Biomedical subjects

H Weissbach

Publications and source records attributed to H Weissbach.

At least 55 records · Page 3Linked to original sources

PubMed1991

Transcriptional start and MetR binding sites on the Escherichia coli metH gene.

The 5' upstream region of the Escherichia coli metH gene has been sequenced. Primer extension analysis revealed a transcription start site at 324 bases upstream of the initiator codon. An 8 base sequence homologous to the MetR binding region on the E. coli metE gene is present 217 bp downstream of the transcription start site. Gel retardation experiments showed that purified MetR protein could bind to a 30 base oligonucleotide containing the putative MetR binding region. No "met box" was present which explains the relative lack of regulation of the expression of the metH gene by methionine.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1991

Calcium-dependent autophosphorylation of the glucose-regulated protein, Grp78.

The characteristics of phosphorylation of the 78-kDa glucose-regulated protein (Grp78), also known as the immunoglobulin heavy chain binding protein, were studied in vitro and in vivo. The purified protein from either calf liver or bovine kidney cells (MDBK) could be phosphorylated in vitro with [gamma-32P]ATP, in a reaction that is stimulated by Ca2+ and inhibited by the Ca(2+)-chelator ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA). In the presence of EGTA, excess Ca2+ increased the rate of phosphorylation about 18-fold. Based on EGTA/Ca2+ titrations, the optimal Ca2+ concentration for phosphorylation was estimated to be 10-50 microM. Other divalent cations such as Mg2+, Mn2+, and Zn2+ were found to be inhibitory as was the Ca2+ antagonist lanthanum (La3+). The in vivo phosphorylation of Grp78 was studied in MDBK cells labeled with 32Pi. In the presence of inducers of Grp78 synthesis, such as ionomycin, tunicamycin, or 2-deoxyglucose, there was a large increase in the level of Grp78 in the cells but a decrease in the amount of phosphorylated protein. Two-dimensional gel analysis of Grp78 purified from bovine liver and MDBK cells identified at least four isoforms. After in vivo and in vitro phosphorylation of Grp78 all the acidic isoforms contained radioactivity but not the most basic isoform. Phosphoamino acid analysis of Grp78 showed that serine and threonine were phosphorylated in vivo and only threonine was phosphorylated in vitro.

Adenosine Triphosphate↗

PubMed1991

Regulation of methionine synthesis in Escherichia coli.

The biosynthesis of methionine in Escherichia coli is under complex regulation. The repression of the biosynthetic pathway by methionine is mediated by a repressor protein (MetJ protein) and S-adenosyl-methionine which functions as a corepressor for the MetJ protein. Recently, a new regulatory locus, metR, has been identified. The MetR protein is required for both metE and metH gene expression, and functions as a transactivator of transcription of these genes. MetR is a unique prokaryotic transcription activator in that it possesses a leucine zipper motif, first described for eukaryotic DNA-binding proteins. The transcriptional activity of MetR is modulated by homocysteine, the metabolic precursor of methionine. Finally, it is known that vitamin B12 can repress expression of the metE gene. This effect is mediated by the MetH holoenzyme, which contains a cobamide prosthetic group.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1991

Biochemistry of methionine sulfoxide residues in proteins.

The oxidation of methionine to methionine sulfoxide constitutes one of the many post-translational modifications that proteins undergo. This non-enzymatic reaction has been shown to occur both in vivo and in vitro, and has been associated with the loss of biological activity in a wide variety of proteins and peptides. The presence of methionine sulfoxide residues in proteins is implicated in a variety of pathological conditions. An enzyme that is present in all organisms tested specifically catalyzes the reduction of the methionine sulfoxide residues in proteins. The physiological reductant for this enzyme appears to be thioredoxin.

Bacterial Proteins↗

PubMed1990

Interaction of DnaK with ATP: binding, hydrolysis and Ca+2-stimulated autophosphorylation.

The autophosphorylation of DnaK from Escherichia coli using ATP as phosphate donor is markedly stimulated by Ca+2 and to a lesser degree by Mn+2. Mg+2 and other divalent ions are without effect in this reaction. Lanthanum, an agonist/antagonist of Ca+2, is also effective in stimulating the autophosphorylation. In contrast, Mg+2 but not Ca+2, markedly stimulates the hydrolysis of ATP catalyzed by DnaK. Also at 0 degrees, ATP forms a stable complex with DnaK without hydrolysis that is independent of cations. About 15% of the DnaK in E. coli is associated with membrane vesicles where it also can be phosphorylated in the presence of Ca+2.

Adenosine Triphosphate↗

PubMed1990

Central nervous system function in systemic lupus erythematosus.

Autoantibodies to three eukaryotic 60S ribosomal phosphoproteins P0, P1 and P2 have been found in the sera of 10-20% of patients with systemic lupus erythematosus (SLE). These antibodies inhibit protein synthesis in vitro and when microinjected into cultured human fibroblasts. The three proteins share a common epitope contained within the carboxyl terminal 22 amino acids of each protein. Because a significant number of SLE patients have central nervous system disturbances with major behavioral disorders, the antiribosomal protein autoantibodies were measured in this subset of SLE individuals to determine whether or not there was an association. These antibodies are present in 90% of SLE patients who were diagnosed as having psychosis, secondary to the disease.

Amino Acid Sequence↗

PubMed1990

Structure-function studies on Escherichia coli MetR protein, a putative prokaryotic leucine zipper protein.

The Escherichia coli metR gene has been sequenced. The sequence predicts a protein of 317 amino acids and a calculated molecular weight of 35,628. This is about 15% larger than the protein from Salmonella typhimurium reported previously [Plamann, L.S. & Stauffer, G.V. (1987) J. Bacteriol. 169, 3932-3937]. The protein is a homodimer and contains a leucine zipper motif characteristic of many eukaryotic DNA-binding proteins. Replacement of two of the leucines in the leucine zipper region of the MetR protein, or substitution of proline for one of the leucines, results in loss of biological activity of the protein. In addition, truncation studies have identified a region on MetR that may be involved in the homocysteine activation of metE expression.

Amino Acid Sequence↗

PubMed1990

Hsp70 proteins, similar to Escherichia coli DnaK, in chloroplasts and mitochondria of Euglena gracilis.

The heat-shock response of Euglena gracilis was studied by pulse-labeling cells with [35S]sulfate at both the normal growth temperature (21 degrees C) and an elevated temperature (36 degrees C). Analysis of the labeled proteins by polyacrylamide gel electrophoresis indicated that the rate of synthesis of at least 3 major and 15 minor polypeptides increased in cells grown at the higher temperature. Three of the proteins appear to be immunologically related to the ubiquitous approximately 70-kDa heat-shock protein (Hsp70) family. One protein of 68 kDa was found in the cytoplasm (P68cyt) and was the major heat-shock protein in Euglena gracilis. Two other proteins, 68 and 70 kDa, were localized in mitochondria (P68mit) and chloroplasts (P70chl), respectively, and they crossreacted with a polyclonal antibody raised against the Escherichia coli heat-shock protein DnaK. Like DnaK, P68mit and P70chl could be phosphorylated in vitro with [gamma-32P]ATP in a reaction that was stimulated by Ca2+. A protein with characteristics similar to those of P70chl was also found in chloroplasts isolated from maize and spinach.

Animals↗

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↗

PubMed1989

A member of the Hsp70 family is localized in mitochondria and resembles Escherichia coli DnaK.

A 71-kDa protein (P71) with properties similar to those of the Escherichia coli heat shock protein DnaK has been found in extracts of HeLa cells. P71 was copurified by ATP-agarose affinity chromatography with three additional proteins of the Hsp70 family. Of these proteins, only P71 crossreacted strongly with antiserum raised against purified DnaK, and both DnaK and P71 could be phosphorylated in vitro with [gamma-32]ATP in a reaction that was markedly stimulated by Ca2+. In HeLa cells, P71 was found to be concentrated in mitochondria. A protein similar to P71 was also found in calf liver and yeast mitochondria.

Calcium↗

PubMed1989

Quantification of lupus anti-ribosome P antibodies using a recombinant P2 fusion protein and determination of the predicted amino acid sequence of the autoantigen in patients' mononuclear cells.

The cDNA encoding the ribosomal protein P2 antigen was cloned from a human cDNA library constructed in the lambda gt11 expression vector. A beta-galactosidase-P2 fusion protein was purified to near homogeneity and used to develop an ELISA which was highly specific for anti-P antibodies produced in murine and human SLE. The median concentration of human IgG anti-P antibodies in serum was estimated to be 100 micrograms/ml (range 6-450 micrograms/ml). Pre-incubation of human anti-P sera with a synthetic peptide, corresponding to the C-terminal 22 amino acids of P2, completely inhibited reactivity with the fusion protein in the ELISA. These findings confirm that lupus anti-P sera show a striking restriction in epitope specificity and indicate that the P2 fusion protein is a useful alternative to the synthetic peptide antigen for detection and quantification of anti-P antibodies. To investigate the possibility that anti-P antibodies were induced by 'altered-self', cDNA encoding P2 were also cloned from lupus patients and control mononuclear cells. The predicted amino acid sequences of the patients' P2 were identical to that of the normal controls indicating that a primary structural abnormality of the P2 autoantigen was unlikely.

Amino Acid Sequence↗

PubMed1988

The inhibition of protein synthesis by IgG containing anti-ribosome P autoantibodies from systemic lupus erythematosus patients.

Autoantibodies found in approximately 15% of patients with systemic lupus erythematosus recognize three 60 S ribosomal phosphoproteins P0, P1, and P2. Fab fragments obtained from sera of these patients inhibited globin mRNA translation in an in vitro protein synthesizing system which was reversed by the addition of excess ribosomes. Further studies suggested that these antibodies bind to ribosomes in the intact cell. Thus, when IgG fractions from these sera were microinjected into cultured human fibroblasts [35S]methionine incorporation into cellular proteins was inhibited.

Animals↗

PubMed1988

Frequency and epitope recognition of anti-ribosome P antibodies from humans with systemic lupus erythematosus and MRL/lpr mice are similar.

Autoantibodies reactive against a shared, conserved epitope on the ribosomal phosphoproteins P0, P1, and P2 occur in approximately 15% of patients with SLE and are relatively specific for this disease. To determine whether anti-P antibodies occur in murine lupus, serum from MRL/lpr and NZB/W F1 mice were analyzed by immunoblotting as well as by ELISA using a synthetic peptide Ag. Of those analyzed, 4 of 35 (11%) MRL/lpr, 0 of 25 NZB/W F1 and 0 of 13 control NIH/Swiss mice had anti-P antibodies. Anti-P specificity was confirmed by immunoblotting of ribosomal proteins separated by two-dimensional gel electrophoresis and by inhibition of anti-P reactivity on immunoblots with the synthetic peptide Ag. These findings indicate a striking similarity in the frequency and fine epitope specificity of anti-P antibodies in humans and MRL/lpr mice with SLE.

Animals↗

PubMed1988

Properties of the ribosomal P2 protein autoantigen are similar to those of foreign protein antigens.

Approximately 15% of patients with systemic lupus erythematosus have autoantibodies that bind to a shared epitope previously shown to be located on the carboxyl-terminal 22 amino acids of three 60S ribosomal proteins, P0, P1, and P2 ("P proteins"). A hydrophilicity plot and fine epitope mapping with seven synthetic peptides revealed that the properties of the antigenic site were similar to certain properties of epitopes on foreign protein antigens--namely, the epitope was located in the most hydrophilic portion of the P2 protein and also in the terminal region of the molecule. However, this site has been highly conserved during evolution. A mouse monoclonal antibody induced by immunization with ribosomal proteins had a fine specificity similar to the lupus antibodies. This finding indicates that a highly conserved region of a lupus autoantigen may also be antigenic in some normal animals. Therefore, lupus autoantibodies may be similar in most, if not all respects, to antibodies produced by immunization.

Amino Acid Sequence↗

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↗

PubMed1988

Regulation of ribulose bisphosphate carboxylase expression in Rhodospirillum rubrum: characteristics of mRNA synthesized in vivo and in vitro.

The synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in Rhodospirillum rubrum was regulated by the CO2 concentration in the culture medium. The specific activity of RuBPCase in cells grown photolithotrophically in low concentrations of CO2 (1.5%) was five to ten times higher than that in cultures grown at high concentrations of CO2 (10%). Increased enzyme activity was reflected by an increase in both RuBPCase mRNA and RuBPCase protein. RuBPCase expression was also studied in vitro with a plasmid-borne genomic clone (pRR117) as the template in a partially defined Escherichia coli system containing either E. coli or R. rubrum RNA polymerase. With both enzymes there was excellent synthesis of RuBPCase mRNA, but no significant synthesis of RuBPCase was detected. The promoter region of the RuBPCase gene was sequenced, and mRNA start sites were mapped. A single major in vivo transcriptional start site was detected in RuBPCase mRNA extracted from R. rubrum. However, transcripts synthesized from pRR117 in vitro or from E. coli transformed with pRR117 started at upstream sites that were different from the in vivo transcription site. Two major features of the RuBPCase promoter region are three 6-base-pair direct repeats and a 31-base-pair region of dyad symmetry.

Base Sequence↗