Adenosine 3‘,5‘-Phosphate in Biological Materials

Abstract

The detection and measurement of adenosine 3') 5'-phosphate are complicated by the extremely low levels present in most biological materials and by the sensitivity of the standard assay to activation or inhibition by substances native to the tissue or fluid under study (l-3).These problems made the availability of a specific means of destroying adenosine 3',5'-phosphate very desirable.An enzymatic activity capable of destroying adenosine 3',5'phosphate was detected in various mammalian tissues several years ago (4).Investigation showed that this activity was due to a magnesium-dependent phosphodiesterase that catalyzed the hydrolysis of the cyclic nucleotide at the 3'-position, yielding adenosine 5'-phosphate.It was found to be inhibited by the methyl xanthines (4, 5) and stimulated by imidazole (5).No other physiological mechanism has been found by which the action of adenosine 3') 5'-phosphate could be terminated.Hence, these data indicated a significant role for the enzyme in the control of the levels of adenosine 3',5'-phosphate present in biological systems.This report deals with the purification, properties, and distribution of the cyclic 3', 5'-nucleotide phosphodiesterase, the occurrence of adenosine 3',5'-phosphate in human urine, and the use of purified samples of the enzyme to aid in the identification and measurement of this adenosine 3') 5'-phosphate.EXPERIMENTAL PROCEDURE Jlaterials-Cyclic 3') 5'-AMP1 was purchased from Sigma Chemical Company and from Schwarz BioResearch, Inc.It was standardized by comparison of the molar absorbancy index in acid at 260 rnp, and of the biological assay with a known sample of the cyclic nucleotide on supernatant fractions of liver (4).Imidazole was purchased from the Mann Biochemical Corporation and from the Eastman Organic Chemicals Division of Eastman Kodak Company.Crotalus atrox venom was purchased in the form of lyophilized crystals from Ross Allen's Reptile Institute, Silver Springs, Florida.Methyl xanthines were obtained from the Nutritional