Affinity chromatography of peptidylarginine deiminase from rabbit skeletal muscle on a column of soybean trypsin inhibitor (Kunitz)-Sepharose.
We designed a simple procedure for the purification of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle using substrate affinity chromatography. Of the immobilized substrate ligands tested, i.e. protamine and soybean trypsin inhibitor (Kunitz) (STI), STI-Sepharose was found to be an effective affinity adsorbent for purification of the enzyme. The specific binding of peptidylarginine deiminase to STI-Sepharose was observed in the presence of calcium ion, and the enzyme could be selectively eluted from the affinity adsorbent by washing with chelator. A 1,800-fold purification with a 50% yield was achieved in the three-step procedure, which involved DEAE-Sephacel ion-exchange and STI-Sepharose affinity chromatography. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity and the recovery were considerably higher than have been obtained by any procedures previously reported. The specific interaction of peptidylarginine deiminase with STI immobilized on Sepharose was also investigated quantitatively by frontal affinity chromatography. In this method, a peptidylarginine deiminase solution was applied continuously to an STI-Sepharose column and the retardation of the elution front was measured as a parameter of the strength of the interaction. The dissociation constant for the enzyme with STI was found to be 2.3 X 10(-7)M. This value was in good agreement with that obtained by kinetic analysis in our previous studies. Peptidylarginine deiminase required millimolar Ca2+ for the binding to STI-Sepharose. The Ca2+ dependence of the enzyme binding was quite similar to that of the enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Takahara
H
H
Okamoto
H
H
Sugawara
K
K
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002384:Catalysis; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006867:Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D009132:Muscles / Q000201:enzymology*; D011485:Protein Binding; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D011817:Rabbits; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000378:metabolism
D014361:Trypsin Inhibitors; D014360:Trypsin Inhibitor, Kunitz Soybean; D006867:Hydrolases; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D002118:Calcium