Involvement of superoxide in the prolyl and lysyl hydroxylase reactions

Four superoxide dismutase active copper chelates, Cu(acetylsalicylate)₂, Cu(salicylate)₂, Cu(lysine)₂ and Cu(tyrosine)₂, proved to be inhibitors of prolyl and lysyl hydroxylase. The kinetics of the inhibition are consistent with the proposal that these compounds dismutated ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{staggered?}}$ at the active site of the enzymes. The data strongly suggest that ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{staggered?}}$ is the active form of O₂ in the prolyl and lysyl hydroxylase reactions.     

Activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase in the liver of rats with hepatic injury

The activities of four enzymes catalysing post-translational modifications of the collagen polypeptide chains were assayed in the livers of rats with experimental hepatic injury. The liver injury was induced by injecting carbon tetrachloride twice weekly, and assays of the enzymic activities were carried out 2 and 4 weeks after commencement of administration of carbon tetrachloride. The liver homogenates were preincubated with Triton X-100 before the assays, because such treatment was found to increase the activities of all four enzymes in the supernatants of liver homogenates. The activities of all four enzymes had increased by 2 weeks after commencement of carbon tetrachloride administration. No increase was found in the collagen content of the livers at this stage and thus an increase in all four enzyme activities preceded an increase in the collagen content of the liver. A further slight increase was found in three of the enzyme activities during the subsequent 2 weeks of the experiment, whereas no further increase was found in the collagen galactosyltransferase activity. A statistically significant correlation was found between all four enzyme activities, but the magnitude of the increases varied considerably. The largest increase was found in lysyl hydroxylase activity, and at 4 weeks the magnitude of this was about three times that of the collagen galactosyltransferase activity. The results thus indicate that the increased enzyme activities cannot be explained simply by an increase in the number of collagen-producing cells having similar enzyme activity patterns to those of the cells initially present in the liver.     

Depressor effect of diabetes in spontaneously hypertensive rat: role of vascular reactivity and prolyl hydroxylase and lysyl oxidase activities

Streptozotocin (STZ)-induced diabetes (8 weeks) produced a marked depressor effect in the spontaneously hypertensive rat (SHR), confirming earlier studies, but had no effect on arterial pressure of normotensive controls (WKY). We investigated the phenomenon further by examining the effects of diabetes on the activities of aortic prolyl hydroxylase (PH) and lysyl oxidase (LO), marker enzymes for collagen biosynthesis, and on the reactivity of isolated mesenteric arteries to vasoactive agents. PH and LO activities of nondiabetic SHR were greater than those of the WKY controls. Diabetes markedly reduced PH and LO activities of SHR aortae, but had no significant effect on PH and LO activities of the WKY strain. The effects of diabetes on vascular collagen biosynthetic enzymes of SHR were not associated with reductions in mesenteric arterial responsiveness or sensitivity to norepinephrine, methoxamine, serotonin or KC1. These results suggest that the depressor effect of diabetes in SHR is associated with a reduction in vascular collagen biosynthesis but not a reduction in vascular reactivity.     

Inhibition of prolyl hydroxylase activity by bradykinin analogs containing a prolyl-like residue

A number of substituted bradykinin analogs were prepared in which the proline in position 3 was replaced by analogs of proline. All of the bradykinin analogs, with the exception of l-azetidine-2-carboxyl³-bradykinin showed significant ability to inhibit prolyl hydroxylase activity. Addition of an l-glutamyl residue to the amino terminus of 3,4-dehydro-l-prolyl³-bradykinin and trans-4-hydroxy-l-prolyl³-bradykinin resulted in competitive inhibitors of increased effectiveness with K_i, values approximately 10^?4m. One of the peptides, l-3,4-dehydro-l-prolyl³-bradykinin, appeared to serve as a substrate for prolyl hydroxylase.     

Regulation of prolyl and lysyl hydroxylase activities in cultured human skin fibroblasts by ascorbic acid

Studies with confluent human skin fibroblasts maintained in 0.5% serum supplemented medium have given new insight into the regulatory influences of ascorbate. These include a reduction of prolyl hydroxylase activity, a stimulation of lysyl hydroxylase activity, and an acceleration of collagen production. The lack of parallel between prolyl hydroxylase activity and collagen production indicates that the rate of collagen synthesis is not controlled by the level of prolyl hydroxylase.     

Association of prolyl hydroxylase activity with membranes

Addition of ionic and nonionic detergents to whole homogenates of liver, kidney and lung prepared by a mild homogenization technique resulted in a two- to three-fold increase of prolyl hydroxylase activity. After subcellular fractionation of whole homogenates of liver, particulate and supernatant fractions were incubated in the presence and absence of triton X-100 and assayed for prolyl hydroxylase activity. All particulate fractions tested were able to release significant amounts of prolyl hydroxylase activity in the presence of triton. The release of enzyme activity by triton was observed with the 1000 × g and 17,000 × g supernatants but not with the 105,000 × g supernatant; thus indicating that detergent does not activate soluble enzyme nor make the substrate more accessible to hydroxylation by the enzyme during incubation. Rigorous homogenization of the 17,000 × g particulate fraction with the Polytron ST system resulted in a substantial loss of the amount of prolyl hydroxylase activity released by treatment with triton. These data suggest that a significant amount of prolyl hydroxylase activity is associated with membranes under physiological conditions.     

Inhibition of prolyl hydroxylase by poly(ADP-ribose) and phosphoribosyl-AMP. Possible role of ADP-ribosylation in intracellular prolyl hydroxylase regulation

Poly(ADP-ribose) prepared by incubating NAD+ with rat liver nuclei inhibited the hydroxylation reaction catalyzed by purified prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) in vitro. Near complete inhibition of the enzyme was seen in the presence of 6 nM (ADP-Rib)18 with a Ki(app) of 1.5 nM. The monomer unit of poly(ADP-ribose), adenosine diphosphoribose (ADP-Rib), was found to be a weak inhibitor. On the other hand, poly(ADP-ribose)-derived phosphoribosyl-AMP (PRib-AMP) and its dephosphorylated product, ribosyl-ribosyl-adenine (Rib-RibA), inhibited the enzyme in nanomolar concentrations (Ki(app) 16.25 nM). The order of inhibition was (ADP-Rib)18 greater than PRib-AMP, Rib-RibA much greater than ADP-Rib. These results suggested that the 1"----2' ribosyl-ribosyl moiety in these compounds was involved in the inhibition of the enzyme. The possibility that intracellular prolyl hydroxylase is regulated by the involvement of ADP-ribosylation reactions was examined in confluent cultures of skin fibroblast treated with 20 mM lactate. The activity of prolyl hydroxylase was stimulated by 145% over that of untreated cultures. In the lactate-treated cells, the level of NAD+ was lowered and the total ADP-ribosylation of cellular proteins reduced by 40%. These observations imply that the lactate-induced activation of cellular prolyl hydroxylase is mediated by a reduction in ADP-ribosylation and that the synthesis and degradation of ADP-ribose moiety(ies) may possibly regulate prolyl hydroxylase activity in vivo.     

Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase

The hydroxylation of proline and lysine residues by the collagen hydroxylases is coupled with a stoichiometric decarboxylation of 2-oxoglutarate. Ascorbate is virtually a specific requirement for these enzymes, but previous studies have demonstrated that it is not consumed during most catalytic cycles. Prolyl 4-hydroxylase and lysyl hydroxylase are known also to catalyze an uncoupled decarboxylation of 2-oxoglutarate in the absence of the peptide substrate. It is shown here that, unlike the complete hydroxylation reaction, the uncoupled decarboxylation reaction involves stoichiometric ascorbate consumption. This stoichiometric ascorbate consumption was also seen when the rate of the uncoupled prolyl 4-hydroxylase reaction was enhanced by the addition of poly(L-proline). Since collagen hydroxylases may catalyze occasional uncoupled reaction cycles even in the presence of the peptide substrates, the main function of ascorbate in these reactions in vivo is suggested to be that of reactivating the enzymes after such uncoupled cycles.     

A microassay for lysyl oxidase activity.

Pinnell and Martin's [(1968) Proc. Nat. Acad. Sci. USA61, 708–716] standard assay for lysyl oxidase is modified to determine activity in small samples. Data collected by both methods are comparable, and the microassay has the advantages of being economical and rapid.     

Simplified assay for lysyl oxidase activity.

A simplified method for the assay of lysyl oxidase activity was developed. The method is based on the measurement of tritiated water released by enzyme action from labeled protein-bound lysine and hydroxylysine. Trichloroacetic acid (TCA) supernates of the incubation mixtures are passed through small Dowex 50 (H⁺) columns and the effluents are counted. For rapid screening purposes an indication of the presence of enzyme activity in enzyme preparations can be obtained by measuring the radioactivity present in aliquots of the TCA supernates as such and by measuring the radioactivity after drying at 60°C, taking the difference between the two as a measurement of enzyme activity.     

Suppression of fibroblast proliferation and lysyl hydroxylase activity by minoxidil

The effect of minoxidil on lysyl hydroxylase activity and proliferation of human skin fibroblasts in culture was examined. Exposure of cells to minoxidil resulted in a specific loss of lysyl hydroxylase activity, the extent of which was dependent on the concentration of minoxidil from 25 to 500 microM and the duration of the treatment from 6 to 48 h. This phenomenon was unaffected by culture conditions, i.e. ascorbic acid status, serum concentration, and cell density. Minoxidil added directly to cell extracts had no effect on lysyl hydroxylase activity, showing a requirement for intact cells. Mixing experiments with extracts of minoxidil-treated cells and controls gave additive results which rule out the possibility that a metabolite derived from minoxidil could be inhibiting the enzyme activity. The effect of minoxidil on fibroblast lysyl hydroxylase activity disappeared in the presence of cycloheximide, an inhibitor of protein synthesis. Moreover, the recovery of the enzyme activity that occurred after removal of minoxidil from the culture medium could be prevented by actinomycin D, an inhibitor of RNA synthesis. These results indicate that minoxidil may inhibit the synthesis of lysyl hydroxylase in the cell. In addition to suppressing fibroblast lysyl hydroxylase activity, minoxidil caused inhibition of cell growth within 48 h in a manner dependent on the concentration from 10 to 1000 microM, the latter resulting in almost complete cessation of cell proliferation. This effect was not accompanied by cytotoxicity as judged by the criteria of dye exclusion, plating efficiency, growth recovery, and protein synthesis. The inhibition of fibroblast proliferation by minoxidil appeared to be related to its ability to inhibit DNA synthesis measured by incorporation of tritiated thymidine into acid-precipitable material.     

Stimulation of prolyl hydroxylase activity by chelating agents.

The activity of purified prolyl hydroxylase was enhanced several fold by addition of some chelating agents to the assay medium. Chelating agents could be classified into three groups. The chelating agents of Group I such as α, α′-dipyridyl were inactive until they reached equimolar concentration with ferrous ion in the assay mixture. The Group II agents, EDTA, diethylenetriaminepentaacetic acid, etc., stimulated the enzymatic activity 1.5- to 3-fold at equimolar concentration with ferrous ion. But the agents of both groups precipitously inhibited the enzymatic activity at concentrations greater than ferrous ion. On the other hand, Group III chelating agents, such as nitrilotriacetic acid, enhanced the enzymatic activity 5- to 10-fold at concentrations greater than ferrous ion. Nucleoside triphosphates, which also stimulate the enzymatic activity several fold and whose optimal concentrations are 1–3 × 10^?m, may be analogous to nitrilotriacetic acid of Group III.     

Genomic structure and embryonic expression of zebrafish lysyl hydroxylase 1 and lysyl hydroxylase 2.

Collagen biosynthesis in both invertebrates and vertebrates is critically dependent upon the activity of lysyl hydroxylase (LH) enzymes. In humans, mutations in the genes encoding LH1 and LH2 have been shown to cause two distinct connective tissue disorders, Ehlers-Danlos (Type VIA) and Bruck syndromes. While the biochemical properties of these enzymes have been intensively studied, their embryonic patterns of expression and developmental roles remain unknown. We now present the cloning and analyses of the genes encoding LH1 and LH2 in the zebrafish, Danio rerio. We find these genes to be similarly organized to other vertebrate lh (plod) genes, including the presence of an alternatively spliced exon in lh2. We also examine the mRNA expression patterns of lh1 and lh2 during embryogenesis and find them to exhibit unique and dynamic patterns of expression. These results strongly suggest that LH enzymes are not merely housekeeping enzymes, but play distinct developmental roles. The identification of these genes in the zebrafish, a genetic model organism whose development is well characterized, now provides the basis for the establishment of the first animal models for both Ehlers-Danlos (Type VIA) and Bruck syndromes.     

Developmental changes in prolyl hydroxylase activity and protein in chick embryo.

Changes in prolyl hydroxylase activity and immunoreactive protein were studied in various chick embryo tissues during the embryonic development. Both the enzyme activity and the amoung of immunoreactive protein increased till the 16th day of development and declined thereafter in all tissues studied. Comparison of the enzyme activity to the content of the total immuno-reactive protein indicated that there are distinct differences in the degree of enzyme activity between different chick embryo tissues, and in the same tissue between different stages of embryonic development. The highest relative enzyme activities were found in cartilage and skin, in which about 60% of the enzyme was active on the 16th day of development and only 20-30% was active on the 20th day of development; the lowest values were observed in spleen and large vessels, in which below 10% of the enzyme protein was in the active form on the 20th day of development Gel filtration studies demonstrated that in cartilage of 16-day-old chick embryos about 60% of the total immunoreactive enzyme in the tissue was present in the form of active prolylhydroxylase tetramer, whereas on the 20th day of development only 30% of the enzyme protein in cartilage was in the tetramer form. By contrast, in large vessels of the 16-day-old chick embryos, essentially all the enzyme was in the form of prolyl hydroxylase monomers.     

Changes in lung lysyl oxidase activity in streptozotocin-diabetes and in starvation.

The effects of streptozotocin-induced diabetes and of starvation on the lysyl oxidase activity of rat lung were investigated. Enzyme activity was elevated 2--3 fold in the lungs of streptozotocin-diabetic rats. In contrast, starvation of rats produced a rapid loss of lung lysyl oxidase activity, with levels approximating 25% of control values after 48--72 h of starvation. Enzyme activity was essentially fully restored to control values upon refeeding the 48-h starved animals for 3 h. These studies demonstrate the responsiveness of lysyl oxidase to these physiological states and suggest a component, enzymatic basis of change in lung function known to occur in the diabetic state.