Identification, purification and characterization of membrane-associated glutamic acid decarboxylase from porcine brain.

摘要

Three forms of membrane associated glutamate decarboxylase (MGAD) referred to as MGAD I, II and III were identified and purified to apparent homogeneity from porcine brain. MGADs were purified by using a combination of column chromatography on DE 52, Ultragel AcA 34, hydroxylapatite and Sephadex G-200 and native gradient polyacrylamide gel electrophoresis (PAGE). The purified MGAD preparations thus obtained migrated as single protein bands in native gradient PAGE and SDS PAGE. Furthermore, in native PAGE the protein bands correlated with GAD activity. The molecular weights of MGAD I, II and III were calculated to be 96 +/{dollar}-{dollar} 3, 120 +/{dollar}-{dollar} 5 and 120 +/{dollar}-{dollar} 3 kDa respectively, from native PAGE. Based on the subunit molecular weights of these proteins from SDS PAGE, 48 +/{dollar}-{dollar} 2, 60 +/{dollar}-{dollar}2 and 60 +/{dollar}-{dollar} 4 kDa respectively, they are concluded to be homodimers. The biochemical characteristics of MGADs such as pH optima, substrate specificity, thermal stability and sensitivity towards inhibitor and kinetic parameters were similar to that of SGAD reported in the literature. MGAD I and II were established as integral membrane proteins based on the following experiments: phase partitioning in Triton X-114, charge shift electrophoresis, chromatography on a hydrophobic interaction column and membrane solubilization studies. MGAD III was established as a protein associated with the membrane in a Ca{dollar}sp{lcub}2+{rcub}{dollar}-dependent fashion. This conclusion is based on liposome binding experiments and membrane extraction studies. Immunoprecipitation and immunoblotting tests using sera from Insulin Dependent Diabetes Mellitus (IDDM) patients revealed the presence of antibodies against MGADs particularly MGAD I and II in IDDM. The role of MGAD in the pathogenesis of IDDM and related autoimmune disorders is also discussed.