Purification and characterization of dipeptidyl aminopeptidase III from human placenta.

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Abstract
Dipeptidyl aminopeptidase II (DAP III) from a human placental post-microsomal supernatant was purified 760-fold by means of ammonium sulfate fractionation and successive chromatographies on diethylaminoethyl (DEAE)-cellulose, Sephadex G-200, Butyl-Toyopearl 650 and DEAE-Toyopearl columns. The enzyme showed a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 84000. The enzyme was strongly inhibited by metallochelators, and the activity lost was most effectively restored by the addition of Zn2+. The enzyme was also extremely inhibited by some sulfhydryl reagents such as p-chloromercuribenzoic acid (PCMB) and 5,5''-dithiobis(2-nitrobenzoic acid) (DTNB), and the activity of the enzyme inhibited by DTNB was restored by the addition of thiol reagents. Among various .beta.-naphthylamides examined. Arg-Arg-.beta.-naphthylamide (Arg-Arg-.beta.NA) was most rapidly hydrolyzed.