Ian G. Giles

SALMONELLA TYPHIMURIUM RESPONSES TO A BACTERICIDAL PROTEIN FROM HUMAN NEUTROPHILS

Bactericidal/permeability‐increasing protein [BPI] is a cationic antimicrobial protein from neutrophils that specifically binds to the surfaces of Gram‐negative bacteria via the lipid A component of lipopolysaccharide. To obtain information about the responses of Salmonella typhimurium to cell‐surface damage by BPI, two‐dimensional gel electrophoresis and N‐terminal microsequencing were used to identify proteins that were induced or repressed following BPI treatment. The majority of the affected proteins are involved in central metabolic processes. Upon addition of BPI, the β‐subunit of the F1 portion of Escherichia coli ATP synthase was repressed threefold whereas six proteins were induced up to 11‐fold. Three of the latter were identified as lipoamide dehydrogenase, enoyl‐acyl carrier protein reductase, and the heat‐shock protein HtpG. Additionally, a novel protein, BipA, was identified that is induced over sevenfold by BPI; sequence analysis suggests that it belongs to the GTPase superfamily and interacts with ribosomes. A conserved direct‐repeat motif is present in the regulatory regions of several BPI‐inducible genes, including the bipA gene. Only one of the BPI‐responsive proteins was induced when cells were treated with polymyxin B, which also binds to lipid A. We therefore conclude that BPI and polymyxin B affect different global regulatory networks in S. typhimurium even though they bind with high affinity to the same cell‐surface component.

The sequence of an antibiotic resistance gene from an antibiotic-producing bacterium: Homologies with transposon genes

The APH gene of a butirosin‐producing Bacillus circulans has been cloned and sequenced; a comparison of the translated protein sequence with those from TN5 and TN903 indicates that they may have a common origin.

The use of bacteriophage M13 carrying defined fragments of the Escherichia coli gltA gene to determine the location and structure of the citrate synthase promoter region

SummaryThe gltA gene from Escherichia coli, which encodes citrate synthase, has been located on a 3.24 Kb HindIII/EcoRl restriction fragment. This region contains one restriction site for BamHl and two for BglII. Defined restriction fragments from this region were cloned into suitably cleaved replicative form M13mp8 and M13mp9. The recombinants (M13gltA1 → 10) were isolated as single stranded DNA and characterised on the basis of molecular weight and DNA sequence. The single stranded DNA was converted to the double stranded replicative form and used to transform E. coli strain JM103 from which bacteriophage were isolated. Infection of JM103 with different bacteriophage followed by measurement of expressed citrate synthase activity showed that the complete gltA gene must span the BamHl restriction site, that the control region was on the 5′-terminal side of this restriction site and that the coding region for citrate synthase protein commenced on the 3′-terminal side. Analysis of the DNA sequence of this region allowed us to confirm this model, to identify the start sequence for translation of the structural gene and a number of sequences controlling the initiation of transcription. Of special interest is the fact that there must be an extensive leader sequence (305 nucleotides) separating the predicted sites for initiation of transcription and translation.

An investigatin into the apparent inhibition by arginine phosphate of the activity of Carcinus maenas type-M pyruvate kinase

An enzymic synthesis utilising arginine kinase for preparing arginine phosphate in a high state of purity is described. The dissociation constant of magnesium arginine phosphate, determined by gel filtration, was 30.0 +/- 0.9 mM. That for potassium arginine phosphate was calculated to be 63.0 +/- 4.0 mM measured by the effect of potassium on the apparent magnesium dissociation constant. The effect of KCl on the reaction catalysed by the type-M pyruvate kinase from Carcinus maenas (the common shore crab) pincer and leg muscle was investigated. No effect was seen on the C. maenas pyruvate kinase activity, apart from that due to alteration of the K+ concentration, on adding up to 70 mM potassium arginine phosphate to the reaction medium. The less pure form of arginine phosphate was found to give an apparent noncompetitive inhibition of the enzyme when phosphoenolpyruvate was the varied substrate. This apparent inhibition can be accounted for by the removal of ADP from the assay medium in a side reaction involving arginine kinase and arginine phosphate. These results are discussed in terms of the possible physiological control of the type-M pyruvate kinase from C. maenas.

The regulation of pyruvate kinase in the hepatopancreas of Carcinus maenas.

Abstract 1. 1. Estimation of the activity of pyruvate kinase and pyruvate carboxylase in the hepatopancreas of Carcinus maenas gave values of 28.5 and 7.4 units/g wet wt tissue respectively. 2. 2. The concentrations of substrates, products and allosteric effectors of these enzymes in hepatopancreas were measured. 3. 3. The activities of pyruvate kinase and pyruvate carboxylase were redetermined using the approximate physiological range of substrate and effectors. 4. 4. Under these conditions the effective activity of pyruvate kinase could be decreased to less than 2 units/g wet wt tissue whereas that for the pyruvate carboxylase was 2.6 units/g wet wt tissue indicating that a net synthesis of phosphoenolpyruvate could occur in vivo .

Biochemical data processing with microcomputers : Part 2. A BASIC program for the Detection, Integration and Area Assignment of Chromatographic Elution Profiles

Abstract A BASIC program which can be used for routine area calculations is summarised. It accommodates most of the problems encountered during peak integration and area assignment, e.g., sloping baselines, overlapping peaks and shoulders on either edge of a major peak. Various levels of user interaction are incorporated so as to permit the parameters used in the calculations of an individual trace to be modified at the discretion of an experienced operator.

Phosphorylation of type-L pyruvate kinase in intact hepatocytes Localisation of the phosphorylation site in response to both glucagon and the Ca2+-linked agonist phenylephrine

Pyruvate kinase is one of the enzymes which can be phosphorylated by stimulation of the cell with either glucagon or Ca2+‐linked hormones. Whether these two classes of hormones phosphorylate the same site on the enzyme is unclear. Our results demonstrate that isolation of [32P]phosphorylated type‐L pyruvate kinase from glucagon‐treated hepatocytes followed by aspartyl‐prolyl cleavage yields a [32P]phosphorylated peptide of M r 17000. This fragment is also phosphorylated in response to the Ca2+‐mediated agonist phenylephrine.

An introduction to microcomputers in biochemistry

Abstract In this review I hope to give a short account of the development and present state of the art of microcomputers. In addition I will describe some of the uses that microcomputers have already been put to in the biochemistry laboratory and what they may be used for in the future. Finally and perhaps more importantly, for anyone who has (or is) thinking about purchasing a microcomputer but finds the jargon and acronyms in manufacturers catalogues meaningless a short glossary is provided. As this review is of a general nature few specific references are supplied. Instead a selected bibliography containing introductory texts is given. This list is not exhaustive and is not meant to be; rather it contains works that this author has found to be particularly useful.

Biochemical data processing with microcomputers : Part 1. On-line Data Acquisition from an Amino-Acid Analyser with a Microcomputer

Abstract A program written in BASIC is described, together with the necessary electronic interfacing, to allow sequential on-line data capture from an automated amino-acid analyser by means of a commercial microcomputer. The data from two concurrent analytical channels can be handled, allowing the measurement of amino acids by spectrophotometry at 440 nm and 570 nm after reaction with ninhydrin. The procedure uses a box-car averaging technique to increase the signal-to-noise ratio, with a concurrent partial integration. This approach preserves area information whilst compressing the data into a manageable number of points for subsequent integration and area apportionment.

Biochemical data-processing with microcomputers : Part 3. On-line data acquisition from a continuous flow analyzer

Abstract A program, written in BASIC, is described which allows data acquisition from a continuous flow analyzer. The program was developed for a readily available microcomputer, but should be easily modified for use on similar machines. Once the peak height has been measured, the concentration of the analyte is calculated by reference to a previously defined calibration. The program is designed to handle data from more than one channel, although there is a practical limit of 3–4 simultaneously active channels. The results of the separate assays are collated and printed as a group for each specimen, even when the analytical methods require different times for completion.