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 inhib ition of soluble F-1-ATPase by the purified bovine heart inhibitor. Ho wever, beginning with the report of Horstman and Racker in 1970 and fo r approximately 17 years thereafter, workers in many laboratories rout inely 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 subm itochondrial particles prepared from a species naturally poor in IF1 f or this purpose. Thus, rat heart SMP which are largely depleted of IF1 in their native state were found to allow the performance of particul arly linear and reproducible IF1 titration assays regardless of the sp ecies source of the IF1 titrated on them. The present study presents t he first systematic comparison of the effects of a variety of factors upon functional assays for IF1. These include variations in IF1 functi onal assays due to seasonal effects on bovine A particles as well as t o 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 weathe r. Moreover, the more active cold-weather particles allowed the perfor mance of better IF1 assays. Also, the larger the species of origin of the IF1-depleted heart muscle SMP, the less IF1 was required to produc e a given amount of ATPase inhibition regardless of both seasonal effe cts and the species source of the IF1. Effects due to SMP preparation procedure and SMP concentration employed in the IF1 assays are also di scussed. In our hands, all factors considered, rat heart SMP preparati ons produced more linear and more reproducible IF1 titrations than bee f heart A particles. The main drawback of rat heart SMP is their cost. (C) 1994 Academic Press, Inc.