Characterization of cyclic nucleotide phosphodiesterases with multiple separation techniques

• Published in: Archives of biochemistry and biophysics Vol. 175; no. 2; pp. 700 - 709
• Main Authors: Van Inwegen, R.G., Pledger, W.J., Strada, S.J., Thompson, W.J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.1976
• Subjects: 3',5'-Cyclic-AMP Phosphodiesterases - isolation & purification; 3',5'-Cyclic-AMP Phosphodiesterases - metabolism; Animals; Dithiothreitol - pharmacology; Enzyme Activation; Isoelectric Focusing; Kidney - enzymology; Kinetics; Magnesium - pharmacology; Male; Manganese - pharmacology; Phosphoric Diester Hydrolases - isolation & purification; Phosphoric Diester Hydrolases - metabolism; Rats; Subcellular Fractions - enzymology
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/0003-9861(76)90562-2

Studies of various conditions and techniques used to separate cyclic nucleotide phosphodiesterases of rat kidney have demonstrated that the cationic cofactor requirements, apparent kinetic constants, number, size, and net charge of separated enzyme forms can be altered by a variety of factors. Dithiothreitol affects the number and kinetic properties of enzyme forms fractionated by isoelectric focusing and the degree of cooperativity of a low K m cyclic AMP-specific enzyme separated on Sephadex G-150. In contrast to results obtained by sucrose gradient analyses, isoelectric focusing in glycerol gradients resolved cyclic nucleotide phosphodiesterase into a single peak of activity. Inclusion of ethylene glycol in the buffers used for DEAE-cellulose chromatography greatly enhanced the yields of eluted enzymes, and the pH of the salt gradients markedly affected cyclic nucleotide phosphodiesterase elution profiles. Our results suggest (a) that in addition to protein sulfhydryl reactions, hydrophobic interactions of enzyme subunits may play an important role in the regulation of this enzyme system, (b) that cautious interpretation of results obtained from a single separation technique is required since relatively slight modifications in any one isolation procedure can result in markedly different data, and (c) that the oligomeric nature of cyclic nucleotide phosphodiesterase requires physical analysis by a variety of techniques to avoid biochemical anomalies.