Biomedical subjects

T M Huang

Publications and source records attributed to T M Huang.

9 recordsLinked to original sources

PubMed1992

Characterization of the tetramer-dimer-monomer equilibrium of the enzymatically active subunits of pigeon liver malic enzyme.

The tetrameric malic enzyme from pigeon liver was reversibly dissociated in the sequence of tetramer-dimer-monomer in an acidic environment (pH 4.5) or when the ionic strength or temperature of the solution was perturbed (0.2 M ammonium sulfate or < 10 degrees C). The dissociated monomer was enzymatically active according to the following criteria: (a) separation and direct activity staining of the monomer in the native gradient polyacrylamide gel, (b) activity staining of the monomer at its pI region in the isoelectric focusing gel, and (c) the enzyme showing lower but definite enzyme activity under conditions where only monomer existed in the solution. The catalytic constant (kcat) and specificity constant (kcat/KmMal) for the monomer were found to be 19 +/- 6 s-1 and 58 x 10(3) s-1.M-1, respectively, only one-seventh and one-seventeenth of those for the tetramer. Different types of interactions are involved in the monomer-monomer and dimer-dimer associations: (a) Two dissociation processes showed different pH dependences. The monomer-monomer interactions involve an amino acid with a side chain pKa value around 5.7, and an amino acid with a side chain pKa value of 7.2 is involved in the dimer-dimer association. (b) Ammonium sulfate up to 0.2 M only affects the monomer-monomer but not the dimer-dimer interactions. The Gibb's free energy, enthalpy, and entropy all have negative values for the above subunits' dissociations. The overall dissociation is an enthalpy-driven process. Association of the subunits to form dimers and tetramers involves salt-bridge, van der Waals, and hydrogen-bonding interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Human placental alkaline phosphatase. An improved purification procedure and kinetic studies.

An improved method for the purification of human placental alkaline phosphatase is described. The partially purified enzyme from Sigma was further purified by successive Concanavalin A-Sepharose and Q-Sepharose chromatography. The whole procedure may be completed in one working day. Highly purified enzyme was obtained with a 39% yield. The intrinsic fluorescence of the enzyme decreased at elevated temperature. The conformation of the enzyme molecule was studied by the fluorescence quenching technique. Upward Stern-Volmer plots were obtained for the quenching data which suggested that, in addition to collisional quenching, static quenching was involved in the quenching mechanism. The dynamic and static quenching constants were found to be 0.7 +/- 0.16 M-1 and 0.44 +/- 0.1 M-1, respectively, using acrylamide as the quenching agent. The corresponding values were 0.43 +/- 0.23 M-1 and 0.84 +/- 0.18 M-1, respectively, with KI as the quenching agent. Mg2+ and PO4(3-) induced protein conformational changes which altered both the dynamic and static quenching constants. Mg2+ was found to be a non-essential activator for the placental alkaline-phosphatase-catalyzed hydrolysis of 4-nitrophenyl phosphate. At pH 9.8, Mg2+ increased Vmax by 1.2-fold without affecting the Kd of the substrate. The tetranitromethane-modified enzyme showed slower migration toward the anode on electrophoresis and increased Kd for Mg2+.

Alkaline Phosphatase↗

PubMed1992

Kinetic mechanism of the cytosolic malic enzyme from human breast cancer cell line.

The kinetic mechanism of the cytosolic NADP(+)-dependent malic enzyme from cultured human breast cancer cell line was studied by steady-state kinetics. In the direction of oxidative decarboxylation, the initial-velocity and product-inhibition studies indicate that the enzyme reaction follows a sequential ordered Bi-Ter kinetic mechanism with NADP+ as the leading substrate followed by L-malate. The products are released in the order of CO2, pyruvate, and NADPH. The enzyme is unstable at high salt concentration and elevated temperature. However, it is stable for at least 20 min under the assay conditions. Tartronate (2-hydroxymalonate) was found to be a noncompetitive inhibitor for the enzyme with respect to L-malate. The kinetic mechanism of the cytosolic tumor malic enzyme is similar to that for the pigeon liver cytosolic malic enzyme but different from those for the mitochondrial enzyme from various sources.

Animals↗

PubMed1991

Purification and characterization of the cytosolic NADP(+)-dependent malic enzyme from human breast cancer cell line.

Cytosolic NADP(+)-dependent malic enzyme from a cultured human breast cancer cell line was purified to near homogeneity by two highly efficient chromatography systems: Pharmacia-LKB Q-Sepharose anion-exchange chromatography and adenosine-2',5'-bisphosphate-agarose affinity chromatography. The overall yield was 27%. The enzyme is presumably a tetramer composed of four probably identical subunits of Mr 65,000, which is similar to the enzyme from other sources. The pI and optimum reaction pH values for the tumor malic enzyme are 5.5 and 7.2, respectively. At pH 6.9, most of the enzyme exists as monomers. Activation energy for the enzyme-catalyzed oxidative-decarboxylation reaction is 57.4 kJ/mol. The enzyme is strictly NADP+ dependent, as NAD+ cannot support the oxidative-decarboxylation reaction. ATP at low concentration inhibits the enzyme activity. Fumarate at concentrations up to 5 mM does not affect the enzymatic reaction rate. Therefore the tumor cytosolic malic enzyme, unlike the mitochondrial malic enzyme, is not an allosteric regulatory enzyme.

Breast Neoplasms↗

PubMed1990

[Micellar chromatographic study of amines].

Aqueous solutions of the cationic surfactant, hexadecyl-trimethyl ammonium bromide (CTAB), and the anionic surfactant, sodium dodecylsulfate (SDS) were used as mobile phase in HPLC to study the micellar chromatographic retention mechanism of seven amines and weak organic acids with various distribution coefficients and to study the influence of different pH values mobile phase on the solute retention. An exponential model equation for micellar chromatographic retention mechanism was put forward, which gives a reasonable explanation of the effects of electrostatic interaction and hydrophobic interaction between the solute and the three phases (micelle phase, surfactant modified stationary phase and bulk water phase). This equation can be used to estimate the micellar chromatographic retention pattern of the solute.

Amines↗

PubMed1989

Enzyme immunoassay system for panel testing.

An immunoassay system based on enzyme immunoassay technology has been developed for quantitative panel testing. The system includes test card disposables, reagents, and an instrument. Patients' samples are processed semiautomatically in the instrument with minimum user intervention. The test card has multiple test areas at individual locations on a membrane solid phase so that simultaneous determinations from a single specimen are possible. Each panel also includes positive and negative reagent procedural controls. Factory-determined calibration curves for each analyte are provided in barcode form with each test kit. The reagents include a specimen dilution buffer, enzyme conjugate, and precipitogenic substrate. Up to 10 test cards at a time can be processed in random-access and continuous-access modes, with automated agitation of sample and reagents over the solid phase, temperature-controlled incubation, and membrane washing and reading, data reduction, and printout of results. The optical reader measures diffuse reflectance and features source intensity and wavelength compensation.

Chemistry, Clinical↗

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↗

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↗

PubMedDate not supplied

Oxidation and cleavage of 3-aminopyridine adenine dinucleotide phosphate with periodate.

Treatment of 3-aminopyridine adenine dinucleotide phosphate with sodium periodate in the neutral pH resulted in oxidation of the ribose linked to 3-aminopyridine and cleavage of the dinucleotide into adenosine- and 3-aminopyridine-containing moieties. Separation of these moieties was afforded by thin-layer chromatography, high-performance liquid chromatography, and fast protein liquid chromatography. From fast atom bombardment mass spectra and nuclear magnetic resonance spectra, the adenosine-containing moiety was identified as 2'-phosphoadenosine 5'-phosphate while the aminopyridine moiety was present in a mixture of the hydrated 3-aminopyridine mononucleotide/nucleoside dialdehyde. Separation of the completely oxidized product by Pharmacia fast protein liquid chromatography gave three major peaks corresponding to 2'-phosphoadenosine 5'-phosphate, 2'-phosphoadenosine 5'-diphosphate and oxidized 3-aminopyridine nucleoside, with minor amount of oxidized 3-aminopyridine mononucleotide. Thus the oxidized 3-aminopyridine adenine dinucleotide phosphate was shown to cleave by two pathways: it may either undergo beta-elimination to give 2'-phosphoadenosine 5'-diphosphate and oxidized 3-aminopyridine nucleoside; or the phosphodiester linkage may be hydrolyzed to give 2'-phosphoadenosine 5'-phosphate and oxidized 3-aminopyridine mononucleotide. The latter compound may further undergo beta-elimination and eventually give oxidized 3-aminopyridine nucleoside. Hydrolysis could be prevented by storing the sample as lyophilized powder, while beta-elimination was diminished by lowering the storage temperature. We found that the lyophilized powder of oxidized 3-aminopyridine adenine dinucleotide phosphate can be stored at -50 degrees C for several months with minimum decomposition.

Chromatography, High Pressure Liquid↗