ENZYMATIC-SYNTHESIS OF [ALPHA-P-32]ATP OF HIGH SPECIFIC ACTIVITY

被引：4

作者：

KIHARA, K ^{[1
]}

NOMIYAMA, H ^{[1
]}

YUKUHIRO, M ^{[1
]}

MUKAI, JI ^{[1
]}

机构：

[1] KYUSHU UNIV, INST AGR CHEM, FUKUOKA 812, JAPAN

来源：

ANALYTICAL BIOCHEMISTRY | 1976年 / 75卷 / 02期

关键词：

D O I：

10.1016/0003-2697(76)90128-7

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

An improved enzymatic preparation of [.alpha.-32P]ATP that is both rapid and economical is described. 3''-AMP is phosphorylated at the 5''-position using polynucleotide kinase and [.gamma.-32P]ATP to give 5''-labeled adenosine-5'',3''-diphosphate (pAp) without lowering the molar specific activity. Nuclease P1 (Penicillium citrinum) is then added to the reaction mixture. Since nuclease P1 is specific towards 3''-phosphate esters it hydrolyzes the product pAp to 5''-AMP and Pi. The unreacted 3''-AMP is hydrolyzed to yield adenosine and Pi. The mixture is then electrophoresed to separate [32P]AMP, which is eluted and incubated with myokinase and pyruvate kinase to form [.alpha.-32P]ATP. The method should be applicable to the preparation of the other 3 .alpha.-32P-labeled ribonucleoside triphosphates by replacing 3''-AMP by other appropriate 3''-ribomononucleotides.

引用

页码：672 / 673

页数：2

共 10 条

[1] STUDIES ON A NUCLEASE FROM PENICILLIUM-CITRINUM .3. SUBSTRATE-SPECIFICITY OF NUCLEASE-P1 [J].