Tsushima H, Sumi H, Mihara H, Joronen I, Hopsu-Havu V K
Department of Physiology, Miyazaki Medical College, Japan.
Biol Chem Hoppe Seyler. 1988 May;369 Suppl:243-50.
Cysteine proteinase inhibitors in the human melanoma tissue transplanted into nude mice were found to increase in concentration during tumor growth. The activity (unit/g) at 8 weeks was about 3 times higher than the activity at 4 weeks after transplantation. The inhibitors were separated into two main forms (Mr about 76,000 and 10,000) with Sephacryl S-200 and/or Sephadex G-75 gel chromatography. The activities of the inhibitors of both molecular masses increased parallely during tumor growth. The high molecular mass inhibitor fractions reacted with antisera made against alpha-cysteine proteinase inhibitor (alpha-CPI, human kininogen) and against neutral low-molecular mass proteinase inhibitor (cystatin B). Free cystatin B appeared to be liberated in SDS-polyacrylamide gel electrophoresis following electroimmunoblotting with an antiserum to cystatin B. Similarly, free cystatin B was detected in gel chromatography on Sephadex G-75 after alkali treatment at pH 11.5. It may thus represent a cystatin B--cysteine proteinase complex mixed with alpha-CPI. The low molecular mass inhibitor fractions reacted with antisera made against cystatin A and cystatin B. When the low-molecular mass inhibitor fraction was subjected to isoelectric focusing, it was separated into three peaks with pIs 8.0, 7.4, and 6.0. The inhibitors with pI 8.0 and 7.4 reacted with antisera made against cystatin B, while the inhibitor with pI 6.0 reacted with antisera made against cystatin B and cystatin A.
将人黑色素瘤组织移植到裸鼠体内后，发现半胱氨酸蛋白酶抑制剂的浓度在肿瘤生长过程中会升高。移植后8周时的活性（单位/克）约为4周时活性的3倍。通过Sephacryl S - 200和/或Sephadex G - 75凝胶色谱法将抑制剂分离为两种主要形式（分子量约为76,000和10,000）。在肿瘤生长过程中，两种分子量抑制剂的活性平行增加。高分子量抑制剂组分与针对α - 半胱氨酸蛋白酶抑制剂（α - CPI，人激肽原）和中性低分子量蛋白酶抑制剂（胱抑素B）制备的抗血清发生反应。在用抗胱抑素B抗血清进行电免疫印迹后，在SDS - 聚丙烯酰胺凝胶电泳中似乎释放出游离的胱抑素B。同样，在pH 11.5进行碱处理后，在Sephadex G - 75凝胶色谱中检测到游离的胱抑素B。因此，它可能代表与α - CPI混合的胱抑素B - 半胱氨酸蛋白酶复合物。低分子量抑制剂组分与针对胱抑素A和胱抑素B制备的抗血清发生反应。当低分子量抑制剂组分进行等电聚焦时，它被分离为三个峰，其等电点分别为8.0、7.4和6.0。等电点为8.0和7.4的抑制剂与针对胱抑素B制备的抗血清发生反应，而等电点为6.0的抑制剂与针对胱抑素B和胱抑素A制备的抗血清发生反应。
