Lysyl-tRNA synthetase from Bacillus stearothermophilus. Formation and isolation of an enzyme-lysyladenylate complex and its analogue.

The formation of an enzyme.lysyladenylate complex was studied with a highly purified lysyl-tRNA synthetase [L-lysine:tRNALYS ligase (AMP-forming); EC 6.1.

Characterization of monoclonal antibodies against (1R,2R)-cyclohexanediamine platinum(II)-DNA adduct.

Two monoclonal antibodies raised against Pt(II)(1R,2R-cyclohexanediamine)-DNA were prepared, and the specificity of the monoclonal antibodies against Pt(II)(cyclohexanediamine)-DNA derivatives was determined by competitive enzyme-linked immunosorbent assay. The binding affinity of the monoclonal antibodies is apparently influenced by the cyclohexanediamine moiety of Pt(II)(cyclohexanediamine)-DNA adducts, but the monoclonal antibodies can not bind to the low molecular analogue, Pt(II)(1R,2R-cyclohexanediamine)-d(GpG), which is the intrastrand binding model compound of Pt(II)(1R,2R-cyclohexanediamine)-DNA. Therefore, the monoclonal antibodies recognize the macromolecular parts, including DNA duplex, in addition to the cyclohexane moieties of the platinum complexes on Pt(II)(cyclohexanediamine)-DNA adducts.

Copper binding selectivity of N- and C-sites in serum (human)- and ovo-transferrin.

Copper binding selectivity of the N- and C-sites in serum (human)- and ovo-transferrin was investigated through copper binding constants, copper dissociation rate constants, and EPR spectra. At pH 7.4, stepwise copper binding constants of serum (human)-transferrin were K1 = 1.

Novel beta-D-galactofuranose-containing high-mannose type oligosaccharides in ascorbate oxidase from Acremonium sp. HI-25.

Ascorbate oxidase from the fungus Acremonium sp. HI-25 is a copper-containing glycoprotein that catalyzes the oxidation of ascorbic acid to dehydroascorbic acid. Monosaccharide composition analysis showed that the enzyme contains exclusively N-linked oligosaccharide chains.

Lysyl-tRNA synthetase from Bacillus stearothermophilus. Purification, and fluorometric and kinetic analysis of the binding of substrates, L-lysine and ATP.

Lysyl-tRNA synthetase [L-lysine:tRNA(Lys)ligase (AMP forming); EC 6.1.1.

Elucidation of the subsite structure of bacterial saccharifying alpha-amylase and its mode of degradation of maltose.

The subsite structure of bacterial saccharifying alpha-amylase (BSAm) was elucidated by two methods using a series of maltooligosaccharides labeled with [14C]D-glucose at the reducing end. The rate parameter k0/Km and the cleavage frequency were obtained using the labeled substrates at sufficiently low concentrations to eliminate transglycosylation and condensation. This evaluation showed that the active center is composed of five subsites, with the catalytic site located between the 3rd and the 4th subsites from the nonreducing end.

Comparison of the binding of beta-cyclodextrin and alpha- and gamma-cyclodextrins with pullulanase from Klebsiella pneumoniae as studied by equilibrium and kinetic fluorometry.

The change in fluorescence spectra of crystalline pullulanase from Klebsiella pneumoniae caused by the addition of alpha-, beta-, and gamma-cyclodextrins and 6-O-alpha-glucosyl-alpha-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin was investigated at 25 degrees C and pH 5.6. The fluorescence intensity at around 325 nm (excitation at 280 nm) was increased by the addition of all the cyclodextrins studied.

Analysis of internal motion of single tryptophan in Streptomyces subtilisin inhibitor from its picosecond time-resolved fluorescence.

A mode of internal motion of single tryptophan, Trp 86, of Streptomyces subtilisin inhibitor, was analyzed from its time-resolved fluorescence. The intensity and anisotropy decays of Trp 86 were measured in the picosecond range. These decays were analyzed with theoretical expressions derived assuming that the indole ring of tryptophan as an asymmetric rotor rotates around covalent bonds connecting indole with the peptide chain and an effective quencher of fluorescence of Trp 86 is the nearby SS bond of Cys 35-Cys 50.

Characterization of ascorbate oxidase from Acremonium sp. HI-25.

The ascorbate oxidase obtained from a microorganism, Acremonium sp. HI-25 (molecular weight, 80 kDa; monomeric protein), was studied with respect to atomic absorption, EPR, absorption spectra, circular dichroism (CD) spectra, and steady-state kinetics. The enzyme was found to be a multicopper protein, containing four copper atoms of three kinds, types 1, 2, and 3 copper, in the ratio of 1:1:2.

Identification of amino acid residues responsible for the changes of absorption and fluorescence spectra on the binding of subtilisin BPN' and Streptomyces subtilisin inhibitor.

An ultraviolet absorption difference spectrum characteristic of the ionization change of a tyrosyl residue was observed on the binding of subtilisin BPN' with Streptomyces subtilisin inhibitor (SSI) at alkaline pH. This difference spectrum was considered to be induced by a pKa shift (from 9.7 to > or = 11.

Purification and characterization of alpha-glucosidase from Torulaspora pretoriensis YK-1.

alpha-Glucosidase was partially purified 103-fold from a cell-free extract of Torulaspora pretoriensis YK-1 by column chromatography on Toyopearl HW55F, DEAE-Toyopearl 650M, hydroxylapatite and phenyl-Toyopearl 650M. Further purification by preparative polyacrylamide gel electrophoresis (PAGE) gave the homogenous protein, but the specific activity was reduced. The molecular weight of the enzyme was estimated to be 69,000 by SDS-PAGE and 60,000 by gel filtration.

Interaction between pullulanase from Klebsiella pneumoniae and cyclodextrins.

The interaction between pullulanase from Klebsiella pneumoniae and alpha-, beta-, and gamma-cyclodextrins and 6-O-alpha-glucosyl-alpha-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin was examined by means of inhibition studies of the enzyme activity, UV difference spectroscopy, and flow calorimetry. All the above cyclodextrins were found to be competitive inhibitors, but beta-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin showed strong inhibition, the inhibitor constants being two orders of magnitude less than those of alpha- and gamma-cyclodextrins. The difference spectra of beta-cyclodextrin were slightly but significantly different from those of the other cyclodextrins, showing blue shift of a few nanometers.

Zinc deficient bovine erythrocyte superoxide dismutase has low specific activity.

Zinc deficient bovine superoxide dismutase (Cu2E2SOD (E = empty)) was prepared and purified by high performance liquid chromatography (HPLC). Each peak was characterized as to protein, copper content and specific activity. The Cu2E2SOD peak fractionated by HPLC has a low specific activity at pH 7.

Novel oligomannose-type sugar chains derived from glucose oxidase of Aspergillus niger.

The primary structure of the N-linked sugar chains of glucose oxidase from Aspergillus niger was investigated. These sugar chains were released from the polypeptide backbone by hydrazinolysis, and the reducing ends of the sugar chains were pyridylaminated. HPLC of the pyridylamino sugar chains with an amide-silica column showed at least seven sugar chain peaks.

The role of His143 in the catalytic mechanism of Escherichia coli aspartate aminotransferase.

In aspartate aminotransferase (AspAT), His143 is located within a hydrogen-bonding distance to Asp222 that forms a strong ion pair with the ring nitrogen of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP). His143 of Escherichia coli AspAT was replaced by Ala or Asn. The mutant enzyme H143A showed a slight increase in the maximum velocity of the overall transamination reaction between aspartate and 2-oxoglutarate, while H143N AspAT showed a decrease to 60% in the maximum rate of the overall reactions in both directions.

Effect of maltotriitol on the action pattern of porcine pancreatic alpha-amylase using amylose as a substrate.

The effect of the oligosaccharide analog maltotriitol (G3OH) on the action pattern of porcine pancreatic alpha-amylase (PPA) was examined using amylose as a substrate. Fluorescence titration indicated that two molecules of G3OH can bind to one molecule of PPA. The slope in the blue value versus extent-of-reaction plot was shifted by G3OH from that for multiple attack in the direction of that for random attack as the G3OH concentration increased.

In vitro action of human and porcine alpha-amylases on cyclomalto-oligosaccharides.

The vitro action of human and porcine pancreatic alpha-amylases on cyclomalto-oligosaccharides (cyclodextrins) was investigated both by a high-performance liquid chromatographic analysis and a quantitative analysis of the reducing power of cyclodextrin hydrolyzates. Cyclomalto-octaose (gamma-cyclodextrin) was hydrolyzed to produce mainly maltose, but cyclomalto-hexaose and -heptaose were little affected both by human and porcine alpha-amylases. Quantitative analysis of reducing power revealed that the ring-opening rate of gamma-cyclodextrin catalyzed by human pancreatic alpha-amylase was 2.

Binding of isomaltose and maltose to the glucoamylase from Aspergillus niger, as studied by fluorescence spectrophotometry and steady-state kinetics.

The binding of maltose, isomaltose, and D-glucono-1,5-lactone to the glucoamylase [E.C.3.

Pre-steady-state kinetics of Escherichia coli aspartate aminotransferase catalyzed reactions and thermodynamic aspects of its substrate specificity.

The four half-transamination reactions [the pyridoxal form of Escherichia coli aspartate aminotransferase (AspAT) with aspartate or glutamate and the pyridoxamine form of the enzyme with oxalacetate or 2-oxoglutarate] were followed in a stopped-flow spectrometer by monitoring the absorbance change at either 333 or 358 nm. The reaction progress curves in all cases gave fits to a monophasic exponential process. Kinetic analyses of these reactions showed that each half-reaction is composed of the following three processes: (1) the rapid binding of an amino acid substrate to the pyridoxal form of the enzyme; (2) the rapid binding of the corresponding keto acid to the pyridoxamine form of the enzyme; (3) the rate-determining interconversion between the two complexes.

Fluorescence labeled and cross-linked subtilisin: kinetic characteristics and binding to Streptomyces subtilisin inhibitor.

In the preceding paper, the preparation of fluorescent cross-linked subtilisin was described. In this paper we present the catalytic and binding properties of the modified enzyme. Kinetic analysis showed that the cross-linked dimeric subtilisin retained both catalytic activity and binding affinity toward synthetic substrates.

Stopped-flow chemical modification with N-bromosuccinimide: a good probe for changes in the microenvironment of the Trp 62 residue of chicken egg white lysozyme.

The stopped-flow chemical modification with N-bromosuccinimide (NBS) of Trp 62 of hen (chicken) egg white lysozyme (EC 3.2.1.

Substitution of a lysyl residue for arginine 386 of Escherichia coli aspartate aminotransferase.

Substitution of a lysyl residue for Arg-386 of Escherichia coli aspartate aminotransferase resulted in an extensive decrease in Vmax values (0.8% with the aspartate-2-oxoglutarate pair and 0.2% with the glutamate-oxalacetate pair, compared with the corresponding values for the wild-type enzyme).

Kinetic study on the interaction of Rhizopus chinensis aspartic protease with Streptomyces pepsin inhibitor (acetylpepstatin).

The fluorescence of tryptophan residues of Rhizopus chinensis aspartic protease was quenched about 25% upon binding with an inhibitor, Streptomyces pepsin inhibitor (acetylpepstatin). The kinetics of binding between the enzyme and the inhibitor was studied by the fluorescence stopped-flow method. The concentration dependence of apparent rate constants was consistent with a two-step mechanism involving a fast bimolecular association followed by a slow unimolecular process.