Peterkofsky B, Assad R
J Biol Chem. 1976 Aug 10;251(15):4770-7.
A fraction greatly enriched in microsomes was prepared from chick embryo limb bone tissue homogenates by differential centrifugation in a high density solution of Metrizamide. This fraction was used to determine the submicrosomal localization of prolyl hydroxylase. At a low concentration (0.05%) of the non-ionic detergents Triton X-100 and Brij-35, 90 to 93% of prolyl hydroxylase activity was released from microsomes. Concentrations of Triton X-100 greater than 0.1% were required to solubilize the intrinsic membrane enzyme NADH-ferricyanide reductase and to release membrane-bound ribosomes, while Brij-35 did not extensively solubilize membrane components even at concentrations up to 0.4%. In addition, prolyl hydroxylase activity which could subsequently be released from microsomes by Brij-35 was relatively resistant to trypsin proteolysis at concentrations which removed more than 50% of the ribosomes and approximately 40% of the protein from microsomes. These results suggest that 90 to 93% of prolyl hydroxylase activity in connective tissue is located within the cisternae of the endoplasmic reticulum. Gel filtration of prolyl hydroxylase released from microsomes or found in the soluble fraction of limb bone homogenates revealed two peaks of activity corresponding to molecular weights of 230,000 and 450,000 to 500,000. The latter is twice the value reported for purified chick embryo prolyl hydroxylase. A fraction of the total prolyl hydroxylase activity (generally 20 to 35%) in microsome preparations could be measured in the absence of detergent, although the microsomal membrane should be impermeable to the large unhydroxylated collagen chains used as substrate. On the basis of experimental data, it was concluded that detergent-independent activity was most likely due to damaged microsomal membranes and that this damage was sufficient to allow substrate and trypsin to enter the cisternae but not to allow prolyl hydroxylase to be released.
通过在高密度的甲泛葡胺溶液中进行差速离心，从鸡胚肢体骨组织匀浆中制备出了富含微粒体的组分。该组分用于确定脯氨酰羟化酶的亚微粒体定位。在非离子去污剂Triton X-100和Brij-35浓度较低（0.05%）时，90%至93%的脯氨酰羟化酶活性从微粒体中释放出来。需要大于0.1%的Triton X-100浓度才能溶解内在膜酶NADH-铁氰化物还原酶并释放膜结合核糖体，而即使在浓度高达0.4%时，Brij-35也不会大量溶解膜成分。此外，随后可被Brij-35从微粒体中释放出来的脯氨酰羟化酶活性，在能去除微粒体中50%以上核糖体和大约40%蛋白质的浓度下，对胰蛋白酶水解相对具有抗性。这些结果表明，结缔组织中90%至93%的脯氨酰羟化酶活性位于内质网的潴泡内。对从微粒体释放或在肢体骨匀浆可溶性组分中发现的脯氨酰羟化酶进行凝胶过滤，显示出两个活性峰，分别对应分子量为230,000和450,000至500,000。后者是纯化鸡胚脯氨酰羟化酶报道值的两倍。尽管微粒体膜对用作底物的未羟化大胶原链应是不可渗透的，但在微粒体制剂中，总脯氨酰羟化酶活性的一部分（通常为20%至35%）在无去污剂的情况下也可被检测到。根据实验数据得出结论，不依赖去污剂的活性很可能是由于微粒体膜受损，且这种损伤足以使底物和胰蛋白酶进入潴泡，但不足以使脯氨酰羟化酶释放出来。
