Martiniuk F, Honig J, Hirschhorn R
Arch Biochem Biophys. 1984 Jun;231(2):454-60. doi: 10.1016/0003-9861(84)90408-9.
Acid alpha-glucosidase has been purified from human placenta to a specific activity of approximately 6800, (4-methylumbelliferyl-alpha-D-glucoside as a substrate) or 55,400 mumol g-1 min-1 (glycogen or maltose as substrate). The purified enzyme gives rise to multiple protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), i.e., a major doublet of 82K and 69K , a minor doublet of 25K and 21K , and a faint band of 100K. All of the molecular weight species stained as glycoproteins with an intensity apparently proportional to their protein content, and were present in enzyme from individuals homozygous for the allozyme alpha-Glu 1. Isoelectric focusing revealed only enzymatically active proteins which, when analysed by SDS-PAGE, gave rise to multiple molecular weight species. Chromatography of I125-labeled, purified enzyme on Bio-Gel P-100 revealed only a radiolabeled, high-molecular-weight species which corresponded with enzyme activity. These findings suggest that, in the native state, the mature enzyme exists as a high-molecular-weight species, which is dissociable in SDS to several low-molecular-weight species. These results are consistent with reports that a 100K primary product of translation is post-translationally modified to yield polypeptides of lower molecular weights, and that all of the molecular species are absent in cells genetically deficient for acid alpha-glucosidase. The possibility that the low-molecular-weight (20- 25K ) protein bands in SDS-gels corresponded to a previously reported low-molecular-weight species generated by treatment with guanidine-HCl was investigated. The I125-labeled, purified acid maltase was dissociated by guanidine into two equal peaks of approximately 64K and 28K molecular weight. Surprisingly, both peaks, when analyzed on SDS-gels, yielded identical and equally intensely staining bands of 64K molecular weight. These results suggest that the mature acid alpha-glucosidase is made up of polypeptides which are bonded in the native state by at least two different types of interaction, one type which is dissociable in SDS and one type which is dissociable in guanidine but not in SDS. The nature and possible function of the 25K polypeptide generated only by guanidine-HCl remains to be determined.
酸性α-葡萄糖苷酶已从人胎盘中纯化出来，其比活性约为6800（以4-甲基伞形酮基-α-D-葡萄糖苷为底物）或55400 μmol g⁻¹ min⁻¹（以糖原或麦芽糖为底物）。纯化后的酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上产生多条蛋白带，即一条主要的82K和69K双峰、一条次要的25K和21K双峰以及一条微弱的100K条带。所有分子量的条带均被染成糖蛋白，其强度显然与其蛋白质含量成正比，并且存在于α-葡萄糖苷酶同工酶α-Glu 1纯合个体的酶中。等电聚焦仅显示出具有酶活性的蛋白质，当通过SDS-PAGE分析时，会产生多种分子量的条带。用¹²⁵I标记的纯化酶在Bio-Gel P-100上进行色谱分析，仅显示出一种与酶活性相对应的放射性标记的高分子量条带。这些发现表明，在天然状态下成熟酶以高分子量形式存在，在SDS中可解离为几种低分子量形式。这些结果与以下报道一致：翻译的100K初级产物经过翻译后修饰产生较低分子量的多肽，并且在酸性α-葡萄糖苷酶基因缺陷的细胞中所有分子量形式均不存在。研究了SDS凝胶中低分子量（20 - 25K）蛋白带是否对应于先前报道的用盐酸胍处理产生的低分子量形式的可能性。用¹²⁵I标记的纯化酸性麦芽糖酶被胍解离成两个分子量约为64K和28K的相等峰。令人惊讶的是，当在SDS凝胶上分析时，两个峰均产生相同且染色强度相同的64K分子量条带。这些结果表明，成熟的酸性α-葡萄糖苷酶由多肽组成，这些多肽在天然状态下通过至少两种不同类型的相互作用结合在一起，一种在SDS中可解离，另一种在胍中可解离但在SDS中不可解离。仅由盐酸胍产生的25K多肽的性质和可能的功能仍有待确定。
