Nω-Phosphoarginine Phosphatase (17 kDa) and Alkaline Phosphatase as Protein Arginine Phosphatases

Seven synthetic polymers,(Glu4,Tyr)n, (Arg)n, (Arg, Pro, Thr)n (Arg-Gly-Glu)6, (Arg-Gly-Phe)5, (Glu-Arg-Gly-Phe)5, and (Ala-Leu-Arg-Arg-Ile-Arg-Gly-Glu-Arg)2, were treated with phosphoryl chloride to phosphorylate their Tyr, Thr, and Arg residues. Protamines and histories were phosphorylated similarly. These phosphorylated peptides were examined as to whether or not they serve as substrates for intestinal alkaline phosphatase [EC 3.1.3.1] and liver Nω-phosphoarginine phosphatase [Kuba, M., Ohmori, H., and Kumon, A. (1992)Eur. J. Biochenu 208, 747-752]. Phosphorylated polyarginine was hydrolyzed with a lower Km with alkaline phosphatase than with Nω-phosphoarginine phosphatase, while the phosphorylated forms of (Arg-Gly-Phe)6 and culpeine were better substrates for Nω-phosphoarginine phosphatase. When (Arg, Pro, Thr)n and culpeine were phosphorylated chemically after treatment with phenylglyoxal, these phosphorylated peptides were worse substrates for Nω-phosphoarginine phosphatase than for alkaline phosphatase. Moreover, the results of proton-decoupled 31P NMR analysis indicated that Nω-phosphoar-ginine phosphatase released P1 from Nω-phosphoarginine residues of phosphopeptides. These results indicate that both phosphatases function as protein arginine phosphatases in different manners, and thatNω-phosphoarginine phosphatase is useful for selectively detecting Nω-phosphoarginine residue in peptides containing various kinds of phosphorylated amino acids.