Lah T T, Kokalj-Kunovar M, Kastelic L, Babnik J, Stolfa A, Rainer S, Turk V
Department of Biochemistry, Jozef Stefan Institute, Ljubljana, Slovenia.
Cancer Lett. 1992 Jan 31;61(3):243-53. doi: 10.1016/0304-3835(92)90295-7.
Cysteine proteinase inhibitors (CpI) of all three families were found in ascites fluid from patients with ovarian carcinoma. CPIs were isolated by affinity chromatography on carboxymethylated papain Sepharose, followed by gel filtration, anti-stefin-Sepharose and ion exchange chromatography. The highest apparent inhibition against cathepsin B (Cat B) was found in the low molecular mass (LMM) CPI fraction. Immunochemical analysis of this fraction revealed the presence of cystatin C and both stefins A and B while the high molecular mass (HMM) CPI fraction contained kininogens. We demonstrated that CPIs were not completely associated with cysteine proteinases (CPs): about 20% of HMM CPIs and 50% of LMM CPIs were free in native ascites fluid. Affinity chromatography on anti-Cat B-Sepharose revealed that the major LMM CPI, associated with Cat B in native ascites fluid, was the full length form of cystatin C, pI 9.3, and not its truncated form, pI 7.85. The latter was isolated and found to inhibit Cat B in vitro with apparent Ki 0.18 +/- 0.2 nM. Stefin A was isolated from alkaline activated ascites fluid in its two isoforms, pI 4.6 and 4.9. In native ascites, the pI 4.9 isoform was mostly associated with Cat B. Ki for Cat B was 3.55 +/- 1.7 nM, not significantly different from the Ki values measured for stefin A, isolated from other human tissues and biological fluids.
在卵巢癌患者的腹水液中发现了所有三个家族的半胱氨酸蛋白酶抑制剂（CpI）。通过羧甲基化木瓜蛋白酶琼脂糖凝胶亲和层析，随后进行凝胶过滤、抗丝抑蛋白琼脂糖凝胶和离子交换层析，从中分离出了CpI。在低分子量（LMM）CpI组分中发现了对组织蛋白酶B（Cat B）的最高表观抑制活性。对该组分进行免疫化学分析发现存在胱抑素C以及丝抑蛋白A和B，而高分子量（HMM）CpI组分中含有激肽原。我们证明CpI并非完全与半胱氨酸蛋白酶（CP）结合：在天然腹水液中，约20%的HMM CpI和50%的LMM CpI是游离的。抗组织蛋白酶B琼脂糖凝胶亲和层析显示，在天然腹水液中与组织蛋白酶B结合的主要LMM CpI是pI为9.3的全长胱抑素C，而非其截短形式pI 7.85。分离出截短形式并发现其在体外对组织蛋白酶B的抑制活性表观Ki为0.18±0.2 nM。从碱性激活的腹水液中分离出了两种同工型的丝抑蛋白A，其pI分别为4.6和4.9。在天然腹水中，pI为4.9的同工型主要与组织蛋白酶B结合。对组织蛋白酶B的Ki为3.55±1.7 nM，与从其他人体组织和生物体液中分离出的丝抑蛋白A所测得的Ki值无显著差异。
