HYDROLYSIS OF PHOSPHONATE ESTERS CATALYZED BY 5'-NUCLEOTIDE PHOSPHODIESTERASE
HYDROLYSIS OF PHOSPHONATE ESTERS CATALYZED BY 5'-NUCLEOTIDE PHOSPHODIESTERASE
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DOI：
10.1021/bi00693a030
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发表时间：
1975-01-01
期刊：
影响因子：
2.9
通讯作者：
BUTLER, LG
中科院分区：
文献类型：
--
作者：
KELLY, SJ;DARDINGER, DE;BUTLER, LG
4-合成了硝基苯基和2-萘基苯基膦酸单酯，并通过离子交换层析、L-苯丙氨酰-Sepharose梯度洗脱和凝胶过滤，从牛小肠碱性磷酸酶中分离出催化其水解的酶。新鲜牛小肠去溶酶经（NH_4）_2SO_4分级分离后，用同样的方法纯化。纯化的酶经聚丙烯酰胺凝胶电泳和沉降平衡离心均为均一酶。它的分子量为108，000，含有约21%的碳水化合物，氨基酸组成与来自相同组织的碱性磷酸酶的报道有很大不同。均相肠酶是膦酸酯水解的有效催化剂，但不是磷酸单酯水解的有效催化剂，通过其水解5 ′-TMP的4-硝基苯基酯而不是3 ′-TMP的4-硝基苯基酯的能力，将其鉴定为5 ′-核苷酸磷酸二酯酶。也与这种鉴定一致的是，已经显示（Kelly和Butler，1975; Kelly等人，1975），能够水解膦酸单酯的酶活性广泛分布于整个自然界。从小牛肠中获得的这种酶的一些性质与肠碱性磷酸酶的性质相似，这增加了这两种活性是同一种蛋白质性质的可能性。本文描述了从碱性磷酸酶中分离出磷酸酯酶活性，从商业制剂和新鲜牛肠中纯化出具有磷酸酯酶活性的酶，并鉴定出它是一种能水解单链核酸为5 ′-磷酸核苷（5 ′-核苷酸磷酸二酯酶）的核酸外切酶。
4-Nitrophenyl and 2-naphthyl monoesters of phenylphosphonic acid have been synthesized, and an en-zyme catalyzing their hydrolysis was resolved from alkaline phosphatase of a commercial calf intestinal alkaline phosphatase preparation by extensive ion-exchange chromatography, chromatography on L-phenylalanyl-Sepharose with a decreasing gradient of (NLLhSO^ and gel filtration. De-tergent-solubilized enzyme from fresh bovine intestine was purified after (NH4) 2S04 fractionation by the same tech-nique. The purified enzyme is homogeneous bypolyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. It has a molecular weight of 108,000, con-tains approximately 21% carbohydrate, and has an amino acid composition considerably different from that reported from alkaline phosphatase from the same tissue. The homo-geneous intestinal enzyme, an efficient catalyst of phosphonate ester hydrolysis but not of phosphate monoester hydrolysis, was identified as a 5'-nucleotide phosphodiesterase by its ability to hydrolyze 4-nitrophenyl esters of 5'-TMP but not of 3'-TMP. Also consistent with this identification have shown (Kelly and Butler, 1975; Kelly et al., 1975) that an enzymic activity capable of hydrolyzing monoesters of phosphonic acids1 is broadly distributed throughout nature. Several properties of this enzyme ob-tained fromcalf intestine resemble those of intestinal alkaline phosphatase, raising the possibility that both activities are properties of the same protein. In this paper we describe the resolution of phosphonate esterase activity from alkaline phosphatase, the purification to homogeneity, from the commercial preparations as well as from fresh bovine intes-tine, of the enzyme responsible for phosphonate esterase ac-tivity, and its identification as an exonuclease capable of hydrolyzing single-stranded nucleic acids to nucleoside 5'-phosphates (5'-nucleotide phosphodiesterase).