An Anticlumping Factor for Mitochondria in the Supernatant Fraction of Rat-Liver Homogenates

Abstract

IN a rat-liver homogenate that has not been strongly diluted the mitochondria remain substantially unclumped for several hours even at room temperature ; but if it is diluted more than about five-fold with a medium containing electrolyte, serious clumping may occur in a few minutes. If it is diluted with the supernatant from another homogenate which has been only slightly diluted, clumping is almost completely inhibited. This shows that a natural antidumping factor is present in the supernatant. The factor is thermo-labile, non-dialysable and precipitable from supernatant by half-saturated ammonium sulphate. Its activity is lost gradually at 0° C. but is retained indefinitely at − 15° C. The lability suggests that it is not identical with heparin, which is known to prevent mitochondrial clumping. This is confirmed by the fact that our factor does not, like heparin, produce nuclear haloes¹ by combining with histone and liberating deoxyribonucleic acid. On the other hand, all its properties known so far correspond with those of the ribonuclease inhibitor discovered by Roth^2,3, which is also not identical with heparin although the latter inhibits ribonuclease. We therefore tested crystalline pancreatic ribonuclease for clumping activity and found it very potent in absence but not in presene of supernatant, as expected. Thus there is good reason for accepting as a working hypothesis that our antidumping factor and Roth's ribonuclease inhibitor are identical, though this can be established with certainty only by isolation of the two substances. Our factor, like Roth's, varies greatly in concentration in apparently similar preparations. One supernatant had perceptible antidumping activity at a dilution of 1/180, suggesting that even crude concentrates of the factor will be useful protectors in any experiments where mitochondria need to be kept in good condition.