Yoshinari M, Taurog A, Krupp P P
Endocrinology. 1985 Aug;117(2):580-90. doi: 10.1210/endo-117-2-580.
A procedure was devised for fractionating crude thyroid lysosomal particles (P750-15,000) by self-forming density gradient centrifugation with colloidal silica. Two discrete particle-containing peaks were observed, based on 131I-labeling and acid phosphatase activity: a heavy peak (density, 1.11-1.12) and a light peak (density, 1.05). Ultrastructural analysis revealed that the heavy peak consisted almost entirely of lysosomes, whereas the light peak represented a heterogeneous mixture of small vesicles and fragments of other intracellular organelles. In thyroids removed from rats 30 min after 131I injection, almost all of the 131I was present in the low density peak. This 131I appeared on sucrose density gradient centrifugation as a 19S peak, and it was almost completely insoluble in trichloroacetic acid. This was interpreted as indicating that the low density peak contained pinocytotic vesicles. In thyroids removed 4 days after 131I injection, the radioactivity appeared largely in the high density peak. Both the trichloroacetic acid solubility and the pattern on sucrose density gradient centrifugation indicated that the [131I] thyroglobulin had undergone extensive proteolysis. Thyroglobulin proteolytic activity was found primarily in the high density particles and to only a small extent in the low density particles. Studies performed at intervals after 131I injection combined with double labeling (131I and 125I) experiments provided evidence that radioactivity was transferred from the low density to the high density particles. Heterogeneity existed within the dense peak, related to the degree of thyroglobulin degradation, as it was observed that thyroid lysosomes become denser with increasing proteolysis of thyroglobulin. The acid phosphatase in the low density particles could be distinguished from that in the high density (lysosomal) particles by its elution pattern on Sephadex G-200 column chromatography, its response to freezing and thawing, and its reactivity with p-nitrophenylphosphate. It was concluded, therefore, that the acid phosphatase in the low density fraction was derived from prolysosomal structures such as vesiculated Golgi-endoplasmic reticulum-lysosomes. The prolysosomal acid phosphatase associated with the low density fraction appeared to be a large membrane-bound molecule which could be transformed into lysosomal acid phosphatase by incubation at pH 5.0.
设计了一种通过用胶体二氧化硅进行自形成密度梯度离心来分离粗甲状腺溶酶体颗粒（P750 - 15,000）的方法。基于¹³¹I标记和酸性磷酸酶活性观察到两个离散的含颗粒峰：一个重峰（密度为1.11 - 1.12）和一个轻峰（密度为1.05）。超微结构分析表明，重峰几乎完全由溶酶体组成，而轻峰代表小泡和其他细胞内细胞器碎片的异质混合物。在¹³¹I注射30分钟后从大鼠体内取出的甲状腺中，几乎所有的¹³¹I都存在于低密度峰中。这种¹³¹I在蔗糖密度梯度离心中呈现为一个19S峰，并且几乎完全不溶于三氯乙酸。这被解释为表明低密度峰包含胞饮小泡。在¹³¹I注射4天后取出的甲状腺中，放射性主要出现在高密度峰中。三氯乙酸溶解度和蔗糖密度梯度离心图谱均表明，[¹³¹I]甲状腺球蛋白经历了广泛的蛋白水解。甲状腺球蛋白蛋白水解活性主要存在于高密度颗粒中，在低密度颗粒中仅占很小比例。在¹³¹I注射后的不同时间间隔进行的研究以及双标记（¹³¹I和¹²⁵I）实验提供了证据，表明放射性从低密度颗粒转移到了高密度颗粒。致密峰内存在异质性，这与甲状腺球蛋白的降解程度有关，因为观察到随着甲状腺球蛋白蛋白水解程度的增加，甲状腺溶酶体变得更致密。通过其在Sephadex G - 200柱色谱上的洗脱图谱、对冻融的反应以及与对硝基苯磷酸的反应性，可以将低密度颗粒中的酸性磷酸酶与高密度（溶酶体）颗粒中的酸性磷酸酶区分开来。因此得出结论，低密度部分中的酸性磷酸酶源自前溶酶体结构，如泡状高尔基体 - 内质网 - 溶酶体。与低密度部分相关的前溶酶体酸性磷酸酶似乎是一种大的膜结合分子，通过在pH 5.0下孵育可转化为溶酶体酸性磷酸酶。
