Characterization of ribonucleases and ribonuclease inhibitor in subcellular fractions from rat adrenals

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Abstract
The presence of 2 RNA-degrading enzymes, 1 with optimum activity at pH5[center dot]6 (acid ribonuclease) and the other with optimum activity at pH 7[center dot]8 (alkaline ribonuclease), in rat adrenals has been demonstrated. The acid ribonuclease was localized in the mitochondrial fraction whereas the alkalineribonuclease was present in mitochondria as well as in the supernatant fraction. Freezing and thawing of mitochondria and treatment with Triton X-100 gave a 3 to 4-fold increase in acid-ribonuclease activity, whereas the mitochondrial alkaline-ribonuclease activity was practically unaffected. The amount of free ribonuclease in the adrenal supernatant was small. Treatment of the supernatant fraction with N-ethylmaleimide resulted in release of large amounts of ribonuclease activity, indicating the presence of a ribonuclease inhibitor having reactive thiol groups. Considerable amounts of free ribonuclease inhibitor in excess over the bound alkaline ribonuclease are present in the rat-adrenal supernatant fraction. The inhibitor is heat-labile and non-diffusible. A 400-500-fold purification of the ribonuclease inhibitor was achieved by (NH4)2SO4 fractionation, treatment with calcium phosphate gel and diethylaminoethyl cellulose chromatography. It is concluded that the adrenal inhibitor is protein in nature, similar to the inhibitor present in rat liver.