PubMed Health⌕ Search

Biomedical subjects

B Redfield

Publications and source records attributed to B Redfield.

At least 19 recordsLinked to original sources
PubMed1996

ATP hydrolysis is not required for the dissociation of a substance P.BiP complex.

BiP is a member of the hsp70 family of proteins that is present in the endoplasmic reticulum where it functions as a molecular chaperone. Rapid quantitative assays have been used to study the effect of mutating BiP residue 229, located in the ATP binding site, from threonine to glycine. Although binding of ATP to the mutant BiP was not affected, the mutant protein possessed 10-20% of the wild-type BiP ATPase activity. Binding to a model peptide substrate, substance P (Brot et al. (1994) Proc. Natl. Acad. Sci. USA 91, 12120-12124), was twofold higher with mutant BiP at 4 degrees C than with wild-type BiP, and was ATP dependent. Under these conditions the substance P that was bound to mutant BiP, but not the wild-type, could be released by higher levels of ATP (5-10 microM), and the ratio of substance P released to ATP hydrolyzed was greater than 10. These results suggest that stoichiometric ATP hydrolysis is not required for release of a chaperone from its substrate.

Adenosine Triphosphate↗
PubMed1995

Interaction of BiP with substance P and nucleotides.

A rapid and simple spin column assay has been used to study interactions of BiP with substance P (SP) and ATP. At 4 degrees C, the binding of SP to BiP requires ATP and a stable SP-BiP.ATP complex is formed. Nonhydrolyzable ATP analogues or ADP cannot replace ATP. Although ATP converts BiP dimers to monomers, the requirement for ATP for SP binding is not solely due to BiP dissociation, because purified BiP monomers also require ATP for peptide binding. At 37 degrees C, there is rapid binding of SP to BiP even in the absence of ATP and, in fact, ATP at concentrations above 5 microM causes release of SP from BiP. At this higher temperature, there is also rapid hydrolysis of ATP bound to BiP. These results extend our previous results (Brot et al., 1994) that indicated the formation, at low ATP concentrations, of a labile SP.BiP.ATP complex that, after ATP hydrolysis, resulted in a stable SP.BiP.ADP complex.

Adenosine Triphosphate↗
PubMed1994

Similarity of nucleotide interactions of BiP and GTP-binding proteins.

BiP is a member of the Hsp70 heat shock protein family found in the lumen of the endoplasmic reticulum, that binds to a variety of proteins destined to be secreted. Substance P (SP) has been used as a model peptide to study the interaction of BiP with protein substrates. SP stimulates BiP ATPase activity and forms a stable complex with BiP that is dissociated in the presence of levels of ATP > 50 microM. At lower concentrations of ATP, the SP remains bound to BiP, and the results are consistent with the view that a BiP-ATP complex is initially formed that reacts with SP to form a ternary complex, SP-BiP-ATP. Hydrolysis of ATP in this complex yields a SP-BiP-ADP complex. An exchange of ATP with ADP bound to BiP has also been demonstrated, and the results suggest that the interactions of BiP with ATP resemble those seen with GTP-binding proteins and GTP.

Adenosine Diphosphate↗
PubMed1994

BiP is a substrate for src kinase in vitro.

In a study to investigate the ability of chaperones to modulate src kinase activity, it was observed that BiP, a member of the HSP70 family found in the endoplasmic reticulum, is an excellent substrate for src kinase in vitro. The reaction requires polylysine and the results suggest that two tyrosine residues are phosphorylated. Although there is no evidence for this reaction in vivo, it does provide a very efficient method to label BiP.

Carrier Proteins↗
PubMed1993

Dissociation of glucose-regulated protein Grp78 and Grp78-IgE Fc complexes by ATP.

Recent studies have shown that ATP can dissociate dimers of the glucose-regulated protein Grp78 to monomers. In the present study, we have used purified recombinant Grp78 from Escherichia coli to investigate this reaction in more detail. During the course of the Grp78 dimer-monomer conversion, a stable Grp78 monomer-ATP complex is formed. Upon removal of the ATP, the Grp78 dimer is reformed. ADP, nonhydrolyzable ATP analogues, and GTP do not effect the dissociation of Grp78 dimers. A cell line that overproduces IgE Fc has been used to examine the nature of the Grp78-IgE Fc complexes present and the effect of ATP on them. Grp78-IgE Fc complexes ranging from 100 kDa to 300 kDa were observed by sucrose gradient analysis, suggesting that aggregate forms of Grp78 may be present in some of these complexes. Treatment of the extracts with ATP resulted in release of a Grp78 monomer from the complex. These results suggest that the dissociation of Grp78 oligomers by ATP may be involved in the function of Grp78 in protein translocation through the endoplasmic reticulum.

Adenosine Triphosphate↗
PubMed1992

Role of the metF and metJ genes on the vitamin B12 regulation of methionine gene expression: involvement of N5-methyltetrahydrofolic acid.

The repression of MetE synthesis in Escherichia coli by vitamin B12 is known to require the MetH holoenzyme (B12-dependent methyltransferase) and the metF gene product. Experiments using trimethoprim, an inhibitor of dihydrofolate reductase, show that the MetF protein is not directly involved in the repression, but that N5-methyltetrahydrofolic acid (N5-methyl-H4-folate), the product of the MetF enzymatic reaction is required. Since the methyl group from N5-methyl-H4-folate is normally transferred to the MetH holoenzyme to form a methyl-B12 enzyme, the present results suggest that a methyl-B12 enzyme is involved in the vitamin B12 repression of metE expression. Other results argue against the possibility that a methyl-B12 enzyme functions in this repression solely by decreasing the cellular level of homocysteine, which is required for MetR activation of metE expression. Experiments with metJ mutants show that the MetJ protein mediates about 50% of the repression of metE expression by B12 but is totally responsible for the regulation of metF expression by vitamin B12.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1989

The effect of homocysteine on MetR regulation of metE, metR and metH expression in vitro.

An Escherichia coli S-30 DNA directed protein synthesis system was used to study the effect of homocysteine on the in vitro expression of the metE, metH and metR genes. In the presence of purified MetR protein, which is known to regulate the expression of these genes, homocysteine activates metE expression and inhibits both metR and metH expression. These findings support the recent in vivo results of Urbanowski, M.L. and Stauffer, G.V. (1989), J. Bacteriol. 171, 3277-3281.

Escherichia coli↗
PubMed1989

Regulation of methionine synthesis in Escherichia coli: effect of the MetR protein on the expression of the metE and metR genes.

A plasmid (pRSE562) containing the metE and metR genes of Escherichia coli was used to study the expression of these genes and the role of the MetR protein in regulating metE expression. DNA sequence analysis of the 236-base-pair region separating these genes showed the presence of seven putative met boxes. When this plasmid was used to transform either wild-type E. coli, metE mutant, or metR mutant, MetE enzyme activity increased 5- to 7-fold over wild-type levels. The metR gene was subcloned from pRSE562, and this plasmid, pMRIII, relieved the methionine auxotrophy of a metR mutant after transformation. The metR gene was also cloned into a vector containing the lambda PL promoter, and the MetR protein was overexpressed and purified to near homogeneity. This protein, when added to an in vitro DNA-dependent protein synthesis system in which the MetE and/or MetR proteins were synthesized, caused a large increase in the expression of the metE gene but a decrease in the expression of the metR gene. The in vitro expression of both genes was inhibited by the MetJ protein and S-adenosylmethionine in the presence or absence of MetR protein. These results provide evidence that the product of the metR gene is a trans-activator of the expression of the metE gene and that the expression of the metR gene is under autogenous regulation and is repressed by the MetJ protein.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1989

Methionine synthesis in Escherichia coli: effect of the MetR protein on metE and metH expression.

Studies by Urbanowski et al. [Urbanowski, M. L., Stauffer, L. T., Plamann, L. S. & Stauffer, G. V. (1987) J. Bacteriol. 169, 1391-1397] have identified a regulatory locus, called metR, required for the expression of the metE and metH genes. We recently purified the MetR protein from Escherichia coli and showed that it could stimulate the in vitro expression of the metE gene and autoregulate its own synthesis. In the present study, the purified MetR protein has been shown to stimulate the in vitro expression of the metH gene. Also, the in vitro synthesized MetE, MetH, and MetR proteins were shown to be biologically active. The transcription start sites for the metE and metR genes have been determined, and DNA footprinting experiments have identified regions in the metE-metR intergenic sequence that are protected by either the MetR or MetJ proteins.

Bacterial Proteins↗
PubMed1988

Antibody to sigma 32 cross-reacts with DnaK: association of DnaK protein with Escherichia coli RNA polymerase.

A polyclonal antibody to sigma 32, the heat shock sigma factor, has been used to show the presence of low levels of sigma 32 in Escherichia coli RNA polymerase preparations (E sigma 70), which explains the observed in vitro activity of E sigma 70 towards heat shock genes. The sigma 32 antibody cross-reacts with DnaK, and DnaK has been found associated with purified preparations of both E sigma 70 and the heat shock RNA polymerase, E sigma 32.

Bacterial Proteins↗
PubMed1985

Regulation of methionine synthesis in Escherichia coli: effect of metJ gene product and S-adenosylmethionine on the in vitro expression of the metB, metL and metJ genes.

The regulation of the expression of three Escherichia coli met genes, metB, which codes for cystathionine gamma-synthetase (EC 4.2.99.9), metL, which codes for aspartokinase II-homoserine dehydrogenase II (EC 2.7.2.4-EC 1.1.1.3) and metJ, which codes for the methionine regulon aporepressor, has been studied using highly purified DNA-directed in vitro protein synthesis systems. In a system where the entire gene product is synthesized, the expression of the metB and metL genes is specifically inhibited by MetJ protein (repressor protein) and S-adenosylmethionine (AdoMet). In a simplified system that measures the formation of the first dipeptide of the gene product (fMet-Ala for the metJ gene), MetJ protein and AdoMet partially repress (approximately 40-60%) metJ gene expression. Thus, the metJ gene can be partially autoregulated by its gene product.

Bacterial Proteins↗
PubMed1985

Regulation of methionine synthesis in Escherichia coli: Effect of metJ gene product and S-adenosylmethionine on the expression of the metF gene.

The regulation of the expression of the Escherichia coli metF gene, which codes for 5,10-methylenetet-rahydrofolate reductase (EC 1.1.99.15), has been investigated by using a simplified DNA-directed in vitro system that measures the formation of the first dipeptide (fMet-Ser) of the gene product. The synthesis of fMet-Ser directed by a plasmid containing the metF gene is specifically inhibited by metJ protein (repressor protein). S-Adenosylmethionine enhances the inhibition by the metJ protein of metF gene expression. The inhibition by the metJ protein is at the level of transcription and the results suggest that S-adenosylmethionine is functioning as an allosteric effector.

Journal Article↗
PubMed1983

Simplified in vitro system for study of eukaryotic mRNA translation by measuring di- and tripeptide formation.

An in vitro system for measurement of rabbit globin mRNA translation has been developed based on the formation of the NH2-terminal dipeptide, fMet-Val. The basic components include a partially purified initiation factor preparation from rabbit reticulocytes supplemented with eukaryotic initiation factor 4A, purified and formylated yeast Met-tRNAi, and rabbit liver or Escherichia coli Val-tRNA1Val. Picomole quantities of fMet-Val are synthesized, dependent on mRNA, and the dipeptide is readily assayed by a simple extraction procedure. In the presence of Leu-tRNA or His-tRNA, the tripeptides fMet-Val-Leu and fMet-Val-His are synthesized, corresponding to the NH2-terminal sequence of alpha- and beta-globin, respectively. Therefore, tripeptide synthesis provides a simple means to distinguish between the expression of the alpha- and beta-globin mRNA species.

Animals↗
PubMed1980

L factor that is required for beta-galactosidase synthesis is the nusA gene product involved in transcription termination.

The DNA-dependent in vitro synthesis of Escherichia coli beta-galactosidase requires the presence of a soluble protein referred to as L factor [Kung, H., Spears, C. & Weissbach, H. (1975) J. Biol. Chem. 250, 1556-1562]. In the present study, comparison of physical, immunological, and biological properties shows that L factor is the product of the E. coli nusA gene. The nusA gene product is known to interact with bacteriophage lambda N gene protein and to prevent premature termination of transcription from the early lambda promoters. Our results suggest that premature transcription termination in the lac operon of E. coli may also be overcome by the nusA protein.

Bacterial Proteins↗
PubMed1979

DNA-directed in vitro synthesis of beta-galactosidase. Purification and characterization of stimulatory factors in an ascites extract.

Previous studies have described a partially defined system for the DNA-directed in vitro synthesis of beta-galactosidase (Kung, H.F., Redfield, B., Treadwell, B.V., Eskin, B., Spears, C., and Weissbach, H. (1977) J. Biol. Chem. 252, 6889-6894). An Ehrlich ascites extract was shown in these in vitro studies to acylate Escherichia coli tRNA with 13 amino acids, and the ascites extract was used in place of the corresponding 13 E. coli aminoacyl-tRNA synthetases. The present studies indicate that the ascites extract is supplying an additional protein factor, besides the aminoacyl-tRNA synthetases, that stimulates the DNA-directed synthesis of beta-galactosidase. The protein factor has been highly purified and may be functioning by protecting mRNA against degradation. In addition, NAD or T4 DNA ligase stimulates the synthesis of beta-galactosidase in the partially defined system.

Amino Acyl-tRNA Synthetases↗