Ca²⁺ was not affected by clonidine. Half-maximal activation was observed at –20 mV. In addition, when Ca²⁺-currents were recorded from intact -cells, using the perforated patch whole-cell configuration, clonidine (1 M) also failed to detectably affect the Ca²⁺-current. It is therefore suggested that the inhibition of -cell electrical activity and insulin-secretion produced by ₂-adrenoreceptor stimulation does not result from suppression of the L-type Ca²⁺-current.     

β-1-3- and β-1-4-glucan synthesis by membrane fractions from the fungus Saprolegnia

The -glucan synthetase activity of the fungus Saprolegnia monoica was assayed by supplying UDP-glucose to membrane fractions of mycelial homogenate. The analysis of glucan products by hydrolysis with various -glucanases and by chromatography show that both -1-3- and -1-4-linkages are formed at high substrate concentrations. In the absence of MgCl₂, -1-3-linked glucans are mainly produced. By increasing MgCl₂ concentrations the total synthesis activity and -1-3-linkages production are reduced. At low substrate concentrations in the presence of MgCl₂, -1-4-linked glucans are the only polysaccharide synthesized. Electron microscopy of radioactive products, synthesized by original membrane fractions or by membrane fractions isolated from continuous sucrose density gradients, shows microfibrils when the assays are conducted at high substrate concentrations in the absence of MgCl₂.Abbreviations G.S. I glucan synthetase I - G.S. II glucan synthetase II - Dol. P dolichol phosphate