Purification and characterization of pyrophosphatase from developing embryos of Hyalomma dromedarii

Abstract

The enzyme inorganic pyrophosphatase (E.C. 3.6.1.1.) was purified to apparent homogeneity from Hyalomma dromedarii developing embryos. Its molecular weight from the elution volume on Sephadex G-200 was estimated to be 48,400 ± 2000, and by SDS-polyacrylamide gel electrophoresis to be 49,800 ± 2000. The enzyme exhibited an absolute requirement for Mg 2+. and was unaffected by Zn 2+, Mn 2+, Ca 2+, Fe 2+, Co 2+ or Cu 2+. The enzyme can be protected against heat denaturation by the presence of Mg 2+ (5 mM) and mercaptoethanol (0.1 mM). H. dromedarii pyrophosphatase is specific for pyrophosphate ( K m = 0.52 mM). No hydrolytic activity was detected in the presence of ATP, CTP, UTP, IDP, creatine phosphate, d-glucose-6-phosphate and phenylphosphate. However, a slight pyrophosphatase activity was detected with ADP (7%), GDP (4%) and II-P (5%). The optimal activity of the enzyme was around pH 7.5. The effect of pH on K m and V max was examined. Results suggest the involvement of two ionizable groups with pK values 7.5 and 8.8 in the ionization of enzyme-substrate complex and another two ionizable groups with pK values 6.9 and 9.1 either in the free enzyme or free substrate. Arrhenius plots for H. dromedarii pyrophosphatase at pH 7.2 were biphasic and exhibited discontinuity at 30°C. The activation energies were 4300 and 11,200 cal above and below the transition temperature respectively. No discontinuity was obtained at pH values 6.2, 8.4 and 9.0 and the calculated activation energies were 6500, 8900 and 7400 cal respectively.