Developmental chanegs in protocollagen lysyl hydroxylase activity in the chick embryo

121

Biochimica et Biophysica Acta, 343 (1974) 121--128

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA

27353

DEVELOPMENTAL CHANGES IN P R O T O C O L L A G E N LYSYL H Y D R O X Y L A S E ACTIVITY IN THE CHICK EMBRYO

LASSE RYH:~NEN and KARI I. KIVIRIKKO Department of Medical Biochemistry, University of Oulu, Oulu (Finland)

(Received September 20th, 1973)

Summary Developmental changes in protocollagen lysyl hydroxylase activity were studied in whole chick embryos and in several tissues of chick embryos. In whole chick embryos, the enzyme activity increased between the 7th and 15th day of development, and decreased thereafter. In bone and skin the enzyme activities likewise had maximum values on the 15th day of development, whereas in lung, kidney, heart, spleen and liver the enzyme activities showed relatively small changes between the 9th and 21st day of development. Comparison of lysyl hydroxylase activities in several bones and other tissues of 15-day-old embryos indicated that the enzyme activity in all bones studied was considerably higher than that in skin. However, values even higher than those in bone were found in leg tendon. The lowest enzyme activities were measured in heart, spleen and liver. Attempts to influence the development of lysyl hydroxylase activity by administration of lactate or ascorbate on to the chorioallantoic membrane caused no significant changes in the enzyme activity. The changes in lysyl hydroxylase activity observed in the present study agree with previous data on changes in prolyl hydroxylase activity and in collagen biosynthesis in the developing chick embryo. It seems possible that changes in lysyl hydroxylase activity like those in prolyl hydroxylase activity may correlate with changes in collagen biosynthesis in several situations.

Introduction Collagen is the major c o m p o n e n t of animal connective tissues and the most abundant protein in the body. It contains two amino acids, hydroxyproline and hydroxylysine which are absent in most other proteins. The hydroxyproline and hydroxylysine in collagen are synthesized by hydroxylations of certain prolyl and lysyl residues already incorporated into peptide linkages (for reviews, see refs 1,2). These hydroxylations are catalyzed by two separate enzymes [3- 6 ] , prolyl hydroxylase and lysyl hydroxylase, and both reactions

122 require molecular oxygen, ferrous iron, a-ketoglutarate and a reducing agent which can be ascorbate [ 1 , 2 , 7 - 1 0 ] . Prolyl hydroxylase has been isolated in a highly purified form from two sources [3,11,12], and a number of studies have been reported on changes in the activity of the enzyme in animal and human tissues in various experimental and clinical conditions [1,2]. Most of such studies indicate that increases in the rate of collagen biosynthesis are accompartied by increased prolyl hydroxylase activity, and in many reports the increased enzyme activity was noted prior to an increase in the collagen content of the tissue studied [1,2]. Lysyl hydroxylase has not y e t been obtained in pure form, but recently the enzyme was purified up to 300-fold from chick embryo extracts [5], and was found to resemble prolyl hydroxylase in many of its properties [5,13]. The activity of lysyl hydroxylase in h u m a n skin was found to be highest in foetuses, and higher in infants than in adult subjects [14]. The enzyme activity was markedly reduced in cultured skin fibroblasts from two patients with a deficiency of hydroxylysine in collagen [15]. No other data are available on changes in lysyl hydroxylase activity in various conditions. In the present study attempts were made to gain some information about the relationship of changes in lysyl hydroxylase activity to collagen biosynthesis by studying the developmental course of lysyl hydroxylase activity in various tissues of chick embryos. Collagen has been detected by assays on hydroxyproline in chick embryos as early as 60 h after fertilization [16], and marked changes have been reported to take place in collagen biosynthesis [16--19] and prolyl hydroxylase activity [20] during embryonic development. Material and Methods

Extraction of lysyl hydroxylase from whole chick embryos and from various tissues of the embryos Fertilized eggs of white Leghorn chickens were purchased from Siipikarjanhoitajien Liitto r.y., Hameenlinna, Finland, and they were incubated at 37°C in humidified incubators. Embryos were dissected free of surrounding membranes, and various tissues were carefully dissected out at 0°C. The embryos or tissues were homogenized in a cold (0°C) solution consisting of 0.2 M NaC1, 0.1 M glycine, 50 pM dithiothreitol, and 20 mM Tris--HC1 buffer adjusted to pH 7.5 at 4°C (10 ml of solution per g of embryo) [5]. Soft tissues from all embryos, and bone and whole embryos up to 15 days of development were homogenized with a Teflon and glass homogenizer. In other instances the homogenization was carried out with an Ultra-Turrax homogenizer 3 times for 5 s. No difference in the enzyme activity was observed, when 15-day-old embryos or bones from 15-day-old embryos were homogenized with a Teflon and glass homogenizer or with an Ultra-Turrax homogenizer. The homogenates were centrifuged at 15000X g for 30 min at 4°C, and aliquots of the supernatant were used for the assay of lysyl hydroxylase activity. Previous studies indicated that about 90% of the enzyme activity in the homogenates of whole chick embryos [5] or human skin [14] was recovered in the 15000 X g supernatant with the buffer solution used in the present experiments. The addition of 0.1% Trition X-100 did not increase the enzyme activity extracted [5,14].

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Assay of lysyl hydroxylase activity In order to assay lysyl hydroxylase activity, aliquots of the 15000 X g supernatant were incubated for 30 min at 37°C with 200000 disint./min of [Lys-14C]protocollagen substrate in a final volume of 1 ml containing 0.05 mM FeSO4, 0.5 mM a-ketoglutarate, 0.5 mM ascorbic acid, 0.1 mg of catalase (Calbiochem), 0.1 mM dithiothreitol (Calbiochem), 1.5 mg of bovine serum albumin (Sigma), and 50 mM Tris--HC1 buffer adjusted to pH 7.8 at 25°C (ref. 5). The volume of the 15000 X g supernatant used in the incubation was dependent on the age of the embryo, and on the tissues studied. The 15000 × g supernatant from liver was diluted with an equal volume of 20 mM Tris- HC1 buffer adjusted to pH 7.8 at 25°C, and the supernatant from other tissues with 4 volumes of the same buffer. An aliquot of 0.05 to 0.10 ml of the dilution was then used in the assays with the exception of bone, where the aliquot was 0.02 to 0.05 ml. The enzymic reaction was stopped by adding 10 ml of cold acetone [21], and after the samples had stood in the cold for 30 min, they were centrifuged at about 3 000 X g for 30 min and the supernatants were removed by suction. The pellets were dried by blowing N2 into the tubes, and suspended in 1 ml of distilled water to which 6 ml of 0.3 M c i t r a t e - p h o s p h a t e buffer (pH 6.4) was then added. H y d r o x y [ 14 C]lysine in the samples was assayed by periodate oxidation with a specific chemical procedure [22] using half the volumes originally suggested. The lysine-labelled protocollagen used as a substrate was prepared as described previously [5], and stored frozen in aliquots of 200000 disint./min. Each substrate preparation could usually be divided into about a hundred such aliquot. Because of variation in the properties of biologically prepared protocollagen substrate, only enzyme activities assayed with samples of the same substrate preparation are directly comparable with one another. Therefore, all final experiments reported in the study were carried out using only three different substrate preparations. The experiments were designed so that embryos from different ages were dissected and analyzed on the same day, and the levels of the experiments carried out with different substrate preparations were adjusted by using a crude a m m o n i u m sulphate enzyme [5] (17 to 45 per cent saturation) prepared from chick embryos, and stored frozen in aliquots. One such aliquot was included as a standard in every assay. The variation in the activity of the standard enzyme observed in h y d r o x y l a t i o n experiments with the same substrate preparation was about _+ 5 per cent. All counting of ~4 C was performed in a Wallac liquid Scintillation spectrometer with an efficiency of 85 per cent and a background of 25 cpm. Results

In order to study developmental changes in lysyl hydroxylase activity in whole chick embryos, the enzyme activity was determined in the 15000 X g supernatants of the embryo homogenates between the 4th and 19th day of embryonic development (Fig. 1). The activity of lysyl hydroxylase was found to increase between the 7th and 15th day of development. A rapid decline in the enzyme activity was noted thereafter. The m a x i m u m in the enzyme activity on the 15th day was observed whether the activity was calculated per unit wet weight or per unit dry weight of the embryo.

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125 TABLE I RELATIVE ACTIVITIES OLD CHICK EMBRYO

OF LYSYL HYDROXYLASE

IN D I F F E R E N T

TISSUES OF THE 15-DAY-

T h e e n z y m e a c t i v i t i e s in d i f f e r e n t t i s s u e s w e r e d e t e r m i n e d as d i s i n t . / m i n p e r m g w e t w e i g h t o f t h e tissue, a n d t h e y w e r e t h e n c o m p a r e d to t h e e n z y m e a c t i v i t y in t i b i a e , w h i c h w a s t a k e n as 1 0 0 . T h e d a t a w e r e o b t a i n e d in t h r e e s e p a r a t e e x p e r i m e n t s , e a c h o f t h e m i n c l u d i n g a s s a y s f o r t h e e n z y m e a c t i v i t y in t i b i a e . V a l u e s f o r o t h e r t i s s u e s w e r e d e t e r m i n e d in t w o o f t h e t h r e e e x p e r i m e n t s , a n d t h e a s s a y s in t h e e x p e r i m e n t w e r e c a r r i e d o u t w i t h d u p l i c a t e s a m p l e s c o n t a i n i n g t i s s u e f r o m 5 to 20 e m b r y o s d e p e n d i n g on t h e t i s s u e s t u d i e d . T h e v a l u e s h o w n f o r a tissue is t h e m e a n o f all t h e s e values.

Tissue

Relative enzyme activity per m g w e t wt. o f t i s s u e

Leg t e n d o n Humerus Femur Tibia Sternum Skull Skin L a r g e vessels Lung Kidney Heart Spleen Liver

145.9 122.6 103.1 100.0 88.1 69.7 39.7 23.6 18.4 1 5.9 12.5 12.3 7.2

Assays of lysyl hydroxylase activity in several tissues of the embryo indicated that only the enzyme activity in bone and skin among the tissues studied had a definite m a x i m u m value on the 15th day of development (Fig. 2). The enzyme activities in lung, kidney, heart, spleen and liver showed relatively small changes between the 9th and 21st day, although a small maximum on the 15th day was noted in lung and kidney. Comparison of lysyl hydroxylase activities in several bones and other tissues of 15-day-old chick embryos indicated that enzyme activities in all bones studied were considerably higher than those in skin (Table I). However, values even higher than those in bone were observed in embryonic leg tendon. Developmental changes in the enzyme activity in leg tendon were n o t studied further due to difficulties in preparing sufficient quantities of tendons from younger embryos. The lowest enzyme activities were found in heart, spleen and liver. In order to study whether it was possible to influence the development of lysyl hydroxylase activity in chick embryos by lactate or ascorbate, these substances were administered on to the chorioallantoic membrane. The results indicated that administration of sodium lactate in amounts ranging from 40 to 200 mg or sodium ascorbate in amounts ranging from 20 to 100 mg either as a single dose 3 h or as two doses 3 and 12 h before the removal of embryos from the eggs did not significantly affect the enzyme activity in the 15000 × g supernatant of whole embryos on the 10th day of development.

126 Discussion Previous studies on collagen metabolism in chick embryos indicated the presence of an in flexion point around the midpoint of embryonic development. The contents of free hydroxyproline and hydroxyproline in soluble collagen in whole chick embryos and in the skin of chick embryos [16], and the accumulation of collagen in chick embryo corium [18] had m a x i m u m values on the 14th to 16th day of development. The activity of prolyl hydroxylase likewise showed a clear maximum on the 14th day [20]. The present results demonstrate a similar inflexion point in lysyl hydroxylase activity. Accordingly, the developmental changes in lysyl hydroxylase activity seem to correlate in chick embryos with changes in collagen biosynthesis, and it seems possible that levels of this enzyme activity like those of prolyl hydroxylase activity [1,2] correlate with collagen biosynthesis in several other instances, too. This suggestion is supported by recent studies on changes in lysyl hydroxylase activity in human skin with age [14] and in rat liver in experimental fibrosis (Risteli, J. and Kivirikko, K.I., to be published). Assays of lysyl hydroxylase activity in various tissues of chick embryos indicated that a definite maximum value on the 15th day is present in bone and skin only. Similar findings were previously reported on prolyl hydroxylase activity [20]. The differences in lysyl hydroxylase activity between various tissues on the 15th day are similar to differences in prolyl hydroxylase activity [20] except for embryonic bones. The activity of prolyl hydroxylase in tibiae was on the 14th day about 1.2 times that in skin, and about 4 times that in liver. On the 15th day there were no differences in prolyl hydroxylase activities between tibiae and skin [20]. On the contrary, the lysyl hydroxylase activity in tibiae was on the 15th day about 2.5 times that in skin and about 14 times that in liver, and remained markedly higher in tibiae than in skin at all develop= mental ages studied. The lysyl hydroxylase activity in other bones was likewise higher than that in skin. The hydroxylysine c o n t e n t of collagens from different tissues shows considerable variation ranging from about 5 to 40 residues per 1000 amino acids [1]. Collagens from basement membranes [23] and cartilage [24] are particularly rich in hydroxylysine. Furthermore, the level of hydroxylation of lysyl residues in collagen from the same tissue may vary with age [25--27], and a marked elevation in the level of h y d r o x y l a t i o n of lysyl residues was reported in bone collagen from rachitic animals [28,29]. Recent studies also indicated that the hydroxylysine contents of collagens from tissues such as skin and tendon do not reflect the maximal extent to which lysyl hydroxylase can h y d r o x y l a t e these proteins, and the hydroxylysine contents of these collagens could be doubled by a reaction with lysyl hydroxylase in vitro [30]. The present data on differences in lysyl hydroxylase activity between various tissues are in agreement with these studies. The embryonic bones studied here were rich in cartilage, and the high lysyl hydroxylase activity agrees with the high hydroxylysine c o n t e n t of cartilage collagen. The high lysyl hydroxylase activity in embryonic leg tendons is likewise not surprising. Although collagen from Achilles tendon from adult animals has a low turnover rate and the hydroxylysine c o n t e n t of this collagen is not elevated, cells isolated from embryonic leg

127

tendons were reported to synthesize collagen at a rapid rate, and a high proportion of the lysyl residues in this collagen was found to be h y d r o x y l a t e d [31]. An increased h y d r o x y l a t i o n of lysyl residues in embryonic leg tendon compared to tendon from adult animals was also found by analysis of specific peptides isolated from the collagen [26]. The data do not indicate whether all changes observed in the present study are due to differences in the activity of a single enzyme, or whether they partly reflect changes in the proportions of two or more lysyl hydroxylases with different activities. Partially purified lysyl hydroxylase from whole chick embryos was recovered in gel filtration in at least two forms with different molecular weights [5], but the enzyme has not been isolated as a pure protein. It is not known whether these forms represent different forms of the same protein or whether they represent different proteins. Because lactate [32] and ascorbate [33] are known to activate prolyl hydroxylase in cultured fibroblasts, experiments were carried out to show whether it was possible to influence the development of lysyl hydroxylase activity in chick embryos by administration of these substances. No increase in the enzyme activity was observed in these experiments. These results do not necessarily indicate differences between the activation of prolyl and lysyl hydroxylases, because no data are available to show whether the lactate activation of prolyl hydroxylase observed in fibroblasts in culture can be demonstrated in chick embryos, and because attempts to activate prolyl hydroxylase by ascorbate in chick embryos caused only slight increases in the enzyme activity [20]. Acknowledgements The authors gratefully acknowledge the expert technical assistance of Miss Marja Tasala and Mrs Pirkko Tolonen. The work was supported in part by grants from the National Research Council for Medical Sciences, Finland, and from the Sigrid Jusdlius Foundation.

Note added in proof (Received January 9th, 1973) We have recently found that the activity of lysyl hydroxylase in the 15 000 X g supernatant of the homogenates of whole chick embryos and various tissues of chick embryos, increases by about 30--40%, when the homogenates are preincubated with 0.1% triton X-100 at 4°C for 2 h. The magnitude of this increase was found to be similar in tissues with a high lysyl hydroxylase activity and low lysyl hydroxylase activity, e.g. in bone and in liver. Accordingly, the magnitude of all changes and differences reported in the present study would have been the same, if the homogenates had been preincubated with Triton X-100. References 1 G r a n t , M.E. a n d P r o c k o p , D.J. ( 1 9 7 2 ) N e w Engl. J. Meal. 2 8 6 , 1 9 4 - - 1 9 9 ~ 2 4 2 - - 2 4 9 ~ 2 9 1 - - 3 0 0 2 K i v i r i k k o , K.I. ( 1 9 7 3 ) Proc. 2 n d Eur. Syrup. o n C o n n e c t i v e Tissue R e s e a r c h , H a n n o v e r , in the press

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