Min-1 Mg-1 Research Articles

Exchange reactions catalyzed by group‐transferring enzymes oppose the quantitation and the unravelling of the identity of the pentose pathway

1. The distributions and rates of transfer of carbon isotopes from a selection of specifically labelled ketosugar-phosphate substrates by exchange reactions catalyzed by the pentose and photosynthetic carbon-reduction-pathway group-transferring enzymes transketolase, transaldolase and aldolase have been measured using 13C-NMR spectroscopy. 2. The rates of these exchange reactions were 5, 4 and 1.5 mumol min-1 mg-1 for transketolase exchange, transaldolase exchange and aldolase exchange, respectively. 3. A comparison of the exchange capacities contributed by the activities of these enzymes in three in vitro liver preparations with the maximum non-oxidative pentose pathway flux rates of the preparations shows that transketolase and aldolase exchanges exceeded flux by 9-19 times in liver cytosol and acetone powder enzyme preparations and by 5 times in hepatocytes. Transaldolase was less effective in the comparison of exchange versus flux rates: transaldolase exchange exceeded flux by 1.6 and 5 in catalysis by liver cytosol and acetone powder preparations, respectively, but was only 0.6 times the flux in hepatocytes. 4. Values of group enzyme exchange and pathway flux rates in the above three preparations are important because of the feature role of liver and of these particular preparations in the establishment, elucidation and measurement of a proposed reaction scheme for the fat-cell-type pentose pathway in biochemistry. 5. It is the claim of this paper that the excess of exchange rate activity (particularly transketolase exchange) over pathway flux will overturn attempts to unravel, using isotopically labelled sugar substrates, the identity, reaction sequence and quantitative contribution of the pentose pathway to glucose metabolism. 6. The transketolase exchange reactions relative to the pentose pathway flux rates in normal, regenerating and foetal liver, Morris hepatomas, mammary carcinoma, melanoma, colonic epithelium, spinach chloroplasts and epididymal fat tissue show that transketolase exchange may exceed flux in these tissues by factors ranging over 5-600 times. 7. The confusion of pentose pathway theory by the effects of transketolase exchange action is illustrated by the 13C-NMR spectrum of the hexose 6-phosphate products of ribose 5-phosphate dissimilation, formed after 30 min of liver enzyme action, and shows 13C-labelling in carbons 1 and 3 of glucose 6-phosphate with ratios which range over 2.1-6.4 rather than the mandatory value of 2 which is imposed by the theoretical mechanism of the pathway.

Synthesis of pyrophosphate and ATP by soluble mitochondrial F1

Soluble F1 from beef heart that has been depleted of adenine nucleotides to values of 0.4 mol of ADP and 0.1 mol ATP/mol of enzyme has the capacity to synthetize about 0.1 mol of ATP/mol of enzyme from medium phosphate in the presence of Mg2+ and 30% dimethyl sulfoxide. Under the same conditions, native and adenine nucleotide depleted F1 can also synthesize pyrophosphate to values that range from 0.03 to 0.05 mol/mol of F1. The formation of pyrophosphate requires Mg2+ and dimethyl sulfoxide. The formed pyrophosphate remains bound to F1 during filtration through Sephadex centrifugation columns. In all water media, adenine nucleotide depleted, but not native, F1 can hydrolyze pyrophosphate to values of about 0.2 nmol min-1 mg-1. This activity is inhibited or stimulated by agents (adenylyl imidodiphosphate, aurovertin, and methanol) that produce such effects on the ATPase activity of F1; NaN3 stimulated the activity. Therefore, F1 from bovine heart mitochondria has the capacity to catalyze synthesis and hydrolysis of ATP. Synthesis of pyrophosphate by the soluble F1 appears to follow the same energetic considerations that have been postulated for ATP synthesis by the soluble enzyme [de Meis (1989) Biochim. Biophys. Acta 973, 339-349].

Purification and some properties of 11-hydroxythromboxane B2 dehydrogenase from porcine kidney.

A protein with NAD-dependent 11-hydroxythromboxane B2 dehydrogenase activity was purified to apparent homogeneity from porcine kidney using a relatively simple purification procedure, involving precipitation, anion-exchange chromatography (diethylaminoethyl-cellulose), affinity chromatography (5'-AMP-Sepharose) and gel-filtration chromatography (Protein Pak 125). The dehydrogenase was found to have a molecular mass of 50-55 kDa as determined by comparison with standards on SDS/PAGE. The molecular mass on gel-filtration chromatography was dependent on the ionic strength of the buffer. The apparent Km and Vmax values for thromboxane B2 were also dependent of the ionic strength with a Vmax of 214 nmol min-1 mg-1 using 250 mM Tris/HCl, pH 8.0, and a corresponding Km of 2.9 mM. The enzyme was NAD dependent and was clearly separated from the proteins with 15-hydroxyprostaglandin dehydrogenase activity also present in the kidney. Furthermore, it was found that 11-hydroxythromboxane B2 dehydrogenase did not utilize prostaglandin D2, prostaglandin E2, prostaglandin F2 alpha or cholic acid as substrate, and that the enzyme did not catalyse the reverse reaction, conversion of 11-dehydrothromboxane B2 to thromboxane B2.

Thymidine phosphorylase activity of platelet-derived endothelial cell growth factor is responsible for endothelial cell mitogenicity.

Recombinant human platelet-derived endothelial cell growth factor, expressed in the yeast Saccharomyces cerevisiae was purified to greater than 98% purity by anion-exchange and hydroxyapatite chromatography. It was shown to possess thymidine phosphorolytic activity in vitro (pH optimum, pH 5.3; Km, 0.11 mM; Vmax, 12.5 mmol min-1 mg-1; turnover number, 9.4 s-1). Covalent modification simultaneously inhibited the enzymatic and mitogenic properties of the protein, while interaction with a cell-surface receptor was not required to stimulate mitogenesis. Purified Escherichia coli thymidine phosphorylase was also mitogenic toward endothelial cells. It is proposed that platelet-derived endothelial cell growth factor is human thymidine phosphorylase which promotes endothelial cell proliferation by reducing thymidine levels that would otherwise be inhibitory to endothelial cell growth.

Characterization of Maize Acetyl-Coenzyme A Carboxylase.

Maize (Zea mays L.) leaf acetyl-CoA carboxylase (ACCase) was purified about 500-fold by ammonium sulfate fractionation and gel filtration and blue Sepharose affinity and anion-exchange chromatography. Most ACCase activity (85%) recovered from the anion-exchange column was found in a highly purified fraction (specific activity 5.5 [mu]mol acid-stable product min-1 mg-1) that consisted primarily of a single 227-kD biotinylated polypeptide. The fraction represented 29% of the original activity and was designated ACCase I. A second partially purified ACCase activity (ACCase II) eluted earlier during anion-exchange chromatography, contained a single biotinylated polypeptide of 219 kD, was poorly recognized by antiserum raised against the ACCase I polypeptide, and was less inhibited by the herbicides haloxyfop or sethoxydim than was ACCase I. ACCase I and II both utilized propionyl-CoA as substrate about 50% as effectively as acetyl-CoA, and neither utilized methylcrotonyl-CoA. Immunoprecipitation with antiserum and protein blotting of crude extracts of leaf, embryo, and endosperm tissue and suspension cells indicated that most ACCase activity in these tissues was immunologically similar and consisted of ACCase I. Only leaves contained significant amounts of the ACCase II polypeptide; however, no ACCase II polypeptide was found in isolated mesophyll chloroplasts. The ACCase I and II polypeptides appear to be subunits of distinct ACCase isoforms.

A Simple Technique to Determine Glutathione (GSH) Levels and Synthesis in Ocular Tissues as GSH-bimane Adduct: Application to Normal and Galactosemic Guinea-pigs

A fluorimetric technique previously described for other tissues has been applied to determine levels of glutathione and its synthetic rates in ocular tissues of Hartley guinea-pigs. Monochlorobimane forms a stable, fluorescent adduct with glutathione in a reaction catalyzed by glutathione-S-transferase. The fluorescent signal recorded over time is directly proportional to the synthetic rate of glutathione. Lens, cornea and retina were homogenized and cytosolic fractions dialyzed overnight to deplete endogeneous glutathione. Glutathione synthetic rates were determined from a mixture of glutathione precursors and co-factors, viz. cysteine+dithiothreitol, glutamate+glycine, ATP and Mg++ in the presence of monochlorobimane. The mixture was supplemented with glutathione-S-transferase to catalyze the formation of the fluorescent adduct. Glutathione synthetic rates were determined in the absence and presence of buthionine sulfoximine, an inhibitor of γ-glutamyl cysteine synthetase. The difference in fluorescence change over time in the presence and absence of buthionine sulfoximine was used to estimate glutathione synthesis. Basal levels of glutathione in pre-dialyzed cytosolic fractions of the lens, cornea, and retina were 21·8±2·2, 36·5±4·1 and 38·6±2·8 nmol mg-1 protein, respectively. The maximal glutathione synthetic rates in these tissues were 0·52±0·04, 2·25±0·67 and 3·35±0·65 nmol min-1 mg-1 protein, respectively. When γ-glutamyl cysteine is used as a precursor instead of cysteine, the glutathione synthetase activities from lenses and retinas were 0·19 ±0·08 and 1·54± 0·76 nmol-1 min mg-1 protein. The present technique when applied to galactosemic guinea-pigs, showed that galactose feeding for 14 days caused a significant depletion (65 %) of lens glutathione levels, but the rates of glutathione synthesis in those lenses were not altered. The present technique permits measurements of glutathione levels and synthesis in different ocular tissues. It is suggested that the monochlorobimane method may be of value in delineating the role of glutathione in various ocular disorders such as in the galactose-induced cataract.

Insensitivity of barley endosperm ADP-glucose pyrophosphorylase to 3-phosphoglycerate and orthophosphate regulation.

Crude extracts of starchy endosperm from barley (Hordeum vulgare cv Bomi) contained high pyrophosphorolytic activity (up to 0.5 mumol of glucose-1-P formed min-1 mg-1 of protein) of ADP-glucose pyrophosphorylase (AGP) when assayed in the absence of 3-phosphoglycerate (3-PGA). This high activity was observed regardless of whether AGP had been extracted in the presence or absence of various protease inhibitors or other protectants. Western blot analysis using antibodies specific for either the small or large subunit of the enzyme demonstrated that the large, 60-kD subunit was prone to proteolysis in crude extracts, with a half-time of degradation at 4 degrees C (from 60 to 53 to 51 kD) on the order of minutes. The presence of high concentrations of protease inhibitors decreased, but did not prevent this proteolysis. The small, 51-kD subunit of barley endosperm AGP was relatively resistant to proteolysis, both in the presence or absence of protease inhibitors. For the crude, nonproteolyzed enzyme, 3-PGA acted as a weak activator of the ADP-glucose synthetic reaction (about 25% activation), whereas in the reverse reaction (pyrophosphorolysis) it served as an inhibitor rather than an activator. For both the synthetic and pyrophosphorolytic reactions, inorganic phosphate (Pi) acted as a weak competitive or mixed inhibitor of AGP. The relative insensitivity to 3-PGA/Pi regulation has been observed with both the nonproteolyzed crude enzyme and partially purified (over 60-fold) AGP, the latter characterized by two bands for the large subunit (molecular masses of 53 and 51 kD) and one band for the small subunit (51 kD). Addition of 3-PGA to assays of the partially purified, proteolyzed enzyme had little or no effect on the Km values of all substrates of AGP, but it reduced the Hill coefficient for ATP (from 2.1 to 1.0). These findings are discussed with respect to previous reports on the structure and regulation of higher plant AGP.

Human insulin receptor beta-subunit transmembrane/cytoplasmic domain expressed in a baculovirus expression system: purification, characterization, and polylysine effects on the protein tyrosine kinase activity.

We have expressed, purified, and characterized the insulin receptor protein tyrosine kinase (PTK) retaining the transmembrane and downstream domains. The proteins expressed in insect cells using a baculovirus expression system were identified as membrane-bound by immunofluorescence staining and biochemical characterization. One-step purification by immunoaffinity chromatography from Triton X-100 cell extracts resulted in a approximately 360-fold increase in the specific kinase activity with a yield of approximately 50%. An appMr = approximately 60,000 protein was the major component identified by both silver staining of the purified enzyme and immunostaining of the crude extracts after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Using nondenaturing conditions, the molecular weight was estimated to be approximately 250,000 and approximately 500,000 by glycerol gradient centrifugation and gel permeation chromatography, respectively, suggesting that oligomers of the beta-subunit domains such as tetramers and octamers are formed. The basal PTK activity of this enzyme was much higher than those of previously reported soluble-form insulin receptor PTKs expressed in insect cells or the native receptor. Km and Vmax for two substrates, src-related peptide and poly(Glu, Tyr) (4:1), were 2.4 mM and 2.5 mumol min-1 mg-1 and 0.26 mM and 1.2 mumol min-1 mg-1, respectively. Specific activities measured under two previously reported conditions using histone H2B as a substrate were 100 or 135 nmol min-1 mg-1, in contrast to those of soluble PTKs which were reported to be 20 or 70 nmol min-1 mg-1, respectively. The purified enzyme was autophosphorylated at Tyr residues. Autophosphorylation activated the enzyme approximately 3-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of Bacillus cereus phosphatidylinositol-specific phospholipase C with thiophosphate and fluorescent analogs of phosphatidylinositol.

Thiophosphate analogs (C-S-P bond) of phosphatidylinositol (Cn-thio-PI: racemic hexadecyl-, dodecyl-, and octylthiophosphoryl-1-myo-inositol) and a fluorescent analog (pyrene-PI: rac-4-(1-pyreno)-butylphosphoryl-1-myo-inositol) were all substrates for phosphatidylinositol-specific phospholipase C from Bacillus cereus. Hydrolysis of thio-PI was followed by coupling the production of alkylthiol to a disulfide interchange reaction with dithiobispyridine. Hydrolysis of pyrene-PI was followed using a HPLC-based assay with fluorescence detection. The activity of PI-PLC with thio-PI analogs showed an interfacial effect. C16-Thio-PI, which had a critical micelle concentration (CMC) of 7 microM, gave a hyperbolic activity versus concentration curve between 0 and 2 mM, while C8-thio-PI, which had a CMC above 10 mM, showed very low activity which increased greatly upon introduction of an interface in mixed micelles with hexadecylphosphocholine (HDPC). Pyrene-PI, which aggregates above 0.3 mM, gave a sigmoidal activity curve with much higher activity above the CMC. All three thio-PI homologs as mixed micelles with HDPC gave hyperbolic activity curves with PI-PLC that were a function of bulk concentration of substrate at constant surface concentration and surface concentration of substrate at constant bulk concentration. The maximal activity of PI-PLC with pure C16-thio-PI micelles was 6.25 mumol min-1 mg-1, while that with pyrene-PI was estimated to be 68 mumol min-1 mg-1. With pure C16-thio-PI micelles, 0.022 mM substrate gave half Vmax, similar to that in mixed micelles with HDPC.

Inorganic pyrophosphatase from bovine retinal rod outer segments.

Rod outer segments from bovine retina contain a higher level of intracellular inorganic pyrophosphatase (EC 3.6.1.1) activity than has been found in any other mammalian tissue; the specific activity in extracts of soluble outer segment proteins is more than 6-fold higher than in extracts from bovine liver and more than 24-fold higher than in skeletal muscle extracts. This high activity may be necessary to keep inorganic pyrophosphate concentrations low in the face of the high rates of pyrophosphate production that accompany the cGMP flux driving phototransduction. We have begun to explore the role of inorganic pyrophosphatase in photoreceptor cGMP metabolism by 1) studying the kinetic properties of this enzyme and its interactions with divalent metal ions and anionic inhibitors, 2) purifying it and studying its size and subunit composition, and 3) examining the effects of pyrophosphate on rod outer segment guanylyl cyclase. Km for magnesium pyrophosphate was 0.9-1.5 microM, and the purified enzyme hydrolyzed > 885 mumol of PPi min-1 mg-1. The enzyme appears to be a homodimer of 36-kilodalton subunits when analyzed by gel electrophoresis and density gradient centrifugation, implying that kcat = 10(3) s-1, and kcat/Km = 0.7-1 x 10(9) M-1 s-1. The enzyme was inhibited by Ca2+ at submicromolar levels: 28% inhibition was observed at 138 nM [Ca2+], and 53% inhibition at 700 nM [Ca2+]. Imidodiphosphate acted as a competitive inhibitor, with Ki = 1.2 microM, and fluoride inhibited half-maximally approximately 20 microM. Inhibition studies on rod outer segment guanylyl cyclase confirmed previous reports that pyrophosphate inhibits guanylyl cyclase, suggesting an essential role for inorganic pyrophosphatase in maintaining cGMP metabolism.

Purification and partial characterization of two feruloyl esterases from the anaerobic fungus Neocallimastix strain MC-2.

Two extracellular feruloyl esterases (FAE-I and FAE-II) produced by the anaerobic fungus Neocallimastix strain MC-2 which cleave ferulic acid from O-(5-O-[(E)-feruloyl]-alpha-L- arabinofuranosyl)-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) were purified. The molecular masses of FAE-I and FAE-II were 69 and 24 kDa, respectively, under both denaturing and nondenaturing conditions. Apparent Km and maximum rate of hydrolysis with FAXX were 31.9 microM and 2.9 mumol min-1 mg-1 for FAE-I and 9.6 microM and 11.4 mumol min-1 mg-1 for FAE-II. FAE-II was specific for FAXX, but FAE-I hydrolyzed FAXX and PAXX, the equivalent p-coumaroyl ester, at a maximum rate of metabolism ratio of 3:1.

Stereoselective inhibition of renal organic cation transport in human kidney.

Amantadine was found to be concentrated by human cortical slices, with apparent Km and Vmax values of 187 +/- 11 microM and 1.37 +/- 0.28 nmol mg-1 min-1 respectively (mean +/- s.e. mean, n = 4). Addition of quinine (8S, 9R-(-)-isomer) or quinidine (8R, 9S-(+)-isomer) competitively inhibited this accumulation (apparent Ki values of 261 +/- 44 (n = 4) and 586 +/- 68 microM (n = 3), respectively). The stereoselectivity of this inhibition is the reverse of that seen with respect to the renal clearances of the diastereoisomers.

Histamine N-methyl transferase: inhibition by drugs.

1. Histamine N-methyl transferase activity was measured in samples of human liver, brain, kidney, lung and intestinal mucosa. The mean (+/- s.d.) rate (nmol min-1 mg-1 protein) of histamine N-methylation was 1.78 +/- 0.59 (liver, n = 60), 1.15 +/- 0.38 (renal cortex, n = 8), 0.79 +/- 0.14 (renal medulla, n = 8), 0.35 +/- 0.08 (lung, n = 20), 0.47 +/- 0.18 (human intestine, n = 30) and 0.29 +/- 0.14 (brain, n = 13). 2. Inhibition of histamine N-methyl transferase by 15 drugs was investigated in human liver. The IC50 for the various drugs ranged over three orders of magnitude; chloroquine was the most potent inhibitor. 3. The average IC50 values for chloroquine were 12.6, 22.0, 19.0, 21.6 microM in liver, renal cortex, brain and colon, respectively. These values are lower than the Michaelis-Menten constant for histamine N-methyltransferase in liver (43.8 microM) and kidney (45.5 microM). Chloroquine carried a mixed non-competitive inhibition of hepatic histamine N-methyl transferase. Some side-effects of chloroquine may be explained by inhibition of histamine N-methyl transferase.

Cloning and in vitro expression of the human selenoprotein, type I iodothyronine deiodinase.

The type I 5' iodothyronine deiodinase (5' DI) catalyzes the deiodination of T4 to the biologically active hormone T3 and accounts for a significant fraction of its production. We have recently cloned the complementary DNA (cDNA) for the rat 5' DI, which contains the rare amino acid selenocysteine, and used this to screen human liver and kidney cDNA libraries to identify a human 5' DI cDNA clone. From these, we constructed a cDNA encoding a functional 5' DI. The 2222 base pair human 5' DI cDNA is approximately 200 nucleotides shorter than the 2.4-kilobase hybridizing band in Northern blots of human liver, kidney, and thyroid, because of missing 5' untranslated sequence and the poly A tail. The deduced amino acid sequence codes for a protein of 28.7 kilodaltons assuming the UGA codon at position 382 encodes selenocysteine, and is highly homologous (88% similarity) to the rat. We transiently expressed the 5' DI in COS-7 cells to establish that it encodes a functional enzyme and to study its kinetics. These show saturable deiodination of rT3 (Ka 0.52 +/- 0.04 mumol/L and Vmax 63.2 +/- 16.4 pmol min-1 mg-1). T4 and gold thioglucose are competitive inhibitors of rT3 deiodination. 6-n-Propylthiouracil (PTU) is an uncompetitive inhibitor (with rT3) and competitive inhibitor (with dithiothreitol) of rT3 deiodination. 6-n-Propylthiouracil inhibits T4 to T3 conversion. Labeling of COS-7 cells transiently transfected with the human 5' DI cDNA with bromoacetyl-125I-T3 demonstrates a 28-kilodalton protein. This indicates that in the human, as well as in the rat messenger RNA, the UGA encodes selenocysteine and translation terminates at the UAA codon at nucleotides 754 to 756. Reverse T3 and gold thioglucose (100 nmol/L) block bromoacetyl-125I-T3 labeling of the transiently expressed human and rat 5' DI proteins. These results demonstrate that the human 5' DI is a selenoprotein, analogous to the rat enzyme. Given the previously demonstrated critical role of the selenium atom in catalyzing deiodination by this protein, we conclude that this trace element is essential for normal thyroid hormone action in man.

Purification and properties of 3-hydroxybutyryl-coenzyme A dehydrogenase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593.

The enzyme 3-hydroxybutyryl-coenzyme A (CoA) dehydrogenase has been purified 45-fold to apparent homogeneity from the solvent-producing anaerobe Clostridium beijerinckii NRRL B593. The identities of 34 of the N-terminal 35 amino acid residues have been determined. The enzyme exhibited a native M(r) of 213,000 and a subunit M(r) of 30,800. It is specific for the (S)-enantiomer of 3-hydroxybutyryl-CoA. Michaelis constants for NADH and acetoacetyl-CoA were 8.6 and 14 microM, respectively. The maximum velocity of the enzyme was 540 mumol min-1 mg-1 for the reduction of acetoacetyl-CoA with NADH. The enzyme could use either NAD(H) or NADP(H) as a cosubstrate; however, kcat/Km for the NADH-linked reaction was much higher than the apparent value for the NADPH-linked reaction. Also, NAD(H)-linked activity was less sensitive to changes in pH than NADP(H)-linked activity was. In the presence of 9.5 microM NADH, the enzyme was inhibited by acetoacetyl-CoA at concentrations as low as 20 microM, but the inhibition was relieved as the concentration of NADH was increased, suggesting a possible mechanism for modulating the energy efficiency during growth.

Protein kinase C activity in rat renal proximal tubule cells.

The presence of protein kinase C (PKC) in proximal tubule cells of the rat kidney is established by means of immunodetection and by the demonstration of calcium- and phospholipid-dependent, staurosporine-inhibitable histone phosphorylation. The calcium-dependence of renal PKC is described. Maximal activation of the enzyme (178.2 and 258.8 pmol P1 mg-1 min-1 for cytosol and membrane respectively) was achieved with 5 microM of Ca2+. Phorbol 12, 13 dibutyrate (PDBu) translocated PKC from cytosol to membrane in a dose- and time-dependent fashion, while 4 alpha-phorbol 12,13-didecanoate produced no significant effect on translocation. Cytosolic PKC activity was compared in immature and mature tissues (10- and 40-day-old kidneys). Basal activity was found to be significantly higher (P less than 0.05) in immature cells (272.8 vs. 157.5 pmol Pi mg-1 min-1). PDBu at 10(-6) M for 15 min reduced immunoreactivity in the soluble fraction of both groups, which was accompanied by a significant decrease in kinase activity. We speculate that the high PKC activity in the infant kidney plays a role in cell growth.

Pyruvate oxidation by Methanococcus spp.

In the absence of H2, Methanococcus spp. utilized pyruvate as an electron donor for methanogenesis. For Methanococcus voltae A3, Methanococcus maripaludis JJ1, and Methanococcus vannielii, typical rates of pyruvate-dependent methanogenesis were 3.4, 2.8, and 3.9 nmol min-1 mg-1 cell dry wt, respectively. These rates were 1–4% of the rates of H2-dependent methanogenesis. For M. voltae, the concentration of pyruvate required for one-half the maximum rate of methanogenesis was 7 mM, and pyruvate-dependent methanogenesis was linear for 3 days. Radiolabeled acetate was formed from [3-14C]pyruvate, and the stoichiometry of pyruvate consumed per acetate produced was 1.12±0.27. The stoichiometry of pyruvate consumed per CH4 produced was 3.64±0.34. These values are close to the expected values of 1 acetate and 4 CH4. Although 10–30% of total cell carbon could be obtained from exogenous pyruvate during growth with H2, pyruvate did not replace the nutritional requirement for acetate in Methanococcus voltae A3 or two acetate auxotrophs of Methanococcus maripaludis, JJ6 and JJ7. These results suggest that pyruvate was not oxidized in the presence of H2. The inability to oxidize pyruvate during H2-dependent methanogenesis would prevent a futile cycle of pyruvate oxidation and biosynthesis during autotrophic growth.

Supply of O2 regulates O2 demand during utilization of reserves of poly-beta-hydroxybutyrate in N2-fixing soybean bacteroids.

A liquid reaction medium containing dissolved air and oxyleghaemoglobin, but no energy-yielding substrate, was supplied to bacteroids confined in a stirred flow reaction chamber. The relative oxygenation of the leghaemoglobin in the chamber was determined automatically by spectrophotometry of the effluent solution, and the concentrations of free, dissolved O2 ([O2]) and rates of O2 consumption were calculated. Dissolved CO2 and NH3 from N2 fixation were determined in fractions of the effluent solution. Bacteroids utilized endogenous reserves of poly-beta-hydroxybutyrate (PHB), which were depleted by 9.2% during a typical 5 h-long experiment. Stepwise increases in flow rate (increasing supply of O2) initially produced a drop in O2 demand and resulted in a rise in [O2] and a decline in N2 fixation. Subsequently, O2 demand rose (presumably because of increased mobilization of substrate from PHB) and [O2] declined to a low level. N2 fixation was fully restored, or even enhanced, within 15-20 min of establishment of a new, steady [O2]. This pattern of regulation by O2 supply was completely eliminated by adding low concentrations (20-50 microM) of oxidizable substrate (succinate, malate, ethanol) to the reaction medium. During endogenous activity, rates of CO2 evolution were proportional to, but less than, rates of O2 consumption up to 5.4 nmol O2 min-1 mg-1, above which CO2 evolution exceeded O2 consumption. These and other features of endogenous activity are discussed in relation to sustaining N2 fixation by nodules in vivo.

Effect of atrial natriuretic factor on renal prostaglandin E2 release in the anaesthetized dog.

Experiments were undertaken in two groups of barbiturate anaesthetized dogs to examine whether atrial natriuretic factor (ANF) exerts an effect on renal release of prostaglandin E2 (PGE2). In the first group, intravenous infusion of ANF (50 ng min-1 kg-1 body wt) reduced basal PGE2 release from 4.4 +/- 0.8 pmol min-1 to 1.8 +/- 0.7 pmol min-1. In the second group, intrarenal infusion of an alpha 1-adrenoceptor agonist, phenylephrine (2.5-6.75 micrograms min-1), raised PGE2 release from 2.7 +/- 0.5 pmol min-1 to 7.5 +/- 1.3 pmol min-1. During continuous alpha 1-adrenergic stimulation, intravenous infusion of ANF (100 ng min-1 kg-1 body wt) reduced PGE2 release to 3.5 +/- 1.0 pmol min-1. These results demonstrate that ANF reduces basal and alpha 1-adrenergic stimulated renal PGE2 release.

Control of glycoprotein synthesis: substrate specificity of rat liver UDP-GlcNAc:Man alpha 3R beta 2-N-acetylglucosaminyltransferase I using synthetic substrate analogues.

UDP-GlcNAc: Man alpha 3R beta 2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) is the key enzyme in the synthesis of complex and hybrid N-glycans. Rat liver GlcNAc-T I has been purified more than 25,000-fold (M(r) 42,000). The Vmax for the pure enzyme with [Man alpha 6(Man alpha 3)Man alpha 6](Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc beta-Asn as substrate was 4.6 mumol min-1 mg-1. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds an N-acetylglucosamine (GlcNAc) residue in beta 1-2 linkage to the Man alpha 3Man beta-terminus of the substrate. Several derivatives of Man alpha 6(Man alpha 3)Man beta-R, a substrate for the enzyme, were synthesized and tested as substrates and inhibitors. An unsubstituted equatorial 4-hydroxyl and an axial 2-hydroxyl on the beta-linked mannose of Man alpha 6(Man alpha 3)Man beta-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the alpha 3-linked mannose (Man) of the substrate increases the KM 20-fold. Modifications on the alpha 6-linked mannose or on the core structure affect mainly the KM and to a lesser degree the Vmax, e.g., substitutions of the Man alpha 6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower the KM, whereas various other substitutions at the 3-position increase the KM slightly. Man alpha 6(Man alpha 3)4-O-methyl-Man beta 4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.