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Biomedical subjects

J G Dong

Publications and source records attributed to J G Dong.

5 recordsLinked to original sources
PubMed1995

Structural studies of vinblastine alkaloids by exciton coupled circular dichroism.

SCF-CI-dipole velocity MO calculations have shown that the bisignate circular dichroic curves of vinblastine/vincristine alkaloids at ca 210 and 220-230 nm are due to exciton coupling between the indoline and indole moieties. Furthermore, a combination of X-ray crystal structure data with MM2 local energy minimization provides a convenient means for estimation of the preferred solution conformation.

Circular Dichroism↗
PubMed1993

Activation of 1-aminocyclopropane-1-carboxylate oxidase by carbon dioxide.

1-Aminocyclopropane-1-carboxylate (ACC) oxidase requires CO2/HCO3- as an essential activator for its activity. Taking advantage that the equilibrium concentrations of CO2 and HCO3- vary with pH and that the interconversions of CO2 and HCO3- are slower at low temperature, we identified CO2 rather than HCO3- as the active species involved in the activation process. Preincubation of the enzyme with a saturating concentration of CO2 resulted in increased activation of the enzyme when preincubation pH was raised, indicating that CO2 reacted with an enzyme group having an alkaline pKa. It is suggested that the CO2 activation of ACC oxidase involves the formation of a carbamate. CO2 increases the Vmax of the reaction but decreases the affinity of the enzyme toward its substrate ACC. A plausible reaction scheme accounting for the CO2 activation process is presented.

Amino Acid Oxidoreductases↗
PubMed1992

Purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from apple fruit.

1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene. Following conventional column fractionation, the enzyme was purified 180-fold to near homogeneity with a specific activity of 20 nmol/(mg.min). This purified enzyme preparation migrated as a single protein band with an apparent molecular mass of 35 kDa on SDS/PAGE and 39 kDa on gel filtration. As in vivo, the purified enzyme required CO2 for activity. Removal of CO2 from the reaction mixture completely abolished the enzyme activity, while 0.5% atmospheric CO2 (0.15 mM in the medium) gave half-maximal activity. The purified enzyme displayed an absolute requirement for Fe2+ and ascorbate. The stoichiometry of the enzymatic reaction was determined: ACC + ascorbate + O2-->C2H4 + HCN + CO2 + dehydroascorbate + 2 H2O. A polyclonal antibody was raised against a synthetic tridecapeptide (PDLEEEYRKTMKE) whose sequence was deduced from the apple pAE12 cDNA [Dong, J. G., Olson, D., Silverstone, A. & Yang, S. F. (1992) Plant Physiol. 98, 1530-1531], which is homologous to tomato cDNAs encoding ACC oxidase. On a Western blot, this antibody specifically recognized the purified ACC oxidase protein. The amino acid composition of the purified enzyme agreed well with that deduced from the pAE12 sequence. When the protein was cleaved with CNBr and one of the peptide fragments was isolated and sequenced for 20 cycles, its sequence (KEFAVELEKLAEKLLDLLCE) precisely matched that predicted from pAE12 (residues 115-134). When preclimacteric apple fruit was treated with ethylene, a parallel increase in in vivo and in vitro ACC oxidase activities was observed, and this increase was accompanied by a concomitant increase in the level of pAE12 transcript. These observations support the conclusion that the isolated ACC oxidase protein is encoded by pAE12.

Amino Acid Oxidoreductases↗
PubMed1990

Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase.

The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14), the key enzyme in ethylene biosynthesis, is inactivated by its substrate S-adenosylmethionine (AdoMet). Apple ACC synthase was purified with an immunoaffinity gel, and its active site was probed with NaB3H4 or Ado[14C]Met. HPLC separation of the trypsin digest yielded a single radioactive peptide. Peptide sequencing of both 3H- and 14C-labeled peptides revealed a common dodecapeptide of Ser-Leu-Ser-Xaa-Asp-Leu-Gly-Leu-Pro-Gly-Phe-Arg, where Xaa was the modified, radioactive residue in each case. Acid hydrolysis of the 3H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the 14C-labeled peptide indicated a protonated molecular ion at m/z 1390.6, from which the mass of Xaa was calculated to be 229, a number that is equivalent to the mass of a lysine residue alkylated by the 2-aminobutyrate portion of AdoMet, as we previously proposed. These results indicate that the same active-site lysine binds the PLP and convalently links to the 2-aminobutyrate portion of AdoMet during inactivation. The active site of tomato ACC synthase was probed in the same manner with Ado[14C]Met. Sequencing of the tomato active-site peptide revealed two highly conserved dodecapeptides; the minor peptide possessed a sequence identical to that of the apple enzyme, whereas the major peptide differed from the minor peptide in that methionine replaced leucine at position 6.

Amino Acid Sequence↗