Purification and properties of human placental dipeptidyl peptidase IV

Dipeptidyl peptidase IV (EC 3.4.14.5; Gly-Pro-p-nitroanilidase) was purified 1840-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100 and subjected to zinc acetate fractionation, hydroxylapatite chromatography, Sephacryl S-300 chromatography and then affinity chromatographies with Lentil Lectin-Sepharose 4B and Gly-Pro-NH-(CH2)6-NH-Sepharose 4B. Dipeptidyl peptidase IV was completely separated from leucine aminopeptidase by the final affinity chromatography. The apparent molecular weight of the enzyme was estimated to be 350,000 by gel filtration. The purified enzyme gave a single band with a weight of 124,000 with sodium dodecyl sulfate (SDS) gel-electrophoresis, suggesting that the enzyme consists of three subunits. The isoelectric point of the enzyme was 4.4. The purified enzyme was most active at pH 8.0 with Gly-Pro-p-nitroanilide as substrate and the Km value for this substrate was 2.27mM.