Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.11.2 (prolyl hydroxylase)
 1,814 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A purification of up to 4000-fold is reported for lysyl hydroxylase (EC 1.14.11.4) from extract of chick-embryo homogenate and one of about 300-fold from extract of chick-embryo cartilage. Multiple forms of the enzyme were observed during purification from whole chick embryos. In gel filtration the elution positions of the two main forms corresponded to average molecular weights of about 580000 and 220000. These two forms could also be clearly separated in hydroxyapatite chromatography. In addition, some enzyme activity was always eluted between the two main peaks both in gel filtration and in hydroxyapatite chromatography. The presence of the two main forms was also observed when purifying enzyme from chick embryo cartilage. Both forms of the enzyme hydroxylated lysine in arginine-rich histone, which does not contain any -X-Lys-Gly- sequence. No difference was found between the enzyme from whole chick embryos and from chick embryo cartilage in this respect. Lysyl hydroxylase was found to have affinity for concanavalin A, indicating the presence of some carbohydrate residues in the enzyme molecule. Lysyl and prolyl hydroxylase activities increased when the chick embryo homogenate was assayed in the presence of lysolecithin. Preincubation of the homogenate either with lysolecithin or with Triton X-100 increased lysyl hydroxylase activity in homogenate, and in the 1500 x g and 150000 x g supernatants, suggesting that the increase in the enzyme activity was due to liberation of the enzyme from the membranes. Divalent cations were found to inhibit the activity of lysyl and prolyl hydroxylases in vitro. An inhibition of about 50% was achieved with 15 mM calcium 60 muM copper and 3 muM zinc concentrations. The mode of inhibition was tested with Cu2+, and was found to be competitive with Fe2+.
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PMID:Lysyl hydroxylase. Further purification and characterization of the enzyme from chick embryos and chick embryo cartilage. 18 Oct 88

The effect of diabetes and insulin on the activities of both prolyl hydroxylase (trivial name; proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) and lysyl hydroxylase (trivial name; lysine,2-oxoglutarate dioxygenase, EC 1.14.11.4) in isolated rat renal glomeruli was determined. Three groups of experimental animals were used: age-matched controls, streptozotocin-diabetic, and insulin-treated streptozotocin-diabetic. Using 14C-labeled lysine or proline hydroxylase substrate prepared from chick embryo tibiae, glomerular 17 000 X g supernatant enzyme was incubated in a complete hydroxylating system for 60 and 120 min Lysyl hydroxylase activity was significantly increased in diabetic preparations, but prolyl hydroxylase activity did not differ from control. Administration of insulin to streptozotocin-injected animals completely restored glomerular lysyl hydroxylase to normal levels. The results suggest that the specific elevation of lysyl hydroxylase relates to the biochemical changes contributory to diabetic nephropathy, and that insulin may reverse this process.
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PMID:Effect of diabetes and insulin on rat renal glomerular protocollagen hydroxylase activities. 18 35

Lysyl hydroxylase (LH) is an essential enzyme in collagen biosynthesis that catalyzes the formation of hydroxylysine required for intermolecular crosslinking of collagen. We have isolated a partial (2.2-kb) cDNA for LH from human skin fibroblasts using PCR. DNA sequencing revealed 72% homology of the human coding sequence with the chick LH sequence at the nucleotide level and 76% homology predicted at the amino acid level. The LH cDNA hybridized strongly with two mRNA species of 2.4 and 3.4 kb on Northern blots of normal fibroblast RNA. Administration of minoxidil decreased both mRNA species without affecting levels of the mRNAs for the beta subunit of prolyl 4-hydroxylase (PH) or alpha 1(I) collagen. Two derivatives of minoxidil (3' hydroxyminoxidil and 4' hydroxyminoxidil) produced similar decreases in LH mRNAs. In contrast hydralazine increased the mRNAs for LH in parallel with its previously reported effect on the mRNA for the beta subunit of PH. This effect is accompanied by virtual elimination of the alpha 1(I) collagen mRNAs. These results on the action of minoxidil and hydralazine at the pretranslational level correlate well with their previously reported effect on enzyme activity and collagen biosynthesis and indicate that changes in steady-state mRNA levels can account directly for changes at the protein level. Moreover, the unique action of minoxidil in specifically decreasing LH mRNAs contrasts with the less specific stimulatory effects of hydralazine and suggests that these pharmaceuticals are regulating expression of LH at a pretranslational level by different mechanisms.
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PMID:Characterization of a partial cDNA for lysyl hydroxylase from human skin fibroblasts; lysyl hydroxylase mRNAs are regulated differently by minoxidil derivatives and hydralazine. 133 16

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

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 X-Lys-Gly sequences. We report here on the isolation of cDNA clones coding for the enzyme from a chick embryo lambda gt11 library. Several overlapping clones covering all the coding sequences of the 4-kilobase mRNA and virtually all the noncoding sequences were characterized. These clones encode a polypeptide of 710 amino acid residues and a signal peptide of 20 amino acids. The polypeptide has four potential attachment sites for asparagine-linked oligosaccharides and 9 cysteine residues, at least one of which is likely to be involved in the binding of the Fe2+ atom to a catalytic site. A surprising finding was that no significant homology was found between the primary structures of lysyl hydroxylase and prolyl 4-hydroxylase in spite of the marked similarities in kinetic properties between these two enzymes. A computer-assisted comparison indicated only an 18% identity between lysyl hydroxylase and the alpha-subunit of prolyl 4-hydroxylase and a 19% identity between lysyl hydroxylase and the beta-subunit of prolyl 4-hydroxylase. Visual inspection of the most homologous areas nevertheless indicated the presence of several regions of 20-40 amino acids in which the identity between lysyl hydroxylase and one of the prolyl 4-hydroxylase subunits exceeded 30% or similarity exceeded 40%. Southern blot analyses of chick genomic DNA indicated the presence of only one gene coding for lysyl hydroxylase.
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PMID:Molecular cloning of chick lysyl hydroxylase. Little homology in primary structure to the two types of subunit of prolyl 4-hydroxylase. 170 64

The anthracyclines doxorubicin and daunorubicin were found to act as irreversible inhibitors of prolyl 4-hydroxylase. The reaction rate for enzyme from both chick and human origin was first order, the concentration of inhibitor giving 50% inhibition being 60 microM for both compounds after 1 h. The effect was dependent on the presence of iron ions in the reaction mixture. Inactivation could be prevented by addition of high concentrations of ascorbate, but not 2-oxoglutarate, before the inactivation period. The same results were obtained with competitive analogues of these cosubstrates. Lysyl hydroxylase from chick embryos was also susceptible to inactivation. Its activity was decreased by 50% after incubation for 1 h with a 150 microM concentration of the inhibitors. When chick-embryo prolyl 4-hydroxylase was incubated with [14-14C]doxorubicin, both enzyme subunits were radioactively labelled, about 70% of the total radioactivity being found in the alpha-subunit. Since the anthracyclines are known to undergo a redox reaction generating semiquinone radicals with Fe3+ only, the results suggest that the enzyme-bound iron ion is oxidized to a tervalent intermediate in uncoupled reaction cycles. The data also suggest that both enzyme subunits contribute to the catalytic site of prolyl 4-hydroxylase.
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PMID:Syncatalytic inactivation of prolyl 4-hydroxylase by anthracyclines. 284 Aug 91

Peptides containing the unphysiological amino acid 5-oxaproline (Opr) in the sequence R1-Xaa-Opr-Gly-OR2 were found to inactivate prolyl 4-hydroxylase from chick and human origins. Of the substances investigated, compounds with aromatic substituents R1 and R2 were particularly effective when compared with those with an aliphatic group or without a C-terminal blocking group. Both affinity of the individual peptides for the enzyme and partition ratio contributed to the differences in efficiency. Benzylcarbonyl-Phe-Opr-Gly-benzyl ester was the most effective substance tested, its concentration giving 50% inactivation in 1 h being 0.8 microM. Inactivation was only observed in the presence of 2-oxoglutarate and Fe2+. The Opr peptides enhanced the decarboxylation of 2-oxoglutarate by prolyl 4-hydroxylase, the Vmax values obtained with the individual peptides being positively correlated with their inactivating efficiency. Inactivation was prevented by high concentrations of peptide substrate and ascorbate. Lineweaver-Burk kinetics experiments suggested noncompetitive inhibition with respect to peptide substrate and ascorbate. Lysyl hydroxylase was not affected by Opr peptides in concentrations of up to 1.5 mM in either the presence or absence of prolyl 4-hydroxylase. The results suggest that the oxaproline compounds are specific syncatalytic inactivators of prolyl 4-hydroxylase.
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PMID:Syncatalytic inactivation of prolyl 4-hydroxylase by synthetic peptides containing the unphysiologic amino acid 5-oxaproline. 284 31

This study was performed to compare the extractability of dwarf growth plate collagen and hexosamine and that of homozygous nonaffected Malamutes and to measure the activity of three of the enzymes involved in the post-translational modifications of the collagen molecule. No significant differences were found in the activity of prolyl hydroxylase or lysyl oxidase in the dwarf growth plates. Lysyl hydroxylase activity in the dwarf was decreased to 22% and 33% that of the activity present in the homozygous nonaffected growth plates. Amino acid analysis of the collagen isolated from dwarf growth plates failed to reveal any decrease in hydroxylysine content. Growth plates were extracted with either 1 M sodium chloride or 4 M guanidine hydrochloride. The extracts were applied to a DEAE-cellulose column. Amino acid analyses of the material which did not bind to DEAE revealed a slight decrease in the amount of guanidine-extractable hydroxyproline in the dwarf but a 60-fold increase in the amount of salt-extractable hydroxyproline in the dwarf growth plates. Material which eluted with 1 M sodium choloride was analyzed for hexosamine. There was a 10-fold increase in the amount of salt-extractable hexosamine present in the dwarf growth plates, whereas no significant differences were observed in the guanidine-extracted material. Hexosamine analysis of the growth plates revealed a significant increase in the total amount of hexosamine present in the dwarf growth plates. SDS-polyacrylamide gels of the material which did not bind to DEAE as well as the pepsin digested, 0.9M sodium chloride precipitated collagen demonstrated the presence of only type II collagen.
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PMID:Studies of the intercellular matrix of growth plates from dwarf and homozygous nonaffected Alaskan Malamutes: collagen and hexosamine. 625 32

Prolyl and lysyl hydroxylase activities in cultures of human skin fibroblasts from fetal to 94-yr-old donors were measured. In contrast to earlier studies with whole skin, neither prolyl nor lysyl hydroxylase activity was found related to donor age. Prolyl hydroxylase activity increased 3- to 6-fold when cell extracts were incubated with ascorbate and other hydroxylation cofactors before assay. A similar increase in prolyl hydroxylase activity occurred when cells were incubated with ascorbate. Lysyl hydroxylase activity remained unaltered under these conditions.
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PMID:Prolyl and lysyl hydroxylase activities of human skin fibroblasts: effect of donor age and ascorbate. 625 74

The kinetics of the lysyl hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) reaction were studied using enzyme from chick embryos by varying the concentration of one substrate in the presence of different fixed concentrations of the second substrate, while the concentrations of the other substrates were held constant. Intersecting lines were obtained in double-reciprocal plots for all possible pairs involving Fe2+, alpha-ketoglutarate, O2 and the peptide substrate, whereas parallel lines were obtained for pairs comprising ascorbate and each of the other substrates. The pair composed of Fe2+ and alpha-ketoglutarate gave an asymmetrical initial veolcity pattern, indicating binding of these two reactants in this order, that of Fe2+ being at thermodynamic equilibrium. The initial velocity patterns are identical with those reported for prolyl 4-hydroxylase, and the apparent Km and Kd values calculated from these data are also very similar. The largest difference was fo-nd in Km and Kd for alpha-ketoglutarate, which were about 4 times the corresponding values for prolyl 4-hydroxylase. Ascorbate was found to be a quite specific requirement for lysyl hydroxylase, but the enzyme catalyzed its reaction for a short time at a high rate in the complete absence of this vitamin, suggesting that the reaction with ascorbate does not occur during each catalytic cycle. Lysyl hydroxylase catalyzed an uncoupled decarboxylation of alpha-ketoglutarate in the absence of the peptide substrate, the rate being about 4% of that observed in the presence of a saturating concentration of the peptide substrate. This uncoupled decarboxylation required the same cosubstrates as the complete reaction.
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PMID:Studies on the lysyl hydroxylase reaction. I. Initial velocity kinetics and related aspects. 676 66


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