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Biomedical subjects

C S Jones

Publications and source records attributed to C S Jones.

At least 109 records · Page 6Linked to original sources
PubMed1985

The structure of the mer operon.

The DNA sequence has been determined for a 3.8-kb region which encodes the mercury-resistance (mer) operon of the IncFII plasmid NR1. The sequence reveals 4 open reading frames which could encode proteins of 12,522, 9,429, 14,965, and 58,912 d corresponding to the 4 previously described Hg-inducible proteins detected in minicells carrying mer+ plasmids. The Hg(II) reductase protein sequence is about 90% homologous to that of Tn501, but the DNA sequence shows a homology of 60-70% to that of Tn501 except for short regions of very high homology. The entire mer region is 63.4% G-C overall. The region encoding the merR (positive regulatory) function has 3 possible open reading frames, 2 of which overlap in one direction and the third of which reads in the opposite direction. Attempts to visualize the polypeptide(s) encoded by the merR cistron were unsuccessful.

Bacteria↗
PubMed1985

PSE-4 beta lactamase: a serotype-specific enzyme in Pseudomonas aeruginosa.

PSE-4 enzyme is the most common plasmidic beta lactamase in Pseudomonas aeruginosa but its production is invariably non-transferable by conjugation. Of 20 PSE-4+ isolates from 10 separate sources, 16 were serotype O:16 and two belonged to the related O:2(b) serotype. One of the two remaining organisms was not O-typable and the other was agglutinated by several unrelated antisera. Examination of additional O:16 strains confirmed the unusual frequency of PSE-4 enzyme in this serotype. None of the PSE-4+ strains was able to transfer carbenicillin resistance in mating experiments and none contained extrachromosomal DNA. Two explanations of the relationship between enzyme production and O antigenicity are proposed. PSE-4 production may be transmissible, perhaps by transduction, between strains of O:16 or related serotypes, or PSE-4+ P. aeruginosa may represent a disseminated subtype. A third hypothesis, that the PSE-4 coding element carried serotype determinants, was discounted. PSE-4+ and PSE-4- P. aeruginosa strains of O:16 and related serotypes were found to represent a definite cluster by their phage-susceptibility pattern and pyocin type (type 1). The only characters linked to PSE-4 production were resistance to spectinomycin, streptomycin and sulphonamide and the genes responsible for these characters seemed to occur on the PSE-4 coding element.

Anti-Bacterial Agents↗
PubMed1984

Human placental beta-galactosidase: structural and immunological observations.

beta-Galactosidase purified to apparent homogeneity from human placenta occurred in two separable fractions. A low molecular mass form (relative mass (Mr) 170 000) is composed of a single polypeptide chain (Mr 70 000). This was derived from a larger form by molecular sieve chromatography at both low (10 mM) and high (500 mM) NaCl concentration. The larger form of beta-galactosidase also contains small amounts of two polypeptides with apparent Mr values of 23 000 and 35 000 daltons. Both forms of the enzyme hydrolyze synthetic aryl galactosides and natural glycolipid substrates at comparable rates. Antibodies raised in rabbits against the low Mr beta-galactosidase also cross-reacts with the high Mr enzyme. The antibody preparation also cross-reacted with beta-hexosaminidase even though the latter was found at very low levels in the antigen, as judged by lack of detection of representative protein bands on sodium dodecyl sulfate - polyacrylamide gel electrophoresis and enzyme activity measurements. A portion of this cross-reactivity (35%) against beta-hexosaminidase could not be absorbed from the preparation without the simultaneous loss of beta-galactosidase activity, suggesting that the two enzymes show a degree of antigenic identity.

Amino Acids↗
PubMed1984

The DNA sequence of the mercury resistance operon of the IncFII plasmid NR1.

The DNA sequence has been determined for a 3.8-kilobase region which encodes the mercury resistance (mer) operon of the IncFII plasmid NR1. The sequence reveals four open reading frames which could encode proteins of 12,391, 9,429, 14,965, and 58,781 daltons. On the basis of their sizes, amino acid compositions, hydropathicities, and estimated isoelectric points, the peptides encoded by these open reading frames correspond to the four previously described Hg-inducible proteins detected in minicells carrying mer+ plasmids. The NR1 mer locus is 63.4% GC overall, and the Hg(II) reductase protein sequence is 90% homologous to that of Tn501. The region encoding the merR (positive regulatory) function has three open reading frames. The smallest of these possible merR peptides (6,457 daltons) begins approximately 280 bp to the right of the adjacent IS1b and reads towards the structural genes of the mer operon. The next largest reading frame (13,139 daltons) in the merR region begins 37 bp to the left of the beginning of the smallest peptide and also reads towards the structural genes. The largest reading frame (15,907 daltons) in the merR region lies on the complementary strand and reads away from the structural genes towards IS1b. Although attempts to visualize the merR gene product were not successful, in vitro mutagenesis allows us to eliminate the largest reading frame as a merR candidate. We were also able to show that approximately 50% of the smallest detectable mer peptide (9,429 daltons) is located in the periplasm.

Bacterial Proteins↗
PubMed1983

Enzymatic hydrolysis of sphingomyelin liposomes by normal tissues and tissues from patients with Niemann-Pick disease.

Liposomes of [3H]sphingomyelin are readily hydrolyzed by extracts of human spleen, liver, cultured skin fibroblasts and purified placental sphingomyelinase in the absence of detergents. The pH optimum for hydrolysis by liver and spleen extracts was 6.5-7.0 while the fibroblast activity showed an optimum at pH 4.0-4.3. However, the pH optimum for purified placental sphingomyelinase in the presence of Triton X-100 (pH 5.0) is only slightly different from that displayed with liposomes (pH 5.3). The data clearly show that hydrolysis of liposomal sphingomyelin by sphingomyelinase is affected by the composition and purity of the enzyme source.

Female↗
PubMed1983

Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing.

Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100.

Amino Acids↗
PubMed1983

The active site of lysosomal sphingomyelinase: evidence for the involvement of hydrophobic and ionic groups.

The natural substrate for sphingomyelinase contains hydrophobic and polar moieties. In this study, we have employed pH rate studies and examined hydrophobic compounds and phosphorylated esters for their effect on sphingomyelinase activity in an attempt to determine some of the kinetic properties of this enzyme. Sphingomyelinase, purified from human placentae, undergoes noncompetitive inhibition by octylglucoside and Nonidet P-40, two nonionic detergents containing terminal octyl groups. The effect of these detergents at the hydrophobic binding site is somewhat different from that of Triton X-100, which contains an isooctyl terminal group, and this may serve to identify a structural basis for the effects. Sphingomyelinase activity is also modulated by several nucleotides. Inhibition by 5'-adenosine monophosphate (5'-AMP) is also noncompetitive. Other nucleotide monophosphates (such as 5'-uridine monophosphate (5'-UMP), 5'-cytidine monophosphate (5'-CMP), 2'-adenosine monophosphate (2'-AMP), and 3'-adenosine monophosphate (3'-AMP) and phosphorylated intermediates (such as phosphorylcholine, phosphorylethanolamine and hexose phosphates) have a lower inhibitory effect. The data suggest that the inhibition by 5'-AMP involves the combined effect of the phosphate group and the purine ring, structural requirements which may also be satisfied by bis(4-methylumbelliferyl)phosphate, a synthetic enzyme substrate. Studies of pH rate indicate that the maximal velocity for the hydrolysis of sphingomyelin is independent of pH over the range 3.5-6.2 while the Km value shows a pH dependence. The Km value is lowest from pH 4.0-5.2 and rises at pH values outside this range. The log Vmax/Km and pKm relationships, when plotted as a function of pH, have been used to identify the dissociation constants for the binding of sphingomyelin by the enzyme. These occur at pK values of 4.1 and 5.5. The activity of sphingomyelinase is also reduced when the enzyme is photooxidized in the presence of methylene blue or rose bengal and carbamylated by diethylpyrocarbonate (DEPC). These results are interpreted to show that 1). the enzyme contains a hydrophobic binding site which involves linear aliphatic moieties containing at least eight carbon atoms; 2) two ionic groups are involved in formation of the enzyme substrate complex, one of which is presumed to be the carboxylate group of aspartate or glutamate (represented by pK 4.1) and the second may be the protonated imidazolium group of histidine (represented by pK 5.5); and 3) since the maximal velocity shows no pH dependence, the interactions involving the hydrophobic and ionic groups affect only the binding of the substrate to the enzyme and formation of the enzyme-substrate complex.

Adenosine Monophosphate↗
PubMed1983

Prolactin receptors in the lactating rabbit mammary gland during initiated involution.

The binding of prolactin to receptors in the mammary gland, liver and kidney was studied during initiated involution in the rabbit. The decline in prolactin receptor numbers at the mammary gland was significant after 10 days of involution although affinity for the hormone remained constant. There were no changes in either receptor number or affinity in the liver and kidney tissue. There was no significant differences in plasma prolactin levels in arterial and venous blood samples taken across the mammary gland.

Animals↗
PubMed1983

The effect of initiated involution on enzyme activity and substrate uptake by the mammary gland of the lactating rabbit.

1. Arteriovenous difference studies across the lactating rabbit mammary gland for glucose, acetate, triacylglycerol and non-esterified fatty acids during initiated involution are reported. 2. A significant reduction in substrate utilisation is paralleled by a decrease in the activities of fatty acid synthetase, acetyl CoA synthetase, citrate synthase and glutamate dehydrogenase in biopsy samples taken from the gland. 3. Results from the analysis of lipid fractions within the gland during this period are discussed in relation to lipid resorption.

Acetate-CoA Ligase↗
PubMed1982

Complex kinetics of bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine hydrolysis by purified sphingomyelinase in the presence of Triton X-100.

We have examined the hydrolysis of the synthetic phosphodiesters, bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine, by purified placental sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) in the presence of Triton X-100. Triton X-100 enhanced activity with bis(4MU)phosphate at all concentrations tested. At very low concentrations of detergent, bis(4MU)phosphate hydrolysis approached zero. Our results indicate that bis(4MU)phosphate does not form a micelle with Triton X-100. The observed enhancement of bis(4MU)phosphate activity with Triton X-100 is likely due to a direct effect of detergent on the enzyme itself. HDNP-phosphorylcholine formed its own micelle (or liposome) in the absence of Triton X-100 and, at substrate concentrations below 4 mM, hydrolysis was inhibited by Triton X-100. The extent of this inhibition varied with detergent concentrations but could be totally eliminated at substrate values above 4 mM. For theoretical reasons kinetic constants which could be obtained with the HDNP-phosphorylcholine substrate at concentrations above 4 mM are not considered to be truly representative of the real values. We conclude that neither substrate is recommended to describe the true kinetic parameters pertaining to purified sphingomyelinase. In addition, bis(4MU)phosphate may not be suitable as an aid for diagnosis of sphingomyelinase deficiency states.U

Animals↗
PubMed1981

Purification of sphingomyelinase to apparent homogeneity by using hydrophobic chromatography.

Placental sphingomyelinase has been purified to apparent homogeneity by a procedure that makes extensive use of hydrophobic interaction chromatography on sphingosylphosphocholine-CH-, octyl-, hexyl- and Blue-Sepharoses. Enzyme purification is about 10000- 14000-fold over starting extract with excellent yield (usually greater than 28%). Purification of bis-4-methylumbelliferyl phosphate phosphodiesterase activity generally paralleled that of sphingomyelinase during the final stages of the procedure. The enzyme also hydrolysed bis-p-nitrophenyl phosphate, but at a lower rate compared with bis-4-methylumbelliferyl phosphate. A single major protein was observed under non-denaturing conditions. Sphingomyelinase, denatured by reduction and alkylation, is composed of a major polypeptide chain with an apparent molecular weight of 89 100 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Two minor lower-molecular-weight components were consistently obtained at 47 500 and 30 700. These results were also obtained after maleoylation of the reduced and alkylated sample. The enzyme contains a blocked-N-terminal amino acid. An extensive search for contaminating enzymes revealed the presence of minor amounts of acid phosphatase, which were removed from the final enzyme sample. The highly purified enzyme is stable for several weeks when stored with Triton X-100 at 4 degrees C. The pure enzyme aggregates under denaturing and electrophoretic conditions and special care must be taken to ensure that hydrophobic bonding of the protein is decreased as much as possible. The reproducibility and large scale of this procedure should facilitate further study on the structure and kinetic properties of the enzyme.

Chemical Phenomena↗