Mathur R, Panneerselvam K, Balasubramanian A S
Department of Neurological Sciences, Christian Medical College and Hospital, Vellore, India.
Biochem J. 1988 Aug 1;253(3):677-85. doi: 10.1042/bj2530677.
Neutral alpha-D-mannosidase from monkey brain was purified by Co2+-chelate affinity chromatography and immunoadsorbent affinity chromatography. The purified enzyme, with subunit Mr 45,000, was essentially homogeneous with only traces of two contaminant proteins as revealed by SDS/polyacrylamide-gel electrophoresis and AgNO3 staining. The purified enzyme, on preincubation with Co2+ at 37 degrees C or 60 degrees C followed by assay, showed a time-dependent enhancement in activity. The enhanced activity of the enzyme persisted even after removal of the Co2+. Bacitracin could partially prevent the activation. An aminopeptidase activity that was stimulated by Co2+ both at 37 degrees C and at 60 degrees C was present in the purified enzyme. After preincubation of the enzyme with Co2+ there was evidence for the release of amino acids, as revealed by t.l.c., but the Mr determined by SDS/polyacrylamide-gel electrophoresis was not appreciably altered. It is suggested that a Co2+-stimulated thermostable aminopeptidase, inseparable from the neutral mannosidase, may be involved in the stimulation of neutral mannosidase activity during its preincubation with Co2+.
通过钴离子螯合亲和层析和免疫吸附亲和层析对猴脑中性α-D-甘露糖苷酶进行了纯化。纯化后的酶亚基分子量为45,000，经SDS/聚丙烯酰胺凝胶电泳和硝酸银染色显示，基本上是纯的，仅含有痕量的两种污染蛋白。纯化后的酶在37℃或60℃与钴离子预孵育后再进行活性测定，结果显示活性随时间增强。即使去除钴离子后，酶的增强活性仍持续存在。杆菌肽可部分阻止这种激活。纯化后的酶中存在一种氨肽酶活性，在37℃和60℃时均受钴离子刺激。酶与钴离子预孵育后，经薄层层析显示有氨基酸释放的迹象，但经SDS/聚丙烯酰胺凝胶电泳测定的分子量没有明显改变。有人提出，一种与中性甘露糖苷酶不可分离的钴离子刺激的热稳定氨肽酶，可能在其与钴离子预孵育期间参与了中性甘露糖苷酶活性的刺激过程。
