Roswit W T, McCourt D W, Partridge N C, Jeffrey J J
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63130.
Arch Biochem Biophys. 1992 Feb 1;292(2):402-10. doi: 10.1016/0003-9861(92)90009-l.
Two protein inhibitors of metalloproteinases (TIMP) were isolated from medium conditioned by the clonal rat osteosarcoma line UMR 106-01. Initial purification of both a 30-kDa inhibitor and a 20-kDa inhibitor was accomplished using heparin-Sepharose chromatography with dextran sulfate elution followed by DEAE-Sepharose and CM-Sepharose chromatography. Purification of the 20-kDa inhibitor to homogeneity was completed with reverse-phase high-performance liquid chromatography. The 20-kDa inhibitor was identified as rat TIMP-2. The 30-kDa inhibitor, although not purified to homogeneity, was identified as rat TIMP-1. Amino terminal amino acid sequence analysis of the 30-kDa inhibitor demonstrated 86% identity to human TIMP-1 for the first 22 amino acids while the sequence of the 20-kDa inhibitor was identical to that of human TIMP-2 for the first 22 residues. Treatment with peptide:N-glycosidase F indicated that the 30-kDa rat inhibitor is glycosylated while the 20-kDa inhibitor is apparently unglycosylated. Inhibition of both rat and human interstitial collagenase by rat TIMP-2 was stoichiometric, with a 1:1 molar ratio required for complete inhibition. Exposure of UMR 106-01 cells to 10(-7) M parathyroid hormone resulted in approximately a 40% increase in total inhibitor production over basal levels.
从克隆大鼠骨肉瘤细胞系UMR 106-01条件培养基中分离出两种金属蛋白酶（TIMP）蛋白抑制剂。采用硫酸葡聚糖洗脱的肝素-琼脂糖层析，随后进行DEAE-琼脂糖和CM-琼脂糖层析，对30 kDa抑制剂和20 kDa抑制剂进行初步纯化。用反相高效液相色谱法将20 kDa抑制剂纯化至同质。20 kDa抑制剂被鉴定为大鼠TIMP-2。30 kDa抑制剂虽未纯化至同质，但被鉴定为大鼠TIMP-1。对30 kDa抑制剂的氨基末端氨基酸序列分析表明，其前22个氨基酸与人类TIMP-1的同源性为86%，而20 kDa抑制剂的序列在前22个残基上与人类TIMP-2相同。用肽：N-糖苷酶F处理表明，30 kDa大鼠抑制剂是糖基化的，而20 kDa抑制剂显然未糖基化。大鼠TIMP-2对大鼠和人类间质胶原酶的抑制是化学计量的，完全抑制需要1:1的摩尔比。将UMR 106-01细胞暴露于10^(-7) M甲状旁腺激素中，导致总抑制剂产量比基础水平增加约40%。
