The preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130,000-140,000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50 X 10(-3) M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.

Peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, was identified and partially purified from rabbit skeletal muscle. The specific activity of the crude extract from the muscle was aboutt 120 times that of the extract from newborn rat epidermis. The enzyme was purified abou 250-fold over the initial extract of rabbit skeletal muscle in a yield of about 53%. The enzyme requires Ca2+ as an essential cofactor and the activity was not inhibited by monoiodoacetate or PCMB. The molecular weight of the enzyme was found to be about 115,000 by gel chromatography on Bio-Gel P-300. The enzyme catalyzed the deimination of arginyl residue when both the alpha-amino and alpha-carboxyl groups were substituted, and showed low activity when substrates had either a free alpha-amino or a free alpha-carboxyl group. The action of the enzyme on free L-arginine was negligible. Proteins such as protamine, histone, and ribonuclease A were better substrates than the other small synthetic peptides tested.

4.

Introduction: Although several studies suggest beneficial effects of low-dose estrogen-progestins (LEPs) and progestins on dysmenorrhea in Japanese women, the difference in efficacy between drugs remains unknown. Methods: We identified studies by searching the MEDLINE, Cochrane Library, and ICHUSHI databases and included randomized controlled trials (RCTs) that used total dysmenorrhea score and visual analogue scale (VAS) as outcome measures to evaluate LEPs and progestins for primary and secondary dysmenorrhea. We analyzed results by meta-analysis and network meta-analysis (NMA). Results: We identified 10 articles on eight RCTs and included seven drugs (six LEPs and one progestin, i.e., dienogest) and placebo in the analysis. Meta-analysis showed improvements in total dysmenorrhea score and VAS for almost all drugs compared with placebo. In NMA, VAS in secondary dysmenorrhea improved more with dienogest than with norethisterone/ ethinylestradiol (mean difference -25.84 [95% CrI -44.46 to -7.15]). In the comparison of administration regimens, VAS improved more with progestin-continuous than LEP-cyclic and the surface under the cumulative ranking (SUCRA) of LEP-extended and progestincontinuous appeared to be higher than that of LEP-cyclic. Conclusions: We confirmed that LEPs and dienogest are effective for primary and secondary dysmenorrhea and suggest that continuous regimens may be more effective than cyclic regimens in improving outcomes.