Kowalczyk T H, Phillips N F
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4935.
Anal Biochem. 1993 Jul;212(1):194-205. doi: 10.1006/abio.1993.1312.
A method has been developed for determining endopolyphosphatase (polyphosphate depolymerase, EC 3.6.1.10) activity. The enzyme catalyzes the hydrolysis of inorganic polyphosphates [poly(Ps)] by cleaving internal phosphoanhydride bonds without removal of terminal phosphate residues. During the reaction, shorter poly(P) chains are formed and the molar concentration of poly(P) increases. This enzymatic activity is difficult to quantitate, because the substrates and products of the reaction are chemically identical. The commonly used viscometric method lacks sensitivity and cannot be used with shorter poly(P) substrates. The method described here overcomes these problems, and in addition is rapid, simple, and can be used for distinguishing between the endopolyphosphatase and exopolyphosphatase (EC 3.6.1.11) activities. It is based on monitoring the increase in the number of poly(P) chains generated by endopolyphosphatase. For this purpose, the method takes advantage of the specific property of poly(P) glucokinase (EC 2.7.1.63) which utilizes poly(Ps) of different sizes present in the endopolyphosphatase reaction mixture and reduces them to fairly uniform very short-chain product, poly(P)m. The concentration of poly(P)m is expressed in terms of acid-labile phosphorus and is proportional to the duration of the endopolyphosphatase reaction (i.e., the number of original poly(P) chains) and to protein concentration. The increase in the poly(P)m concentration is a relative measure of the endopolyphosphatase activity. Under certain conditions, m equals 3.5 and the activity can be expressed in standard units, since the exact number of poly(P) chains formed by endopolyphosphatase can be calculated from the increase in molar concentration of poly(P)3.5. Accuracy and advantages of the assay are discussed.
已开发出一种用于测定内聚磷酸酶（多聚磷酸解聚酶，EC 3.6.1.10）活性的方法。该酶通过裂解内部磷酸酐键催化无机多聚磷酸盐[聚（P）]的水解，而不除去末端磷酸残基。在反应过程中，会形成较短的聚（P）链，且聚（P）的摩尔浓度会增加。这种酶活性难以定量，因为反应的底物和产物在化学上是相同的。常用的粘度测定法缺乏灵敏度，不能用于较短的聚（P）底物。本文所述的方法克服了这些问题，此外还具有快速、简单的特点，可用于区分内聚磷酸酶和外聚磷酸酶（EC 3.6.1.11）的活性。它基于监测内聚磷酸酶产生的聚（P）链数量的增加。为此，该方法利用了聚（P）葡萄糖激酶（EC 2.7.1.63）的特殊性质，该酶利用内聚磷酸酶反应混合物中存在的不同大小的聚（P），并将它们还原为相当均匀的非常短链产物聚（P）m。聚（P）m的浓度以酸不稳定磷表示，与内聚磷酸酶反应的持续时间（即原始聚（P）链的数量）和蛋白质浓度成正比。聚（P）m浓度的增加是内聚磷酸酶活性的相对度量。在某些条件下，m等于3.5，且活性可以用标准单位表示，因为可以根据聚（P）3.5摩尔浓度的增加来计算内聚磷酸酶形成的聚（P）链的确切数量。讨论了该测定方法的准确性和优点。
