Affinity Chromatography of Peptidylarginine Deiminase from Rabbit Skeletal Muscle on a Column of Soybean Trypsin Inhibitor (Kunitz)-Sepharose

Abstract

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)

Peptidylarginine Deiminase Isoforms 1–3 Are Expressed in the Epidermis and Involved in the Deimination of K1 and Filaggrin

“…We showed that PAD1 and 2 extracted from human epidermis bind onto immobilized STI in the presence of calcium, as described previously for PAD2 from rabbit skeletal muscle (Takahara et al, 1983(Takahara et al, , 1986. In contrast, PAD3 does not show any affinity toward STI.…”

“…Enrichment of PAD by affinity chromatography on a column of immobilized STI A TE-NP40 buffer extract of human epidermis was diluted in one volume of 2-fold concentrated buffer B (40 mM Tris-HCl (pH 7.5), 10% glycerol, 0.1 M NaCl, 10 mM 2-mercaptoethanol, 10 mM DTT) containing 20 mM CaCl 2 , and applied to a column of STI-agarose (Pierce Chemical) as described previously (Takahara et al, 1986). The column was washed with buffer B containing 10 mM CaCl 2 , and adsorbed proteins were eluted with buffer B containing 10 mM EGTA.…”

Vimentin Is Involved in Peptidylarginine Deiminase 2-Induced Apoptosis of Activated Jurkat Cells

“…A typical concentration of cytosolic Ca 2+ is approximately 10 −7 M, which is too low for PADI2 activity (Asaga et al, 1998; Vossenaar et al, 2004b). The minimum Ca 2+ concentration required for PADI2 activity is approximately 100-fold higher than that required for normal cytosolic Ca 2+ (Takahara et al, 1986). Although nonactivated Jurkat cells expressed endogenous and exogenous PADI2, most of the enzymes were inactivated because of the low cytosolic Ca 2+ concentration.…”

Discovery of a new class of inhibitors for the protein arginine deiminase type 4 (PAD4) by structure-based virtual screening

“…The enzymatic activity of purified PAD4 was determined by citrulline colorimetric assay and the protein content was checked by Bradford protein assay. One unit of PAD4 was defined as the amount of PAD4 needed to produce 1 μM of Nα-benzoylcitrulline ethyl ester from N-α-benzoylarginine ethyl ester (BAEE) per hour [24]. …”