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.

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.

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.

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.

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.

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.

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.

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

Studies by Urbanowski et al. [Urbanowski, M. L.

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.

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.

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.

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.

Synthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase in anin vitro partially definedE. coli system.

Thein vitro DNA- or RNA-directed synthesis of the large subunit (LS) of spinach chloroplast ribulose-1,5-biphosphate carboxylase (RuP2C) has been examined in a highly definedE. coli transcription-translation system. Spinach chloroplast DNA, RNA and recombinant plasmids containing the spinach chloroplast LS gene (rbcL) have been used as templates in thein vitro system and a quantitative assay has been developed to measure LS formation.

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.

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.

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.

Binding of reticulocyte elongation factor 1 to ribosomes and nucleic acids.

The present study has examined the requirements for the binding of rabbit reticulocyte elongation factor 1 (EF-1) to ribosomes under different assay conditions. When a centrifugation procedure was used to separate the ribosome EF-1 complex, the binding of EF-1 to ribosomes required GTP and Phe-tRNA, but not poly(U). The results suggested that undr these conditions a ternary complex, EF-1 .

DNA-directed in vitro synthesis of beta-galactosidase. Studies with purified factors.

The phage DNA-directed synthesis of beta-galactosidase has been examined in a system containing the following purified Escherichia coli factors: RNA polymerase; cyclic AMP receptor protein; N10-formyltetrahydrofolate Met-tRNAf transformylase; initiation factors 1, 2, and 3; elongation factors Tu, Ts, and G; release factors 1 and 2; 20 aminoacyl-tRNA synthetases; L factor (Kung, H. F., Spears, C.

Elongation factor 1 from Artemia salina: properties and disaggregation of the enzyme.

Elongation factor 1 has been purified from undeveloped embryos of Artemia salina. The purified enzyme appears to be an aggregate (molecular weight approximately to 200 000) which on sodium dodecylsulfate gels shows the presence of two major protein bands whose estimated molecular weights are 52 000 and 47 000. Lipid material appears to be associated with the purified protein.