Assay of mannose-6-phosphatase in untreated and detergent-disrupted rat-liver microsomes for assessment of integrity of microsomal preparations.
Assay of mannose-6-phosphatase in untreated and detergent-disrupted rat-liver microsomes for assessment of integrity of microsomal preparations.
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测定未经处理和去污剂破坏的大鼠肝微粒体中的甘露糖 6 磷酸酶，以评估微粒体制剂的完整性。
DOI：
10.1111/j.1432-1033.1986.tb09550.x
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Blanckaert,N
中科院分区：
文献类型：
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作者：
Vanstapel,F;Pua,K;Blanckaert,N
1开发了一种准确、精密和方便的方法，用于测量低甘露糖-6-磷酸酶活性的潜伏期，以评估微粒体中的膜通透性屏障。该方法基于Arion等人的先前工作[J. Biol. Chem.（1976）251，4901-4907]，并包括测量未处理的微粒体部分和相应微粒体中的甘露糖-6-磷酸酶活性，所述微粒体被完全破坏以消除膜渗透性屏障。2通过在0 ℃下在4 mM两性离子洗涤剂3-[（3-胆酰胺基-丙基）二甲基-铵基]-2-羟基-1-丙烷磺酸盐（Chapso）。通过该预处理产生的酶活化（高达50倍）至少与任何其他先前报告的破坏性程序的作用一样大，表明在这些条件下微粒体膜渗透性屏障被消除。3通过Chapso或超声处理破坏微粒体显著增强了甘露糖-6-磷酸酶活性的热不稳定性。此外，微粒体暴露于高浓度Chapso会产生酶失活，在测定酶活性之前，通过稀释洗涤剂可部分逆转酶失活。在Chapso破坏微粒体的各种孵育条件下研究这些酶失活现象，以及随后在Chapso存在下测定甘露糖-6-磷酸酶活性，使我们能够确定无法检测到酶不稳定性的条件。4使用这些优化的微粒体破坏和己糖-6-磷酸水解酶测定程序，我们发现未处理大鼠肝微粒体的低甘露糖-6-磷酸酶活性始终低于5%，完全破坏的微粒体中的总酶活性。5必须在以下条件下准确和精确测定膜制备物中甘露糖-6-磷酸酶的结构潜伏期：良好控制的条件，特别注意在存在去污剂的情况下酶的显著热不稳定性，并且是使用该方法评估微粒体制备物完整性的先决条件。
1An accurate, precise, and convenient procedure was developed for measurement of the latency of the low‐Kmmannose‐6‐phosphatase activity for the purpose of assessment of the membrane permeability barrier in microsomes. This approach is based on previous work of Arion et al. [J. Biol. Chem.(1976)251, 4901–4907] and consists of measurement of mannose‐6‐phosphatase activity in the untreated microsomal fraction and in the corresponding microsomes that are fully disrupted in order to eliminate the membrane permeability barrier.2Complete disruption of rat liver microsomes was achieved by incubation for 60 min at 0°C in the presence of 4 mM zwitterionic detergent 3‐[(3‐cholamido‐propyl)dimethyl‐ammonio]‐2‐hydroxy‐1‐propane sulphonate (Chapso). That the microsomal membrane permeability barrier was eliminated under those conditions was suggested by the fact that the enzyme activation (up to 50‐fold) produced by this pretreatment was at least as large as the effect of any other previously reported disruptive procedure.3Disruption of the microsomes by Chapso or by ultrasonication markedly enhanced the thermolability of the mannose‐6‐phosphatase activity. In addition, exposure of the microsomes to high concentrations of Chapso produced enzyme inactivation that could be partially reversed by dilution of the detergent prior to assaying the enzymic activity. Investigation of these enzyme inactivation phenomena under various incubation conditions for disruption of the microsomes by Chapso and for subsequent assay of mannose‐6‐phophatase activity in the presence of Chapso enabled us to define conditions under which instability of the enzyme was undetectable.4Using these optimized procedures for disruption of microsomes and assay of hexose‐6‐Pphosphohydrolase, we found that the low‐Kmmannose‐6‐phosphatase activity of untreated rat liver microsomes consistenly was less than 5% of the total enzyme activity in the fully disrupted microsomes.5Accurate and precise assay of the structural latency of mannose‐6‐phosphatase in membrane preparations must be performed under well‐controlled conditions, with special attention to the marked thermolability of the enzyme in the presence of detergent, and is a prerequisite for using this approach for the purpose of assessing intactness of microsomal preparations.