Bracco F, Banay-Schwartz M, Deguzman T, Lajtha A
Center for Neurochemistry, Rockland Research Institute, Ward's Island, NY 10035, U.S.A.
Neurochem Int. 1982;4(6):541-9. doi: 10.1016/0197-0186(82)90043-2.
The breakdown of cytoplasmic tubulin from brain (purified by ammonium sulfate fractionation and DEAE cellulose chromatography) by cathepsin D from brain (purified by ammonium sulfate fractionation and pepstatin Sepharose chromatography) was studied; changes in the intensity of tubulin gel bands were determined. The pH optimum of hemoglobin breakdown by cathepsin D was 3.2; the pH optimum for tubulin breakdown was 5.8; at pH 5.8 there was no significant hemoglobin breakdown by the enzyme. Tubulin breakdown had an apparent K(m) of 1.8 x 10(?5)M and a V(max) of 0.56 ?g tubulin (?g enzyme per min). The rate of breakdown was heterogeneous and studied on length of incubation; the major portion of tubulin was rapidly broken down and a smaller portion was more stable. The rate under our experimental conditions was 18%/h in the 1-4 h period and 2%/h after 4 h. This was not due to enzyme instability: after 4 h of inhibition freshly added tubulin was rapidly broken down, whereas freshly added enzyme did not increase the rate of breakdown. Thus breakdown heterogeneity was due to substrate (tubulin) heterogeneity. Pepstatin inhibited cathepsin D breakdown of tubulin at acid pH; at pH 7.6 it had no effect. Leupeptin was not inhibitory. We calculated that the cathepsin D content in brain, if fully active, could break down cytoplasmic tubulin with a half-life of 24 h, but it is likely that under in vivo conditions enzyme activity is greatly modified.
研究了来自脑的组织蛋白酶D（通过硫酸铵分级分离和胃蛋白酶抑制剂琼脂糖凝胶色谱法纯化）对来自脑的细胞质微管蛋白（通过硫酸铵分级分离和二乙氨基乙基纤维素色谱法纯化）的降解；测定了微管蛋白凝胶条带强度的变化。组织蛋白酶D降解血红蛋白的最适pH为3.2；降解微管蛋白的最适pH为5.8；在pH 5.8时，该酶对血红蛋白没有明显降解作用。微管蛋白降解的表观米氏常数（K(m)）为1.8×10⁻⁵M，最大反应速度（V(max)）为0.56μg微管蛋白/（μg酶·分钟）。降解速率是不均匀的，并研究了孵育时间；大部分微管蛋白迅速降解，一小部分更稳定。在我们的实验条件下，1 - 4小时期间的降解速率为18%/小时，4小时后为2%/小时。这不是由于酶的不稳定性：在抑制4小时后，新添加的微管蛋白迅速降解，而新添加的酶并没有增加降解速率。因此，降解的不均匀性是由于底物（微管蛋白）的不均匀性。胃蛋白酶抑制剂在酸性pH下抑制组织蛋白酶D对微管蛋白的降解；在pH 7.6时没有作用。亮抑酶肽没有抑制作用。我们计算出，如果脑内的组织蛋白酶D完全有活性，其可以在24小时的半衰期内降解细胞质微管蛋白，但在体内条件下，酶的活性很可能会发生很大改变。
