Alkaline ribonuclease and phosphodiesterase activity in rat liver plasma membranes

• Published in: Biochemical journal Vol. 132; no. 3; pp. 449 - 458
• Main Authors: Prospero, T D, Burge, M L, Norris, K A, Hinton, R H, Reid, E
• Format: Journal Article
• Language: English
• Published: England 01-03-1973
• Subjects: Animals; Cell Membrane - drug effects; Cell Membrane - enzymology; Centrifugation, Density Gradient; Centrifugation, Zonal; Edetic Acid - pharmacology; Hydrogen-Ion Concentration; Liver - cytology; Liver - drug effects; Liver - enzymology; Magnesium - pharmacology; Male; Phosphoric Diester Hydrolases - metabolism; Rats; Ribonucleases - metabolism; Solubility; Spectrophotometry; Spectrophotometry, Ultraviolet; Subcellular Structures
• ISSN: 0264-6021; 0306-3283; 1470-8728
• DOI: 10.1042/bj1320449

The ribonuclease and phosphodiesterase activities of rat liver plasma membranes, purified from the crude nuclear fraction by centrifugation in an A-XII zonal rotor and flotation, were examined and compared. The plasma membrane is responsible for between 65 and 90% of the phosphodiesterase activity of the cell and between 25 and 30% of the particulate ribonuclease activity measured at pH8.7 in the presence of 7.5mm-MgCl(2). Both enzymes were most active between pH8.5 and 8.9. Close to the pH optimum, both enzymes were more active in Tris buffer than in Bicine or glycine buffer. Both plasma-membrane phosphodiesterase and ribonuclease were strongly activated by Mg(2+), there being at least a 12-fold difference between the activity in the presence of Mg(2+) and of EDTA. There is, however, a difference in the response of the enzymes to Mg(2+) and EDTA in that the phosphodiesterase is fully activated by 1.0mm-MgCl(2) and fully inhibited by 1.0mm-EDTA, whereas the ribonuclease requires 7.5mm-MgCl(2) for full activation and 5mm-EDTA for full inhibition. Density-gradient centrifugation has indicated that on solubilization in Triton X-100 most of the ribonuclease activity is released into a small fragment of the same size as that containing the phosphodiesterase activity. The relationship between the two activities is discussed in view of these results.