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J Ozols

Publications and source records attributed to J Ozols.

At least 37 records · Page 2Linked to original sources
PubMed1994

Isolation and structure of a third form of liver microsomal flavin monooxygenase.

Flavin-containing monooxygenases (FMOs) catalyze NADPH-dependent oxygenation of nucleophilic nitrogen, sulfur, and phosphorous atoms in various drugs, pesticides, and xenobiotics. Two forms of this enzyme have been isolated and characterized from rabbit liver microsomes [Ozols, J. (1989) Biochem. Biophys. Res. Commun. 163, 49-55]. The isolation and the structure of a third isoform (FMO3) is presented here. The isolation procedure for FMO3 included solubilization of liver microsomes with cholate, poly(ethylene glycol) precipitation, chromatography on anion- and cation-exchange and hydroxyapatite columns in the presence of nonionic detergents and glycerol. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, FMO3 exhibited a distinct, single band with a M(r) higher than those of FMO1 and FMO2. FMO3 copurified with a polypeptide complex of high FMO activity. This complex consisted of three polypeptides, named FMO3, FMO1a, and FMO2a. The column chromatographic behavior of FMO1 a and 2a was distinct from that of FMO1 and 2. The electrophoretic mobility of FMO1a was identical to that of FMO1. Automated sequence analysis of this polypeptide complex indicated the presence of only one predominant peptide with an open N-terminus. The derived N-terminal amino acid sequence of some 20 residues was identical to the N-terminus of FMO2. The FMO complex, however, did not contain a polypeptide corresponding to the electrophoretic mobility of FMO2. The N-terminus of FMO3 was blocked by an acetyl residue. Automated Edman degradation of peptides obtained from chemical and enzymatic digests established the amino acid sequence of some 514 residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Lipovitellin 2 beta is the 31 kD Ni(2+)-binding protein (pNiXb) in Xenopus oocytes and embryos.

An Ni(2+)-binding protein (pNiXb, 31 kD) present in mature Xenopus laevis oocytes and in embryos from fertilization in N/F stage 42, was isolated and characterized. After oocytes or embryos were fractionated by PAGE, electroblotted onto nitrocellulose, and probed with 63Ni2+, pNiXb was detected by autoradiography. pNiXb, a yolk protein located in the embryonic gut, was purified from yolk platelets by ammonium sulfate precipitation, delipidation, gel filtration chromatography, and HPLC analysis. During these steps, pNiXb copurified with lipovitellin 2. The N-terminal sequence of purified pNiXb exactly matched that of Xenopus lipovitellin 2 beta, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Since pNiXb and lipovitellin 2 beta agree in N-terminal sequence, amino acid composition, and apparent molecular weight, they appear to be identical. Based on a metal-blot competition assay, the abilities of metal ions to compete with 63Ni2+ for binding to pNiXb were ranked: Zn2+ approximately Cu2+ approximately Co2+ > Cd2+ approximately Mn2+ > Sn2+. This study shows that Xenopus lipovitellin 2 beta is a metal-binding protein in vitro, and raises the possibility that it may function similarly in vivo.

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

Microassay for oligosaccharyltransferase: separation of reaction components by partitioning in detergent solution followed by ultrafiltration.

Oligosaccharyltransferase catalyzes the transfer of oligosaccharide from a lipid dolichol to asparagine acceptor sites on nascent polypeptides. We have developed an assay for this enzyme which is based on the specific distribution of the substrate and product of the reaction in detergent solution. GlcNAc-[3H]GlcNAc-PP-Dol was synthesized for use as a carbohydrate donor. Benzoyl-Asn-Leu-Thr-amide, a commercially available peptide, was used as the oligosaccharyltransferase glycan acceptor substrate. In the presence of Triton X-100, GlcNAc-[3H]GlcNAc-PP-Dol partitions into detergent micelles while glycosylated acceptor peptide partitions into the intermicellar aqueous compartment. Separation of GlcNAc-[3H]GlcNAc-PP-Dol and glycopeptide was achieved by ultrafiltration. With this method oligosaccharyltransferase activity in microsomal preparations could be measured with as little as 1 microgram of protein.

Amino Acid Sequence↗
PubMed1993

Interaction of non-myristoylated NADH-cytochrome b5 reductase with cytochrome b5-dimyristoylphosphatidylcholine vesicles.

An expression vector for NADH-cytochrome b5 reductase containing a thrombin cleavage site directly before the N-terminal glycine residue of the flavoprotein was used to isolate the non-myristoylated enzyme by thrombin cleavage of the initial fusion protein of a short segment of the multiple cloning site of the plasmid vector and the reductase. This flavoprotein preparation, containing only the 28-residue N-terminal peptide segment of the membrane-binding domain of the mammalian enzyme, binds to phospholipid vesicles and interacts with membrane-bound cytochrome b5. The effect of N-myristoylation of the enzyme therefore appears to be limited to facilitating and stabilizing interactions with phospholipid vesicles. However, the relatively short intervening peptide sequence that separates the crucial peptide membrane-binding domain from lysine 41, which has been implicated in the active-site interaction with cytochrome b5 (Strittmatter, P., Kittler, J. M., Coghill, J. E., and Ozols, J. (1992) J. Biol. Chem. 267, 2519-2523), provides some limitation of the distance from the membrane surface for the interactions required for rapid electron transfer from the flavin of the reductase to the heme of cytochrome b5.

Base Sequence↗
PubMed1993

Isolation and the complete amino acid sequence of lumenal endoplasmic reticulum glucose-6-phosphate dehydrogenase.

I have isolated glucose-6-phosphate dehydrogenase from rabbit liver microsomes and determined its complete amino acid sequence. Sequence determination was achieved by automated Edman degradation of peptides generated by chemical and enzymatic cleavages. The microsomal enzyme consists of 763 residues and is quite dissimilar from the previously characterized cytosolic enzymes. The N terminus of the microsomal enzyme is blocked by a pyroglutamyl residue. Carbohydrate is attached at Asn-138 and Asn-263, implying that the bulk of the protein is oriented on the lumenal side of the endoplasmic membrane. The amino acid sequence of the microsomal protein shows limited homology to the extensively sequenced cytosolic glucose-6-phosphate dehydrogenases. Clusters of up to six identical residues can be identified in four regions: peptide segments at residues 10-21, 154-163, and 173-261. In addition, another array of identical residues, requiring a 100-residue deletion in the sequence of the microsomal enzyme, spans residues 436-462 and corresponds to residues 348-373 of the cytosolic protein. Two segments with a Gly-Xaa-Gly-Xaa-Xaa-Gly motif, related to a coenzyme binding fold, were identified at Gly-399 and Gly-491. In the cytosolic enzymes, a variation of this sequence motif occurs at Gly-37 and Gly-241. The 300-residue C-terminal segment of the microsomal enzyme is unique and has no counterpart in the cytosolic or the bacterial enzymes. An unexpected finding with regard to the microsomal enzyme is that it lacks an identifiable membrane-spanning region or the lumenal-protein C-terminal consensus sequences Lys-Asp-Glu or His-Ile/Thr-Glu-Leu. Thus, the mode of transport and retention of this protein in the lumen of endoplasmic reticulum remains to be determined.

Amino Acid Sequence↗
PubMed1992

Characterization of lysyl residues of NADH-cytochrome b5 reductase implicated in charge-pairing with active-site carboxyl residues of cytochrome b5 by site-directed mutagenesis of an expression vector for the flavoprotein.

An expression vector for bovine NADH-cytochrome b5 reductase was constructed from two DNA fragments that were derived from beef liver poly(A+) RNA using the polymerase chain reaction. Site-directed mutagenesis of the 3 lysine residues of the reductase, previously implicated in the formation of active-site charge pairs with carboxylate residues of cytochrome b5, was then used to obtain the purified catalytic domains of flavoproteins modified at each of these sites. The observed marked decreases in catalytic efficiencies of substitutions of a negative charge at the normally positively charged residues with the catalytic domain of cytochrome b5 are consistent with their participation in the formation of charge pairs with carboxylate groups of the hemeprotein to optimize rapid electron transfer from the reductase flavin to the heme of the cytochrome.

Amino Acid Sequence↗
PubMed1992

pNiXa, a Ni(2+)-binding protein in Xenopus oocytes and embryos, shows identity to Ep45, an estrogen-regulated hepatic serpin.

A Ni(2+)-binding protein (pNiXa, 45 kD, pI 8.5) discovered in Xenopus embryos, was isolated from oocytes. Based on amino acid sequences, pNiXa belongs to the serpin superfamily and shows identity to the cDNA sequence of Ep45, an estrogen-regulated hepatic serpin that contains an (HX)n-motif found in eukaryotic transcription factors. Nondenatured pNiXa, purified by Ni-affinity chromatography, inhibited bovine alpha-chymotrypsin. The presence of pNiXa in embryos when they are susceptible to Ni2+, the high avidity of pNiXa for Ni2+, and the (HX)n-motif point to pNiXa as a molecular target of Ni(2+)-teratogenesis.

Amino Acid Sequence↗
PubMed1991

Multiple forms of liver microsomal flavin-containing monooxygenases: complete covalent structure of form 2.

Hepatic flavin-containing monooxygenases catalyze NADPH-dependent oxygenation of a wide variety of drugs that possess a nucleophilic heteroatom. Two forms of these enzymes (form 1 and 2) have been isolated from rabbit liver microsomes and partially characterized (Ozols, J., 1989, Biochem. Biophys. Res. Commun. 163, 49-55). The complete amino acid sequence of form 2 is presented here. Sequence determination was achieved by pulsed liquid-phase and solid-phase sequencing of 40 peptides generated by chemical and enzymatic cleavages, including CNBr cleavage of tryptophanyl residues. Form 2 monooxygenase contains 533 amino acid residues and has a molecular weight of 60,089. The COOH terminus of this enzyme is very hydrophobic and presumably functions to anchor the protein to the membrane. Form 2 is readily degraded, since a form lacking residues 1 to 278 and a form without the COOH-terminal segment were also isolated from solubilized membrane preparations. The amino acid sequence of form 2 is 52% identical to that of form 1 and shows 55% identity to the sequence of rabbit lung monooxygenase derived from the cDNA data. The putative FAD and NADP binding segments around residues 9 and 190 are conserved in all three forms. Three variable segments can also be identified in these isoforms. These are residues 308 to 321, residues 408 to 421, and the membrane binding domain, residues 505 to 533. A comparison of the presently limited amino acid sequence data of flavin-containing monooxygenases (FMOs) implies that a particular FMO in different mammalian species may be very similar, but isozymes within a species may exhibit more extensive variability with respect to homology and catalytic activity. This study documents the structural diversity of a second hepatic FMO from rabbit liver and establishes this class of drug-metabolizing enzymes as a family of related proteins.

Amino Acid Sequence↗
PubMed1990

Characterization of the covalent cross-links of the active sites of amidinated cytochrome b5 and NADH:cytochrome b5 reductase.

Preparations of amidinated cytochrome b5 and cytochrome b5 reductase, cross-linked by using a soluble carbodiimide to promote the formation of covalent bonds between carboxyl groups of the hemeprotein and nucleophilic residues of the flavoprotein at the surfaces involved in protein-protein contacts during electron transfer, have been used to characterize the charge pair interactions that occur during electron transfer between the free proteins. Sequence analyses of tryptic, V8 protease-, and Asp-N protease-generated peptides show that the heme propionyl carboxyl group at the surface of the cytochrome forms an ester bond with Ser162 of the reductase, thus implicating Lys163 as the normal participant in ionic bonding between the active sites of the two proteins. Moreover, Lys41 and Lys125 directly form amide bonds with carboxyl residues on the active-site surface of the cytochrome. In the case of Lys41, this involves Glu52 and/or Glu60, and Glu47 and/or Glu48 for Lys125, again implicating these residues as the groups that form charge pairs during normal interactions between the active sites of the two proteins.

Amides↗
PubMed1990

Covalent structure of liver microsomal flavin-containing monooxygenase form 1.

Liver microsomal, flavin-containing monooxygenases catalyze NADPH- and oxygen-dependent oxidation of a wide variety of antipsychotic and narcotic drugs. Two forms of these enzymes have been isolated and partially characterized (Ozols, J. (1989) Biochem. Biophys. Res. Commun. 163, 49-55). The amino acid sequence of form 1 is presented here. Sequence determination has been achieved by automated Edman degradation of peptides generated by chemical and enzymatic cleavages. The NH2 terminus of form 1 oxygenase is blocked. Partial acid hydrolysis of the blocked peptides removed acetyl groups and permitted their analysis by Edman degradation. Form 1 monooxygenase contains 536 residues. A peptide of 32 residues at the COOH terminus of the protein could not be sequenced in a gas-phase or pulsed liquid-phase sequenator, due to its extreme hydrophobicity. Covalent coupling of this peptide to an aryl amine membrane by means of carbodiimide, followed by automated solid-phase sequencing, established the order of 30 amino acid residues. The hydrophobic segment at the COOH terminus presumably functions to anchor the monooxygenase to the microsomal membrane. The amino acid sequence of form 1 monooxygenase, despite overlapping substrate specificity, is not related to the cytochrome P-450 superfamily. Comparison of the sequence of form 1 oxygenase with other known sequences, except for some short segments similar to those in the bacterial flavin-containing monooxygenases, did not reveal significant sequence similarities that would suggest a structural or evolutionary relationship.

Amino Acid Sequence↗
PubMed1989

Liver microsomes contain two distinct NADPH-Monooxygenases with NH2-terminal segments homologous to the flavin containing NADPH-monooxygenase of Pseudomonas fluorescens.

Two NADPH-reductase preparations (FAD-containing monooxygenases) were isolated from rabbit liver microsomes, referred to as from 1 and from 2. Purification was achieved by means of anion-exchange, cation-exchange and hydroxylapatite chromatography in the presence of cholate and Nonidet P-40. Affinity chromatography on 2', 5'-ADP Sepharose was used to increase the purity and to concentrate the enzyme. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, form 1 exhibited a single band at Mr 58,500 and form 2 at Mr 58,000. The NH2- terminus of form 1 is blocked, whereas the NH2-terminus of form 2 is homologous to the NADPH-phydroxybenzoate hydrolase from Pseudomonas fluorescens. The latter and the form 2 enzyme share 11 identical residues in the NH2-terminal segment of 15 residues. Both forms were subjected to tryptic cleavages and peptide mapping. Sequence analysis of the peptides obtained indicated that forms 1 and 2 are similar but not identical proteins. A tryptic peptide, homologous to residues 3 to 32 of form 2 enzyme was isolated from the form 1 protein. This segment has 24 residues that are identical to the form 2 and contains the consensus sequence Gly-X-Gly-X-X-Gly, found in most FAD binding proteins. These results indicate that the NADPH-monooxygenase system consists of at least two distinct proteins representing different gene products.

Amino Acid Sequence↗
PubMed1989

Structure of cytochrome b5 and its topology in the microsomal membrane.

The complete amino acid sequence of human and chicken liver microsomal cytochrome b5 was determined. The amino termini of cytochrome b5 from four other mammalian species were examined in order to determine their complete covalent structure. As in the rat species, cytochrome b5 preparations from man, rabbit, calf and horse had an acetylated alanine as the first residue. In contrast, the pig cytochrome had alanine at the amino terminus. The amino terminus of the chicken cytochrome b5 was also unmodified, and extended three residues absent in the mammalian species. In order to investigate whether the carboxy-terminal segment of cytochrome b5 is located on the cytosolic or the luminal side of the microsomal membrane, rabbit liver microsomes were treated with trypsin and subjected to gel filtration and high-pressure liquid chromatography. The nonpolar peptide isolated from these microsomes lacked the terminal hexapeptide, indicating that when cytochrome b5 is bound to intact microsomes, the carboxy terminus is located on the cytosolic side of the membrane and does not extend in the lumen of the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1989

Isolation, properties, and the complete amino acid sequence of a second form of 60-kDa glycoprotein esterase. Orientation of the 60-kDa proteins in the microsomal membrane.

A second form (form 2) of glycosylated esterase was isolated from liver microsomal membranes and characterized. The subunit molecular weight of form 2 is identical to that of the 60-kDa protein previously reported (Ozols, J. (1987) J. Biol. Chem. 262, 15316-15321). The NH2 terminus of the form 2 enzyme is blocked. Digestion of form 2 with pyroglutamyl aminopeptidase, followed by electroblotting and sequence analysis of the blotted protein, indicated that a pyroglutamyl residue was located at the NH2 terminus. Sequence analysis of the deblocked protein as well as characterization of the peptides obtained from enzymatic and chemical cleavages of the intact protein led to the elucidation of its complete amino acid sequence. The protein is a single polypeptide consisting of 532 residues. Carbohydrate is attached at asparaginyl residue 249. The sequence of form 2 esterase is 50% identical to the sequence of form 1 enzyme. The amino acid sequence of the first 26 residues of form 1 enzyme from human liver microsomes shows that 23 residues are identical to that of rabbit form 1, but only 8 residues that are identical to form 2. Treatment of the forms 1 and 2 isozymes with N-glycosidase F or endo-N-acetylglucosaminidase H resulted in a decrease of their subunit molecular weights, indicating that the carbohydrate attached is of the high mannose type. To determine whether the 60-kDa proteins are located on the cytoplasmic or luminal side of the endoplasmic membrane, microsomes were treated with proteolytic enzymes and the two 60-kDa isozymes were isolated and characterized. Sequence analysis of both proteins indicated that their NH2 termini were unaffected by proteolysis. Form 1 isozyme isolated from trypsin-treated microsomes, however, lacked the COOH-terminal heptapeptide (residues 533-539). These results, in addition to the finding of an N-linked carbohydrate, suggest that the two 60-kDa proteins are oriented on the luminal side of the endoplasmic membrane.

Amino Acid Sequence↗
PubMed1989

cDNA sequence for the alpha M subunit of the human neutrophil adherence receptor indicates homology to integrin alpha subunits.

The receptor on human neutrophils (polymorphonuclear leukocytes) that mediates cellular adherence consists of two noncovalently associated subunits, designated alpha M (Mac-1 alpha, Mol alpha, or CD11b; Mr, 170,000) and beta (Mac-1 beta, Mol beta, or CD18; Mr, 100,000). We isolated a cDNA clone for the human neutrophil alpha M subunit by screening a lambda gt 11 cDNA library made from chronic myelogenous leukemia neutrophils by using an affinity-purified rabbit polyclonal antibody directed against the alpha M subunit. We used this cDNA clone to obtain additional clones from cDNA libraries made from differentiated HL-60 promyelocytic leukemia cells. Together these cDNAs constitute the complete 1137-amino acid sequence for the mature human alpha M subunit protein. The deduced amino acid sequence indicates the presence of an extensive extracellular domain with three putative metal-binding regions, (i) an amino acid region that is homologous to the A domain of von Willebrand factor, (ii) a 26-amino acid hydrophobic sequence that is a potential transmembrane domain, and (iii) a 19-amino acid cytoplasmic region. The amino acid sequence for the human neutrophil alpha M subunit contains regions that are closely related to amino acid sequences of adhesion receptors belonging to the integrin family.

Amino Acid Sequence↗