Extrinsic and intrinsic factors that influence inactivation and purification of the unstable adenosine triphosphatase solubilized from membranes of an Escherichia coli K 12 strain

Azocar, O.; Muñoz, E.

Biochimica et Biophysica Acta 482(2): 438-452


ISSN/ISBN: 0006-3002
PMID: 18189
DOI: 10.1016/0005-2744(77)90258-3
Accession: 068518093

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Washing with EDTA at pH 9.0 was an obligatory step to solubilize completely the ATPase (ATP phosphohydrolase, EC from membranes of E. coli K 12 strain 414. After solubilization, the enzyme was highly unstable (half-life at 4.degree. C < 4 h) and this instability hindered its purification. Alkaline pH and EDTA were among the extrinsic factors responsible for the instability of the enzyme because the half-life of the ATPase was increased to > 30 days by lowering its pH to 7.5 and adding 3-5 mM Mg2+. Inactivation of the ATPase of E. coli K 12 strain 414 was associated with the appearance of components of higher and lower mobility than an ATPase band of relative mobility 0.22 .+-. 0.02 (Tris/glycine system, pH 8.5 .+-. 0.2). The ATPase of E. coli K 12 strain 414 was purified and, under conditions of maximal stability, it yielded a homogeneous protein (by gel electrophoresis in Tris/acetate buffers, pH 7.5) which had a basal specific activity of 20 .mu.mol substrate transformed .cntdot. min-1 .cntdot. mg protein-1 and was stimulated by trypsin to 50 .mu.mol .cntdot. min-1 .cntdot. mg-1. The nature and/or pH of the buffer electrophoretic system strongly influenced the protein profile of purified ATPase. The Tris/glycine system induced the appearance of several components as a likely result of dissociation of purified ATPase. This increase in the number of bands induced by external factors is termed extrinsic heterogeneity. Electrophoresis in sodium dodecyl sulfate (Tris/glycine, pH 8.5) revealed the presence of 5 subunits: .alpha. (MW; 54,000 .+-. 2000); .beta. (48,000 .+-. 3000), .gamma. (small proportion: 30,000 .+-. 3000), .delta. (21,000 .+-. 2000) and .epsilon. (13,000), together with a component x (MW .apprx. 120,000) whose significance as a true subunit is questionable. By dodecyl sulfate electrophoresis in Tris/acetate (pH 7.5), only 4 subunits (.alpha., .beta., .gamma. and .epsilon.) and component x were detected. In this system, the MW of the major subunits (.alpha., 66,000; .beta., 60,000) were slightly higher. These results represent small molecular differences with regard to ATPase from other E. coli strains and are discussed in relationship to the stability of the ATPase from E. coli K 12 strain 414.