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I R Phillips

Publications and source records attributed to I R Phillips.

At least 37 records · Page 2Linked to original sources
PubMed1995

Evidence for the importance of weakly bound water for matrix metalloproteinase activity.

The effects of organic cosolvents on the kinetic characteristics of two matrix metalloproteinases, gelatinase A and stromelysin 1, were investigated. In each case, addition of the cosolvent resulted in a decrease in the apparent kcat/Km for the catalyzed hydrolysis of fluorogenic peptide substrates. Two factors were identified as being responsible for this decrease in catalytic activity: hydrophobic partitioning of the substrate in favor of the bulk solvent and decrease in the water content of the enzyme. The former reflects the hydrophobic nature of the enzyme-substrate interaction and the effect can be corrected for by using the solvent to water partition coefficient of the substrate in the mixed solvent systems. The catalyzed hydrolysis of substrate, corrected for the effect of hydrophobic partitioning, was demonstrated to be sixth order in water for gelatinase A and third order in water for stromelysin 1. Variation in water concentration did not produce saturation even at concentrations close to 55.5 M. The results indicate that weakly bound water molecules are essential to mediate the interaction between substrate and enzyme. The sensitivity of these enzymes to water concentration could be an important mechanism for regulating catalytic activity in vivo.

Amino Acid Sequence↗
PubMed1995

Effects of the anticonvulsant, valproate, on the expression of components of the cytochrome-P-450-mediated monooxygenase system and glutathione S-transferases.

It has been shown previously that the anticonvulsant agent, sodium valproate, induces certain cytochrome P-450 monooxygenase activities and decreases glutathione S-transferase activity. We have used Western blotting, RNase protection assays and Northern blot hybridization to determine the effects of valproate on the abundance of individual components of the cytochrome P-450 monooxygenase and of glutathione S-transferase subunits. Due to the short half-life of the drug in rats we have used an in vitro experimental system comprised of rat hepatocytes co-cultured with rat primitive biliary epithelial cells. Valproate was shown to be a potent inducer of two members of the cytochrome P-450 (CYP)2B subfamily, CYP2B1 and 2B2. The induction of the proteins was mediated at the level of the mRNAs, with the mRNA for CYP2B1 being more highly induced than that for CYP2B2. The drug also induced, but to a much lesser extent, two important components of the cytochrome-P-450-mediated monooxygenase system, NADPH-dependent cytochrome P-450 reductase and cytochrome b5, and their corresponding mRNAs. Thus, the effects of valproate on cytochromes P-450 and other components of the cytochrome-P450-mediated monooxygenase system mimic those of another, structurally diverse, antiepileptic drug, phenobarbital. However, in contrast to phenobarbital, which induces glutathione S-transferase subunits 1, 2, 3, 4 and 7, valproate selectively decreases the abundance of subunits 3 and/or 4. It has been shown previously that CYP2B1 is involved in the production of metabolites of valproate implicated in hepatotoxicity. The induction of this protein by valproate would thus contribute substantially to the hepatotoxic effects associated with the drug.

Animals↗
PubMed1995

The molecular biology of the flavin-containing monooxygenases of man.

cDNA clones encoding five distinct members of the FMO family of man (FMOs 1, 2, 3, 4 and 5) were isolated by a combination of library screening and reverse transcription-polymerase chain reaction techniques. The deduced amino acid sequences of the human FMOs have 82-87% identity with their known orthologues in other mammal but only 51-57% similarity to each other. The hydropathy profiles of the proteins are very similar. From the calculated rate of evolution of FMOs (a 1% change in sequence per 6 million years) it would appear that individual members of the FMO gene family arose by duplication of a common ancestral gene some 250-300 million years ago. Each of the FMO genes was mapped by the polymerase chain reaction to the long arm of human chromosome 1. The localization of the FMO1 gene was further refined to 1q23-q25 by in situ hybridization of human metaphase chromosomes. RNase protection assays demonstrated that in man each FMO gene displays a distinct developmental and tissue-specific pattern of expression. In the adult, FMO1 is expressed in kidney but not in liver, whereas in the foetus its mRNA is abundant in both organs. FMO3 expression is essentially restricted to the liver in the adult and the mRNA is either absent, or present in low amounts, in foetal tissues. FMO4 is expressed more constitutively. Human FMO1 and FMO3 cDNAs were functionally expressed in prokaryotic and eukaryotic cells. FMO1 and FMO3, expressed in either system, displayed product stereoselectivity in their catalysis of the N-oxidation of the pro-chiral tertiary amines, N-ethyl-N-methylaniline (EMA) and pargyline. Both enzymes were stereoselective with respect to the production of the (-)-S-enantiomer of EMA N-oxide. But in the case of pargyline, the enzymes displayed opposite stereoselectivity, FMO1 producing solely the (+)-enantiomer and FMO3 predominantly the (-)-enantiomer of the N-oxide.

Amino Acid Sequence↗
PubMed1995

The second zinc atom in the matrix metalloproteinase catalytic domain is absent in the full-length enzymes: a possible role for the C-terminal domain.

Domain deletion mutants of the matrix metalloproteinases consisting of the catalytic domain only contain two zinc atoms per molecule. One is essential for catalysis, while the other may fulfil a structural role. We have analysed the zinc contents of both the full-length and the truncated mutants of prostromelysin-1 and progelatinase A and report that the second zinc atom is not present in the full-length form of the proenzymes. Thus it seems likely that the role proposed for this zinc atom in maintaining the structure of the enzyme catalytic domain is performed by the C-terminal domain in the full-length enzyme.

Amino Acid Sequence↗
PubMed1994

A nomenclature for the mammalian flavin-containing monooxygenase gene family based on amino acid sequence identities.

A nomenclature based on comparisons of amino acid sequences is proposed for the members of the mammalian flavin-containing monooxygenase (FMO) gene family. This nomenclature is based on evidence of a single gene family composed of five genes. The percentage identities of the amino acid sequences of the five known forms of mammalian FMO are between 52 and 57% in rabbit and between 50 and 58% across species lines. The identities of all orthologs are greater than 82%. There is no evidence for multiple, highly related forms of the enzyme or for more than one mammalian FMO gene family. In the proposed system, the mammalian flavin-containing monooxygenase gene family is designated as "FMO" and the individual genes are distinguished by an Arabic numeral. The FMOs known as the "liver" and "lung" enzymes become FMO1 and FMO2, and the more recently described forms of the enzymes become FMO3, FMO4, and FMO5. Human FMO gene designations, FMO1 and FMO3, remain unchanged, but the gene designated FMO2 becomes FMO4. Following convention, the genes and cDNA designations will be italicized and the mRNA and protein designations will be nonitalicized. The purpose of the proposed nomenclature is to provide for the unambiguous identification of orthologous forms of mammalian FMOs, regardless of the species or tissue in question. The proposed classification considers only members of the mammalian flavin-containing monooxygenase gene family and has no bearing on the generally accepted definition of a multisubstrate flavin-containing monooxygenase.

Amino Acid Sequence↗
PubMed1994

The pyruvate dehydrogenase complex: cloning of the rat somatic E1 alpha subunit and its coordinate expression with the mRNAs for the E1 beta, E2, and E3 catalytic subunits in developing rat brain.

We report the isolation of cDNA clones encoding the somatic form of the E1 alpha subunit of the pyruvate dehydrogenase complex of rat. The deduced amino acid sequence has 99.5, 98, and 97% identity, respectively, with the orthologous proteins of mouse, human, and pig and 98.5% identity with a rat E1 alpha sequence reported previously. The cDNAs isolated in this and earlier studies predict different E1 alpha subunit mRNA sizes and amino acid sequences. These differences have been investigated by PCR, northern blot hybridization, and RNase protection. We have used our E1 alpha cDNA, in conjunction with cDNA probes to the E1 beta, E2, and E3 catalytic subunits of rat pyruvate dehydrogenase complex and also to rat citrate synthase, to perform RNase protection assays of developing rat whole brain RNA. The results show a 2.5-fold increase in the concentration of each of the subunit mRNAs and a 1.2-fold increase in citrate synthase mRNA from late foetal stage to 5 days post partum. Thereafter, the mRNA levels remained constant. These data indicate that the respective six- and threefold increases in the amounts of pyruvate dehydrogenase complex and citrate synthase found to occur in rat brain between birth and adulthood are mediated principally by translational and/or posttranslational mechanisms.

Aging↗
PubMed1994

Interaction of proteins with a cytochrome P450 2B2 gene promoter: identification of two DNA sequences that bind proteins that are enriched or activated in response to phenobarbital.

Cytochromes P450 (CYPs) are of central importance in the metabolism of foreign hydrophobic compounds. Members of the CYP2B subfamily are inducible at the transcriptional level by the barbiturate, phenobarbital. Owing to the lack of a suitable phenobarbital-responsive cell line, very little is known regarding the mechanisms by which phenobarbital induces the expression of these genes. We report the use of gel retardation and DNase I footprinting to investigate the presence of regulatory protein binding sites within a CYP2B2 gene promoter. Two DNA sequences, located between -183 to -199 and -31 to -72, have been identified that bind rat liver nuclear proteins that are enriched or activated in vivo by phenobarbital. Gel retardation competition experiments demonstrated that the two sequences bound different proteins. In vitro transcription competition experiments demonstrated that the sequences and the proteins with which they interact are involved in regulating CYP2B2 gene transcription. These two DNA sequences and their cognate binding proteins may play a role in the induction of CYP2B2 gene expression in response to phenobarbital.

Animals↗
PubMed1993

Characterization of cDNAs encoding the rat testis-specific E1 alpha subunit of the pyruvate dehydrogenase complex: comparison of expression of the corresponding mRNA with that of the somatic E1 alpha subunit.

cDNA clones encoding the testis-specific form of the rat pyruvate dehydrogenase complex E1 alpha subunit have been isolated. Comparison of the predicted amino acid sequence with those of the somatic and testis-specific E1 alpha forms of man and mouse and the somatic E1 alpha form of rat indicates the change of a serine residue, believed to be phosphorylated in vivo by pyruvate dehydrogenase E1 alpha-specific kinase, to an alanine at position 233. The implications of this change are discussed. Northern blot analysis and RNase protection assays indicate that the expression of mRNA encoding testis-specific E1 alpha subunit is restricted to testis whereas mRNA for the somatic form is found in all tissues analyzed, albeit in very small amounts in testis.

Amino Acid Sequence↗
PubMed1993

The activity of the tissue inhibitors of metalloproteinases is regulated by C-terminal domain interactions: a kinetic analysis of the inhibition of gelatinase A.

The cloning and expression of the full-length tissue inhibitor of metalloproteinase 2 (TIMP-2), delta 187-194TIMP-2, and delta 128-194TIMP-2 and the purification of these inhibitors and a cleaved version of TIMP-2 lacking nine C-terminal amino acids (delta 186-194TIMP-2) are described. The mechanism of inhibition of gelatinase A by the TIMPs was investigated by comparing the kinetics of association of TIMP-1, TIMP-2, the C-terminal deletions, and the mutants of both TIMPs which consisted of the N-terminal domain only. The full-length TIMPs inhibited gelatinase A rapidly with association constants of 3.2 x 10(6) M-1 s-1 for TIMP-1 and 2.1 x 10(7) M-1 s-1 for TIMP-2 at I = 0.2. The C-terminal peptide of TIMP-2 is proposed to exist as an exposed "tail" responsible for binding to progelatinase A and for increasing the rate of inhibition of active gelatinase A through electrostatic interactions with the C-terminal domain of the enzyme. The C-terminal domains of both TIMP-1 and TIMP-2 participate in low-affinity interactions with the C-terminal domain of gelatinase A which increase the rate of association by a factor of about 100 in both cases.

Amino Acid Sequence↗
PubMed1993

Maintenance and induction in co-cultured rat hepatocytes of components of the cytochrome P450-mediated mono-oxygenase.

Hepatocytes grown in culture rapidly lose many of the cytochromes P450 (CYP) responsible for metabolizing foreign compounds. Among the proteins most readily lost are members of the CYP2B subfamily. We have investigated, by RNase protection assays, the ability of rat hepatocytes, cultured conventionally or co-cultured with rat liver epithelial cells, to maintain the expression of genes encoding members of the CYP2B subfamily, and the inducibility of this expression by phenobarbital. After 4 days of conventional hepatocyte culture CYP2B mRNAs were undetectable, but remained inducible by phenobarbital. In co-cultured hepatocytes the abundance of the mRNAs remained relatively constant from 4-14 days. After 7 days of co-culture the concentration of the mRNAs was increased 12-15-fold by phenobarbital. RNase protection assays with probes capable of distinguishing between CYP2B1 and 2B2 mRNAs demonstrated that the ratios of the abundance and inducibility of the two mRNAs were the same in co-culture as in vivo. Co-cultured hepatocytes also maintained the expression of genes coding for two other components of the cytochrome P450-mediated mono-oxygenase, namely cytochrome P450 reductase and cytochrome b5.

Animals↗
PubMed1993

Localization of genes encoding three distinct flavin-containing monooxygenases to human chromosome 1q.

We have used the polymerase chain reaction to map the gene encoding human flavin-containing monooxygenase (FMO) form II (N. Lomri, Q. Gu, and J. R. Cashman, 1992, Proc. Natl. Acad. Sci. USA 89: 1685-1689) to chromosome 1. We propose the designation FMO3 for this gene as it is the third FMO gene to be mapped. The two other human FMO genes identified to date, FMO1 and FMO2, are also located on chromosome 1 (C. Dolphin, E. A. Shephard, S. Povey, C. N. A. Palmer, D. M. Ziegler, R. Ayesh, R. L. Smith, and I. R. Phillips, 1991, J. Biol. Chem. 266: 12379-12385; C. Dolphin, E. A. Shephard, S. F. Povey, R. L. Smith, and I. R. Phillips, 1992, Biochem. J. 286: 261-267). The localization of FMO1, FMO2, and FMO3 has been refined to the long arm of chromosome 1. Analysis of human metaphase chromosomes by in situ hybridization confirmed the mapping of FMO1 and localized this gene more precisely to 1q23-q25.

Base Sequence↗
PubMed1992

Cloning, primary sequence and chromosomal localization of human FMO2, a new member of the flavin-containing mono-oxygenase family.

We have previously reported the cloning of cDNAs for a flavin-containing mono-oxygenase (FMO) of man, designated FMO1 [Dolphin, Shephard, Povey, Palmer, Ziegler, Ayesh, Smith & Phillips (1991) J. Biol. Chem. 266, 12379-12385], that is the orthologue of pig and rabbit hepatic FMOs. We now describe the isolation and characterization of cDNA clones for a second human FMO, which we have designated FMO2. The polypeptide encoded by the cDNAs is 558 amino acid residues long, has a calculated M(r) of 63337, and contains putative FAD- and NADP-binding sites that align exactly with those described in other mammalian FMOs. Human FMO2 has 51-53% primary sequence identity with human FMO1, rabbit pulmonary FMO and rabbit liver FMO form 2, and thus represents a fourth, distinct, member of the mammalian FMO family. The corresponding mRNA is present in low abundance in adult human liver. Southern blot hybridization with single-exon probes demonstrated that human FMO2 and FMO1 are the products of single genes. The gene encoding FMO2 (designated FMO2) was mapped, by the polymerase chain reaction, to human chromosome 1, the same chromosome on which FMO1 is located.

Amino Acid Sequence↗