Purification and characterization of dipeptidylpeptidase IV from goat brain

Abstract

Dipeptidylpeptidase IV (DPP IV) proteinase was purified ∼133-fold with specific activity 29.2 units/mg to an apparent electrophoretic homogeneity. It was isolated from goat brain by homogenization, acid-autolysis at pH 6.0, 20-50% (NH4)2SO4 fractionation, ion-exchange chromatography on CM-Sephadex C-50 at pH 5.87, Sephadex G-150 gel filtration and anion-exchange chromatography on DEAE-Sephadex A-50 at pH 8.0. The molecular weight of the enzyme estimated by gel-filtration chromatography was ∼200000 Da. The pH optimum of the enzyme for the hydrolysis of Gly-Pro-4mβNA. was 8.0. Of the synthetic chromogenic substrates tested, Ala-Pro-4mβNA was hydrolyzed maximally by DPP-IV enzyme followed by Gly-Pro-4mβNA. K m value for the hydrolysis of Gly-Pro-4mβNA was 0.5 mM. The enzyme activity of the purified preparation was inhibited by PMSF, TLCK, puromycin and heavy metal ion i.e. Zn2+ but was not inhibited by p-hydroxy-mercuriphenylsulphonic acid, EDTA, iodoacetic acid, indicating that goat brain DPP IV is a serine protease. The dipeptides, His-Pro and Ala-Pro, competitively inhibited the hydrolysis of Gly-Pro-4mβNA. The enzyme was stable between pH 6.8-9.0 and upto 50°. The optimum temperature for the hydrolysis of Gly-Pro-4mβNA. was ∼50° with an activation energy Ea of ∼11 kcal mol-1. These properties are similar to those of DPP IV purified from other tissues. The purification and characterization of DPP IV is of great value for the identification of its natural substrates and for the study of its physiological significance in the brain tissue.