5'-ribonucleotide phosphohydrolase from bovine pituitary gland.

Abstract

1.A new method for the purification of pituitary 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) is presented in which an 86-fold increase in specific activity was achieved with a 12–15% yield.2.The mean value of the sedimentation coefficient, 6.6 S, and the molecular weight obtained by Sephadex G-200 filtration, 237 000, suggests that the molecules of 5′-nucleotidase are elongated.3.The amino acids involved in the enzymic activity of 5′-nucleotidase are close together in the enzyme molecule.4.The purified enzyme catalysed only the hydrolysis of 5′-mononucleotides. The highest rate of substrate hydrolysis was observed with guanosine 5′-monophosphate, and the lowest with adenosine 5′-monophosphate.5.Oxidation of vicinal −OH groups of the ribose moiety of 5′-nucleotide by NaIO4 makes the nucleotide ineffective as a substrate for 5′-nucleotidase. The oxidized adenosine 5′-monophosphate is a non-competitive inhibitor of the pituitary 5′-nucleotidase.6.Diisopropylfluorophosphate had no effect on, and sodium dodecyl sulphate completely inhibited, the enzyme.7.The only activating amino acid was glycine; other amino acids were inhibitors. Nucleosides and 2′(3′)-nucleotides also inhibited the activity of the pituitary enzyme. The highest inhibition was observed with adenosine. The inhibition was of a non-competitive type.8.The possibility that aromatic amino acids in the enzyme may take part in the binding of adenosine and other nucleosides is suggested. A strong decrease in the molar absorbance coefficient at 260 mμ (4.5·103) accompanied the binding of adenosine to the enzyme.9.The pituitary 5′-nucleotidase can be digested by Streptomyces griseus proteinase (pronase) giving active fragments of lower molecular weight.10.Active fragments obtained after proteolysis had different specificities toward 5′-nucleotides than the untreated enzyme. A new method for the purification of pituitary 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) is presented in which an 86-fold increase in specific activity was achieved with a 12–15% yield. The mean value of the sedimentation coefficient, 6.6 S, and the molecular weight obtained by Sephadex G-200 filtration, 237 000, suggests that the molecules of 5′-nucleotidase are elongated. The amino acids involved in the enzymic activity of 5′-nucleotidase are close together in the enzyme molecule. The purified enzyme catalysed only the hydrolysis of 5′-mononucleotides. The highest rate of substrate hydrolysis was observed with guanosine 5′-monophosphate, and the lowest with adenosine 5′-monophosphate. Oxidation of vicinal −OH groups of the ribose moiety of 5′-nucleotide by NaIO4 makes the nucleotide ineffective as a substrate for 5′-nucleotidase. The oxidized adenosine 5′-monophosphate is a non-competitive inhibitor of the pituitary 5′-nucleotidase. Diisopropylfluorophosphate had no effect on, and sodium dodecyl sulphate completely inhibited, the enzyme. The only activating amino acid was glycine; other amino acids were inhibitors. Nucleosides and 2′(3′)-nucleotides also inhibited the activity of the pituitary enzyme. The highest inhibition was observed with adenosine. The inhibition was of a non-competitive type. The possibility that aromatic amino acids in the enzyme may take part in the binding of adenosine and other nucleosides is suggested. A strong decrease in the molar absorbance coefficient at 260 mμ (4.5·103) accompanied the binding of adenosine to the enzyme. The pituitary 5′-nucleotidase can be digested by Streptomyces griseus proteinase (pronase) giving active fragments of lower molecular weight. Active fragments obtained after proteolysis had different specificities toward 5′-nucleotides than the untreated enzyme.