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P H Ray

Publications and source records attributed to P H Ray.

36 records · Page 2Linked to original sources
PubMed1987

Identification and purification of a recombinant Treponema pallidum basic membrane protein antigen expressed in Escherichia coli.

A recombinant plasmid designated pLVS3 previously was described that harbored a 14-kilobase insert of Treponema pallidum genomic DNA. Escherichia coli maxicells programmed with this plasmid synthesized three treponemal protein antigens of molecular weights 39,000, 35,000, and 25,000 (39K, 35K, and 25K proteins, respectively). In this study, a detailed deletion analysis of pLVS3 demonstrated that the genetic information for all three protein antigens is contained within a 1.5-kilobase EcoRI-HpaI restriction fragment. The DNA sequence of this fragment revealed a single open reading frame of 361 codons that most likely encodes a signal peptide-bearing precursor to the 39K protein that can be transiently detected in E. coli maxicells. Evidence indicated that the 35K and 25K protein antigens are derivatives of the larger protein and are only produced in maxicells. A significant elevation in expression of the 39K treponemal protein antigen in E. coli was obtained by using the E. coli lpp and lac promoters and a genetic construction in which the signal peptide and first four residues of the "mature" 39K protein were replaced by six amino acids encoded by the vector. This hybrid protein exhibited an unusually high pI, which greatly facilitated its purification to homogeneity. By using antibody prepared against the hybrid protein, the native treponemal protein counterpart, also of molecular weight 39,000, was identified as a membrane component of T. pallidum. Since the native protein also exhibited a net positive charge, it has been designated the T. pallidum basic membrane protein.

Antigens, Bacterial↗
PubMed1986

Localization and purification of two enzymes from Escherichia coli capable of hydrolyzing a signal peptide.

The signal peptide generated during the maturation of prolipoprotein by the purified prolipoprotein signal peptidase can be isolated in substrate amounts (Dev, I. K., and Ray, P. H. (1984) J. Biol. Chem. 259, 11114-11120). This signal peptide is degraded predominantly from the carboxyl terminus by cell-free extracts of Escherichia coli. The signal peptide is degraded (at least 300-fold) more rapidly than other cellular proteins in E. coli. Greater than 90% of the signal peptide hydrolase activity is localized in the cytoplasm. Two enzymes from the cytoplasmic fraction responsible for the degradation of the signal peptide have been identified and purified to near homogeneity. The major activity is associated with a monomeric protein with a molecular weight of 68,000 (S.E. 3,400) as determined by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme appears to be similar to the oligopeptidase (Vimr, E. R., Green, L., and Miller, C. G. (1983) J. Bacteriol. 153, 1259-1265) that hydrolyzes N-acetyl tetra alanine. The second protein represents approximately 5% of the total cytoplasmic activity and has been shown to be a dimer with a monomer molecular weight of 81,000 (S.E. 5,300). This enzyme is similar to protease So (Chung, H. C., and Goldberg, A. L. (1983) J. Bacteriol. 154, 231-238).

Chromatography, DEAE-Cellulose↗
PubMed1985

Inhibition of prolipoprotein signal peptidase by globomycin.

Globomycin inhibits the prolipoprotein-specific signal peptidase activity by binding to the enzyme in a noncompetitive manner (Ki = 36 nM). The Km of prolipoprotein signal peptidase for the prolipoprotein substrate is 6 (+/- 1) microM.

Anti-Bacterial Agents↗
PubMed1985

In vivo and in vitro synthesis of Escherichia coli maltose-binding protein under regulatory control of the lacUV5 promoter-operator.

It has not been possible to obtain in vitro expression of the positively regulated malE gene encoding the periplasmic maltose-binding protein (MBP) of Escherichia coli. To facilitate in vitro malE expression, we constructed plasmids that place the malE gene under transcriptional control of the lacUV5 promoter-operator. These plasmids could be grouped into three classes, based upon their ability to complement in vivo a chromosomal malE deletion in the presence or absence of isopropyl thiogalactoside. In the one class I plasmid analyzed, the lacUV5-malE junction was just 3' to the malE ATG initiation codon, and this plasmid did not complement the malE deletion. Class II and class III plasmids retained various amounts of the malE promoter. MBP synthesis was solely under control of the lacUV5 promoter in the class II plasmids, and MBP synthesis was under control of both the lacUV5 and malE promoters in the class III plasmids. A malE mutation that renders the MBP signal peptide export defective was genetically recombined onto one of the class II plasmids. The in vivo synthesis and export of plasmid-encoded MBP were studied in the presence and absence of isopropyl thiogalactoside and maltose and in a strain harboring a prlA mutation that suppresses the malE signal sequence mutation and is thought to alter the export machinery of cells. In addition, both class II and class III plasmids programmed the synthesis of precursor MBP in an in vitro-coupled transcription-translation system. When precursor MBP was synthesized in vitro in the presence of E. coli membrane vesicles, a significant portion of wild-type precursor MBP, but not export-defective precursor MBP, was converted to a form that migrated on sodium dodecyl sulfate-polyacrylamide gels identically to mature MBP synthesized in vivo.

ATP-Binding Cassette Transporters↗
PubMed1984

Rapid assay and purification of a unique signal peptidase that processes the prolipoprotein from Escherichia coli B.

A simple and accurate assay for prolipoprotein signal peptidase activity has been described that is based on the solubility of the signal peptide in 80% acetone. The unprocessed precursor and the mature form of the lipoprotein are quantitatively recovered in the precipitate. The signal peptide, from the acetone supernatant utilizing the purified signal peptidase, contains labeled methionine at its NH2 terminus and has Mr = 2200 (S.E. = 69). A specific signal peptidase that processes the modified form of Braun's prolipoprotein to its correct mature form has been purified. This enzyme is globomycin sensitive and has been purified 35,000-fold from the membranes of Escherichia coli by extraction at pH 4.0 with 2% Triton X-100 and heating, followed by conventional column chromatography at room temperature. This prolipoprotein signal peptidase has a pH optimum at 6.0, is not inhibited by EDTA, and requires 1 mM dithiothreitol for stability. The monomer molecular weight of this specific signal peptidase is 17,800 (S.E. = 900) as determined by sodium dodecyl sulfate-gel electrophoresis.

Bacterial Outer Membrane Proteins↗
PubMed1984

Inhibition of arabinose 5-phosphate isomerase. An approach to the inhibition of bacterial lipopolysaccharide biosynthesis.

Arabinose 5-phosphate ( A5P ) isomerase is a key enzyme in the biosynthesis of lipopolysaccharide, an essential component of the outer membrane of Gram-negative bacteria. The mechanism of the isomerase is envisioned to involve an enediol intermediate. A series of compounds, which are analogues of the substrates or intermediate, were tested as inhibitors of A5P isomerase with the belief that a good inhibitor would stop bacterial growth or render the cells more susceptible to other antibiotics or natural defenses. In a series of phosphorylated sugars, the order of isomerase inhibitory activity was as follows: aldonic acids greater than alditols greater than aldoses. Nonphosphorylated sugars were much less inhibitory. The best inhibitor was erythronic acid 4-phosphate (54), which had Km/Ki = 29. None of the compounds displayed antibacterial activity in vitro.

Aldose-Ketose Isomerases↗
PubMed1981

Purification and characterization of cytidine 5'-triphosphate:cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase.

Cytidine 5'-triphosphate:cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) was purified 2,300-fold from frozen Escherichia coli B cells. The enzyme catalyzed the formation of CMP-KDO, a very labile product, from CTP and KDO. No other sugar tested could replace KDO as an alternate substrate. Uridine 5'-triphosphate at pH 9.5 and deoxycytidine 5'-triphosphate at pH 8.0 and 9.5 could be used as alternate substrates in place of CTP. CMP-KDO synthetase required Mg2+ at a concentration of 10.0 mM for optimal activity. The pH optimum was determined to be between 9.6 and 9.3 in tris(hydroxymethyl)aminomethane-acetate or sodium-glycine buffer. This enzyme had an isoelectric point between pH 4.15 and 4.4 and appeared to be a single polypeptide chain with a molecular weight of 36,000 to 40,000. The apparent Km values for CTP and KDO in the presence of 10.0 mM Mg2+ were determined to be 2.0 X 10(-4) and 2.9 X 10(-4) M, respectively, at pH 9.5. Uridine 5'-triphosphate and deoxycytidine 5'-triphosphate had apparent Km values of 8.8 X 10(-4) and 3.4 X 10(-4) M. respectively, at pH 9.5.

Cytidine Monophosphate↗
PubMed1980

Purification and characterization of 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli.

3-Deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase has been purified 450-fold from frozen Escherichia coli B cells. The purified enzyme catalyzed the stoichiometric formation of KDO-8-phosphate and Pi from phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate. The enzyme showed no metal requirement for activity and was inhibited by 1 mM Cd2+, Cu2+, Zn2+, and Hg2+. The inhibition by Hg2+ could be reversed by dithiothreitol. The optimum temperature for enzyme activity was determined to be 45 degrees C, and the energy of activation calculated by the Arrhenius equation was 15,000 calories (ca. 3,585 J) per mol. The enzyme activity was shown to be pH and buffer dependent, showing two pH optima, one at pH 4.0 to 6.0 in succinate buffer and one at pH 9.0 in glycine buffer. The isoelectric point of the enzyme was 5.1. KDO-8-phosphate synthetase had a molecular weight of 90,000 +/- 6,000 as determined by molecular sieving through G-200 Sephadex and by Ferguson analysis using polyacrylamide gels. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 90,000-molecular-weight native enzyme was composed of three identical subunits, each with an apparent molecular weight of 32,000 +/- 4,000. The enzyme had an apparent Km for D-arabinose-5-phosphate of 2 X 10(-5) M and an apparent Km for PEP of 6 X 10(-6) M. No other sugar or sugar-phosphate could substitute for D-arabinose-5-phosphate. D-Ribose-5-phosphate was a competitive inhibitor of D-arabinose-5-phosphate, with an apparent Ki of 1 X 10(-3) M. The purified enzyme has been utilized to synthesize millimole quantities of pure KDO-8-phosphate.

Aldehyde-Lyases↗
PubMed1980

Purification and characterization of specific 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase from Escherichia coli B.

A phosphatase specific for the hydrolysis of 3-deoxy-d-manno-octulosonate (KDO)-8-phosphate was purified approximately 400-fold from crude extracts of Escherichia coli B. The hydrolysis of KDO-8-phosphate to KDO and inorganic phosphate in crude extracts of E. coli B, grown in phosphate-containing minimal medium, could be accounted for by the enzymatic activity of this specific phosphatase. No other sugar phosphate tested was an alternate substrate or inhibitor of the purified enzyme. KDO-8-phosphate phosphatase was stimulated three- to fourfold by the addition of 1.0 mM Co(+) or Mg(2+) and to a lesser extent by 1.0 mM Ba(2+), Zn(2+), and Mn(2+). The activity was inhibited by the addition of 1.0 mM ethylenediaminetetraacetic acid, Cu(2+), Ca(2+), Cd(2+), Hg(2+), and chloride ions (50% at 0.1 M). The pH optimum was determined to be 5.5 to 6.5 in both tris(hydroxymethyl)aminomethane-acetate and HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer. This specific phosphatase had an isoelectric point of 4.7 to 4.8 and a molecular weight of 80,000 +/- 6,000 as determined by molecular sieving and Ferguson analysis. The enzyme appeared to be composed of two identical subunits of 40,000 to 43,000 molecular weight. The apparent K(m) for KDO-8-phosphate was determined to be 5.8 +/- 0.9 x 10(-5) M in the presence of 1.0 mM Co(2+), 9.1 +/- 1 x 10(-5) M in the presence of 1.0 mM Mg(2+), and 1.0 +/- 0.2 x 10(-4) M in the absence of added Co(2+) or Mg(2+).

Cations↗
PubMed1972

Effect of glycerol deprivation on the phospholipid metabolism of a glycerol auxotroph of Staphylococcus aureus.

A study of the effects of glycerol deprivation on the content and metabolism of the phospholipids of a glycerol auxotroph of Staphylococcus aureus showed that (i) there was an increase in the proportions of lysylphosphatidylglycerol (LPB) and a concomitant decrease in the proportion of phosphatidylglycerol. The total phospholipid content per sample and the proportion of cardiolipin did not change, but the phosphatidic acid increased transiently and then fell to pretreatment levels. (ii) The loss of (32)P from the lipids during the chase in a pulse-chase experiment was essentially the same in phosphatidylglycerol, cardiolipin, and phosphatidic acid during glycerol deprivation or growth in the presence of glycerol. LPG lost half the radioactivity in slightly more than two doubling times when grown with glycerol. In the absence of glycerol, (32)P accumulated in LPG for about 20 min and then stopped, after which time there was no apparent turnover. (iii) During glycerol deprivation, the initial (32)P incorporation decreased sixfold compared to that of the control with glycerol. The initial incorporation into LPG decreased only 2.5-fold, whereas that of PG decreased 45-fold. (iv) During glycerol deprivation, the free fatty acid content increased from 1.2 to 12.5% of the total extractable fatty acids and then slowly decreased. The increase was largely iso- and anti-iso-branched 21-carbon-atom fatty acids. In glycerol-supplemented cultures, the major fatty acids were branched 14- to 18-carbon fatty acids. The decrease in longer chain free fatty acids after 60 min represented their esterification into lipids. (v) During glycerol deprivation ribonucleic acid synthesis and cell growth continued for 40 min and protein synthesis continued for 90 min. Then synthesis and growth stopped. (vi) After the addition of glycerol to glycerol-deprived cells, (32)P and (14)C-glycerol were incorporated into the phospholipids without lag; ribonucleic acid, protein synthesis, and cell growth began after a 5- to 10-min lag at the pretreatment rate. The initial rate of lipid synthesis after the addition of glycerol was three times greater than the growth rate. This rapid rate continued for about 25 min until the lipid content and proportions of LPG and phosphatidylglycerol were restored.

Bacterial Proteins↗
PubMed1972

Consequences of glycerol deprivation on the synthesis of membrane components in a glycerol auxotroph of Staphylococcus aureus.

In a glycerol auxotroph of Staphylococcus aureus, the deprivation of glycerol affected the formation of certain membrane components. (i) There was synthesis of fatty acids at the predeprivation rate even though the fatty acids synthesized accumulated as free fatty acids rather than as esterified fatty acids; (ii) there was a complete cessation of phospholipid and vitamin K isoprenologue biosynthesis; (iii) there was conservation of the glycerol esters of the complex phospholipids and glucolipids; (iv) there was an immediate decrease in the rate of synthesis of monoglucoslydiglyceride (30%) and diglucosyldiglyceride (60%); (v) there was a 50% decrease in the rate of synthesis of the polar and nonpolar carotenoids; (vi) there was synthesis of protoheme, heme a, and nonspecific membrane protein at the predeprivation rate; and (vii) there was an abrupt cessation in the formation of new, functional glycine transport activity.

Acetates↗
PubMed1971

Effect of temperature on the fatty acid composition of Thermus aquaticus.

Thermus aquaticus contains four major fatty acids, iso-C(15) (28%), iso-C(16) (9%), normal-C(16) (13%), and iso-C(17) (48%), when grown at 70 C, as determined by gas chromatography and mass spectrometry. Small amounts of iso-C(12), normal-C(12:1), iso-C(13), normal-C(14), iso-C(14), and normal-C(15:1) were also detected. A change in growth temperature (50 to 75 C at 5-C intervals) affects a shift in the proportions of some of the fatty acids. The proportions of the monoenoic and branched-C(17) fatty acids decreased and the proportions of the higher-melting iso-C(16) and normal-C(16) fatty acids increased. Cells grown at 75 C contained 70% more total fatty acids than cells grown at 50 C. The largest increases, in absolute amounts, were in the content of iso-C(16) and normal-C(16) fatty acids, with only a 1.6-fold increase in the major iso-C(15) and iso-C(17) fatty acids. There was a 2.5-fold decrease in normal-C(15:1) and at least a 24-fold decrease in anteiso-C(17), which is present at 50 and 55 C but not at higher temperatures. There was no difference in proportion or amount of fatty acids between exponential and stationary-phase cells grown at 70 C. When cells were grown on glutamate instead of yeast-extract and tryptone at 70 C, the total fatty acid content remained constant, but there was an increase in the proportions of iso-C(16) and normal-C(16) fatty acids concomitant with a decrease in the proportions of the iso-C(15) and iso-C(17) fatty acids.

Bacteria↗
PubMed1971

Effect of growth temperature on the lipid composition of Thermus aquaticus.

The complex lipids of Thermus aquaticus include phospholipids, glucolipids, carotenoids, and vitamin K(2) isoprenologues. The phospholipids account for 30% of the total lipids and have been identified as phosphatidylethanolamine (4%), phosphatidylglycerol (3%), phosphatidylinositol (10%), cardiolipin (3%), and phosphatidic acid (1%). The major phospholipid contained three fatty acids, a long-chain unsaturated amine, and one glycerol per phosphate and accounted for 80% of the lipid phosphate. The carotenoids accounted for 60% of the membrane lipid. The majority of the carotenoids were very polar. Mono- and diglucosyldiglyceride and the 35-, 40-, and 45-carbon vitamin K(2) isoprenologues were also identified. All these lipids were localized in the membrane of T. aquaticus. When the growth temperature was increased from 50 to 75 C and measured at 5 C intervals, there was a progressive increase in the total lipid content. The phospholipids increased 2-fold, the carotenoids increased 1.8-fold, and the glucolipids increased 4-fold between cells grown at 50 C and 75 C. The vitamin K(2) level did not change. The proportions of the individual lipids within each lipid class remained constant as the temperature of growth was raised. Metabolic studies indicated turnover of the diacyl phospholipids during pulse-chase experiments at rates comparable with mesophilic bacteria. The major phospholipid and the carotenoids did not turn over.

Acetone↗
PubMedDate not supplied

Identification, cloning, and purification of protein antigens of Treponema pallidum.

Difficulties in culturing the bacterium Treponema pallidum have greatly hindered syphilis research. In recent years, several laboratories have begun applying recombinant DNA technology to the study of this organism. Recent work is summarized concerning the expression of T. pallidum DNA in Escherichia coli. A number of E. coli clones expressing treponemal protein antigens have been identified. In one instance, a recombinant protein was purified to homogeneity and shown to be identical to a highly immunogenic, native T. pallidum membrane protein of molecular weight 39,000, which was designated the basic membrane protein (BMP) of this organism. In addition, recent experiments are described that were designed to identify cell-surface proteins that would serve as the primary focus of our cloning efforts. Results obtained with use of several different approaches strongly suggest that the outer membrane of T. pallidum is an antigenically inert structure largely devoid of protein. However, a class of low-molecular-weight protein antigens have been identified that are actively secreted into the extracellular medium. Attempts currently are being made to clone these secreted proteins and investigate their roles in the pathogenesis and immunobiology of syphilis.

Animals↗