Life Sciences Commons^™

Molecular Genetic Analysis Of Terminal Steps In Bacteriochlorophyll A Biosynthesis: Characterization Of A Rhodobacter Capsulatus Strain That Synthesizes Geranylgeranoil-Esterified Bacteriochlorophyll A, David Bollivar, Shaojie Wang, James P. Allen, Carl E. Bauer

David Bollivar

Site-directed mutational analysis of the Rhodobacter capsulatus photosynthesis gene cluster was undertaken in order to identify and characterize genetic loci involved in bacteriochlorophyll a biosynthesis. A mutant in orf304 was shown to accumulate the tetrapyrrole intermediate "bacteriochlorophyllide a" which is a tetrapyrrole that has a bacteriochlorophyll a ring structure without the presence of an esterifying alcohol. A mutant in orf391 is shown to synthesize acteriochlorophyll a that is esterified with geranylgeraniol rather than the normal phytol. This latter result provides the first genetic confirmation that esterification of bacteriochlorophyllide a initially involves the addition of a geranylgeraniol group followed by sequential …

Heterologous Expression Of The Bchm Gene Product From Rhodobacter Capsulatus And Demonstration That It Encodes S-Adenosyl-L-Methionine: Mg-Protoporhyrin Ix Methyltransferase, David Bollivar, Ze-Yu Jiang, Carl E. Bauer, Samuel I. Beale

David Bollivar

The bacteriochlorophyll biosynthesis gene, bchM, from Rlodobacter capsulatus was previously believed to code for a polypeptide involved in formation of the cyclopentone ring of protochlorophyllide from Mg-protoporphyrin IX monomethyl ester. In this study, R. capsulatus bchM was expressed in Escherichia coli and the gene product was subsequently demonstrated by enzymatic analysis to catalyze methylation of Mg-protoporphyrin IX to form Mg-protoporphyrin IX monomethyl ester. Activity required the substrates Mg-protoporphyrin IX and S-adenosyl-L-methionine. 14C-labeled product was formed in incubations containing 14C-methyl-labeled S-adenosyl-L-methionine. On the basis of these and previous results, we also conclude that the bchH gene, which was previously reported to …

Protein S-Thiolation In Hepatocytes Stimulated By T-Butyl Hydroperoxide, Menadione, And Neutrophils, Yuh-Cherng Chai, S. Hendrich, James Thomas

Yuh-Cherng Chai

In order to examine potentially important S-thiolated proteins, ^3^5S-labeled hepatocytes were exposed to oxidative stress. A similar group of S-thiolated proteins including carbonic anhydrase III was observed in cells treated with t-butyl hydroperoxide, menadione, or stimulated neutrophils. The radioactive thiols bound to hepatocyte proteins were identified by HPLC and more than 85% was glutathione. In menadione-treated hepatocytes, proteins were gradually S-thiolated over 30 min and 25% of the cellular glutathione pool became protein-bound. In t-butyl hydroperoxide-treated cells, S-thiolation was more transient and 11% of the glutathione was protein-bound. Neutrophil-treated hepatocytes had nearly the same amount of protein S-thiolation (8% after …

S-Thiolation Of Individual Human Neutrophil Proteins Including Actin By Stimulation Of The Respiratory Burst: Evidence Against A Role For Glutathione Disulfide, Yuh-Cherng Chai, S. Ashraf, R. Johnson, James Thomas

Yuh-Cherng Chai

Protein S-thiolation, a reversible modification of protein sulfhydryls resulting in formation of mixed-disulfides, was studied in human neutrophils stimulated with phorbol diester to produce superoxide anion. Rapid S-thiolation of several proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Glutathione was identified as the primary protein-bound thiol by HPLC chromatography, contributing considerably more than 85% of the total. Minor amounts of homocysteine and/or cysteine were also detected as protein-bound thiols. During the first 30 min after stimulation, 10% of the cellular glutathione became protein bound (2 nmol/mg of protein). There was no increase in glutathione disulfide suggesting that S-thiolation of …

Altering The Rna Binding Specificity Of A Translational Repressor, F Lim, Marc Spingola, D S. Peabody

Marc Spingola

Irreversible Steps In The Ferritin Synthesis Induction Pathway.., L. Linggoess, David Mascotti, M. Bhattacharyya-Pakrasi, H. Gang, R. Thach

David P. Mascotti

The ability of cells to re-repress ferritin synthesis after removal of an inducing agent (iron or heme) was investigated. Re-repression was found to be a slow process, requiring approximately 4 (after iron removal) to 10 h (after heme removal) for completion. Desferrioxamine mesylate (Desferal) had only a slight effect on the rate of re-repression, whereas cycloheximide was strongly inhibitory, indicating that new protein synthesis is required for re-repression. Re-repression occurred at a slow but significant rate in the presence of both Desferal and cycloheximide. These results indicate that, in the absence of an iron chelator, the induction of ferritin synthesis …

Protein S-Thiolation And Dethiolation, James Thomas, Yuh-Cherng Chai, Che-Hun Jung

Yuh-Cherng Chai

No abstract provided.

S-Thiolation And Irreversible Oxidation Of Sulfhydryls On Carbonic Anhydrase Iii During Oxidative Stress: A Method For Studying Protein Modification In Intact Cells And Tissues, C. Lii, Yuh-Cherng Chai, W. Zhao, James Thomas, S. Hendrich

Yuh-Cherng Chai

No abstract provided.