Large‐scale purification procedure of spinach leaf mitochondria —isolation and immunological studies of the F1–ATPase

Abstract

A large–scale purification procedure for mitochondria from spinach (Spinacia oteracea L, cv Medania) leaves is described. It involves differential centrifugation and density gradient centrifugation on a self–generating gradient of Percoll, From 3 kg of spinach leaves, 150 mg mitochondrial protein are obtained. The thylakoid contamination is lower than 0.2% on a chlorophyll basis. The mitochondria oxidize malate and glycine with state 3 rates of 108 and 140 nmol (mg protein)‐1 min‐1, with respiratory control ratios of 2,7 and 3,8 and ADP/O ratios of 2,0 and 2.1, respectively. The present large–scale purification procedure will facilitate further biochemical and molecular biological studies of leaf mitochondrial proteins.A pure and active catalytic moiety of the F1–ATPase (EC 3,6,1,3) was purified from the isolated mitochondria. The yield was 5 mg of F1–ATPase from 150 mg mitochondria. The F1–ATPase contained five polypeptides of apparent molecular mass 54 kDa (α), 52 kDa (β), 33 kDa (γ), 22 kDa (ω) and 11 kDa (ɛ). An additional component at 24 kDa was present in variable amounts in some preparations and was therefore not ascribed to the ATPase complex. The enzyme catalyzed ATP hydrolysis at a rate of 12.5 nmol (mg protein)‐1 min‐1. Antibodies against the spinach mitochondrial F1–ATPase cross–reacted only with the a and β subunits of F1–ATPases of spinach chloroplasts, photosynthetic bacteria Rhodospirillum rubrum and beef heart mitochondria.