Kao W W, Berg R A
Biochim Biophys Acta. 1979 Sep 3;586(3):528-36. doi: 10.1016/0304-4165(79)90043-6.
Prolyl hydroxylase activity in cultured L-929 cells was found to increase when cells grew from log phase to stationary phase and when cells were harvested at the mid-log phase and replated at higher cell densities. Cycloheximide and actinomycin D inhibited the cell density-dependent increase in prolyl hydroxylase activity indicating that the increase in prolyl hydroxylase activity required de novo synthesis of protein and RNA. Prolyl hydroxylase was purified from cultured L-929 cells and antibodies against the protein were raised in rabbits. The antibodies were used to demonstrate that L-929 cells contained two forms of prolyl hydroxylase: an enzymatically active, tetrameric form consisting of two alpha and two beta polypeptide chains and an enzymatically inactive form containing immunologically cross-reacting protein. The polypeptide chains alpha, beta and cross-reacting protein were obtained by immunoadsorption. Peptide map analysis indicated that cross-reacting protein was similar if not identical to beta in primary structure, and alpha was different from both beta and cross-reacting protein. The results suggested that the prolyl hydroxylase levels in cells or tissues may be regulated by new protein and/or RNA synthesis.
研究发现，当培养的L-929细胞从对数期生长到稳定期，以及当细胞在对数中期收获并以更高细胞密度重新接种时，脯氨酰羟化酶活性会增加。放线菌酮和放线菌素D抑制了脯氨酰羟化酶活性随细胞密度的增加，这表明脯氨酰羟化酶活性的增加需要蛋白质和RNA的从头合成。从培养的L-929细胞中纯化出脯氨酰羟化酶，并在兔体内制备了针对该蛋白的抗体。这些抗体被用于证明L-929细胞含有两种形式的脯氨酰羟化酶：一种由两条α和两条β多肽链组成的具有酶活性的四聚体形式，以及一种含有免疫交叉反应蛋白的无酶活性形式。通过免疫吸附获得了α、β多肽链和交叉反应蛋白。肽图谱分析表明，交叉反应蛋白在一级结构上即使不与β完全相同也很相似，而α与β和交叉反应蛋白都不同。结果表明，细胞或组织中的脯氨酰羟化酶水平可能受新蛋白质和/或RNA合成的调节。
