Purification and characterization of dipeptidylpeptidase IV from goat brain

Department of Biochemistry, Kurukshetra University, Kurukshetra-136 119, India

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'Department of Chemistry, Kurukshetra University, Kurukshetra-136 119, India

Manuscript received 3 September 2003

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 fiactionation, 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 Gly­Pro-4m13NA was 8.0. Of the synthetic chromogenic substrates tested, Ala-Pro-4mßNA was hydrolyzed maximally by DPP-IV enzyme followed by Gly-Pro-4mßNA. K_m value for the hydrolysis of Gly-Pro-4mßNA 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-44NA. The enzyme was stable between pH 6.8-9.0 and upto 50º. The optimum temperature for the hydrolysis of Gly-Pro-4mf3NA was -50º with an activation energy E_a of -11 kcal mol^-1. These properties are similar to those of DPP 1V 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.