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Biomedical subjects

G G Chang

Publications and source records attributed to G G Chang.

At least 73 records · Page 4Linked to original sources
PubMed1990

Kinetic analysis of duck epsilon-crystallin, a lens structural protein with lactate dehydrogenase activity.

Biochemical characterization and kinetic analysis of epsilon-crystallin from the lenses of common ducks were undertaken to elucidate the enzyme mechanism of this unique crystallin with lactate dehydrogenase (LDH) activity. Despite the structural similarities between epsilon-crystallin and chicken heart LDH, differences in charge and kinetic properties were revealed by isoenzyme electrophoresis and kinetic studies. Bi-substrate kinetic analysis examined by initial-velocity and product-inhibition studies suggested a compulsory ordered Bi Bi sequential mechanism with NADH as the leading substrate followed by pyruvate. The products were released in the order L-lactate and NAD+. The catalysed reaction is shown to have a higher rate in the formation of L-lactate and NAD+. Substrate inhibition was observed at high concentrations of pyruvate and L-lactate for the forward and reverse reactions respectively. The substrate inhibition was presumably due to the formation of epsilon-crystallin-NAD(+)-pyruvate or epsilon-crystallin-NADH-L-lactate abortive ternary complexes, as suggested by the product-inhibition studies. The significance and the interrelationship of duck epsilon-crystallin with other well-known LDHs are discussed with special regard to its role as a structural protein with some enzymic function in lens metabolism.

Animals↗
PubMed1990

Inhibition of human cancer cell growth by periodate-oxidized 3-aminopyridine adenine dinucleotide phosphate.

1. Periodate-oxidized 3-aminopyridine-adenine dinucleotide phosphate inhibited the proliferation of oral epithelium cancer and breast cancer cell lines. 2. The fast growing less differentiated embryonic kidney cell was more affected by the reagent then the embryonic lung fibroblast cell. 3. Incorporation of [3H]leucine of the treated cancer cells was inhibited. Incorporation of [3H]uridine was increased. Incorporation of [3H]thymidine was first increased and then decreased. 4. Tumor malic enzyme activity was inhibited by the reagent; but the treated cells did not show any difference in malic enzyme or glucose-6-phosphate dehydrogenase levels.

Adenine Nucleotides↗
PubMed1989

Periodate-oxidized 3-aminopyridine adenine dinucleotide phosphate as a fluorescent affinity label for pigeon liver malic enzyme.

Treatment of 3-aminopyridine adenine dinucleotide phosphate with sodium periodate resulted in oxidation of the ribose linked to 3-aminopyridine ring and cleavage of the dinucleotide into 3-aminopyridine and adenosine moieties. These two moieties were separated by thin layer chromatography and were synergistically bound to pigeon liver malic enzyme (EC 1.1.1.40), causing inactivation of the enzyme. The inactivation showed saturation kinetics. The apparent binding constant for the reversible enzyme-reagent binary complex (KI) and the maximum inactivation rate constant at saturating reagent concentration (kmax) were found to be 1.1 +/- 0.02 mM and 0.068 +/- 0.001 min-1, respectively. L-Malate at low concentration enhanced the inactivation rate by lowering the KI value whereas high malate concentration increased the kmax. Mn2+ or NADP+ partially protected the enzyme from the inactivation and gave additive protection when used together. L-Malate eliminated the protective effect of NADP+ or Mn2+. Maximum and synergistic protection was afforded by NADP+, Mn2+ plus L-malate (or tartronate). Oxidized and cleaved 3-aminopyridine adenine dinucleotide phosphate was also found to be a competitive inhibitor versus NADP+ in the oxidative decarboxylation reaction catalyzed by malic enzyme with a Ki value of 4.1 +/- 0.1 microM. 3-Aminopyridine adenine dinucleotide phosphate or its periodate-oxidized cleaved products bound to the enzyme anticooperatively. Oxidized 3-aminopyridine adenine dinucleotide phosphate labeled the nucleotide binding site of the enzyme with a fluorescent probe which may be readily traced or quantified. The completely inactivated enzyme incorporated 2 mol of reagent/mol of enzyme tetramer. The inactivation was partially reversible by dilution and could be made irreversible by treating the modified enzyme with sodium borohydride. This fluorescent compound and its counterpart-oxidized 3-aminopyridine adenine dinucleotide may be a potential affinity label for all other NAD(P)+-dependent dehydrogenases.

Adenosine Diphosphate↗
PubMed1988

Reversible dissociation of the catalytically active subunits of pigeon liver malic enzyme.

The pH-induced reversible dissociation of pigeon liver malic enzyme (EC 1.1.1.40) was studied by combined use of chemical cross-linking and SDS/polyacrylamide-gel electrophoresis. The tetrameric enzyme showed a pH-dependent dissociation in an acidic environment. At pH values above 8.0 most molecules existed as tetramers. The enzyme was gradually dissociated at lower pH. When the pH was below 5.0 most of the enzyme was present as the monomeric forms. Reassociation of the subunits was accomplished by adjusting the pH to neutrality. The dissociation and reassociation were almost instantaneous. No trimer was detected. The pigeon liver malic enzyme was thus shown to have a double-dimer quaternary structure with D2 symmetry. In the presence of substrates, the monomer-dimer-tetramer equilibrium favours the direction of dissociation. Tartronate, an L-malate analogue, was found to be more effective than L-malate in this process. When the monomeric forms were immobilized, the enzyme subunits were found to be fully active in catalysis. A possible arrangement of the four identical subunits of the enzyme molecule is proposed to account for the results obtained in this investigation. The origin of the half-of-the-sites reactivity of pigeon liver malic enzyme is also discussed.

Animals↗
PubMed1988

Fluorescence studies on the dissociation and denaturation of pigeon liver malic enzyme.

Exposure of pigeon liver malic enzyme [S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) in medium concentrations of guanidine-HCl at 25 degrees C and pH 7.45 caused biphasic conformational changes of the enzyme molecule. Molecular weight determination confirmed that the enzyme tetramers were dissociated to monomers in phase I transition. Enzymatic activity was completely lost in this phase. Recovery of the enzyme activity was only possible in the early stages of the phase I transition. Phase II was due to enzyme unfolding, as judged by circular dichroism and the fluorescence parameters of the enzyme. The steps of the transformation of native malic enzyme into a completely denatured state were in the following sequence: tetramer----monomer----random coil. Extensive denaturation of the enzyme molecule resulted in irreversible aggregation. Dissociation and denaturation were accompanied by a red-shift of the fluorescence spectrum (328----368 nm). Fluorescence quenching studies indicated that tryptophan residues of the enzyme molecule were buried deeply in the interior of the molecule. The tryptophan residues were only partially accessible by acrylamide and almost inaccessible by KI. Dissociation and denaturation were accompanied by exposure of the tryptophan residues, as manifested by the accessibility of the enzyme molecule toward KI or acrylamide.

Acrylamide↗
PubMed1988

High malic enzyme activity in tumor cells and its cross-reaction with anti-pigeon liver malic enzyme serum.

Rabbit antibodies against pigeon liver malic enzyme (EC 1.1.1.40) were prepared. The antiserum gave single precipitation line with crude pigeon liver extract. Cross reaction was observed with partially purified malic enzyme or crude extract from chicken liver. Positive cross reaction was also observed with the concentrated cytosolic fraction of two human carcinoma cell lines which were demonstrated to contain high malic enzyme activity. All other proteins examined did not react with the antibodies. When purified pigeon liver malic enzyme was mixed with the antiserum in vitro, a time-dependent inactivation of the enzyme activity was observed. Protection of the enzyme activity against antiserum inactivation was afforded by NADP+ or L-malate. Metal Mn2+ gave little protection.

Animals↗
PubMed1988

Amino acid concentrations in serum and aqueous humor from subjects with extreme myopia or senile cataract.

With an amino acid analyzer, we measured amino acids and related compounds in serum and aqueous humor from normal Chinese and some patients with extreme myopia or senile cataracts. Forty peaks were well resolved, and their areas were used to quantify each compound. In the myopic patients, glutamate, alpha-aminoadipate, and methionine concentrations in serum were 10-fold those in the normal subjects. Values for most of the other amino acids and related compounds were also higher in myopic patients' sera. In the cataract patients, concentrations of most of these compounds were lower in serum but higher in aqueous humor than for the normal subjects. Tryptophan was present in significant amounts in sera from the normal subjects, but was not detectable in the senile-cataract patients. The ratio of amino acid concentration in aqueous humor to that in the serum (Ch/Cs) was higher in the cataract patients than in normal subjects for almost all of the compounds we measured.

Amino Acids↗
PubMed1987

Interactions of nicotinamide-adenine dinucleotide phosphate analogues and fragments with pigeon liver malic enzyme. Synergistic effect between the nicotinamide and adenine moieties.

The structural requirements of the NADP+ molecule as a coenzyme in the oxidative decarboxylation reaction catalysed by pigeon liver malic enzyme were studied by kinetic and fluorimetric analyses with various NADP+ analogues and fragments. The substrate L-malate had little effect on the nucleotide binding. Etheno-NADP+, 3-acetylpyridine-adenine dinucleotide phosphate, and nicotinamide-hypoxanthine dinucleotide phosphate act as alternative coenzymes for the enzyme. Their kinetic parameters were similar to that of NADP+. Thionicotinamide-adenine dinucleotide phosphate, 3-aminopyridine-adenine dinucleotide phosphate, 5'-adenylyl imidodiphosphate, nicotinamide-adenine dinucleotide 3'-phosphate and NAD+ act as inhibitors for the enzyme. The first two were competitive with respect to NADP+ and non-competitive with respect to L-malate; the other inhibitors were non-competitive with NADP+. All NADP+ fragments were inhibitory to the enzyme, with a wide range of affinity, depending on the presence or absence of a 2'-phosphate group. Compounds with this group bind to the enzyme 2-3 orders of magnitude more tightly than those without this group. Only compounds with this group were competitive inhibitors with respect to NADP+. We conclude that the 2'-phosphate group is crucial for the nucleotide binding of this enzyme, whereas the carboxyamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity. There is a strong synergistic effect between the binding of the nicotinamide and adenosine moieties of the nucleotide molecule.

Adenosine Monophosphate↗
PubMed1987

Computer programs for the analysis of protein fluorescence quenching data.

Educational computer programs for analysis of fluorescence quenching studies of proteins have been developed. The program is written according to the classical Stern-Volmer equation and some modified Stern-Volmer equations. The static and dynamic quenching constants as well as the accessibility of the quencher molecules to the fluorescence groups are calculated. The experimental data are plotted on a high resolution graph. The calculated data or the graphs can be printed out to obtain a hard copy for filing.

Kinetics↗
PubMed1987

Structural identity of the subunits of pigeon liver malic enzyme.

Pigeon liver malic enzyme was found to have arginine, alanine, and tyrosine as the only N-terminal, N-1, and N-2 amino acids, respectively. Hydrolysis of the reduced and carboxymethylated malic enzyme by carboxypeptidase A yielded quantitative evidence for the following C-terminal sequence: -Leu-(Phe-Ala)-Ile-Leu-COOH. Fifty-five trypsin-digested peptides were separated by HPLC, in accordance with the arginine and lysine contents of each subunit. This more direct structural evidence strongly supports the conclusion that pigeon liver malic enzyme is composed of four chemically identical subunits.

Amino Acid Sequence↗
PubMed1986

Serum ornithine levels in some ocular disorders.

The fact that Chinese have a high occurrence of myopia and the toxicity of ornithine to retinal epithelial cells raised our interest to investigate the serum ornithine levels in high myopic patients. After analyzing several hundred patients, no significant difference in the serum ornithine level was found between Chinese and non-Chinese. However, patients with various ocular disorders did show higher ornithine levels. Serum citrulline levels in myobes and non-Chinese groups were decreased. The significance of these findings needs further investigation.

Citrulline↗
PubMed1985

Mechanism of pigeon liver malic enzyme: modification of essential carboxyl groups.

The maximum velocity of the reaction catalyzed by the pigeon liver malic enzyme depends on the ionization of a functional group of pKa 6.7. This pKa value is independent of temperature within the range 30 degrees-49 degrees C, suggesting the ionization of a carboxyl group. The enzyme activity is inactivated by N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward reagent K) at pH 6.0 and 25 degrees C. N-Methylhydroxamine regenerates the enzymatic activity whereas glycine ethyl ester does not. The addition of Mn2+, NADP+, and L-malate to the incubation mixture decreases the inactivation rate, suggesting that the reaction takes place in the active center. The binding capacities of the modified enzyme with NADP+, L-malate, pyruvate, and Mn2+ are not impaired. The kinetic and chemical evidence indicates that the inactivation is due to the modification of a carboxyl group which may be from glutamyl or aspartyl residues of the enzyme. This carboxyl group might function as a general acid-base catalyst. A detailed mechanism in terms of the exact amino acid residues involved is proposed.

Animals↗
PubMed1984

Continuous spectrophotometric assay for aminoacyl-tRNA synthetases.

A simple, continuous assay for aminoacyl-tRNA synthetases utilizing a commercially available pyrophosphate assay reagent kit was demonstrated. The method coupled aminoacyl-tRNA synthetase activity with pyrophosphate-dependent fructose-6-phosphate kinase, aldolase, triosephosphate isomerase, and glycerophosphate dehydrogenase. PPi formation was correlated with the oxidation of NADH, and was monitored continuously by the decrease of absorbance at 340 nm.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Essential tyrosyl residues of human placental alkaline phosphatase.

Human placental alkaline phosphatase was inactivated with tetranitromethane in a biphasic process. Spectral and amino acid analysis demonstrated that the inactivation was due to the conversion of tyrosine residues to 3-nitrotyrosine. The inactivation process showed saturation kinetics. Protection of the enzyme against tetranitromethane inactivation was afforded by inorganic phosphate. The binding affinity between the modified enzyme and inorganic phosphate was decreased. Our results suggest the involvement of tyrosyl residues in the locus of phosphoryl site of the phosphorylated enzyme forms.

Alkaline Phosphatase↗