Kuutti E R, Tuderman L, Kivirikko K I
Eur J Biochem. 1975 Sep 1;57(1):181-8. doi: 10.1111/j.1432-1033.1975.tb02289.x.
Prolyl hydroxylase was purified from human foetal skin and from a mixture of human foetal tissues by the affinity chromatography procedure using poly(L-proline). The enzyme from both sources was pure, when examined by polyacrylamide gel electrophoresis, as a native protein or in the presence of sodium dodecylsulphate, and enzyme activity recovery varied from 38% to 70% with seven enzyme preparations. The enzyme synthesized from 61.0 mumol to 82.7 mumol hydroxyproline mg protein-1 h-1 degrees C with a saturating concentration of (Pro-Pro-Gly)5 as substrate. The molecular weight of the enzyme was identical with that of the chick prolyl hydroxylase when studied by gel filtration, and the molecular weights of the subunits of the enzyme were about 61000 and 64000 as determined by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The amino acid composition of the human enzyme was very similar to that of the chick prolyl hydroxylase. Antisera to human and chick prolyl hydroxylases were prepared in rabbits. A single precipitin line was seen between the antiserum to human prolyl hydroxylase and the human enzyme in double immunodiffusion, and no cross-reactivity was detected between the human chick enzymes by this technique. However, a distinct cross-reactivity was observed between the human and chick enzymes in inhibition experiments.
采用聚（L-脯氨酸）亲和层析法从人胎儿皮肤和人胎儿组织混合物中纯化了脯氨酰羟化酶。通过聚丙烯酰胺凝胶电泳检测，来自这两种来源的酶无论是作为天然蛋白质还是在十二烷基硫酸钠存在的情况下都是纯的，七种酶制剂的酶活性回收率在38%至70%之间。以饱和浓度的（脯氨酸-脯氨酸-甘氨酸）5作为底物时，该酶每毫克蛋白质每小时每摄氏度合成61.0微摩尔至82.7微摩尔羟脯氨酸。通过凝胶过滤研究时，该酶的分子量与鸡脯氨酰羟化酶相同，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，该酶亚基的分子量约为61000和64000。人酶的氨基酸组成与鸡脯氨酰羟化酶非常相似。在兔体内制备了针对人和鸡脯氨酰羟化酶的抗血清。在双向免疫扩散中，人脯氨酰羟化酶抗血清与人酶之间出现了一条单一的沉淀线，通过该技术未检测到人和鸡酶之间的交叉反应。然而，在抑制实验中观察到人和鸡酶之间存在明显的交叉反应。
