A Specific and Nonspecific Alkaline Monophosphatase in the Venom of Bothrops atrox and Their Occurrence in the Purified Venom

Reis (1) discovered 5’-nucleotidase in 1934. Shortly thereafter, Gulland and Jackson (2) showed its presence in venoms of many species of snake. Although several methods of separation of 5’-nucleotidase from phosphodiesterase of venom have been proposed (3-7), the available preparations of 5’-nucleotidase are still rather crude (8-10). Two reasons prompted us to purify venom 5’-nucleotidase. First, this enzyme is one of the most undesirable contaminants of venom phosphodiesterase. Information concerning the properties of this enzyme would be useful in detecting it and avoiding it in the preparation of phosphodiesterase. The second reason was the hope that a highly purified 5’-nucleotidase might prove useful as a reagent in the study of nucleic acid structure, regardless of which of the two alternatives would be correct: (a) the enzyme dephosphorylates all fragments bearing 5’-monophosphate groups; (b) it is completely specific for mononucleotides. During the past few years, several casual observations on possible contaminants of phosphodiesterase suggested the presence of a monophosphatase other than 5’-nucleotidase. In particular (II-13), the use of phosphodiesterase for degradation of fragments bearing 3’-monophosphate groups led to a consistent deficit of 3’,5’-mononucleoside diphosphates. When such experiments were performed on a larger scale, it became evident that the deficit was caused by the dephosphorylation of 3’,5’-diphosphates, first to mononucleotides, then to nucleosides. An assumption that phosphodiesterase itself is capable of this action appeared unlikely. As shown in this paper, the purified 5’-nucleotidase is incapable of hydrolyzing 3’) 5’-diphosphates; therefore, an additional monophosphatase was strongly implicated. This paper describes the purification of venom 5’-nucleotidase and some of its properties, including the specificity, which is limited strictly to 5’-mononucleotides. The purification of the nonspecific alkaline phosphatase is also described. The nonspecific phosphatase as a contaminant of phosphodiesterase causes the observed shortage of 3’,5’-diphosphates. The error