Use of immobilized adenosine deaminase (EC 3.5.4.4) for the rapid purification of native human CD26/dipeptidyl peptidase IV (EC 3.4.14.5).
The leukocyte differentiation antigen CD26 identified as dipeptidyl peptidase IV.(EC 3.4.14.5), cleaves off N-terminal dipeptides from peptides when a proline or alanine is located at the penultimate position. Seminal plasma and especially prostasomes, prostate-derived organelles which occur freely in seminal plasma, contain high amounts of CD26/dipeptidyl peptidase IV and therefore are suitable sources for the purification of the protein. The use of adenosine deaminase (EC 3.5.4.4) affinity chromatography for its purification is described. CD26/dipeptidyl peptidase IV was purified from human seminal plasma and prostasomes by a two step procedure. Ion exchange chromatography on DEAE-Sepharose, followed by affinity chromatography on adenosine deaminase-Sepharose resulted in the pure, native protein with an overall yield ranging from 35 to 55%. The N-terminal sequence of the amphiphilic enzyme purified from human prostasomes was determined to be Met-Lys-Thr-Pro-Trp-Lys-Val-Leu. The preparation obtained was free of contaminating aminopeptidase activity and proved to be very stable (up to 1 month at 37 degrees C). The calf intestinal adenosine deaminase we used is commercially available and can be employed for the purification of human, bovine and rabbit CD26/dipeptidyl peptidase IV. High affinity binding of porcine dipeptidyl peptidase IV was not observed. The availability of a source with high specific activity and the introduction of adenosine deaminase affinity chromatography permits the rapid purification of milligram quantities of natural mammalian CD26/dipeptidyl peptidase IV.
de Meester
I
I
Department of Clinical Biochemistry, University of Antwerp, Wilrijk, Belgium.
Vanhoof
G
G
Lambeir
A M
AM
Scharpé
S
S
D000243:Adenosine Deaminase; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D018819:Dipeptidyl Peptidase 4 / Q000302:isolation & purification*; D004800:Enzymes, Immobilized; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D015388:Organelles / Q000201:enzymology / Q000276:immunology; D011467:Prostate / Q000201:enzymology / Q000276:immunology; D012661:Semen / Q000201:enzymology* / Q000276:immunology*
D004800:Enzymes, Immobilized; D018819:Dipeptidyl Peptidase 4; D000243:Adenosine Deaminase