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 …

Effect Of Charged Residue Substitutions On The Membrane-Interactive Properties Of Signal Sequences Of The Escherichia Coli Lamb Protein., Jeffrey D. Jones, Lila Gierasch

Lila Gierasch

Although the central role of the signal sequence in protein export is well established, the molecular details underlying signal sequence in vivo function remain unclear. As part of our continuing effort to relate signal sequence phenotypes to specific biophysical properties, we have carried out an extensive characterization of the secondary structure and lipid interactions for a family of peptides corresponding to the wild-type E. coli LamB signal sequence, and mutants that harbor charged residue point mutations in the hydrophobic core region. We used membrane-resident fluorescence quenching according to the parallax method to determine the relative depth of insertion of tryptophan-labeled …

Effect Of Charged Residue Substitutions On The Thermodynamics Of Signal Peptide-Lipid Interactions For The Escherichia Coli Lamb Signal Sequence., Jeffrey D. Jones, Lila Gierasch

Lila Gierasch

We have used tryptophan fluorescence spectroscopy to characterize the binding affinities of an Escherichia coli LamB signal peptide family for lipid vesicles. These peptides harbor charged residue substitutions in the hydrophobic core region. Titrations of peptides with vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-3-phosphoglycerol (65:35 mol%), in conjunction with evaluation of peptide dissociation rates from these vesicles, were used to determine binding parameters quantitatively. We find that under low ionic strength conditions, point mutations introducing negatively charged aspartate residues substantially reduce peptide affinity relative to the wild-type peptide. However, the difference between wild-type and mutant peptide affinities was much lower under …

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 …

Reorganization Of Lipid Domain Structure In Membranes By A Transmembrane Peptide: An Esr Spin Label Study On The Effect Of The Escherichia Coli Outer Membrane Protein A Signal Peptide On The Fluid Lipid Domain Connectivity In Binary Mixtures Of Dimyristoyl Phosphatidylcholine And Distearoyl Phosphatidylcholine., M. B. Sankaram, D. Marsh, Lila Gierasch, T. E. Thompson

Lila Gierasch

The effect of a transmembrane peptide on the domain structure of a two-component, two-phase lipid bilayer composed of dimyristoyl phosphatidylcholine (DMPC) and distearoyl phosphatidylcholine (DSPC) was examined by spin label electron spin resonance (ESR) spectroscopy. The peptide, pOmpA, is the hydrophobic, 25-residue signal sequence of the outer membrane protein A from Escherichia coli. Nitroxide derivatives of the phospholipid DSPC, 16-DSPCSL, and of the pOmpA signal peptide, pOmpA-IASL, were used as probes. The first-derivative lineshapes of the ESR spectra were analyzed using a normalized intensity ratio, R, that gives information on the average sizes of the disconnected fluid domains and their …

The Phosphate Transporter From Pea Mitochondria (Isolation And Characterization In Proteolipid Vesicles), Cecilia A. Mcintosh, David J. Oliver

David J. Oliver

The phosphate transporter from mitochondria will exchange matrix phosphate for cytosolic phosphate and facilitate either phosphate/proton symport or phosphate/hydroxyl ion antiport. The phosphate transported into the matrix by this carrier is either used for ATP synthesis or exchanges back out to the cytosol on the dicarboxylate transporter, permitting entry of malate and succinate into the matrix. The phosphate transporter was solubilized from etiolated pea (Pisum sativum L. cv Alaska) mitochondrial membranes with Triton X-114, purified approximately 500-fold by hydroxylapatite chromatography, and reconstituted into azolectin vesicles that were preloaded with 0.1 or 10 mM phosphate. Phosphate transport was measured as the …

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 …

Expression Of Prelamin A Confers Sensitivity Of Dna Biosynthesis To Lovastatin On F9 Teratocarcinoma Cells, Michael Sinensky, T. Mclain, K. Fantle

Michael Sinensky

No abstract provided.

The Processing Pathway Of Prelamin A, Michael Sinensky, K. Fantle, M. Trujillo, T. Mclain, A. Kupfer, M. Dalton

Michael Sinensky

The conversion of mammalian prelamin A to mature lamin A proceeds through the removal of 18 amino acids from the carboxyl terminus. The initial step in this processing is the isoprenylation of a CAAX box cysteine. This proteolytic event is distinctive for prelamin A among the known prenylated mammalian proteins. Since the carboxyl terminus of prelamin A is removed during maturation, it is not obvious that this protein would undergo the two reactions subsequent to prenylation observed in other CAAX box proteins-the endoproteolytic removal of the carboxyl-terminal 3 amino acids and the subsequent methylation of the now carboxyl-terminal cysteine. To …

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.