Isolation of oligomycin-sensitive adenosine triphosphatase from beef heart mitochondria and analysis of its fine structure.

1. An oligomycin -sensitive ATPase was isolated and partially purified from beef heart mitochondria. The specific activity of ATPase sensitive to oligomycin of the fraction was five to eight times that of aged mitochondrial or of DNP-induced mitochondrial ATPase assayed under the same condition. 2. Electron micrographs of the partially purified oligomycinsensitive ATPase reveal a structure in which headpieces are regularly attached by way of stalks to a thread-like structure derived from a superficial portion of base pieces. 3. A high concentration of the structured material coincided with a high activity of oligomycin-sensitive ATPase. When the headpieces were detached from the structure, the ATPase became insensitive to oligomycin. 4. The fraction of oligomycin -sensitive ATPase was essentially free of membrane structure and was contaminated with a small amount of cytochromes b and Cl but no cyt. a. Cytochrome concentrations of the preparations were indifferent to the activity of oligomycin sensitive ATPase. It follows that ATPase does not require cytochromes or membrane structure for its oligomycin sensitivity. 5. From these results it seems that the factor rendering ATPase sensitive to oligomycin should be contained in the stalks and/or the thread-like portion of basepieces of the structure. The structure is the simplest unit of oligomycinsensitive ATPase as yet obtained. 6. The structure was called “oligomycin-sensitive ATPase particles” (abbreviated as OSA particles). A unit of OSA particles consists of a headpiece attached by a stalk to a portion of base piece. ∗PMID: 4230846 [PubMed indexed for MEDLINE] Copyright c ©OKAYAMA UNIVERSITY MEDICAL SCHOOL Acta Med. Okayama 21. 147-160 (1967) ISOLATION OF OLIGOMYCIN-SENSITIVE ADENOSINE TRIPHOSPHATASE FROM BEEF HEART MITOCHONDRIA AND ANALYSES OF ITS FINE STRUCTURE Shuji SEKI, Goki YAMAMOTO, Hideo HAYASHI, Risaburo INOHARA and Takuzo aDA Department of Biochemistry, Cancer Institute, Okayama University Medical School, Okayama, Japan (Director: Prof. T. Oda) Received for publication, June 10, 1967 Regular arrays of uniform particles are seen attached to the surface of the inner membrane of mitochondrial,2. The attached particle (head piece), when viewed from its functional aspect, is a part of the elementary particle in the tripartite form composed of head piece, stalk and base piece• Recently it was suggested in RACKER'S laboratorys.4 and in our laboratory/i,8,9 that the isolated head piece is a soluble ATPase (coupling factor Fl), which was purified by PULLMAN et at. 6. The soluble ATPase greatly differs from the membrane-bound ATPase of mitochondria in physico-chemical charactor. In relation to the coupling mechanism of oxidative phosphorylation of mitochondria, the most important difference is that the membrane-bound ATPase is oligomycin-sensitive while the soluble ATPase is not. Up to the present we have investigated the molecular organization of the mitochondrial membrane by the method of systematic or stepwise microdisection of the mitochondrial membrane by treating with deoxycholate and potassium chloride,8.9. 'Ve have isolated and partially purified oligomycin-sensitive ATPase, which was almost free from membrane structure, by the same method. The oligomycin-sensitive ATPase has such a unique structure with the headpieces combined with stalks to thread-like base pieces. In the following, this structure will be called "oligomycin-sensitive ATPase particles", and will be abbreviated as OSA particles. We also investigated the relationship between oligomycin-sensitive and insensitive ATPase with this fraction, and will discuss what factor may be responsible for the oligomycin-sensitivity. MATERIALS AND METHODS Assay 0/ ATPase activity: A buffered substrate solution, consisted of 50 /..tmoles Tris-chloride, pH 7.4, 6/..tmoles ATP, 3 /..tmoles MgCI2, and water in a final volume of 0.9 ml, was preincubated for 5 min and 0.1 ml of sample was