Kari I. Kivirikko

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.

Isolation and partial characterization of highly purified protocollagen proline hydroxylase

A procedure for the isolation of relatively stable preparations of highly purified protocollagen proline hydroxylase is reported. The most purified enzyme preparation was homogeneous in the analytical eltracentrifuge, and showed only one minor contaminant in disc electrophoresis. This enzyme preparation synthesized about 600 μg of hydroxyproline per mg protein per h under the assay conditions used, with a saturating concentration of the synthetic polytripeptide (Pro-Gly-Pro) molecular weight 6600, as substrate. n nThe enzyme had an S20w of 6.7. However, the elution pattern in gel filtration suggested a molecular weight of about 350 000. Amino acid analysis indicated that the enzyme preparation contained relatively large amounts of aspartic acid, glutamic acid, serine, glycine and lysine. n nThe activity of the purified enzyme was strongly inhibited by mercuribenzoate-p , and the inhibition could be reversed by dithiothreitol. N-Ethylmaleimide was a less effective inhibitor. The results suggests that free sulphydryl groups are required for the activity of the enzyme. n nAlthough partially purified preparation of the enzyme hydroxylate lysine as well as proline residues in protocollagen, the highly purified enzyme preparations did not hydroxylate lysine residues. Thus two separate enzymes, protocollagen proline hydroxylase and protocollagen lysine hydroxylase, are probably involved in the hydroxylation of proline and in protocollagen.

Collagen biosynthesis enzymes in serum and hepatic tissue in liver disease

Abstract. Serum immunoreactive prolyl hydroxylase protein was measured in sixty‐five patients with liver disease, and liver prolyl hydroxylase activity, immunoreactive prolyl hydroxylase protein and collagen hydroxyproline in forty of these patients. Serum immunoreactive prolyl hydroxylase protein was above the 95% confidence limit of the controls in most patients with primary biliary cirrhosis, portal cirrhosis, acute hepatitis and cancer with liver metastases, but below this in most patients with fatty liver, chronic active hepatitis, extrahepatic cholestasis, cholangitis, cancer without liver metastases and other malignant diseases. Elevated serum immunoreactive prolyl hydroxylase protein decreased rapidly with time in acute hepatitis but not in primary biliary cirrhosis. Liver prolyl hydroxylase activity and immunoreactive prolyl hydroxylase protein were elevated in the same diseases as serum immunoreactive prolyl hydroxylase protein, and correlated significantly with the latter whereas no correlation was found between serum immunoreactive prolyl hydroxylase protein and collagen hydroxyproline. Serum immunoreactive prolyl hydroxylase protein correlated highly significantly with serum alkaline phosphatase and weakly with serum aspartate aminotransferase in primary biliary cirrhosis, but not in any other disease. No correlation was found between serum immunoreactive prolyl hydroxylase protein and other tests of liver function. The results suggest that changes in serum immunoreactive prolyl hydroxylase protein in liver disease primarily reflect changes in this enzyme in the hepatic tissue, and that assays of serum immunoreactive prolyl hydroxylase in liver disease may give useful information on the actual hepatic collagen synthesis.

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.

Serum immunoreactive prolyl hydroxylase in liver disease.

Abstract. The concentration of serum immunoreactive prolyl hydroxylase (SIRPH) was measured in thirty patients with chronic active hepatitis, thirteen with primary biliary cirrhosis, four with alcoholic or idiopathic cirrhosis, and four with acute hepatitis; the values were compared with those in twenty‐three control subjects. Increases in SIRPH were found in all the groups with liver diseases, individual values being highest in primary biliary cirrhosis in which about two‐thirds of patients had values more than two standard deviations above the mean value in the control subjects. No correlation was found between SIRPH and other tests of liver function or some routine laboratory tests. SIRPH may reflect some hitherto unknown or unmeasured process in the diseased hepatic cells.

Effect of tetracycline on collagen biosynthesis in cultured embryonic bones.

Abstract The biosynthesis of collagen was studied with 14 C-proline in organ cultures of embryonic ulnae. The rate of conversion of the incorporated 14 C-proline to 14 C-hydroxyproline in cultured ulnae was optimal up to 6 days, provided that the medium contained ascorbate and was replaced daily. When the medium was not replaced daily, the rate of 14 C-proline incorporation was reduced to about 60 per cent, and only a small part of the incorporated 14 C-proline was converted to 14 C-hydroxyproline. In these conditions, a considerable part of the 14 C-proline was found to be in the form of protocollagen, the proline-rich and hydroxyproline-deficient polypeptide precursor of collagen. The addition of 10, 30 or 100 μg ml tetracycline to the culture medium reduced the rate of incorporation of 14 C-proline into protein. The rate of 14 C-hydroxyproline synthesis was reduced even more, and thus a decrease was observed in the conversion of the incorporated 14 C-proline to 14 C-hydroxyproline. Similar changes were observed when embryonic ulnae from pregnant mice receiving tetracycline were cultured in a medium not containing tetracycline. In experiments with purified protocollagen hydroxylase, tetracycline was found to inhibit the hydroxylation of 14 C-proline-labelled protocollagen, the degree of inhibition being dependent on the concentration of ferrous iron in the reaction mixture. Cultivation of the ulnae with tetracycline had no effect on the total amount of protocollagen hydroxylase activity in the bones when the determination was made in the presence of excess of ferrous iron. The results suggest that tetracycline inhibits collagen synthesis by inhibiting both the incorporation of proline into protocollagen and the conversion of protocollagen to collagen. The latter inhibition is probably due to an interaction of tetracycline with ferrous iron, which is required in the hydroxylation of proline by protocollagen hydroxylase.

Studies on the stability of protocollagen hydroxylase

The enzyme protocollagen hydroxylase [ 1,2] synthesises hydroxyproline by the hydroxylation of proline in protocollagen, the large proline-rich and lysinerich polypeptide precursor of collagen (for review, see [3,4]). The enzyme does not hydroxylate free proline, proline in tripeptides, or proline in poly-Lproline [S] , but hydroxylates proline in polytripeptides of the structure (LProGly-LPro), or (Gly-LAla-LPro), [ 1,2, 5-71. Procedures for the partial purification of protocollagen hydroxylase have been reported [ 1,5,7] ; however, the losses of enzyme have been relatively large, and the most highly purified preparations of the enzyme have rapidly lost their activity. As a result, it has been impracticable to work with these preparations, and studies of the characterisation of protocollagen hydroxylase have been made with enzyme preparations of a lower degree of purification [ 1,2,7] . In the present study, it was found that the addition of glycine protects protocollagen hydroxylase from losses of activity; the enzyme retained the highest degree of activity in solutions containing glycine and with pH values around 8. By the application of these conditions, it proved possible to develop a modified procedure for obtaining more purified preparations of the enzyme.

The absence of iron and certain coenzymes in highly purified protocollagen proline hydroxylase

Abstract The possible presence of protein-bound iron and of certain coenzymes in highly purified protocollagen proline hydroxylase was studied. All three enzyme preparations subjected to the study contained far less than 1 mole of iron/mole of enzyme. The results indicate that protocollagen proline hydroxylase does not contain stoichiometric amounts of iron bound firmly. The absorption spectrum of the enzyme showed no absorption maxima between 280 and 600 mμ, suggesting the absence of certain prosthetic groups, such as flavin nucleotides. Inhibition studies suggested that pyridoxal phosphate is not involved in the enzyme reaction, and direct assays for thiamine indicated the absence of thiamine and thiamine pyrophosphate. Thus, the decarboxylation of α-ketoglutarate coupled to the hydroxylation of peptidyl proline residues does not seem to be catalyzed by these prosthetic groups.

Action of growth hormone on the metabolism of collagen in the rat

Abstract The action of growth hormone on the metabolism of collagen was studied with 14 C-proline. (a) When 14 C-proline was injected 20 days after the beginning of growth hormone injections, the total activity of 14 C-hydroxyproline in the collagen fractions of the skin and in the nonfractionated collagen of the femurs was higher in the rats treated with growth hormone than in the controls. The distribution of 14 C-hydroxy-proline between the soluble and insoluble collagen of the skin was similar in the treated rats and the controls. The total activity of urinary 14 C-hydroxyproline was higher in the treated rats than in the controls, and this increase was of the same magnitude as in the 14 C-hydroxyproline of the skin and bone. (b) When 14 C-proline was injected 12 days before the beginning of growth hormone injections, administration of growth hormone caused an increase in the total activity of urinary 14 C-hydroxyproline compared to the controls. The results of the present study suggest that the rate of collagen synthesis was increased by growth hormone, and this effect was followed by an increased urinary excretion of hydroxyproline. No marked changes were found in the rate of conversion of soluble collagen into insoluble collagen or in the rate of catabolism of soluble collagen when expressed per unit of soluble collagen. The rate of catabolism of insoluble collagen was increased by growth hormone, and this effect contributed to the increased urinary hydroxyproline excretion.

Further evaluation of the significance of urinary hydroxyproline determinations in the diagnosis of thyroid disorders.

Abstract The urinary excretion of total hydroxyproline was determined in 262 patients with various thyroid disorders and in 47 euthyroid patients with elevated serum protein-bound iodine (PBI) but without thyroid disease. The values were compared with normal values (limits of ± 2 S.D.) in 92 children and in 72 adults 18 to 55 years of age, reported previously from our laboratory. In addition, normal values were determined in 53 adult euthyroid patients over 55 years of age. In adult patients, the urinary excretion of hydroxyproline was increased in hyperthyroidism above the upper limit of normal values in 107 out of 111 patients 18 to 65 years of age and in 10 out of 14 patients over 65 years of age. In hypothyroidism the urinary excretion of hydroxyproline was decreased below the lower limit of normal values in 22 out of 33 patients, and PBI was decreased in 24 of these patients. In the euthyroid patients with various thyroid disorders the urinary excretion of hydroxyproline was within the limits of normal values in 63 out of 68 patients. In an additional group of 47 patients with elevated PBI without hyperthyroidism, the urinary hydroxyproline values were within the limits of normal values in 46 cases. In children, the hydroxyproline values were above the upper limit of normal values in 10 out of 13 patients with hyperthyroidism, and below the lower limit of normal values in all 23 patients with hypothyroidism. The results support the earlier suggestion that determination of urinary hydroxyproline excretion may be a useful supplement to existing laboratory tests for the diagnosis of disturbances of thyroid function. The reliability of this test is high in cases of hyperthyroidism in a dult patients, and in cases of hypothyroidism in children.