Jouni Uitto

Protocollagen Proline Hydroxylase Activity in the Skin of Normal Human Subjects and of Patients with Scleroderma

The activity of parotocollagen proline hydroxylase was assayed in punch biopsy specimens of human skin. The enzyme in human skin was similar to that from animal sources, in that it was recovered in the soluble protein fraction and in that it required atmospheric oxygen, ascorbate, α-ketoglutarate and ferrous iron. The activity of protocollagen proline hydroxylase was highest in foetuses and newborn infants, and decreased with advancing age. A slight elevation of the enzyme activity was found in three out of five patients with actively progressing scleroderma.

Human leukocyte collagenase: Characterization of enzyme kinetics by a new method

Abstract Human collagenase was partially purified from polymorphonuclear leukocytes, and a new method for assay of the collagenase activity was developed. The assay employs native radioactive collagen in soluble form as a substrate. The enzyme incubations are performed at 25°C which is below the melting temperatures of the cleavage products TCA and TCB, and these peptides are quantitatively recovered by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Employing this method, an apparent K m value of 1.04 × 10 −6 m for human leukocyte collagenase using type I collagen as a substrate was measured.

A method for studying collagen biosynthesis in human skin biopsies in vitro

Abstract Biosynthesis of collagen was studied by incubating slices of human skin in a medium containing [ 14 C]proline. The presence of hydroxy[ 14 C]proline in nondialysable protein of the skin was taken as a measure of collagen formation. The synthesis of radioactive hydroxyproline was studied under various experimental conditions, and an incubation medium (20 mM N -2-hydroxyethylpiperazine- N ′-2-ethanesulphonic acid buffer (pH 7.4), so mM glucose, 0.05 mg/ml ampicillin, I μC [ 14 C]proline, all in phosphate-free Krebs-Ringer solution) for an optimal rate of collagen synthesis is presented. The separate components of the incubation medium had no inhibitory effect on the hydroxylation of [ 14 C]proline when partially purified preparations of protocollagen proline hydroxylase were incubated with radioactive protocollagen substrate. When skin biopsy specimens from living human subjects of various ages were used for incubation, younger subjects demonstrated a relatively high rate of collagen synthesis and a decrease with advancing age was observed.

Protocollagen Proline Hydroxylase Activity in Rat Heart During Experimental Cardiac Hypertrophy

Cardiac hypertrophy was produced in rats by constricting the abdominal aorta subdiaphragmatically. The weight of the left ventricle was significantly elevated 2 days after aortic constriction and was 19% higher than in shamoperated animals at 11 days. The activity of protocollagen proline hydroxylase (PPH), an enzyme participating in collagen biosynthesis, and the hydroxyproline content of the heart muscle were determined. PPH activity was increased at 2 days after aortic constriction and declined thereafter, being still above the control level at 11 days. The hydroxyproline content of the left ventricle was significantly increased at 11 days in hypertrophied heart muscle compared to controls. The present results suggest that in cardiac hypertrophy an early connective tissue activation occurs. Later, this leads to connective tissue accumulation. The increased collagen content may give support to the cardiac muscle contracting against systolic overload.

Corticosteroid-induced inhibition of the biosynthesis of human skin collagen☆

Abstract The effects of hydrocortisone acetate, fluocinolone acetonide, fluclorolone acetonide, betamethasone-17-valerate, fluprednyliden-21-acetate and flumethasone pivalate on the biosynthesis of human skin collagen were studied in vitro . Skin specimens were incubated in a medium containing a test substance and radioactive proline, and the formation of radioactive hydroxyproline in nondialysable proteins was taken as an index of the rate of collagen biosynthesis. Hydroxyproline formation was inhibited by all the corticosteroids tested in concentrations of 30 μg/ml or higher. The effect on hydroxyproline formation was smallest with hydrocortisone acetate and most pronounced with betamethasone-17-valerate in all concentrations used. In general, the corticosteroid-induced inhibition of collagen biosynthesis was found to be dose-dependent. The differences in the degree to which collagen formation is inhibited by the different corticosteroids may have relevance to the extent of the local side-effects, such as atrophy of skin, reported to be produced by fluorinated corticosteroids.

Collagen metabolism of the skin in Marfan's syndrome

Abstract 1. The metabolism of collagen in Marfans syndrome was studied by using punch biopsy specimens of skin from patients with Marfans syndrome and from healthy control subjects. In addition, the urinary hydroxyproline excretion of the patients with Marfans syndrome was measured. 2. 1. The skin specimens were subjected to extraction with 0.14 M and 1.0 M sodium chloride successively. In Marfans syndrome, the mean concentration of 0.14 M sodium chloride-soluble collagen was found to be 2–3-fold that of the control subjects. The mean concentration of 1.0 M sodium chloride-soluble collagen was also higher in Marfans syndrome, but the difference from the controls was smaller than in the 0.14 M sodium chloride-soluble collagen fractions. 3. 2. Despite the increased content of neutral salt-soluble collagen, the concentration of insoluble or total collagen was not significantly changed. 4. 3. The incorporation of [ 14 C]proline into [ 14 C]hydroxyproline of dermal collagen in vitro was greatly increased in Marfans syndrome. The increase in the uptake of the isotope was of about the same magnitude in both the soluble and insoluble collagen fractions of the skin. 5. 4. The urinary excretion of hydroxyproline was elevated in 2 of the 5 patients with Marfans syndrome. The results suggest that an increase in the turnover rate of collagen is the principal change in the metabolism of collagen in Marfans syndrome. However, a possible slight change in the rate of conversion of soluble collagen to insoluble, mature collagen fibres cannot be wholly excluded on the basis of the present results.

Activation of human leukocyte collagenase by compounds reacting with sulfhydryl groups

Abstract Human collagenase was partially purified from the granules of polymorphonuclear leukocytes by gel filtration, and the effects of two sulfhydryl reagents, N -ethylmaleimide and p -aminophenylmercuric acetate, on the enzyme activity were studied. The enzyme activity was assayed by incubating with soluble [ 14 C]proline-labeled type I collagen, and the rate of collagen cleavage was quantitated by isolating the specific cleavage products by SDS-polyacrylamide gel electrophoresis. Results demonstrated that the collagenase, which was at first mostly in a latent form, was rapidly activated by these two sulfhydryl reagents. The enzyme activity, however, returned gradually to the control level in the presence of the sulfhydryl reagent or after removal of an excess of the reagent. The enzyme activity, after activation with N -ethylmaleimide, could be returned to the control level by the addition of a 2-fold molar excess of cysteine. The heat stability of the enzyme activity before and after activation by N -ethylmaleimide was also tested. The results indicated that the initial enzyme activity, before the activation, was stable at 60°C for at least 5 min, and the enzyme could be subsequently activated by N -ethylmaleimide to the same extent as an unheated control. If, however, the enzyme was first activated by incubating in the presence of N -ethylmaleimide and subsequently incubated at 60°C, a marked decrease in the enzyme activity as a result of the 5 min heating was noted. The results of the present study indicate that human leukocyte collagenase can be activated by compounds reacting with thiol groups.

Effect of hydrocortisone acetate, fluocinolone acetonide, fluclorolone acetonide, betamethasone-17-valerate and fluprednyliden-21-acetate on collagen biosynthesis

Abstract The effect of hydrocortisone acetate, fluocinolone acetonide, fluclorolone acetonide, betamethasone-17-valerate and fluprednyliden-21-acetate on collagen biosynthesis was studied both in vivo and in vitro . In experiments in vivo , test substances and [ 14 C]proline were injected on the chorioallantoic membrane of 11-day-old chick embryos. In experiments in vivo , chick embryo tibiae were incubated in a medium containing the corticosteroids to be tested and [ 14 C]proline. In both types of experiments, the formation of [ 14 C]hydroxyproline was taken as a measure of the rate of collagen biosynthesis. In addition, the effect of these corticosteroids on the activity of protocollagen proline hydroxylase was tested. [ 14 C]hydroxyproline formation in vivo was inhibited by all the corticosteroids tested, and no clear difference in the degree of inhibition could be observed between the various corticosteroids. in vitro , betamethasone-17-valerate inhibited hydroxyproline formation more than the other substances tested. The concentration of corticosteroids required to inhibit collagen formation was considerably higher in vivo than in vitro . The corticosteroids tested did not affect the activity of protocollagen proline hydroxylase in the bones. In all these experiments, total protein synthesis, measured as incorporation of total 14 C-radioactivity into the bones, was inhibited by corticosteroids to the same extent as hydroxyproline formation. The results seem to suggest that corticosteroids inhibit collagen biosynthesis by inhibiting the formation of polypeptide precursors of collagen.

Increased protocollagen proline hydroxylase activity in synovial tissue in rheumatoid arthritis

Abstract Protocollagen proline hydroxylase activity was assayed in biopsy specimens of human synovial tissue. The enzyme required ascorbate, α-ketoglutarate, ferrous iron and molecular oxygen for its activity. The activity, determined from synovial tissue of 18 patients with rheumatoid arthritis, was increased as compared with values obtained from control patients.

Effect of d-Penicillamine on collagen biosynthesis in organ culture

Abstract The effect of d -penicillamine on the biosynthesis of collagen was studied in organ cultures, using ulnae of mouse embryos. Less [ 14 C]proline was incorporated as hydroxy[ 14 C]proline into the nondialysable proteins of the cultured bones when 10, 100 or 300 μg/ml d -penicillamine was present in the medium, and the ratio of hydroxy-[ 14 C]proline to total radioactivity incorporated was lower. In addition, 100 or 300 μg/ml d -penicillamine in the medium inhibited the incorporation of total 14 C radioactivity. When purified preparations of protocollagen proline hydroxylase were incubated with [ 14 C]proline-labelled protocollagen substrate in the presence of d -penicillamine, a decrease in the rate of hydroxy[ 14 C]proline formation was observed. This inhibition was dependent on the penicillamine concentration and could be reversed by increasing the amount of Fe 2+ present in the incubation medium. Assay of the total amount of protocollagen proline hydroxylase activity from bones cultivated with d -penicillamine revealed no significant difference from the controls.