Time-of-Day Effect on Cardiac Responses to Progressive Exercise

This study was designed to examine time-of-day effects on markers of cardiac functional capacity during a standard progressive cycle exercise test. Fourteen healthy, untrained young males (mean?±?SD: 17.9?±?0.7 yrs of age) performed identical maximal cycle tests in the morning (08:00–11:00?h) and late afternoon (16:00–19:00?h) in random order. Cardiac variables were measured at rest, submaximal exercise, and maximal exercise by standard echocardiographic techniques. No differences in morning and afternoon testing values at rest or during exercise were observed for oxygen uptake, heart rate, cardiac output, or markers of systolic and diastolic myocardial function. Values at peak exercise for Vo₂ at morning and afternoon testing were 3.20?±?0.49 and 3.24?±?0.55?L min^?1, respectively, for heart rate 190?±?11 and 188?±?15?bpm, and for cardiac output 19.5?±?2.8 and 19.8?±?3.5?L min^?1. Coefficients of variation for morning and afternoon values for these variables were similar to those previously published for test-retest reproducibility. This study failed to demonstrate evidence for significant time-of-day variation in Vo₂max or cardiac function during standard progressive exercise testing in adolescent males. (Author correspondence: thomas.rowland@bhs.org)     

The Purification of Soybean 11S Globulin with ConA-Sepharose 4B and Sepharose 6B

Kinetics of the acyl transfer catalyzed by Xanthomonas α-amino acid ester hydrolase was studied. The enzyme hydrolyzed d-α-phenylglycine methyl ester (d-PG-OMe) to give equimolar amounts of d-α-phenylglycine and methanol. With d-PG-OMe as an acyl donor and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) as an acyl acceptor, the enzyme transferred the acyl group from d-PG-OMe to 7-ADCA in competition with water. The addition of amine nucleophiles (7-ADCA and 6-aminopenicillanic acid) decreased the molecular activity (k_o) of the enzyme-catalyzed hydrolysis of d-PG-OMe, whereas it did not alter the Michaelis constant (K_M), and plots of l/k_o against the initial concentration of a nucleophile (n_o) gave a straight line. These results support the assumptions that the overall process for hydrolysis and acyl transfer proceeds through a common acyl-enzyme intermediate, that the acylation step of the enzyme is rate-limiting, and that the transfer competes with the hydrolysis of the acyl donor.     

Preparation of Optically Active Allothreonine by Separating from a Diastereoisomeric Mixture with Threonine

A simple procedure is described to obtain D- and L-allothreonine (D- and L-aThr). A mixture of N-acetyl-D-allothreonine (Ac-D-aThr) and N-acetyl-L-threonine (Ac-L-Thr) was converted to a mixture of their ammonium salts and then treated with ethanol to precipitate ammonium N-acetyl-L-threoninate (Ac-L-Thr·NH₃) as the less-soluble diastereoisomeric salt. After separating Ac-L-Thr·NH₃ by filtration, Ac-D-aThr obtained from the filtrate was hydrolyzed in hydrochloric acid to give D-aThr of 80% de, recrystallized from water to give D-aThr of >99% de. L-aThr was obtained from a mixture of the ammonium salts of Ac-L-aThr and Ac-D-Thr in a similar manner.     

Enzyme-Substrate Complex of p-Hydroxybenzoate Hydroxylase; One of the Activated Intermediates of the Overall Reaction

The transglucosidation reaction of brewer’s yeast α-glucosidase was examined under the co-existence of l-sorbose and phenyl-α-glucoside. As the transglucosidation products, three kinds of new disaccharide were chromatographically isolated. It was presumed that these disaccharides consisting of d-glucose and l-sorbose were 1-O-α-d-glucopyranosyl-l-sorbose ([α]_D+89.0), 3-O-α-d-glucopyranosyl-l-sorbose ([α]_D+69.1) and 4-O-α-d-glucopyranosyl-l-sorbose ([α]_D+81.0). The principal product formed in the enzyme reaction was 1-O-α-d-glucopyranosyl-l-sorbose.     

Production of 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) and Its Derivatives by Vibrio tyrosinaticus

Six strains of bacteria belonging to Vibrio and Pseudomonas were selected as good producers of L-DOPA from L-tyrosine out of various bacteria. The condition for the formation of L-DOPA by Vibrio tyrosinaticus ATCC 19378 was examined and the following results were obtained. (1) Intermittent addition of L-tyrosine in small portions gave higher titer of L-DOPA than single addition of L-tyrosine. (2) Higher amount of L-DOPA was produced in stationary phase of growth than in logarithmic phase. (3) Addition of antioxidant, chelating agent or reductant such as L-ascorbic acid, araboascorbic acid, hydrazine, citric acid and 5-ketofructose increased the amount of L-DOPA formed. (4) L-Tyrosine derivatives such as N-acetyl-L-tyrosine amide, N-acetyl-L-tyrosine, L-tyrosine amide, L-tyrosine methyl ester and L-tyrosine benzyl ester were converted to the corresponding L-DOPA derivatives.

In the selected condition about 4 mg/ml of L-DOPA was produced from 4.3 mg/ml of L-tyrosine.     

Purification and Some Properties of β-Xylosidase from Emericella nidulans

β-Xylosidase was purified 662 fold from a culture filtrate by ammonium sulfate fractionation, gel filtration on Biogel P-100, DEAE-Sephadex chromatography, and gel filtration on Sephadex G-200. With isoelectric focusing, the purified β-xylosidase found to be homogeneous on SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis. The molecular weight was estimated by gel filtration to be 240,000, and 116,000 by SDS polyacrylamide gel electrophoresis. The purified β-xylosidase had an isoelectric point at pH 3.25, and contained 4% carbohydrate residue. The optimum pH was found to be in the range of 4.5 ~ 5, and the optimum temperature was 55°C. The enzyme activity was inhibited by Hg^{2 +}, SDS, and N-bromosuccinimide at a concentration of 1 × 10^?3 m, and also p-chloromercuribenzoate at a concentration of 1 × 10^?4m. The purified enzyme hydrolyzed phenyl β-d-xyloside (k_o = 302.6 sec^?1),β-nitrophenyl β-d-xyloside (k_o = 438.9 sec^?1), o-nitrophenyl β-d-xyloside (k_o = 431.0 sec^?1), p-chlorophenyl β-d-xyloside (k_o = 207.9 sec^?1), o-chlorophenyl β-d-xyloside (k_o = 211.8 sec^?1), β-methylphenyl β-d-xyloside k_o = 96.5 sec^?1), o-methylphenyl β-d-xyloside (k_o = 83.1 sec^?1), p-methoxyphenyl β-d-xyloside (k_o = 99.3 sec^?1), o-methoxyphenyl β-d-xyloside (k_o= 100.0 sec^?1), xylobiose (k_o = 992A sec^?1), xylotriose (k_o = 1321.9 sec^?1), xylotetraose (k_o = 7S9.1 sec^?1) and xylopentaose (k_o = 508.0 sec^?1). On enzymic hydrolysis of phenyl β-d-xyloside, the reaction product was found to be β-d-xylose with retention of the configuration. The purified β-xylosidase was practically free of a-xylosidase and β-glucosidase activities.     

Substrate Specificity of the Protease from Streptomyces cellulosae

The substrate specificity and the mode of action of the protease from Streptomyces cellulosae were investigated, using many kinds of peptides and proteins as substrates. The protease hydrolyzed peptides consisting of hydrophobic amino acids such as L-Phe-L-Leu-NH₂, L-Pro-L-Phe-NH₂, l-Leu-L-Met, L-Leu-L-Leu, Gly-L-Ile, L-Phe-L-Phe, L-Pro-L-Leu-Gly-NH₂, etc. The protease hydrolyzed zein best among the proteins tested, but weakly hydrolyzed gelatin, myoglobin, bovine serum albumin, γ-globulin, and collagen. The protease mainly hydrolyzed Ser¹²-Leu¹³, Leu¹³-Tyr¹⁴, and Tyr¹⁴-Gln¹⁵ bonds in the oxidized A-chain of insulin and at least the Leu¹⁵-Tyr¹⁶ bond in the oxidized B-chain of insulin.     

Biochemical characteristics of maltose phosphorylase MalE from Bacillus sp. AHU2001 and chemoenzymatic synthesis of oligosaccharides by the enzyme

ABSTRACT

Maltose phosphorylase (MP), a glycoside hydrolase family 65 enzyme, reversibly phosphorolyzes maltose. In this study, we characterized Bacillus sp. AHU2001 MP (MalE) that was produced in Escherichia coli. The enzyme exhibited phosphorolytic activity to maltose, but not to other α-linked glucobioses and maltotriose. The optimum pH and temperature of MalE for maltose-phosphorolysis were 8.1 and 45°C, respectively. MalE was stable at a pH range of 4.5–10.4 and at ≤40°C. The phosphorolysis of maltose by MalE obeyed the sequential Bi–Bi mechanism. In reverse phosphorolysis, MalE utilized d-glucose, 1,5-anhydro-d-glucitol, methyl α-d-glucoside, 2-deoxy-d-glucose, d-mannose, d-glucosamine, N-acetyl-d-glucosamine, kojibiose, 3-deoxy-d-glucose, d-allose, 6-deoxy-d-glucose, d-xylose, d-lyxose, l-fucose, and l-sorbose as acceptors. The k_cat(app)/K_m(app) value for d-glucosamine and 6-deoxy-d-glucose was comparable to that for d-glucose, and that for other acceptors was 0.23–12% of that for d-glucose. MalE synthesized α-(1→3)-glucosides through reverse phosphorolysis with 2-deoxy-d-glucose and l-sorbose, and synthesized α-(1→4)-glucosides in the reaction with other tested acceptors.     

Kinetic Study on the Formation of Tripeptide Amide by the Protease from Streptomyces cellulosae

The protease from Streptomyces cellulosae preferentially catalyzed the condensation reaction producing tripeptide amides in highly concentrated mixture solutions of various dipeptides and amino acid amides, although it weakly hydrolyzed the substrates at the same time. The tripeptide amides formed were l-Leu-Gly-Gly-NH₂ (P_LGGN) from l-Leu-Gly and Gly-NH₂ and l-Leu-Gly-l-Leu-NH₂ (P_LGLN) from l-Leu-Gly and l-Leu-NH₂. Moreover, the ratio of the rate of P_LGLN formation per the proteolytic activity of this enzyme was much larger than those of the other proteases tested.

The formation of P_LGLN was studied at various concentrations of the substrates (l-Leu-Gly and. l-Leu-NH₂). The dependences of the initial velocities of P_LGLN formation on the substrates concentrations could be explained by a two-substrate, one-product reaction mechanism involving a single active center forming the peptide bonds and two substrate-binding sites. The values of the substrate dissociation constants for enzyme-substrate complexes were about 0.6 m for l-Leu-Gly and 0.008 m for l-Leu-NH₂.     

Identification and characterization of d-arabinose reductase and d-arabinose transporters from Pichia stipitis

d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The k_cat/K_m value with d-arabinose (1.27 min^?1 mM^?1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.     

Purification and some Properties of β-Xylosidase from Trichoderma viride

β-Xylosidase was purified 25 fold from a culture filtrate by ammonium sulfate fractionation, DEAE-Sephadex chromatography, column electrophoresis, gel filtration on Biogel P-100, and isoelectric focusing. The purified β-xylosidase was found to be homogeneous on SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis and on disc electrophoresis. A molecular weight of 101,000 was estimated by chromatography on Sephadex G-200, and 102,000 was obtained by SDS polyacrylamide gel electrophoresis. The purified p-xylosidase had an isoelectric point at pH 4.45, and contained 4.5% carbohydrate residue. The optimum activity for the enzyme was found to be at pH 4.5 and 55°C. The enzyme activity was inhibited by Hg^{2 +}, and N-bromosuccinimide at a concentration of 1 x 10^?3 m. The purified enzyme hydrolyzed phenyl β-d-xyloside (k_o—13.0 sec”¹), p-nitrophenyl β-d-xyloside (k_o=2l.3 sec^?1), o-nitrophenyl β-d-xyloside (k_o = 22.2 sec^?1), o-chlorophenyl β-d-xyloside (k_o = 20.0 sec^?1), p-methylphenyl β-d-xyloside (k_o~9.0 sec^?1), o-methylphenyl β-d-xyloside (k_o= 10.7 sec^?1), p-methoxyphenyl β-d-xyloside (k_o=10.3 sec^?1), o-methoxyphenyl β-d-xyloside (&;_o=10.9 sec^?1), xylobiose (k_o = 36A sec^?1), xylotriose (k_o = 34.5 sec^?1), xylotetraose (k_o~HA sec^?1), and xylopentaose (k_o= 13.0 sec^?1). On enzymic hydrolysis of phenyl β-d-xyloside, the reaction product was found to be β-d-xylose with retention of configuration. The purified p-xylosidase was practically free of α-xylosidase and β-glucosidase activities.     

Optimal Conditions for the Enzymatic Production of l-Aromatic Amino Acids from the Corresponding 5-Substituted Hydantoins

The reaction conditions for the production of l-tryptophan from dl-5-indolyl- methylhydantoin by Flavobacterium sp. AJ-3940, and the cultural conditions for the formation of the enzyme involved by this bacterium were investigated. The optimal pH of this reaction was around 8.5 and the optimal temperature was between 45 to 55°C. The amount of l-tryptophan produced was remarkably increased by the addition of inosine, which formed a water insoluble adduct with l-tryptophan, to the reaction mixture because of the release of end-product inhibition by l-tryptophan. This enzyme was inducibly and intracellularly produced by Flavobacterium sp. AJ-3940 in proportion to the increase in cell growth. Cells showing high activity were obtained using a medium containing 5 g glucose, 5 g (NH₄)₂SO₄, 1 g KH₂PO₄, 3 g K₂HPO₄, 0.1 g MgSO₄ · 7H₂O, 0.01 g CaCl₂ · 2H₂O, 50 ml corn steep liquor and 3.5 g dl-5-indolylmethylhydantoin in a total volume of 1 liter (pH 7.0). Under the best conditions, 43 mg/ml of l-tryptophan was produced from 50 mg/ml of dl-5-indolylmethylhydantoin with a molar yield of 97% in the presence of cells of Flavobacterium sp. AJ-3940. In addition, other l-aromatic amino acids such as l-phenylalanine, l-tyrosine, l-DOPA and related l-amino acids were also produced from the corresponding 5-substituted hydantoins by this bacterium containing the l-tryptophan-producing enzyme induced by dl-5-indolylmethylhydantoin.     

L-Tyrosine Production by Analog-resistant Prototrophic Mutants of Glutamic Acid Producing Bacteria

p-Fluorophenylalanine (PFP) and m-fluorophenylalanine were the most effective inhibitors on the growth of Corynebacterium glutamicum ATCC 13032 among the analogs of phenylalanine and tyrosine tested. Their inhibitory effects were released by L-phenylalanine, and slightly by L-tyrosine and L-tryptophan. 3-Aminotyrosine (3AT), p-aminophenylalanine, o-fluorophenylalanine, and β-2-thienylalanine were weak inhibitors.

Resistant mutants of C. glutamicum isolated on the medium containing both PFP and 3AT or PFP and L-tyrosine were found to accumulate both L-tyrosine and L-phenylalanine, while resistant mutants isolated on the medium containing only PFP were found to produce only L-phenylalanine. Resistant mutants from other glutamic acid producing bacteria isolated on the medium containing both PFP and 3AT or both PFP and L-tyrosine were found to accumulate L-tyrosine and L-phenylalanine.     

Purification and Properties of α-d-Galactosidase Produced by Corticium rolfsii

An α-d-galactosidase was purified from the culture filtrate of Corticium rolfsii IFO 6146 by a combination of QAE-Sephadex A-50 and SE-Sephadex C-50 chromatography. The purified enzyme was demonstrated to be free of other possibly interfering glycosidases and glycanases. The maximum activity of the enzyme towards p-nitrophenyl α-d-galactopyrano-side was found to be at pH 2.5 to 4.5, and the enzyme was fairly active at pH 1.1 to 2.0. The enzyme was stable over a pH range 4.0 to 7.0 at 5°C for 72 hr and relatively unstable at pH 1.1 to 2.0 as compared with endo-polygalacturonase, α-l-arabinofuranosidase and β-d-galactosidase produced by C. rolfsii. The enzymic activity was completely inhibited by Hg²⁺ and Ag⁺ ions, respectively. Km values were determined to be 0.16 × 10^?3 m for p-nitrophenyl α-d-galactopyranoside and 0.26 × 10^?3m for o-nitrophenyl α-d-galactopyranoside. The values of V_max were also determined to be 26.6 μmoles and 28.6 μmoles per min per mg for p- and o-nitrophenyl α-d-galactopyranoside, respectively.     

Studies on the Chemical Structure of Konjac Mannan

The electrophoretically homogeneous glucomannan isolated from konjac flour was composed of d-glucose and d-mannose residues in the approximate ratio of 1: 1.6. Controlled acid hydrolysis gave 4-O-β-d-mannopyranosyl-d-mannose, 4-O-β-d-mannopyranosyl-d-glucose_T 4-O-β-d-glucopyranosyl-d-glucose(cellobiose), 4-O-β-d-glucopyranosyl-d-mannose(epicellobiose), O-β-d-mannopyranosyl-(1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose, O-β-d-glucopyranosyl- (1→4)-O-β-d-mannopyranosyl-(1→4)-d-mannose, O-β-d-mannopyranosyl-(1→4)-O-β-d-glucopy- ranosyl-(1→4)-d-mannose and O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-d-mannose.     

Effects of Methionine,Cystine and Taurine on Plasma Cholesterol Level in Rats Fed a High Cholesterol Diet

l-Methionine γ-lyase (EC 4.4.1.11) catalyzes α,β-elimination of l-2-amino-3-(N-methylamino)propionic acid and l-2-amino-3-(N-hydroxyethylamino)propionic acid to yield pyruvate, ammonia, and the corresponding amines. These amino acids also undergo the enzymatic β-replacement reaction with thiols to produce the corresponding S-substituted cysteines. Thus, l-methionine γ-lyase cleaves a C-N bond in addition to C-S, C-Se, and C-O bonds at the β position of amino acids by elimination and replacement reactions. A linear relationship between the reactivity, (log(V_max/Km) and the pKa value of the conjugated acid of the leaving group has been found for Se-methyl-l-selenocysteine, S-methyl-l-cysteine, and O-methyl-l-serine. However, l-2-amino-3-(N-methylamino)propionic acid has shown lower reactivity than that expected from the pKa value of methylammonium ions.     

Structure of Rugulovasine A,B and their Derivatives

Culture conditions for the preparation of cells containing high tyrosine phenol lyase activity were studied with Erwinia herbicola ATCC 21434. Adding pyridoxine to the medium enhanced enzyme formation, suggesting that it was utilized as a precursor of the coenzyme, pyridoxal phosphate. Glycerol plus succinic acid; amino acids, such as, DL-methionine, DL-alanine and glycine; and metallic ion, ferrous ion promoted enzyme formation as well as cell growth. Adding L-tyrosine, as inducer, to the culture medium was essential for enzyme formation. However, when large amounts of L-tyrosine were added, the enzyme formation was repressed by the phenol liberated from L-tyrosine. In fact, formation of the enzyme was enhanced by removing phenol during cultivation. L(D)-Phenylalanine or phenylpyruvic acid had a synergistic effect on the induction of enzyme by L-tyrosine.

Cells with high enzyme activity were prepared by growing cells at 28°C for 28 hr in a medium containing 0.2% L-tyrosine, 0.2% KH₂PO₄, 0.1% MgSO₄7H₂O, 0.001% FeSO_4·7H₂O, 0.01% pyridoxine-HC1, 0.6% glycerol, 0.5% succinic acid, 0.1% DL-methionine, 0.2% DL-alanine, 0.05% glycine, 0.1% L-phenylalanine and 120 ml/liter hydrolyzed soybean protein in tap water with the pH controlled at 7.5 throughout cultivation.     

Simultaneous determination of primary and secondary d- and l-amino acids by reversed-phase high-performance liquid chromatography using pre-column derivatization with two-step labelling method

This work describes a method for the simultaneous determination of primary d- and l-amino acids and secondary amino acids such as d- and l-proline. In order to remove interferences in the simultaneous determination of primary and secondary amines, the primary amines were derivatized with o-phthalaldehyde/N-acetyl-l-cysteine (OPA/NAC) and subsequently with 1-(9-fluorenyl)ethyl chloroformate (FLEC) for secondary amines, in a pre-column separation derivatization technique. These fluorescent diastereomers of the amino acids were obtained within 3 min at room temperature and determined simultaneously by changing wavelengths during analysis in a single eluting run in the high-performance liquid chromatography column. This method, referred to as the “two-step labelling method,” is effective for the simultaneous determination of d- and l-amino acids.     

Studies on the Metabolism of d-Amino Acid in Microorganisms

It is confirmed by a new method for the determination of d-glutamic acid, that Aerobacter strain A rapidly metabolizes d-glutamic acid, while it only shows feeble metabolic activity towards l-glutamic acid when it is grown on a dl-glutamate-K₂HPO₄ medium. A specific d-glutamic oxidase is demonstrated in the cell-free extracts of Aerobacter strain A. This enzyme seems to be different from d-glutamic-aspartic oxidase obtained from Aspergillus ustus by the authors, since the former has no activity towards d-aspartic acid.     

Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium,Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80°C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70°C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and α-D-glucose-1-phosphate. The K _m for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the k _cat was 5.4 s^-1. In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-β-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s^-1) k _cat followed by 6-deoxy-D-glucose (17 s^-1) and 2-deoxy-D-glucose (16 s^-1). The natural substrate, D-glucose with the k _cat of 8.0 s^-1 had the highest (1.1×10⁴ M^-1 s^-1) k _cat/K _m compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, α-D-glucose-1-phosphate, at higher concentrations.