In vitro reassembly of the ribose ATP-binding cassette transporter reveals a distinct set of transport complexes.

Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate.

Topology of RbsC, the membrane component of the Escherichia coli ribose transporter.

The topology of RbsC, the membrane component of the ribose transporter in Escherichia coli, has been determined by using 34 single-cysteine mutants and a modified fluorescence labeling technique designated multiplex labeling. This technique gives topology, expression, and localization information for a membrane protein from a single batch of bacterial cells. The results indicate that RbsC contains 10 transmembrane-spanning helices, with the N and C termini being in the cytosol.

Elutability of Proteins from Aluminum-Containing Vaccine Adjuvants by Treatment with Surfactants.

The elutability of proteins from adjuvants in model vaccines composed of ovalbumin adsorbed by aluminum hydroxide adjuvant or lysozyme adsorbed by aluminum phosphate adjuvant following treatment with surfactant solutions was studied. Nonionic (Triton X-100, lauryl maltoside), zwitterionic (lauryl sulfobetaine), anionic (sodium dodecyl sulfate), and cationic (cetylpyridinium chloride, dodecyltrimethylammonium chloride) surfactants were investigated. Cetylpyridinium chloride produced the greatest degree of elution (60%) of ovalbumin from aluminum hydroxide adjuvant.

The proteins encoded by the rbs operon of Escherichia coli: I. Overproduction, purification, characterization, and functional analysis of RbsA.

The nucleotide-binding component of the high-affinity ribose transport system of Escherichia coli, RbsA, was overproduced from a T7-7 expression vector, and the protein was purified. Biochemical analyses of the purified protein indicated that the ATP analogues, 5'-FSBA and 8-azido ATP, covalently labeled the protein, a reaction that was inhibited by ATP, but not by GTP or CTP. The pure protein exhibited low-level ATPase activity with a K(m) of about 140 microM.

Probing protein-protein interactions. The ribose-binding protein in bacterial transport and chemotaxis.

A number of mutations at Gly134 of the periplasmic ribose-binding protein of Escherichia coli were examined by a combined biochemical and structural approach. Different mutations gave rise to different patterns of effects on the chemotaxis and transport functions. The smallest residue (alanine) had the least effect on transport, whereas large hydrophobic residues had the smallest effect on chemotaxis.

Identical mutations at corresponding positions in two homologous proteins with nonidentical effects.

The x-ray structure of a mutant (Gly72 to Asp) of the Escherichia coli ribose-binding protein with altered transport function has been solved and refined to 2.2-A resolution with a conventional R-factor (R-factor = [formula: see text]) of 16.0% and good stereochemistry.

Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport.

Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose.

Structural homology between rbs repressor and ribose binding protein implies functional similarity.

The deduced amino acid sequence of the rbs repressor, RbsR, of Escherichia coli is homologous over its C-terminal 272 residues to the entire sequence of the periplasmic ribose binding protein. RbsR is also homologous to a family of bacterial repressor proteins including LacI. This implies that the structure of the repressor consists of a two-domain binding protein portion attached to a DNA-binding domain having the four-helix structure of the LacI headpiece.

Structural and functional analyses of the repressor, RbsR, of the ribose operon of Escherichia coli.

The DNA sequence encoding the rbs repressor protein, RbsR, has been determined. Amino acid sequence analyses of the product of an rbsR-lacZ fusion and of affinity-purified RbsR demonstrate that translation begins at an unusual codon, TTG, and that the initial amino acid is removed during maturation of the protein. DNA-binding assays indicate that RbsR binds to a region of perfect dyad symmetry spanning the rbs operon transcriptional start site and that the affinity for the rbs operator is reduced by addition of ribose, consistent with ribose being the inducer of the operon.

A novel cereal storage protein: molecular genetics of the 19 kDa globulin of rice.

A lambda gt11 cDNA library, constructed from poly(A)+ RNA isolated from immature rice seed endosperm, was screened with affinity-purified antibodies against the rice storage protein called alpha-globulin (previously), or the 19 kDa globulin (our term). A positive clone was isolated and sequenced and shown to encode a 21 kDa precursor for the 19 kDa globulin, based on the identity of portions of the inferred amino acid sequence and the sequence of three cyanogen bromide peptides of the 19 kDa globulin. Analysis of genomic DNA by Southern blotting using the cDNA clone probe revealed one hybridizing band in Eco RI, Hind III, and Bam HI digests.

Cloning and structural characterization of porcine heart aconitase.

A full-length cDNA encoding porcine heart aconitase was derived from lambda gt10 recombinant clones and by amplification of the 5' end of the mRNA. The 2700-base pair (bp) cDNA contains a 29-bp 5' untranslated region, a 2343-bp coding segment, and a 327-bp 3' untranslated region. The porcine heart enzyme is synthesized as a precursor containing a mitochondrial targeting sequence of 27 amino acid residues which is cleaved to yield a mature enzyme of 754 amino acids, Mr = 82,754, having a blocked amino terminus.

The amino acid sequence of cytochrome c553 from Microcystis aeruginosa.

Cytochrome c553 is an electron donor to P700 in the photosynthetic electron transfer chain of cyanobacteria and eukaryotic algae. We have purified this cytochrome from the cyanobacterium Microcystis aeruginosa and determined its amino acid sequence. When the amino acid sequence of this protein is compared to sequences of cytochromes c553 from other organisms, one sees that the evolution of net charge is more pronounced than the evolution of overall structure, further documenting a pronounced shift in the isoelectric point of this protein during the evolution of cyanobacteria.

Drosophila insulin degrading enzyme and rat skeletal muscle insulin protease cleave insulin at similar sites.

Insulin degradation is an integral part of the cellular action of insulin. Recent evidence suggests that the enzyme insulin protease is involved in the degradation of insulin in mammalian tissues. Drosophila, which has insulin-like hormones and insulin receptor homologues, also expresses an insulin degrading enzyme with properties that are very similar to those of mammalian insulin protease.

Identification of phosphorylation sites in peptides using a gas-phase sequencer.

A simple procedure is described for determining the location of phosphorylation sites in phosphopeptides. The method employs measurement of 32P-labeled inorganic phosphate release during Edman degradation cycles using a gas-phase sequencer. The procedure is based on extracting peptides and inorganic phosphate from portions of the sample filter at strategic cycles in the sequence analysis followed by determination of the relative amounts of phosphate and phosphopeptide.

Structure of the coding sequence and primary amino acid sequence of acetyl-coenzyme A carboxylase.

Acetyl-coenzyme A carboxylase (Ac-CoA carboxylase; EC 6.4.1.

Mutational analysis of the glutamine phosphoribosylpyrophosphate amidotransferase pro-peptide.

Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase is synthesized as a pro-enzyme having an 11-amino acid leader. Maturation requires insertion of a [4Fe-4S] cluster and processing of the pro-peptide to expose an NH2-terminal active site cysteine residue. Point and deletion mutations were constructed in the leader region.

Degradation products of insulin generated by hepatocytes and by insulin protease.

The enzymatic mechanisms for insulin breakdown by hepatocytes have not been established, nor have the degradation products been identified. Several lines of evidence have suggested that the enzyme insulin protease is involved in insulin degradation by hepatocytes. To identify the products of insulin generated by insulin protease and to compare them with those produced by hepatocytes, we have incubated insulin specifically iodinated at either the B-16 or the B-26 tyrosines with insulin protease and with isolated hepatocytes, separated the products on high performance liquid chromatography (HPLC), and identified the B-chain cleavages.

Isolation of a cDNA encoding the muscle-specific subunit of human phosphoglycerate mutase.

We have isolated a full-length cDNA specifying the muscle-specific subunit of human phosphoglycerate mutase (PGAM-M). The cDNA encodes a deduced protein 253 amino acids in length and contains an unusually short (37 nucleotides) 3'-untranslated region. The deduced human PGAM-M protein is clearly related to yeast PGAM and to human diphosphoglycerate mutase.

Characterization of osmotin : a thaumatin-like protein associated with osmotic adaptation in plant cells.

Cultured tobacco (Nicotiana tabacum var Wisconsin 38) cells adapted to grow under osmotic stress synthesize and accumulate a 26 kilodalton protein (osmotin) which can constitute as much as 12% of total cellular protein. In cells adapted to NaCl, osmotin occurs in two forms: an aqueous soluble form (osmotin-I) and a detergent soluble form (osmotin II) in the approximate ratio of 2:3. Osmotin-I has been purified to electrophoretic homogeneity, and osmotin-II has been purified to 90% electrophoretic homogeneity.

Identification of A chain cleavage sites in intact insulin produced by insulin protease and isolated hepatocytes.

The degradation of insulin by the enzyme insulin protease and by isolated hepatocytes results in proteolytic cleavages in both the A and B chains of intact insulin. Previous studies have shown that one of the A chain cleavages is between A13 leucine and A14 tyrosine and that a second cleavage occurs carboxyl to the A14 residue. In the present study we have used insulin specifically iodinated on the A19 tyrosine and examined the A chain cleavages by the enzyme and by hepatocytes.

Liver isozyme of rabbit glycogen synthase. Amino acid sequences surrounding phosphorylation sites recognized by cyclic AMP-dependent protein kinase.

Glycogen synthase, the rate-limiting enzyme in glycogen biosynthesis, has been postulated to exist as isozymes in rabbit liver and muscle (Camici, M., Ahmad, Z., DePaoli-Roach, A.

An analysis of the structure of the product of the rbsA gene of Escherichia coli K12.

The predicted amino acid sequence of rbsA, a gene from the high affinity ribose transport operon (rbs) of Escherichia coli K12, is homologous to the products of hisP, malK, and pstB, components of the histidine, maltose, and phosphate high affinity transport operons. The recent finding by Hobson et al. (Hobson, A.

Ribokinase from Escherichia coli K12. Nucleotide sequence and overexpression of the rbsK gene and purification of ribokinase.

The nucleotide sequence of a 1455-base pair TaqI-HinfI fragment of the rbs operon of Escherichia coli K12 has been determined. It includes the 3' terminus of rbsB (the gene for ribose-binding protein) and the entire rbsK gene, encoding ribokinase. Potential consensus promoter sequences and a stable stem-loop structure are present in the rbsB-rbsK intercistronic region.

The nucleotide sequences of the rbsD, rbsA, and rbsC genes of Escherichia coli K12.

The nucleotide sequences of rbsD, rbsA, and rbsC have been determined. These genes encode components of the high affinity ribose transport system in Escherichia coli, and together with the sequences of rbsB (Groarke, J.M.

Identification of the cystines which link the acidic and basic components of the glycinin subunits.

The half-cystine residues involved in linking the acidic and basic polypeptides were determined for several glycinin subunits. The cystines were localized with specific cyanogen bromide fragments either by comparing the electrophoretic mobility of nonreduced and reduced fragments, or by co-purifying and then determining the NH2-terminal sequence of the covalently linked fragments. Residues involved in the disulfides were further identified by labeling them with [3H] iodoacetic acid.