Determination of endopolyphosphatase using polyphosphate glucokinase.
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.
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4935.
T H
TH
Kowalczyk
N F
NF
Phillips
D017766:Acid Anhydride Hydrolases; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005069:Evaluation Studies as Topic; D008722:Methods; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis* / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism; D011122:Polyphosphates / Q000032:analysis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity
D011122:Polyphosphates; D010770:Phosphotransferases; C024812:polyphosphate-glucose phosphotransferase; D010744:Phosphoric Monoester Hydrolases; D017766:Acid Anhydride Hydrolases; C022563:endopolyphosphatase