Further studies of the structure of human placental acid alpha-glucosidase.

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.