A highly sensitive high-performance liquid chromatography-- fluorometric method for the assay of peptidylarginine deiminase activity.
The activity of peptidylarginine deiminase (PAD) has generally been assayed by a colorimetric method using N-benzoyl-L-arginine ethyl ester (BAEE) and N-benzoyl-L-arginine (Bz-L-Arg) as the substrates. The widespread occurrence of citrulline and urea in tissues makes use of this method difficult, especially for small samples. We developed a highly sensitive high-performance liquid chromatography method with N-dansyl-glycyl-L-arginine as the substrate. This method was sensitive enough to determine previously undetectable activity of PAD in HL-60 cells. Two types of PAD (HL-60 cell and brain PAD) could be distinguished by differential competition, using either BAEE or Bz-L-Arg as a preferential substrate in the assay. These data indicate that the present method is applicable to many tissues.
Chikuma
T
T
Department of Hygienic Chemistry, Showa Pharmaceutical University, Machida 194-8543, Japan.
Yamada
M
M
Tsuda
A
A
Yamamoto
M
M
Nakashima
K
K
Yajima
R
R
Kato
T
T
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D001921:Brain / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002956:Citrulline / Q000009:adverse effects; D003619:Dansyl Compounds / Q000032:analysis / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D005998:Glycine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D018922:HL-60 Cells / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000032:analysis*; D008297:Male; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D014508:Urea / Q000009:adverse effects
D003619:Dansyl Compounds; D002956:Citrulline; D014508:Urea; D001120:Arginine; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D005998:Glycine