ZoologicalJoumalofthe LinnaanSociety, 68.399-404. With 2 figures

April 1980

NELLY BLUMENKRANTZ AND GUSTAV ASBOE-HANSEN

Department of Dermatology, University of Copenhagen, Iligshospital, Copenhagen, Denmark

Acceptedforpublrcation March 1979

Protocollagen proline hydroxylase activity was studied in skin and muscle of poikilothermic and homoiothermic animals. Lower PPH activity was found in the former than in the latter. Increased PPH activity was found in skin of young Gullus domesticus and Rattus noruegicus. In specialized skin under hormonal control, i.e. comb and wattles, increased activity in relation to sexual maturation was shown. The PPH activity in muscles of poikilotherms was lower than in those of homoiotherms. Some of the muscles had a considerable enzymatic activity. A decreasing PPH activity was noticed in birds and mammals with age. Heart muscle had more hydroxylating activity than skeletal muscle.

KEY WORDS: - protocollagen - proline - hydroxylase - vertebrate muscle - skin

CONTENTS

Introduction . . . . . . . . . . . . . . . . . . . . . 399 Materialandmethods . . . . . . . . . . . . . . . . . . . . 400 Results 40 1 Discussion . . . . . . . . . . . . . . . . . . . . . . . 403 References . . . . . . . . . . . . . . . . . . . . . . . 404

. . . . . . . . . . . . . . . . . . . . . . . .

INTRODUCTION

The activity of protocollagen proline hydroxylase (PPH), an enzyme which catalyzes hydroxylation of certain proline (Pro) residues of protocollagen to hydroxyproline (Hyp), has been used widely as an indicator of collagen biosynthesis. Various tissues of chick embryo, rat and man have been studied for PPH activity by several authors (Blumenkrantz 8c Asboe-Hansen, 1972, 1975a; Salvador & Tsai, 1973; Suominen, Heikkinen & Parakatti, 1977). Due to widely different incubation conditions and methods, the available results are difficult to compare.

A study of the PPH activity in skin and muscle of various fishes, amphibians, birds and mammals is reported below. The purpose of this study was to compare the PPH of two tissues of the same animal, and to follow the activity in relation to evolution in vertebrates.

399 0024-4082/80/040399-06/$02.00/0 0 1980 The Linnean Society of London

400 N. BLUMENKRANTZ AND G. ASBOE-HANSEN

MATERIAL AND METHODS

Samples of skin and muscle were obtained at autopsy of the animals listed in Fig. 1. The tissues were dissected immediately after the animals had been sacrificed, and were kept in ice. The enzyme was extracted immediately after removal. All steps of the procedure were performed at 4OC. Two animals were used as donors of the samples. All determinations were performed in duplicate. The resulting values represent the average of the percentage hydroxylation.

Preparation .f protocollagen proline hydroxylase

Two grams of minced skin or muscle previously soaked in 20 ml 0.01 M KCl was homogenized at 45,000 g for 6 min in a Virtis homogenizer surrounded by crushed ice. The homogenates were centrifuged at 15,000 g for 30 min at 4°C in a Servall centrifuge. The supernatant was used as enzyme.

Preparation of the unhydroxylated substrate (',C) Pro labelled protocollagen

Ten-day-old chicken embryo tibiae were pre-incubated for 30 min at 37OC under aerobic conditions in a medium of salts, buffer phosphate and glucose in the presence of 1 mM of a Fez+ ion chelator such as aa' dipyridyl or dihydralazine. Immediately after this, the tissues were incubated for 2 h under the same aerobic conditions with 50 pCi P'C) Pro diluted in the same medium. At the end of the incubation period, the tissues were homogenized in the medium. The homogenate was then centrifuged at 15,000 g for 1 h at 4OC in a Servall centrifuge. The supernatant was dialysed for 24 h against 4 changes of 4 litres of 1 M KC1-0.02 M Tris buffer pH 7.6. After dialysis, the sample was boiled for 5 min and, after cooling, it was diluted with 1 mM KC1 to give 8O,OO(b 100,000 dpm/ml. One ml aliquots were distributed and stored frozen in 20 ml ampoules.

Preparation of co-factors

A solution of co-factors was prepared immediately before the hydroxylation assay. The co-factor solution was made up of solution A: 11 1.2 mg FeSO, in 10 ml distilled water, and solution B: 29.2 mg a-ketoglutarate and 140.8 mg ascorbic acid dissolved in 30 ml of water; 0.5 ml of solution A was mixed with 30 ml of solution B and 10 ml of 0.5 M Tris-HC1 pH 7.8 at 25OC; 0.8 ml of this solution was used as co-factor solution in the hydroxylation assay. The content of co- factors was for a-ketoglutarate 0.5 mM, for ascorbic acid 2.0 mM, and for FeSO, 0.05 mM.

PPH assay

This was performed according to Blumenkrantz & Asboe-Hansen (1976). After defrosting the substrate, the enzyme assay was performed in the ampoules. One ml of enzyme and 0.8 ml of the co-factor solution was added to the 1 ml of substrate present in the ampoules. While being shaken, the samples were incubated for 1 h at 37 OC. The reaction was stopped by cooling the samples in ice and by adding 15 ml

PROTOCOLLAGEN PROLINE HYDROXYLASE 40 1

cold acetone, which allows the proteins to precipitate. Thereafter, the samples were centrifuged at 2000 rprn for 30 min. The supernatant was discarded and the pellet hydrolyzed with 2 ml of6 N HCI at 1 loo C for 16-18 h. After evaporation of the HCl at 65OC under vacuum, the samples were suspended in buffer and divided into two aliquots (Blumenkrantz & Asboe-Hansen, 197513). One 0.1 ml aliquot was used to determine the total 14C. The (I4C) Hyp synthesized was analyzed on the other aliquot (4.5 ml) according to procedure IIB of Blumenkrantz & Asboe- Hansen (1975b).

The percentage hydroxylation was calculated as follows :

dpm (14C) Hyp

dpm total 14C in substratel x 100 = % hydroxylation

The results were expressed as percentage hydroxylation of protocollagen by 100 mg wet weight of tissue.

RESULTS

Protocollagen proline hydroxylase in skin

Results are presented in Fig. 1.

Poikilotherms Fish. High PPH activity was found in Gadus morrhua. The difference in the

enzymic activity between dorsal and ventral skin of Pleuronectes platessa was noticeable, while Anguilla anguilla showed no topographic differences. Cyprinus carpius had lower activity in dorsal and ventral skin than in the swim bladder (Fig. 1 ) .

Amphibia. The PPH activity was higher in dorsal than in ventral skin of the amphibians studied. The heat adaptation to 20°C of Bufo bufo produced a decrease in the PPH activity.

Homoiotherms Birds. A general pattern of decrease of PPH activity in skin with increasing age

was observed in Gallus domesticus. In specialized skin under hormonal control, e.g. comb and wattles, there was increasing PPH activity with advancing maturation.

Mammals. There was a decrease in enzymic activity in the skin of rat (Rattus norvegicus, Wistar) with advancing age. The PPH activity in the skin of mouse (Mu5 musculus, C3H) was high, while very low in that of the rabbit (Oryctolagus cuniculus, White Danish land race) analyzed.

Protocollagen proline hydroxylase in muscle

PPH activity in muscle of various vertebrates is presented in Fig. 2.

Poikilotherms

was also found in theBufo bufo adapted to 2OOC. Low PPH activity was found in muscle of fishes and amphibians. A low activity

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Figure 1 . Protocollagen proline hydroxylase activity in skin of vertebrates.

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Figure 2. Protocollagen proline hydroxylase activity in muscle of vertebrates.

PROTOCOLLAGEN PROLINE HYDROXYLASE 403

Homoiotherms

Birds. The highest PPH activity was detected in muscle of the 20-day-old chicken embryo (Gallus domesticus) decreasing from then on up to the 14-week- old bird. The activity in heart muscle also decreased with age, although the values kept a higher level than in skeletal muscle.

Mammals. Maximum PPH activity was observed in very young rats, the activity reaching a plateau with age.

The guinea pig (Cauia porcellus, albino) had the lowest PPH activity in skeletal muscle as compared to other mammals. The heart muscle of the guinea pig had higher PPH activity than that of skeletal muscle.

The muscles of rabbit had similar PPH activities.

DISCUSSION

The low PPH activity in skin of fishes and amphibians, as compared to birds and mammals, is in agreement with the low Hyp content which is known to be valid for the two former groups. The low denaturation temperature which is characteristic of the poikilotherms should possibly be explained by this low Hyp content. The contribution of the total content of imino acids to the stabilization of collagen, as correlated with denaturation temperature, has been reported previously (von Hippel & Wong, 1963). Recent experiments have shown that Hyp is crucial to thermal stability of collagen and procollagen (Berg & Prockop, 1973). Jimenez, Harsch, Murphy 8c Rosenbloom (1974) suggested that, under certain conditions, the body temperature of an organism may, in part, regulate the Hyp content in collagen molecules. Partial triple helix formation occurs more readily at lower temperatures (Jimenez et al., 1974). In vitro experiments on cells having accumulated unhydroxylated procollagen molecules at 3 7 * C, and being allowed to hydroxylate at different temperatures, have shown that the degree of hydroxylation increases with the temperature (Jimenez et ad., 1974).

Although quite universal, the rule of low values of Hyp in poikilotherms has been shown to have at least one exception, in so far as higher Hyp and denaturation temperature was found in skin collagen from Cyprinus carpius, a fresh water fish, than in that of Gadus morrhua, a salt water fish (Eastoe, 1967). We found no reasonable correlation with the lower PPH activity shown in the former fish. We cannot consider this lack of relationship to be due to difference in age because, when analyses of Hyp and Hyl were performed on aliquots of skin of the same animals used in the extraction of PPH, lower values were found in Gadus morrhua than in Cyprinus carpius (not shown). In the latter, the difference between skin and swim bladder was noticeable. I t is difficult to explain the lower content of collagen Hyp in Gadus morrhua which has higher PPH activity, unless this activity is not a limiting factor for the hydroxylation. We do not discard the possibility either of an underhydroxylation, perhaps determined by the environment, or the presence of other amino acids in the Gadus morrhua collagen which might render it resistant to hydroxylation.

I t is notable that the PPH activity was relatively higher in the younger than in the older animals studied. It might mean that in the physiological period, where the building up of the collagen is required, the skin PPH activity parallels the known increase of collagen biosynthesis and metabolism. Our finding of increasing PPH activity with the development of the cock’s comb, which is

404 N. BLUMENKRANTZ AND G. ASBOE-HANSEN

directly related to sexual maturation, coincides with the activity of the same enzyme in the testes of rats (Blumenkranrz & Asboe-Hansen, 1976).

We do not disregard age difference as a factor which may have influenced the results in our fishes and amphibians, the ages of which were not known. Our determinations of PPH activity in muscle of bird showed that there was a marked decrease of PPH activity in muscle of chicks with age. I t is remarkable that different levels of activity of PPH were found to be related to such genetically different collagens as those of skin and muscle, i.e. types I and I11 in skin and type V in muscle (Bentz, Glanville & Kuhn, 1978).

REFERENCES

BENTZ, H., GLANVILLE, R. & KuHN, K., 1978. Type V collagen. A molecule with a chain composition aA(aB),. (Abstract). Biochemistry of Normal and Pathological Connective Tissues. 6th Colloquium of the Federation of European Connective Tissue Clubs: 20 1.

BERG, R. A. & PROCKOP, D. J., 1973. The thermal transition of non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen. Biochemical and Biophysical Research Communications, 52: 1 15-1 20.

BLUMENKRANTZ, N. & ASBOE-HANSEN, G. , 1972. Biosynthesis of collagen ("C) hydroxyproline and ("C) hydroxylysine during maturation of chick embryos. Mechanisms of Ageing and Development, I: 445-450.

BLUMENKRANTZ, N. & ASBOE-HANSEN, G., 1975a. Hydroxyproline and hydroxylysine in different tissues of human embryos. Mechanisms of Ageing and Development, 4 : 281-287.

BLUMENKRANTZ, N. & ASBOE-HANSEN, G . , 1975b. Simple procedure for determination of ("C) hydroxyproline. Analytical Biochemittry, 66: 330-339.

BLUMENKRANTZ, N. & ASBOE-HANSEN, G., 1976. Simplified method for determination of protocollagen- proline hydroxylase. Clinical Biochemistry, 9 : 256-259.

EASTOE, J. E., 1967. Composition of collagen and allied proteins. In G. N. Ramachandran (Ed.), Treatise on Collagen, 1. Chemistry of Collagen: 1-72. New York: Academic Press.

VON HIPPEL, P. H. & WONG, K., 1963. Collagen-gelatin phase transition. I. Further studies of the effects of solvent environment and polypeptide chain composition. Biochemistry, 2: 1387-1 398.

JIMENEZ, S. A,, HARSCH, M., MURPHY, L. & ROSENBLOOM, J., 1974. Effects of temperature on conformation hydroxylation and secretion of chick tendon procollagen. The Journal of BiOkJgical Chemistry, 249: 4480-4486.

SALVADOR, R. A. & TSAI, I., 1973. Collagen proline hydroxylase activity in the uterus of the rat during rapid collagen synthesis in viuo. Archives of Biochemistry and Biophysics, 154: 583-592.

SUOMINEN, H., HEIKKINEN, E. & PARAKATTI, T., 1977. Effect of eight weeks physical training on muscle and collagen tissue of the M. Vastus lateralis in 69-year-old men and women. Journal oJGerontofogy, 32: 33-37.