Vick G W, Blum J J
J Protozool. 1979 Aug;26(3):510-8. doi: 10.1111/j.1550-7408.1979.tb04663.x.
Tetrahymena pyriformis strain HSM secrete large quantities of lysosomal acid hydrolases into the medium. The finding that 2 isozymes of beta-N-acetylhexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase; EC 3.2.1.30) could be resolved by DEAE ion exchange chromatography and of possible differences between the secreted mixture and the intralysosomal hexosaminidase activity suggested that Tetrahymena might prove useful for studies of the control of lysosomal hydrolase isozyme secretion. In the present paper, we report a considerable purification of these isozymes and describe a number of their kinetic properties. Four isozymes were isolated into 2 major forms, A1 and B1, and 2 minor forms, A2 and B2, which were similar to the respective major forms in all kinetic properties tested. Hexosaminidase B1 has a molecular weight of approximately 93,000 daltons and is inhibited by high concentrations of p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The inhibition is reversed by ethanol. Hexosaminidase B1 has a molecular weight of approximately 93,000 daltons and is inhibited by high concentrations of p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The inhibition is reversed by ethanol. Hexosaminidase A1 has a molecular weight of approximately 170,000 and is not inhibited by high concentrations of substrate. The A forms are relatively less active against p-nitrophenyl-N-acetyl-beta-D-galactosaminide than the B forms. Neither hexosaminidases A1 or B1 has any endo-beta-N-acetylhexosaminidase activity. Comparison of the properties of the 2 major isozymes suggested that measurements of activity obtained under different assay conditions could be used to quantitate the amount of each isozyme in a mixture of the two. Log- and early stationary-phase cells secrete approximately 20% of isozyme A and 80% of isozyme B into the medium or into a dilute salt solution. With increasing culture age the fraction of isozyme A secreted rises to over 90%. Supplementation of the proteose-peptone growth medium with glucose causes a decrease in total hexosaminidase subsequently secreted but with no change in proportion of each isozyme. Cells suspended in a dilute salt solution containing 0.1 mM L-propranolol secrete slightly more isozyme A than do control cells suspended without L-propranolol. Phenoxybenzamine (0.2 mM) causes a slight decrease in the proportion of isozyme A released.
梨形四膜虫HSM菌株会向培养基中分泌大量溶酶体酸性水解酶。通过DEAE离子交换色谱法可分离出β-N-乙酰己糖胺酶的2种同工酶（2-乙酰氨基-2-脱氧-β-D-葡萄糖苷乙酰氨基脱氧葡糖水解酶；EC 3.2.1.30），且分泌混合物与溶酶体内己糖胺酶活性可能存在差异，这一发现表明四膜虫可能对溶酶体水解酶同工酶分泌的调控研究有用。在本文中，我们报告了这些同工酶的大量纯化，并描述了它们的一些动力学特性。四种同工酶被分离为2种主要形式，A1和B1，以及2种次要形式，A2和B2，在所有测试的动力学特性方面，它们与各自的主要形式相似。己糖胺酶B1的分子量约为93,000道尔顿，会被高浓度的对硝基苯基-N-乙酰-β-D-葡糖胺抑制。乙醇可使这种抑制作用逆转。己糖胺酶B1的分子量约为93,000道尔顿，会被高浓度的对硝基苯基-N-乙酰-β-D-葡糖胺抑制。乙醇可使这种抑制作用逆转。己糖胺酶A1的分子量约为170,000，不会被高浓度的底物抑制。A形式对对硝基苯基-N-乙酰-β-D-半乳糖胺的活性相对低于B形式。己糖胺酶A1或B1均无内切-β-N-乙酰己糖胺酶活性。对这2种主要同工酶特性的比较表明，在不同测定条件下获得的活性测量值可用于定量两种同工酶混合物中每种同工酶的量。对数期和早期稳定期细胞会将约20%的同工酶A和80%的同工酶B分泌到培养基或稀盐溶液中。随着培养时间的增加，分泌的同工酶A的比例会升至90%以上。在蛋白胨生长培养基中添加葡萄糖会导致随后分泌的总己糖胺酶减少，但每种同工酶的比例不变。悬浮在含有0.1 mM L-普萘洛尔的稀盐溶液中的细胞比未添加L-普萘洛尔悬浮的对照细胞分泌的同工酶A略多。苯氧苄胺（0.2 mM）会使释放的同工酶A的比例略有下降。
