Analysis of factors affecting functional assays for estimating IF1, the mitochondrial ATPase inhibitor.
Functional assays for IF1 have been in use for more than 30 years, i.e., since the initial report of Pullman and Monroy in 1963 on the inhibition of soluble F1-ATPase by the purified bovine heart inhibitor. However, beginning with the report of Horstman and Racker in 1970 and for approximately 17 years thereafter, workers in many laboratories routinely used IF1-depleted bovine heart submitochondrial particles (SMP) prepared from slaughterhouse material for the assay of IF1-containing extracts and preparations. Then, in 1987 we introduced the use of submitochondrial particles prepared from a species naturally poor in IF1 for this purpose. Thus, rat heart SMP which are largely depleted of IF1 in their native state were found to allow the performance of particularly linear and reproducible IF1 titration assays regardless of the species source of the IF1 titrated on them. The present study presents the first systematic comparison of the effects of a variety of factors upon functional assays for IF1. These include variations in IF1 functional assays due to seasonal effects on bovine A particles as well as to the species source of the IF1-depleted particles used in the assays. Interestingly, bovine heart A particles prepared during cold weather were considerably more active than those prepared during warmer weather. Moreover, the more active cold-weather particles allowed the performance of better IF1 assays. Also, the larger the species of origin of the IF1-depleted heart muscle SMP, the less IF1 was required to produce a given amount of ATPase inhibition regardless of both seasonal effects and the species source of the IF1.(ABSTRACT TRUNCATED AT 250 WORDS)
Rouslin
W
W
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
Broge
C W
CW
D000818:Animals; D002417:Cattle; D006736:Horses; D066298:In Vitro Techniques; D008297:Male; D008929:Mitochondria, Heart / Q000201:enzymology*; D011506:Proteins / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013367:Submitochondrial Particles / Q000201:enzymology; D013552:Swine; D000097795:ATPase Inhibitory Protein
D011506:Proteins; D006180:Proton-Translocating ATPases; D000097795:ATPase Inhibitory Protein