Purification And Characterization Of Renal Adenosine Deaminase Enzyme And Its Binding Proteins

Objective:To study biochemical properties of the adenosine deaminase(ADA) from the cells of human kindey.Methods:Adenosine deaminase was purified by immtino-affinity chromatography, the enzyme activity was assayed colorimetrically by the measurement of ammonia produced over a fixed timeResults:The purified ADA was shown to be a homogeneity by polyacrylamide gel electrophoresis(PAGE). The yield and specific activity of purified ADA were 26. 59% and 2561. 41 U/mg, respectively. The purified ADA molecule consists of 45.6 KD small subunit ADA and two inactive binding protein (ADBP) with molecular weight of 68. 1 KD and 108. 7 KD on a 10% SDS-PAGE. Using adenosine or 2-deoxyadenosine as substrates the apparent Km of the enzyme is 89 umol / L and 67 umol / L ,respectively.The optimum temperature and optimum pH of the purified enzyme were 37-40 0Cand PH 6.5--7.0 , respectively. The enzyme activity can be inhibited by p-chloromecuribenzoate while partially restored by2dithiothreitol. The isolated ADBP can inhibit small subunit ADA activity by 18. 4% 21. 5% during 50 -45O umol / L of adenosine.Conclusion:Adenosine deaminase from human kidney is a complex consisting of small subunit ADA and inactive ADBP, the hydrosulfuryl is the essential group in the active center of the enzyme and ADBP can inhibit the activity of small subunit ADA.