Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The formation of 3-hydroxyproline was studied with crude rat kidney cortex extract as a source of enzyme and chick embryo tendon protocollagen and procollagen or cartilage protocollagen as a substrate. Synthesis of 3-hydroxyproline was observed with all these substrates and the formation of 3-hydroxyproline ranged up to seven residues per pro-alpha-chain. The highest rate of 3-hydroxylation took place at 20 degrees C and the reaction required Fe2+, O2,2-oxoglutarate and ascorbate. The formation of 3-hydroxyproline was affected by chain length and the conformation of the substrate, in that longer polypeptide chains proved better substrates, while the native triple-helical conformation of protocollagen or procollagen completely prevented the reaction. Formation of 3-hydroxyproline with tendon procollagen as a substrate was not inhibited by antiserum to prolyl 4-hydroxylase or by poly(L-proline) when these substances were used in concentrations which clearly inhibited 4-hydroxyproline formation with tendon protocollagen as a substrate. Furthermore, pure prolyl 4-hydroxylase did not synthesize any 3-hydroxyproline under conditions in which the crude rat kidney cortex enzyme would readily do so. The data thus strongly suggest that prolyl 3-hydroxylase and prolyl 4-hydroxylase are separate enzymes.
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PMID:Prolyl 3-hydroxylase: partial characterization of the enzyme from rat kidney cortex. 19 Dec 55

Cell lines selected in multiple steps for increasing resistance to hydroxyurea have been shown to have corresponding increases in ribonucleotide reductase activity. We have isolated a number of cDNA clones from a cDNA library constructed from a highly hydroxyurea-resistant hamster cell line, 600H, in which the activity of ribonucleotide reductase is elevated more than 80-fold. These clones correspond to genomic DNA sequences amplified in the 600H cell line compared with the V79 parental line. One of these cDNA clones, termed P5, codes for a 50 kDa protein detected by in vitro translation of poly(A)+ RNA isolated by hybridization/selection. The cDNA sequence contains a single open reading frame of 1317 nucleotides which encodes a polypeptide of 439 amino acids. The amino acid sequence deduced from the cDNA insert contains two copies of the 11-amino-acid sequence Val-Glu-Phe-Tyr-Ala-Pro-Trp-Cys-Gly-His-Cys. Duplicate copies of this sequence also occur in the active site of rat and human protein disulphide isomerase (also known as the beta-subunit of human prolyl 4-hydroxylase, tri-iodothyronine-binding protein) and in Form I phosphoinositide-specific phospholipase C, indicating that P5 falls into this newly defined superfamily of proteins. Genomic sequences similar to the cDNA clone are amplified 10-20-fold in hamster cells selected for resistance to increasing concentrations of hydroxyurea, a phenomenon observed earlier with cDNA clones for the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. RNA blots probed with P5 cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells.
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PMID:The gene for a novel protein, a member of the protein disulphide isomerase/form I phosphoinositide-specific phospholipase C family, is amplified in hydroxyurea-resistant cells. 131 Nov 71

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The enzyme can easily be dissociated into its subunits, but all attempts to associate a tetramer from the dissociated subunits in vitro have been unsuccessful. Molecular cloning of the catalytically important alpha subunit has identified two types of cDNA clone due to mutually exclusive alternative splicing. The beta subunit is a highly unusual multifunctional polypeptide, being identical to the enzyme protein disulfide-isomerase (EC 5.3.4.1). We report here on expression of the alpha and beta subunits of prolyl 4-hydroxylase and a fully active enzyme tetramer in Spodoptera frugiperda insect cells by baculovirus vectors. When the beta subunit was expressed alone, the polypeptide produced was found in a 0.1% Triton X-100 extract of the cell homogenate and was a fully active protein disulfide-isomerase. When either form of the alpha subunit was expressed alone, only traces of the alpha subunit could be extracted from the cell homogenate with 0.1% Triton X-100, and 1% SDS was required to obtain efficient solubilization. These alpha subunits had no prolyl 4-hydroxylase activity. When the cells were coinfected with both alpha- and beta-subunit-producing viruses, an enzyme tetramer was formed, but significant amounts of alpha and beta subunits remained unassociated. The recombinant tetramer was indistinguishable from that isolated from vertebrate tissue in terms of its specific activity and kinetic constants for cosubstrates and the peptide substrate. The two alternatively spliced forms of the alpha subunit gave enzyme tetramers with identical catalytic properties. Baculovirus expression seems to be an excellent system for mass production of the enzyme tetramer and for detailed investigation of the mechanisms involved in the association of the monomers.
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PMID:Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system. 132 38

Protein disulphide isomerase (PDI) is a highly unusual multifunctional polypeptide, identical to the beta-subunit of prolyl 4-hydroxylase. It has two -Cys-Gly-His-Cys- sequences which represent two independently acting catalytic sites of PDI activity. We report here on the expression in baculovirus vectors of various mutant PDI/beta-subunits together with a wild-type alpha-subunit of the human prolyl 4-hydroxylase alpha 2 beta 2 tetramer in Spodoptera frugiperda insect cells. When either one or both of the -Cys-Gly-His-Cys- sequences was converted to -Ser-Gly-His-Cys-, a tetramer was formed as with wild-type PDI/beta-subunit. This tetramer was fully active prolyl 4-hydroxylase. The data demonstrate that PDI activity of the PDI/beta-subunit is not required for tetramer assembly or for the prolyl 4-hydroxylase activity of the tetramer, and thus other sequences of the PDI/beta-subunit may be critical for keeping the alpha-subunits in a catalytically active, non-aggregated conformation. Measurements of the PDI activities of tetramers containing the various mutant PDI/beta-subunits demonstrated that the activity of the wild-type tetramer is almost exclusively due to the C-terminal PDI catalytic sites, which explains the finding that the PDI activity of the PDI/beta-subunit present in the tetramer is about half that in the free polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Site-directed mutagenesis of human protein disulphide isomerase: effect on the assembly, activity and endoplasmic reticulum retention of human prolyl 4-hydroxylase in Spodoptera frugiperda insect cells. 132 60

Protein disulfide isomerase (PDI, EC 5.3.4.1) is a highly unusual multifunctional polypeptide, being identical to the beta subunit of prolyl 4-hydroxylase, a cellular thyroid hormone binding protein and a component of the microsomal triglyceride transfer protein complex, and highly similar to a polypeptide acting in vitro as a glycosylation site binding protein. It has two -Cys-Gly-His-Cys- sequences which, it has been proposed, act as catalytic sites for the isomerase activity, but few data have been available to indicate whether one or both of them do indeed act as catalytic sites and whether the two presumed catalytic sites act independently or cooperatively. We report here on the expression of human PDI in Escherichia coli with three different signal sequences. All three polypeptide variants were secreted into the periplasmic space as fully active enzymes. Oligonucleotide-directed mutagenesis was used to convert either one or both of the -Cys-Gly-His-Cys- sequences to -Ser-Gly-His-Cys-. The PDI activity of both polypeptides containing a single modified sequence was about 50% of that of the wild-type polypeptide, whereas the polypeptide with two modified sequences had no isomerase activity. It is thus concluded that both -Cys-Gly-His-Cys- sequences act as catalytic sites for the isomerase activity, and the two catalytic sites appear to operate independently of one another.
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PMID:Expression and site-directed mutagenesis of human protein disulfide isomerase in Escherichia coli. This multifunctional polypeptide has two independently acting catalytic sites for the isomerase activity. 155 65

Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 dimer, catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in peptide linkages. A deficiency in this enzyme activity is known to exist in patients with the type VI variant of the Ehlers-Danlos syndrome, but no amino acid sequence data have been available for the wildtype or mutated human enzyme from any source. We report the isolation and characterization of cDNA clones for lysyl hydroxylase from a human placenta lambda gt11 cDNA library. The cDNA clones cover almost all of the 3.2-kb mRNA, including all the coding sequences. These clones encode a polypeptide of 709 amino acid residues and a signal peptide of 18 amino acids. The human coding sequences are 72% identical to the recently reported chick sequences at the nucleotide level and 76% identical at the amino acid level. The C-terminal region is especially well conserved, a 139-amino-acid region, residues 588-727 (C-terminus), being 94% identical between the two species and a 76-amino-acid region, residues 639-715, 99% identical. These comparisons, together with other recent data, suggest that lysyl hydroxylase may contain functionally significant sequences especially in its C-terminal region. The human lysyl hydroxylase gene (PLOD) was mapped to chromosome 1 by Southern blot analysis of human-mouse somatic cell hybrids, to the 1p34----1pter region by using cell hybrids that contain various translocations of human chromosome 1, and by in situ hybridization to 1p36.2----1p36.3. This gene is thus not physically linked to those for the alpha and beta subunits of prolyl 4-hydroxylase, which are located on chromosomes 10 and 17, respectively.
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PMID:Cloning of human lysyl hydroxylase: complete cDNA-derived amino acid sequence and assignment of the gene (PLOD) to chromosome 1p36.3----p36.2. 157 94

Protein disulfide isomerase (PDI) is a highly unusual multifunctional polypeptide that is identical to the beta-subunit of prolyl 4-hydroxylase, a cellular thyroid hormone-binding protein and a subunit of the microsomal triglyceride transfer protein complex, and very similar to a polypeptide functioning in vitro as a glycosylation site binding protein of oligosaccharyl transferase. The human PDI gene possesses several putative transcriptional control elements, including the highly unusual presence of six CCAAT boxes between -108 and -378 of the 5'-flanking region. We report here on a promoter analysis of this gene. Eleven PDI promoter elements recognized by DNA-binding proteins present in HeLa cell and HT-1080 cell nuclear extracts were identified by DNase I footprinting analysis within the first 630 nucleotides of the 5'-flanking region. Interestingly, these included all six CCAAT elements. Functional 5' deletion analysis suggested that only two or three of the CCAAT elements may contribute significantly to the promoter activity in HeLa cells. Mutations introduced into each of the CCAAT boxes separately indicated, however, that all six appear to contribute to the promoter strength, the largest decreases (approximately 50%) being seen with mutations in the second or fifth CCAAT box. These data thus suggested that efficient expression of the multifunctional PDI polypeptide is secured by multiple CCAAT elements, some of which appear to be functionally redundant. The 5' deletion analysis further suggested that a region between -623 and -518 may contain additional positively and negatively acting elements.
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PMID:Promoter of the gene for the multifunctional protein disulfide isomerase polypeptide. Functional significance of the six CCAAT boxes and other promoter elements. 159 78

Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens and related proteins by hydroxylating proline residues in peptide linkages. The beta-subunit of prolyl 4-hydroxylase (P4HB) is a highly unusual multifunctional polypeptide that is identical to the enzyme protein disulfide isomerase and a major cellular thyroid hormone-binding protein and is highly similar to a glycosylation site-binding polypeptide of oligosaccharyl transferase. We report here the regional assignment of the gene for this multifunctional polypeptide. In situ hybridization mapped the gene to 17q25. Southern blot analyses of restricted DNA from a chromosome-mediated gene transfer transfectant panel suggested that the P4HB gene is located distal to the gene for thymidine kinase, either between the genes for thymidine kinase and galactokinase or on the telomeric side of both these genes.
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PMID:Regional assignment of the human gene coding for a multifunctional polypeptide (P4HB) acting as the beta-subunit of prolyl 4-hydroxylase and the enzyme protein disulfide isomerase to 17q25. 164 89

We have isolated from chicken embryos a novel 53-kDa protein possessing properties which are similar, but not identical to the 55-kDa PDI polypeptide from chicken embryos. The novel 53-kDa polypeptide copurifies with PDI, but is separated by ion-exchange chromatography. The novel 53-kDa polypeptide cross-reacts strongly with antibodies specific for bovine PDI and cross-reacts to varying degrees with six different preparations of antibodies specific for chicken PDI which is identical to the beta-subunit of chicken prolyl 4-hydroxylase. Anti-bovine PDI immunoglobulins selected by the purified 53-kDa polypeptide react with bovine PDI but not with the beta-subunit of prolyl 4-hydroxylase, suggesting that the 53-kDa polypeptide shares epitopes with bovine PDI but not with the chicken prolyl 4-hydroxylase beta-subunit. Amino acid compositional analysis of the purified polypeptide yielded unique data when compared to PDI and other PDI-like polypeptides. Edman degradation from the N terminus of the 53-kDa polypeptide yields a sequence very different from the N terminus of PDI. This sequence is unique when compared to all entries in available databases. A 20-residue sequence of an internal cyanogen bromide fragment of the 53-kDa polypeptide gives a nearly identical match with human beta-endorphin. The 53-kDa polypeptide is capable of cleaving the disulphides of insulin under conditions where PDI is active. The periodic acid-Schiff assay failed to detect bound carbohydrate. These observations support evidence for a family of PDI-like proteins in chicken embryo and suggest that PDI activity is not confined to only one protein.
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PMID:A novel 53-kDa polypeptide from chicken embryo. 166 Aug 84

Excessive accumulation of collagen in the extracellular matrix has a crucial role in fibrosis. Thus pharmacological inhibition of collagen deposition is likely to be beneficial for patients suffering from fibrotic disorders such as liver cirrhosis. Prolyl 4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens and other proteins with collagen-like amino acid sequences by the hydroxylation of proline residues in -X-Pro-Gly- sequences. The reaction products, 4-hydroxyproline residues, serve to stabilize the collagen triple helices under physiological conditions. Conversely, collagen chains that contain no 4-hydroxyproline cannot fold into triple helical molecules that are stable at body temperature. The prolyl 4-hydroxylase reaction therefore seems to be a particularly suitable target for the pharmological regulation of excessive collagen formation. The reaction catalyzed by prolyl 4-hydroxylase requires Fe2+, 2-oxoglutarate, O2 and ascorbate and involves an oxidative decarboxylation of 2-oxoglutarate. The active enzyme is an alpha 2 beta 2 tetramer that consists of two types of inactive monomer and has two catalytic sites. Some parts of the catalytic sites may be built up cooperatively of both the alpha and beta subunits, but the alpha subunit appears to contribute the major part. The beta subunit contains the carboxyl-terminal tetrapeptide sequence -Lys-Asp-Glu-Leu which is essential for the retention of a polypeptide within the lumen of the endoplasmic reticulum. Since the alpha subunit lacks the carboxyl-terminal retention signal, one function of the beta subunit in the prolyl 4-hydroxylase tetramer may be to retain the enzyme within the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Prolyl 4-hydroxylase and its role in collagen synthesis. 166 65


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