Sialoforms of dipeptidylpeptidase IV from rat kidney and liver.
Dipeptidylpeptidase IV (DPP IV, CD26), a serine-type exo- and endopeptidase found in the cell surface membrane of many tissues, was employed as a model membrane glycoprotein to study the expression of sialoforms on cell surface glycoproteins. Native, enzymatically active DPP IV was purified from plasma membranes of kidney and liver by lectin affinity chromatography in conjunction with crown ether anion exchange chromatography. The enzyme was gradient-eluted in continuous fractions, all showing a single polypeptide band of about 100 kDa when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing, denaturing conditions. Analysis of the purified DPP IV by isoelectric focusing (IEF) showed that it consists of several polypeptides of different isoelectric points (IP) ranging from 5.5 to 7.0. In vitro- desialylation of the enzyme and subsequent isoelectric focusing revealed that the differences in isoelectric points were due to differences in the degree of sialylation. Differences in the degree of sialylation between the fractions were also demonstrated by SDS-PAGE under nonreducing and nondenaturing conditions. Increased sialylation of the enzyme as demonstrated by isoelectric focusing resulted in increased migration velocity in nonreducing and nondenaturing SDS-polyacrylamide gels. In vitro -desialylation of the enzyme and its resialylation confirmed that sialylation was responsible for this extraordinary migration behavior. The native enzyme was predominantly sialylated via alpha 2, 6-linkage, as shown by lectin affinity blotting employing Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA). These findings demonstrate that a distinct membrane glycoprotein may exist in various sialoforms, distinguished from each other by a different number of sialic acid residues. Moreover, these sialoforms can be individually purified by crown ether anion exchange chromatography.
Schmauser
B
B
Institut für Molekularbiologie und Biochemie der Freien Universität Berlin, Arnimallee 22, D-14195 Berlin-Dahlem, Germany.
Kilian
C
C
Reutter
W
W
Tauber
R
R
D000818:Animals; D000838:Anions; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007668:Kidney / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008297:Male; D019158:N-Acetylneuraminic Acid / Q000032:analysis*; D051381:Rats; D011915:Rats, Inbred BUF; D017208:Rats, Wistar
D000838:Anions; D018819:Dipeptidyl Peptidase 4; D019158:N-Acetylneuraminic Acid