Blum J J
J Cell Physiol. 1976 Nov;89(3):457-72. doi: 10.1002/jcp.1040890311.
Tetrahymena pyriformis were grown in proteose-peptone medium and then washed and incubated in a dilute salt solution for one hour. The cells were then discarded and the lysosomal hydrolases that had been secreted were subjected to DEAE cellulose column chromatography. At least three isoenzymes of acid phosphatase, three of acid protease, and two of beta-N-acetylhexoseaminidase were found, as well as single peaks of alpha-mannosidase, beta-galactosidase, and beta-fucosidase. The latter two activities were not resolved by the DEAE column and could not be separated in a second chromatographic step on CM-cellulose. Cells were also grown under identical conditions and homogenized in 0.25 M sucrose in order to allow comparison of some of the intracellular lysosomal hydrolases with their secreted counterparts. Two lysosomal populations were resolved by sucrose density gradient sedimentation, a heavy lysosomal fraction, contered at a density of about 1.25 gm/cm3, and a light lysosomal fraction, centered at a density of about 1.16 gm/cm3. These two populations differed in that the light lysosomes did not appear to contain significant amounts of beta-fucosidase, beta-galactosidase, or acid protease, whereas all six of the hydrolase activities studied were present in the heavy lysosomes. The light lysosomal peak occurred in cells grown to transition phase, but was markedly reduced in cells from cultures grown to stationary phase. In addition to these two fractions a third very light particle, containing only alpha-mannosidase activity, was detected just inside the gradient. Measurements were made of the effect of heat (10 minutes at 66 degrees) and of a change in pH from 4.5 (standard assay condition) to 6.0 on the three acid phosphatases and two beta-N-acetylhexoseaminidase isoenzymes resolved by DEAE column chromatography of the secreted hydrolases and on these hydrolyases in the heavy and light lysosomal fractions on the sucrose gradient. Use of the thermostability and pH criteria permitted computation of the expected properties of the intralysosomal acid phosphatase and hexoseaminidase activities if these consisted of the respective isoenzymes in the proportions secreted. It was found that neither the intralysosomal acid phosphatase nor the intralysosomal hexoseaminidase had the properties expected if they consisted of the secreted mixture of the respective isoenzymes, indicating that modification of some of these isoenzymes may have occurred during the 1-hour starvation period or after secretion.
梨形四膜虫在蛋白胨培养基中培养，然后洗涤并在稀盐溶液中孵育1小时。接着弃去细胞，将分泌出的溶酶体水解酶进行DEAE纤维素柱层析。发现了至少三种酸性磷酸酶同工酶、三种酸性蛋白酶同工酶和两种β-N-乙酰己糖胺酶同工酶，以及α-甘露糖苷酶、β-半乳糖苷酶和β-岩藻糖苷酶的单峰。后两种酶的活性在DEAE柱上未被分离，在CM-纤维素的第二次层析步骤中也无法分开。细胞也在相同条件下培养并在0.25M蔗糖中匀浆，以便比较一些细胞内溶酶体水解酶与其分泌的对应物。通过蔗糖密度梯度沉降分离出两个溶酶体群体，一个重溶酶体部分，密度约为1.25g/cm³，一个轻溶酶体部分，密度约为1.16g/cm³。这两个群体的不同之处在于，轻溶酶体似乎不含有大量的β-岩藻糖苷酶、β-半乳糖苷酶或酸性蛋白酶，而所研究的六种水解酶活性都存在于重溶酶体中。轻溶酶体峰出现在生长至过渡期的细胞中，但在生长至稳定期的培养物细胞中明显减少。除了这两个部分外，在梯度内部还检测到第三个非常轻的颗粒，仅含有α-甘露糖苷酶活性。对通过分泌水解酶的DEAE柱层析分离出的三种酸性磷酸酶和两种β-N-乙酰己糖胺酶同工酶以及蔗糖梯度上重溶酶体和轻溶酶体部分中的这些水解酶，测量了加热（66℃ 10分钟）和pH从4.5（标准测定条件）变为6.0的影响。如果溶酶体内酸性磷酸酶和己糖胺酶活性由各自分泌比例的同工酶组成，使用热稳定性和pH标准可以计算出其预期特性。结果发现，溶酶体内酸性磷酸酶和溶酶体内己糖胺酶都不具有如果由各自同工酶的分泌混合物组成时所预期的特性，这表明这些同工酶中的一些可能在1小时饥饿期或分泌后发生了修饰。
