Ca 2+-Mg 2+-ATPase activity in brain coated microvesicles purified on immunosorbents

Abstract

Coated microvesicle fractions isolated from ox forebrain cortex by the ultracentrifugation procedure of Pearse (1) and by the modified, less time consuming method of Keen et al (2) had comparable Ca 2++Mg 2+ dependent ATPase activities (about 9 μmol/h per mg protein). The Na ++K ++Mg 2+ dependent ATPase activity was 3.2 μmol/h per mg (±1.0, S.D., n=3) when microvesicles were prepared according to (1) and 1.5 μmol/h per mg (±1.0, S.D., n=3) when prepared according to (2). Oligomycin, ruthenium red, and trifluoperazine, inhibitors of Ca 2+ transport in mitochondria and erythrocyte membranes had no effect on Ca 2++Mg 2+ dependent ATPase from any of the preparations. As demonstrated both by ATPase assays and electron microscopy, coated microvesicles could be bound to immunosorbents prepared with poly-specific antibodies against a coated microvesicle fraction obtained by the method of Pearse (1). The binding could be inhibited by dissolved coat protein using partially purified clathrin. The fraction of coated vesicles eluted from the immunosorbent was purified relative to the starting material as judged by electron microscopy. The Ca 2++Mg 2+ ATPase activity and calmodulin content was copurified with the coated microvesicles and the specific activity of Na ++K ++Mg 2+ ATPase was decreased. Na ++K ++Mg 2+ dependent ATPase activity in the coated microvesicle fraction could be ascribed to membranes with the appearance of microsomes. These membranes were also bound to the immunosorbents, but the binding was not influenced by clathrin. The capacity of the immunosorbents for these membranes was less than for the coated microvesicles, resulting in a decrease of Na ++K ++Mg 2+ dependent ATPase activity in the eluted coated microvescile fraction. It was concluded that Ca 2++Mg 2+ ATPase activity is not a contamination from plasma membrane vesicles or mitochondrial membranes but seems to be an integral part of the coated vesicle membrane.