Zur Kenntnis der Nucleotidase aus Darmschleimhaut. Nucleinsäuren II

Abstract

To prepare the nucleotidase, 560 g. of calf intestinal mucosa hash were stirred with 4 vols. cold acetone (5[degree]), the mixture filtered, the filter cake washed with acetone and ether and dried in a desiccator. Three g. of the dry powder were extracted with 75 ml. water containing NH3, pH 8, the mixture centri-fugalized, the supernatant soln. saturated with CO2 and centrifugalized after treatment with 15 ml. Oy aluminum hydroxide suspension. The supernatant soln. was treated with 2 vols. of acetone and the precipitated nucleotidase washed with acetone and ether and dried. The crude enzyme was also prepared by the method of H. Albers and E. Albers (Hoppe-Seyler''s Zeitschr. physiol. Chem. 232: 189, 1935). Eight g. of crude enzyme were extracted with 450 ml. H2O, the extract treated with 200 ml. of the aluminum hydroxide suspension, the mixture centrifugalized, and the nucleotidase precipitated from the supernatant soln. with 370 g. (NH4)2SO4. The reaction mixtures contained in 20 ml. soln.; 5 ml. substrate soln., 5 ml. 0.08 N MgSO4 soln. and.5 ml. enzyme soln. The mixtures were incubated at 35[degree], samples removed at 15, 30 and 60 min. intervals and the amt. of inorganic P detd. colorimetrically. The nucleotidase prepn. was the most active yet prepared, 100 g. of ribo-nucleotide being dephosphorylated by 350 mg. of the enzyme in 12 hrs. The enzyme prepns. did not contain polynucleo-tidase nor nucleosidase. The enzyme readily dephosphorylated 3- and 5-nucleotides, glycerophosphate, monophenyl-phosphate, and creatinine but did not attack diphenylpyro-phosphate, diphenylphosphate, and p-chloraniline-phosphoric acid. It hydrolyzed pyrophosphate.