Isolation and determination of the properties of the specific steroid hydroxylating enzymes in animal and human tissue will be undertaken. Determination of the amounts and the properties of the crude membrane-bound hydroxylases in normal, bilateral hyperplastic, benign and malignant human adrenal cortex, in rat adrenal and in mouse adrenal cell cultures will be made. The control of biosynthesis and degradation of adrenal hydroxylating enzymes will be studied. Enzymes and properties will be analyzed by optical and electron spin resonance spectroscopy, chromatography, sucrose gradient centrifugation. Rapid kinetic studies and analyses of intermediates at various steady states will be performed to determine rate limiting steps and the interrelationship of hydroxylases to other cellular processes. Double isotope labelling and disc gel electrophoresis will be employed to study the turnover of adrenal cytochrome P450. The objectives are to determine the mechanism of action of the steroid hydroxylating enzymes, to determine their control characteristics and to determine if abnormalities of these enzymes exist in certain tumors.