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Journal Issue - Volume 1 Issue 7 (July 1992)

Abstract 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...

Abstract 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...

Abstract Escherichia coli transcription termination factor rho is an RNA‐dependent ATPase, and ATPase activity is required for all its functions. We have characterized the binding of ATP to the physiologically relevant hexameric association state of rho in the absence of RNA and have shown that there are six ATP binding sites per rho hexamer. This stoichiometry has been verified by a number of different techniques, including...

Abstract The rho protein of Escherichia coli interacts with the nascent RNA transcript while RNA polymerase is paused at specific rho‐dependent termination sites on the DNA template, and (in a series of steps that are still largely undefined) brings about transcript termination at these sites. In this paper we characterize the interactions of rho with RNA and relate these interactions to the quaternary structure of the functional...

Abstract Two enzymes involved in the biosynthesis of the trypanosomatid‐specific dithiol trypanothione‐glutathionylspermidine (Gsp) synthetase and trypanothione (TSH) synthetase‐have been identified and purified individually from Crithidia fasciculata. The Gsp synthetase has been purified 93‐fold and the TSH synthetase 52‐fold to apparent homogeneity from a single DEAE fraction that contained both activities. This constitutes the first...

Abstract Glucagon, a peptide hormone synthesized and secreted by α islet cells, regulates glucose homeostasis by several mechanisms. Using [γ32P]8N3GTP, a proven photoaffinity probe for GTP, a specific nucleotide binding site on human glucagon was detected that showed preference for GTP. Half‐maximal saturation of photoinsertion into the polypeptide hormone was at 8–12 μM with either [α32P]8N3GTP or [γ32P]8N3GTP. GTP protected photolabeling with an...

Abstract The proposal that the active site vacuole of NAD+‐S‐lactate dehydrogenase is unable to accommodate any imbalance in electrostatic charge was tested by genetically manipulating the cDNA coding for human muscle lactate dehydrogenase to make a protein with an aspartic acid introduced at position 140 instead of the wild‐type asparagine. The Asn 140–Asp mutant enzyme has the same kcat as the wild type (Asn 140) at low pH (4.5), and at...

Abstract An enzymatic procedure for the complete removal of the N‐linked and O‐linked oligosaccharide side chains of the sex steroid‐binding proteins (SBP or SHBG) of human and rabbit plasma under native conditions is described. Deglycosylation was catalyzed by N‐glycanase, neuraminidase, and O‐glycanase and was monitored by SDS‐PAGE, lectin blotting, and molecular weight analyses by electrospray mass spectrometry. Digestion of rabbit SBP with...

Abstract A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60–89). The expressed protein, which contains an additional N‐terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has...

Abstract Ribonuclease A is known to form an equilibrium mixture of fast‐folding (UF) and slow‐folding (US) species. Rapid unfolding to UF is then followed by a reaction in the unfolded state, which produces a mixture of UF, USII, USI, and possibly also minor populations of other US species. The two cis proline residues, P93 and P114, are logical candidates for producing the major US species after unfolding, by slow cis → trans isomerization. Much work has been done in...

Abstract We have performed a computational simulation of the aggregation and chaperonin‐dependent reconstitution of dimeric prokaryotic ribulose bisphosphate carboxylase/oxygenase (Rubisco), based on the data of P. Goloubinoff et al. (1989, Nature 342, 884–889) and P.V. Viitanen et al. (1990, Biochemistry 29, 5665–5671). The aggregation is simulated by a set of 12 differential equations representing the aggregation of the Rubisco folding...

Abstract Capillary HPLC is a very effective means of separating small amounts of peptides and proteins. Capillary columns ranging from 0.01 mm to 0.5 mm in diameter can be constructed using recycled supports and inexpensive fused silica capillary tubing. Commercial pumping systems and UV detectors can be readily converted for operation in the flow rate range of 0.5–50 μL/min. Detailed procedures are given for the construction of...

Abstract We have previously shown that a 34‐residue synthetic peptide representing the calcium‐binding site III of troponin C formed a symmetric two‐site dimer consisting of two helix‐loop‐helix motifs arranged in a head‐to‐tail fashion (Shaw, G.S., Hodges, R.S., & Sykes, B.D., 1990, Science 249, 280–283). In this study the hydrophobicities of the α‐helices were altered by replacing L‐98 and F‐102 in the N‐terminal region and/or I‐121 and...