STIMULATION OF COLLAGEN SECRETION BY ASCORBATE AS A RESULT OF INCREASED PROLINE HYDROXYLATION IN CHICK-EMBRYO FIBROBLASTS
STIMULATION OF COLLAGEN SECRETION BY ASCORBATE AS A RESULT OF INCREASED PROLINE HYDROXYLATION IN CHICK-EMBRYO FIBROBLASTS
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DOI：
10.1016/0003-9861(75)90031-4
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发表时间：
1975-01-01
影响因子：
3.9
通讯作者：
PETERKOFSKY, B
中科院分区：
文献类型：
--
作者：
BLANCK, TJJ;PETERKOFSKY, B
通过在蛋白质中掺入[14C]脯氨酸，然后用纯化的细菌胶原酶分别分析细胞内和培养上清液中的胶原量，来测定鸡胚成纤维细胞的胶原分泌。为了产生不同程度的羟化，细胞与不同浓度的抗坏血酸或在饱和抗坏血酸存在的情况下与不同浓度的α，α‘-联吡啶孵育。抗坏血酸刺激胶原中的脯氨酸的羟基化和胶原的分泌；两个过程的一半最大刺激所需的抗坏血酸的浓度约为4.5x10−7，m。由于细胞可以浓缩10倍的抗坏血酸，这一抗坏血酸羟化的抗坏血酸比纯化的Pro羟基酶的值低100倍(阿伯特，M.T.，和UdenFriend，S.(1974)在《氧气活化的分子机制》(Hayaishi，O.，编)，第173页，学术出版社纽约；Kivirikko K.I.，等(1968)Biochim)。生物群落。学报，151,558-567)。反之，α，ga‘-联吡啶则同时抑制了Pro羟化和胶原蛋白的分泌，在7×10−5，m时，这两个过程的抑制都达到最大值。两种类型的实验结果表明，当胶原中约30%的Pro残基发生羟化时，胶原的分泌与Pro羟化成正比，而最大羟化程度为50%。由于三螺旋胶原蛋白在37°C下的稳定性已被证明依赖于分子中的羟脯氨酸含量(RosenBloom，J.，等人(1973)Arch)。生物化学。生物物理学，158,478-484)，我们认为，观察到的分泌和羟化之间的比例是37℃下稳定的三螺旋胶原量增加的反映，当细胞与饱和浓度的抗坏血酸孵育时，在无细胞提取物中没有测量到显著的Pro羟基酶活性。这些实验表明，抗坏血酸通过作用于Pro羟化部位而不是通过增加Pro羟基酶的活性来影响胶原的分泌。
Collagen secretion by chick embryo fibroblasts was measured by incorporating [14C]proline into proteins and then analyzing the amount of collagen in the cell and medium separately by using purified bacterial collagenase. In order to produce varying levels of hydroxylation, cells were incubated with varying concentrations of ascorbate or with varying concentrations of α,α′-dipyridyl in the presence of saturating ascorbate. Ascorbate stimulated both the hydroxylation of proline in collagen and the secretion of collagen; the concentration of ascorbate required for half-maximal stimulation of both proesses was approximately 4.5 × 10−7,m. Since the cells could concentrate ascorbate 10-fold, thisKMfor proline hydroxylation is 100-fold lower than values reported for purified prolyl hydroxylase (Abbot, M. T., and Udenfriend, S. (1974)inMolecular Mechanisms of Oxygen Activation (Hayaishi, O., ed.), p. 173, Academic Press New York; Kivirikko K. I.,et al.(1968)Biochim. Biophys. Acta,151, 558–567). Conversely, α,ga′-dipyridyl inhibited both proline hydroxylation and collagen secretion; half-maximal inhibition of both processes was observed at 7 × 10−5,m. The results of the two types of experiments show that the secretion of collagen becomes directly proportional to proline hydroxylation when approximately 30% of the proline residues in collagen have been hydroxylated compared to maximal hydroxylation of 50%. Since the stability of triple-helical collagen at 37 °C has been shown to be dependent on the hydroxyproline content of the molecule (Rosenbloom, J.,et al.(1973)Arch. Biochem. Biophys.,158, 478–484), we suggest that the observed proportionality between secretion and hydroxylation is a reflection of the increased amount of stable triple helical collagen at 37 °C. When the cells were incubated with a concentration of ascorbate that was saturating for secretion and hydroxylation, there was no significant activation of prolyl hydroxylase as measured in a cell-free extract. These experiments suggest that ascorbate effects collagen secretion by acting at the site of proline hydroxylation but not by increasing the activity of prolyl hydroxylase.