Alanine dehydrogenase of the β-lactam antibiotic producer Streptomyces clavuligerus

l-Alanine dehydrogenase was found in extracts of the antibiotic producer Streptomyces clavuligerus. The enzyme was induced by ammonia, and the level of induction was dependend on the extracellular concentration. l-Alanine was the only amino acid able to induce alanine dehydrogenase. The enzyme was characterized from a 38-fold purified preparation. Pyruvate (K _m =1.1 mM), ammonia (K _m =20 mM) and NADH (K _m =0.14 mM) were required for the reductive amination, and l-alanine (K _m =9.1 mM) and NAD (K _m =0.5 mM) for the oxidative deaminating reaction. The aminating reaction was inhibited by alanine, serine and NADPH. Alanine inhibited uncompetitively with respect to NADH (K _i =1.6 mM) and noncompetitively with respect to ammonia (K _i =2.0 mM) and pyruvate (K _i =3.0 mM). In the aminating reaction 3-hydroxypyruvate, glyoxylate and 2-oxobutyrate could partially (6–7%) substitute pyruvate. Alanine dehydrogenase from S. clavuligerus differed with respect to its molecular weight (92000) and its kinetic properties from those described for other microorganisms.Abbreviation Alanine-DH l-alanine:NAD oxidoreductase     

Nitrogen assimilation in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system.Glutamine synthetase had a K _m for NH ₄ ⁺ of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a K _m for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: l-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on l-alanine as the sole nitrogen source in the absence of N₂ low levels of a nicotinamide adenine dinucleotide linked l-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In l-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.Abreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulphoximine     

L-alanine:2-oxoglutarate aminotransferase isoenzymes from Arabidopsis thaliana leaves

The occurrence of four l-alanine:2-oxoglutarate aminotransferase (AOAT) isoenzymes (AOAT-like proteins): alanine aminotransferase 1 and 2 (AlaAT1 and AlaAT2, EC 2.6.1.2) and l-glutamate:glyoxylate aminotransferase 1 and 2 (GGAT1 and GGAT2, EC 2.6.1.4) was demonstrated in Arabidopsis thaliana leaves. These enzymes differed in their substrate specificity, susceptibility to pyridoxal phosphate inhibitors and behaviour during molecular sieving on Zorbax SE-250 column. A difference was observed in the electrostatic charge values at pH 9.1 between GGAT1 and GGAT2 as well as between AlaAT1 and AlaAT2, despite high levels of amino acid sequence identity (93 % and 85 %, respectively). The unprecedented evidence for the monomeric structure of both AlaAT1 and AlaAT2 is presented. The molecular mass of each enzyme estimated by molecular sieving on Sephadex G-150 and Zorbax SE-250 columns and SDS/PAGE was approximately 60 kDa. The kinetic parameters: K_m (Ala)=1.53 mM, K_m (2-oxoglutarate)=0.18 mM, k_cat=124.6 s⁻¹, k_cat/K_m=8.1 × 10⁴ M⁻¹·s⁻¹ of AlaAT1 were comparable to those determined for other AlaATs isolated from different sources. The two studied GGATs also consisted of a single subunit with molecular mass of 47.3–70 kDa. The estimated K_m values for l-glutamate (1.2 mM) and glyoxylate (0.42 mM) in the transamination catalyzed by putative GGAT1 contributed to indentification of the enzyme. Based on these results we concluded that each of four AOAT genes in Arabidopsis thaliana leaves expresses different AOAT isoenzyme, functioning in a native state as a monomer.     

Purification and partial characterization of alanine dehydrogenase from Streptomyces aureofaciens

Alanine dehydrogenase was purified to near homogeneity from cell-free extract of Streptomyces aureofaciens, which produces tetracycline. The molecular weight of the enzyme determined by size-exclusion high-performance liquid chromatography was 395 000. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 48 000, indicating that the enzyme consists of eight subunits with similar molecular weight. The isoelectric point of alanine dehydrogenase is 6.7. The pH optimum is 10.0 for oxidative deamination of L-alanine and 8.5 for reductive amination of pyruvate. K _M values were 5.0 mM for L-alanine and 0.11 mM for NAD⁺. K _M values for reductive amination were 0.56 mM for pyruvate, 0.029 mM for NADH and 6.67 mM for NH₄Cl.Abbreviation AlaDH alanine dehydrogenase     

Alanine dehydrogenase of the N2-fixing blue-green alga,Anabaena cylindrica

The l-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270000, has 6 sub-units, each of molecular weight approximately 43000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD⁺ specific and oxaloacetate can partially substitute for pyruvate. The K _m ^app for NAD⁺ is 14 M and 60 M at low and high NAD⁺ concentrations, respectively. The K _m ^app for l-alanine is 0.4 mM, that for pyruvate is 0.11 mM, and that for oxaloacetate is 3.0 mM. The K _m ^app for NH ₄ ⁺ varies from 8–133 mM depending on the pH, being lowest at high pH levels (pH 8.7 or above). Alanine, serine and glycine inhibit ADH activity in the aminating direction. The enzyme is active both in heterocysts and vegetative cells and activity is higher in nitrogen-starved cultures than in N₂-fixing cultures. The data suggest that although alanine is formed by the aminating activity of ADH, entry of newly fixed ammonia into organic combination does not occur primarily via ADH in N₂-fixing cultures of A. cylindrica. Ammonia assimilation via ADH may be important in cultures with an excess of available nitrogen. The deaminating activity of the enzyme may be important under conditions of nitrogen-deficiency.Abbreviations ADH alanine dehydrogenase - DEAE diethylamino ethyl cellulose - EDTA ethylenediamine tetraacetic acid - GDH glutamic dehydrogenase - GS glutamine synthetase - GOT aspartate-glutamate aminotransferase - NAD⁺ nicotinamide adenine dinucleotide - NADH reduced nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH reduced nicotinamide adenine dinucleotide phosphate - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl) aminomethane     

The presence of two serine racemases in Streptomyces garyphalus,a d-cycloserine producer

Two serine racemases (I and II) were isolated from Streptomyces garyphalus. Serine racemase I (molecular weight 93,000) was purified to a single band in an analytical electrofocusing system. Serine racemase II (molecular weight 73,000) was partially purified. Both enzymes used pyridoxal-5-phosphate as cofactor. Besides serine the enzymes utilized alanine as substrate but no other amino acid tested. The K _m values of l-alanine and l-serine for enzyme I were 111 mM and 35 mM respectively. Enzyme I was not inhibited by d-cycloserine but by hydroxylamine. Both substances inhibited enzyme II. The serine racemases may be involved in the biosynthesis of d-cycloserine in S. garyphalus.     

Kinetic and regulatory properties of pyruvate kinase isozymes from flight muscle and fat body of the cockroach,Periplaneta americana

Summary Pyruvate kinases from flight muscle and fat body of the cockroach,Periplaneta americana, were purified to homogeneity. The two tissues contained different forms of the enzyme which were separable by starch gel electrophoresis and isoelectric focusing (pI=5.75 for flight muscle and 6.15 for fat body). Both enzymes had molecular weights of 235,000±20,000.Flight muscle pyruvate kinase displayed Michaelis-Menten kinetics with respect to both ADP and P-enolpyruvate withK _m values of 0.27 and 0.04 mM, respectively.K _m for Mg²⁺ was 0.60 mM andK _a for K⁺ was 15 mM. The enzyme was weakly inhibitied by four compounds, ATP, arginine-P,l-alanine and citrate with apparentK _i values of 3.5, 15, 20 and 24 mM, respectively. Competitive inhibition by 3 mM ATP or 10 mM arginine-P raised theK _m for P-enolpyruvate to 0.067 or 0.057 mM. Fructose-1,6-P₂ did not activate the enzyme but reversed inhibitions by ATP and arginine-P.Fat body pyruvate kinase showed sigmoidal kinetics with respect to P-enolpyruvate with S_0.5=0.32 mM andn _H=1.43.K _m values for ADP and Mg²⁺ were 0.30 and 0.80 mM, respectively with aK _a for K⁺ of 10 mM. ATP andl-alanine were inhibitors of the enzyme; 2 mM ATP raised S_0.5 for P-enolpyruvate to 0.48 mM while 3 mMl-alanine increased S_0.5 to 0.84 mM. Neither citrate nor arginine-P inhibited the enzyme but citrate affected the enzyme by reversingl-alanine inhibition. Fat body pyruvate kinase was strongly activated by fructose-1,6-P₂ with an apparentK _a of 1.5 M. Fructose-1,6-P₂ at 0.1 mM reduced S_0.5 for P-enolpyruvate to 0.05 mM andn _H to 1.0.Flight muscle and fat body pyruvate kinases from the cockroach show properties analogous to those of the muscle and liver forms of mammalian pyruvate kinase. Fat body pyruvate kinase is suited for on-off function in a tissue with a gluconeogenic capacity. Strong allosteric control with a feed-forward activation by fructose-1,6-P₂ is key to coordinating enzyme function with glycolytic rate. The function of flight muscle pyruvate kinase in energy production during flight is aided by a lowK _m for P-enolpyruvate, weak inhibitor effects by high energy phosphates and deinhibition of these effects by fructose-1,6-P₂.     

Purification and characterization of a highly thermostable α-<Emphasis Type="SmallCaps">l</Emphasis>-Arabinofuranosidase from <Emphasis Type="Italic">Geobacillus caldoxylolyticus</Emphasis> TK4

The gene encoding an α-l-arabinofuranosidase from Geobacillus caldoxylolyticus TK4, AbfATK4, was isolated, cloned, and sequenced. The deduced protein had a molecular mass of about 58 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with α-l-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. A histidine tag was introduced at the N-terminal end of AbfATK4, and the recombinant protein was expressed in Escherichia coli BL21, under control of isopropyl-β-D-thiogalactopyranoside-inducible T7 promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 236 kDa, suggesting a homotetrameric structure. AbfATK4 was active at a broad pH range (pH 5.0–10.0) and at a broad temperature range (40–85°C), and it had an optimum pH of 6.0 and an optimum temperature of 75–80°C. The enzyme was more thermostable than previously described arabinofuranosidases and did not lose any activity after 48 h incubation at 70°C. The protein exhibited a high level of activity with p-nitrophenyl-α-l-arabinofuranoside, with apparent K _m and V _max values of 0.17 mM and 588.2 U/mg, respectively. AbfATK4 also exhibited a low level of activity with p-nitrophenyl-β-d-xylopyranoside, with apparent K _m and V _max values of 1.57 mM and 151.5 U/mg, respectively. AbfATK4 released l-arabinose only from arabinan and arabinooligosaccharides. No endoarabinanase activity was detected. These findings suggest that AbfATK4 is an exo-acting enzyme.     

Purification and characterization of dihydroorotase fromPseudomonas putida

Dihydroorotase was purified to homogeneity fromPseudomonas putida. The relative molecular mass of the native enzyme was 82 kDa and the enzyme consisted of two identical subunits with a relative molecular mass of 41 kDa. The enzyme only hydrolyzed dihydro-l-orotate and its methyl ester, and the reactions were reversible. The apparentK _m andV _max values for dihydro-l-orotate hydrolysis (at pH 7.4) were 0.081 mM and 18 μmol min⁻¹ mg⁻¹, respectively; and those forN-carbamoyl-dl-aspartate (at pH 6.0) were 2.2 mM and 68 μmol min⁻¹ mg⁻¹, respectively. The enzyme was inhibited by metal ion chelators and activated by Zn²⁺. However, excessive Zn²⁺ was inhibitory. The enzyme was inhibited by sulfhydryl reagents, and competitively inhibited byN-carbamoylamino acids such asN-carbamoylglycine, with aK _i value of 2.7 mM. The enzyme was also inhibited noncompetitively by pyrimidine-metabolism intermediates such as dihydrouracil and orotate, with aK _i value of 3.4 and 0.75 mM, respectively, suggesting that the enzyme activity is regulated by pyrimidine-metabolism intermediates and that dihydroorotase plays a role in the control of pyrimidine biosynthesis.     

Purification and characterisation of a microbial l-carnitine amidase

A novel enzyme, l-carnitine amidase, was purified about 140-fold from a newly screened microorganism (DSM 6320) to yield a homogeneous protein. The native enzyme has a molecular mass of 125 kDa (gel filtration) and consists of two identical subunits as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Edman degradation. The pH optimum was found around pH 8.5. Out of 60 chemicals tested as substrates (amides of various aliphatic and aromatic acids, nitriles, amino acid amides and dipeptide amides) the amidase hydrolysed only l-carnitine amide. The Michaelis constant (K_m) was found to be 11.6 mm, and the pure protein had a specific activity of 328 units/mg. Complex kinetics were observed with the racemic mixture of d,l-carnitine amide as starting material during enzymatic hydrolysis. Correspondence to: M.-R. Kula     

Purification and characterization of a new type of serine carboxypeptidase from<Emphasis Type="Italic"> Monascus purpureus</Emphasis>

Carboxypeptidase produced by Monascus purpureus IFO 4478 was purified to homogeneity. The purified enzyme is a heterodimer with a molecular mass of 132 kDa and consists of two subunits of 64 and 67 kDa. It is an acidic glycoprotein with an isoelectric point of 3.67 and 17.0% carbohydrate content. The optimum pH and temperature were 4.0 and 40 °C, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 °C for 1 h, and up to 50 °C at pH 5.0 for 15 min. The enzyme was strongly inhibited by piperastatin A, diisopropylfluoride phosphate (DFP), phenylmethylsulfonylfluoride (PMSF), and chymostatin, suggesting that it is a chymotrypsin-like serine carboxypeptidase. Monascus purpureus carboxypeptidase was also strongly inhibited by p-chloromercuribenzoic acid (PCMB) but not by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, indicating that it requires cysteine residue but not metal ions for activity. Benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrate of the enzyme. The K_m, V_max, K_cat, and K_cat/K_m values of the enzyme for Z-Tyr-Glu at pH 4.0 and 37 °C were 0.86 mM, 0.917 mM min^–1, 291 s^–1, and 339 mM^–1 s^–1, respectively.     

A novel <Emphasis Type="SmallCaps">l</Emphasis>-aspartate dehydrogenase from the mesophilic bacterium <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1: molecular characterization and application for <Emphasis Type="SmallCaps">l</Emphasis>-aspartate production

l-aspartate dehydrogenase (EC 1.4.1.21; l-AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative l-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very high specific activity for l-aspartate (l-Asp) and oxaloacetate (OAA) of 127 and 147 U mg⁻¹, respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T _m value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent K _m values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH. The l-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of l-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative production system C released 33 mM of l-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic AspDH and its potential applicability for efficient and attractive production of l-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production.     

Lactobacillus reuteri ATCC 53608 mdh gene cloning and recombinant mannitol dehydrogenase characterization

A gene encoding mannitol-2-dehydrogenase (E.C. 1.1.1.138) (MDH) was cloned from Lactobacillus reuteri and expressed in Escherichia coli. The 1,008-bp gene encodes a protein consisting of 336 amino acids, with a predicted molecular mass of 35,920 Da. The deduced amino acid sequence of L. reuteri MDH (LRMDH) is 77% and 76% similar to the MDHs from Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, respectively. The purified recombinant enzyme appears as a single band of 40 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but gel filtration indicates that the native enzyme is a dimer. The optimum temperature for the recombinant enzyme is 37°C, the pH optima for D-fructose reduction and D-mannitol oxidation are 5.4 and 6.2, respectively. The K_m values for NAD (9 mM) and NADH (0.24 mM) are significantly higher than those for NADP (0.35 mM) and NADPH (0.04 mM). The K_m values of LRMDH for D-fructose and D-mannitol are 34 mM and 54 mM, respectively. Contrary to what the enzyme sequence suggests, recombinant LRMDH contains a single catalytic zinc per subunit.     

Heterologous expression and characterization of <Emphasis Type="Italic">Bacillus coagulans</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase

Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure l-lactic acid from both hexose and pentose sugars including l-arabinose with high yield, titer and productivity under thermophilic conditions. The l-arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn²⁺ was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic pH than at alkaline pH. The kinetic studies showed that the K _m, V _max and k _cat/K _m for the conversion of l-arabinose were 106 mM, 84 U/mg and 34.5 mM⁻¹min⁻¹, respectively. The equilibrium ratio of l-arabinose to l-ribulose was 78:22 under optimal conditions. l-ribulose (97 g/L) was obtained from 500 g/l of l-arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion of 19.4% and a production rate of 65 g L⁻¹ h⁻¹.     

Expression, purification, and characterization of alanine racemase from Pseudomonas putida YZ-26

Alanine racemase catalyzes the interconversion of d- and l-alanine and plays an important role in supplying d-alanine, a component of peptidoglycan biosynthesis, to most bacteria. Alanine racemase exists mostly in prokaryotes and is generally absent in higher eukaryotes; this makes it an attractive target for the design of new antibacterial drugs. Here, we present the cloning and characterization of a new gene-encoding alanine racemase from Pseudomonas putida YZ-26. An open reading frame (ORF) of 1,230 bp, encoding a protein of 410 amino acids with a calculated molecular weight of 44,217.3 Da, was cloned into modified vector pET32M to form the recombinant plasmid pET–alr. After introduction into E.coli BL21, the strain pET-alr/E.coli BL21 expressed His₆-tagged alanine racemase. The recombinant alanine racemase was efficiently purified to homogeneity using Ni²⁺–NTA and a gel filtration column, with 82.5% activity recovery. The amino acid sequence deduced from the alanine racemase gene revealed identity similarities of 97.0, 93, 23, and 22.0% with from P. putida F1, P. putida200, P. aeruginosa, and Salmonella typhimurium, respectively. The recombinant alanine racemase is a monomeric protein with a molecular mass of 43 kDa. The enzyme exhibited activity with l-alanine and l-isoleucine, and showed higher specificity for the former compared with the latter. The enzyme was stable from pH 7.0–11.0; its optimum pH was at 9.0. The optimum temperature for the enzyme was 37°C, and its activity was rapidly lost at temperatures above 40°C. Divalent metals, including Sr²⁺, Mn²⁺, Co²⁺, and Ni²⁺ obviously enhanced enzymatic activity, while the Cu²⁺ ion showed inhibitory effects.     

Purification and characterization of threonine dehydrogenase from Clostridium sticklandii

Threonine dehydrogenase from Clostridium sticklandii has been purified 76-fold from cells grown in a defined medium to a homogeneous preparation of 234 units · mg^-1 protein. Purification was obtained by chromatography on Q-Sepharose fast flow and Reactive green 19-Agarose. The native enzyme had a molecular mass of 67 kDa and consisted of two identical subunits (33 kDa each). The optimum pH for catalytic activity was 9.0. Only l-threo-threo-nine, dl--hydroxynorvaline and acetoin were substrates; only NAD was used as the natural electron acceptor. The apparent K _m values for l-threonine and NAD were 18 mM and 0.1 mM, respectively. Zn²⁺, Co²⁺ and Cu²⁺ ions (0.9 mM) inhibited enzyme activity. The N-terminal amino acid sequence revealed similarities to the class of non-metal short-chain alcohol dehydrogenases, whereas the threonine dehydrogenase from Escherichia coli belongs to the class of medium chain, zinc-containing alcohol dehydrogenases.Abbreviations PMSF phenylmethylsulfonyl fluoride - Dea diethanolamine - Tris tris-(hydroxy-methyl)-aminomethane - Nbs ₂ 5,5-dithiobis-(2-nitrobenzoic acid) - ApADN 3-acetylpyridine adenine diucleotide - thio-NAD thionicotinamide adenine dinucleotide - NBT nitro blue tetrazolium chloride     

Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium <Emphasis Type="Italic">Thermotoga hypogea</Emphasis>

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO₂, H₂, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe²⁺. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K _m values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.     

Characterisation of <Emphasis Type="SmallCaps">l</Emphasis>-alanine and glycine absorption across the gut of an ancient vertebrate

This study utilised an in vitro technique to characterise absorption of two amino acids across the intestinal epithelium of Pacific hagfish, Eptatretus stoutii. Uptake of l-alanine and glycine conformed to Michaelis–Menten kinetics. An uptake affinity (K _m; substrate concentration required to attain a 50% uptake saturation) of 7.0 mM and an uptake capacity (J _max) of 83 nmol cm⁻² h⁻¹ were described for l-alanine. The K _m and J _max for glycine were 2.2 mM and 11.9 nmol cm⁻² h⁻¹, respectively. Evidence suggested that the pathways of l-alanine and glycine absorption were shared, and sodium dependent. Further analysis indicated that glycine uptake was independent of luminal pH and proline, but a component of uptake was significantly impaired by 100-fold excesses of threonine or asparagine. The presence of a short-term (24 h) exposure to waterborne glycine, similar in nature to that which may be expected to occur when feeding inside an animal carcass, had no significant impact on gastrointestinal glycine uptake. This may indicate a lack of cross talk between absorptive epithelia. These results are the first published data to describe gastrointestinal uptake of an organic nutrient in the oldest extant vertebrate and may provide potential insight into the evolution of nutrient transport systems.     

Effects of Na+, H+, and Cl− on alanine transport by lobster hepatopancreatic brush border membrane vesicles

Summary Epithelial brush border membrane vesicles (BBMV) of lobster hepatopancreas were formed by a magnesium precipitation technique previously described (Ahearn et al. 1985).³H-l-alanine transport by these vesicles was sodium and potassium insensitive, in contrast to a strong Na-dependency exhibited by³H-d-glucose transport. Initial alanine entry rates (15 s uptake) were stimulated and transient alanine uptake overshoots were observed when external pH was acidic (e. g. pH 4.0, 5.0 or 6.0) and a Cl gradient was imposed across the vesicular wall; at pH_o=7.4 alanine uptake was reduced in rate and hyperbolic in character. Alanine uptake from an acidic extravesicular environment in the absence of Cl responded to a transmembrane electrical potential difference created by an outwardly-directed, valinomycin-induced, potassium diffusion potential, suggesting that the alanine molecule alone carried sufficient charge under these conditions to respond to the electrical gradient. External 5.0 mMl-lysine andl-serine similarly inhibited the influx and overshoot properties of 0.05 mM³H-l-alanine uptake, whereas 5.0 mMl-leucine had virtually no effect. Trans-stimulation of alanine initial uptake rates and an enhancement of alanine accumulation against a concentration gradient were observed by vesicles preloaded with 1 mMl-lysine, but not by vesicles lacking amino acids or those containing 1 mMl-leucine orl-serine.³H-l-alanine influx from acidic external environments in the presence of a Cl gradient occurred by a combination of carrier-mediated transfer and apparent diffusion. Decreasing pH_o from 6.0 to 4.0 elevated alanineK _t from 0.55 to 2.64 mM, while alanineJ _M increased from 55 to 550 pmol/mg protein· 15 s. Apparent diffusional permeability of the membranes to alanine under these conditions increased slightly. These results suggest, but do not conclusively prove, that alanine transport across BBMV of lobster hepatopancreas may occur by way of a classical y+ transprot protein at acidic pH. The extent of this transport is determined by the magnitude of the transmembrane chloride gradient which serves as a powerful driving force for cationic amino acids in this tissue.     

Purification and characterization of glutamine synthetase from the commerical mushroom Agaricus bisporus

Agaricus bisporus glutamine synthetase, a key enzyme in nitrogen metabolism, was purified to apparent homogeneity. The native enzyme appeared to be a GS-II type enzyme. It has a molecular weight of 325 kDa and consists of eight 46-kDa subunits. Its pI was found at 4.9. Optimal activity was found at 30°C. The enzyme had low thermostability. Stability declined rapidly at temperatures above 20°C. The enzyme exhibits a K _m for glutamate, ammonium, and ATP of 22mm, 0.16mm and 1.25mm respectively in the biosynthetic reaction, with optimal activity at pH 7. The enzyme is slightly inhibited by 10mm concentrations of l-alanine, l-histidine, l-tryptophan, anthranilic acid, and 5-AMP and was strongly inhibited by methionine sulfoximine and phosphinothricine. For the transferase reaction K _i-values were 890 m and 240 m for methionine sulfoximine and phosphinothricine respectively. For the biosynthetic reaction K _i was 17 m for both methionine sulfoximine and phosphinothricine.