Chloroplasts of higher plants contain a nuclear-encoded protein that is a functional homolog of the Escherichia coli chaperonin 10 (cpnlO; also known as groES). In pea (Pisum satvum), chloroplast cpnlO was identified by its ability to (i) assist bacterial chaperonin 60 (cpn60; also known as groEL) in the ATP-dependent refolding of chemically denatured ribulose-1,5-bisphosphate carboxylase and (i) form a stable complex with bacterial cpn6O in the presence ofMg'ATP.The subunit size of the pea protein is -24 kDa-about twice the size of bacterial cpnlO. A cDNA encoding a spinach (Spinacea oleracea) chloroplast cpnlO was isolated, sequenced, and expressed in vitro. The spinach protein is synthesized as a higher molecular mass precursor and has a typical chloroplast transit peptide. Surprisingly, however, attached to the transit peptide is a single protein, comprised of two distinct cpnlO molecules in tandem. Moreover, both halves of this "double" cpnl'0 are highly conserved at a number of residues that are present in all cpnlOs that have been examined. Upon import into chloroplasts the spinach cpnl0 precursor is processed to its mature form of -24 kDa. N-terminal amino acid sequence analysis reveals that the mature pea and spinach cpnlO are identical at 13 of 21 residues.Molecular chaperones play a vital role in protein folding and protein translocation in eukaryotic and prokaryotic cells (for a recent review see ref.

The cloned fur (ferric uptake regulation) gene of Escherichia coli K12 was ligated to an expression vector which was inducible with nalidixic acid. The Fur protein was isolated in a single step by immobilized metal-ion-affinity chromatography over zinc iminodiacetate agarose. The amino acid composition of the isolated protein agreed with that predicted from the gene sequence and indicated post-transcriptional removal of the N-terminal methionine residue. All four cysteines were shown to be present as thiols. Proteolysis with trypsin and chymotrypsin yielded large fragments identifiable on polyacrylamide gel electrophoresis. Various divalent metal ions were found by a nitrocellulose filter binding assay to effect non-specific interaction of the Fur dimer with DNA with a dissociation constant of 7 x 10(-12) M. A much smaller value, 2.5 x 10(-17) M, was measured by gel mobility retardation assay for binding of Fur to a DNA fragment containing the operator sequences of the aerobactin promoter.

No abstract

No abstract

Generation of a fragment-complementing system of the alpha-chain on limited proteolysis with Staphylococcus aureus V8 protease has been investigated. Digestion of the alpha-chain (0.4 mM) of hemoglobin with V8 protease in phosphate buffer at pH 6.0 and 37 degrees C is limited to the peptide bonds of Glu-23, Glu-27, Glu-30, and Asp-47. Gel filtration of a V8 protease digest of the alpha-chain on a Sephadex G-50 column did not release any heme to the low molecular weight region, though some peptides were released from the protein. The filtration studies revealed the presence of two heme-containing components in the digest, the major one eluting at the alpha-chain position and the minor one eluting slightly ahead of the alpha-chain position. Reversed-phase high-performance liquid chromatography and amino-terminal sequence analysis demonstrated that the component eluting at the alpha-chain position contains species generated by the noncovalent interactions of heme and the complementary fragments alpha 1-30 and alpha 31-141. In dilute solutions (0.04 mM) the V8 protease digestion occurred exclusively on the carboxyl side of Glu-30(alpha). This high selectivity was also observed at pH 4.0 and pH 7.8. The visible spectra and the ultraviolet circular dichroic spectra of the digest reflect the native-like structure of the noncovalent fragment system. The dissociation constant of alpha 1-30 appears to be in the range of 10(-8) M. In tetrameric hemoglobin A the peptide bond of Glu-30-Arg-31 of the alpha-chain is not accessible to V8 protease digestion.(ABSTRACT TRUNCATED AT 250 WORDS)