A Study of the Anticoagulatory DNA from the Earthworm, Lumbricus rubellus, and its Regulatory DNA-Binding Protein

  • Kim, Gyoung-Mi (Department of Biochemistry, College of Medicine, Inha University) ;
  • Yu, Kyoung-Hee (Department of Biochemistry, College of Medicine, Inha University) ;
  • Woo, Jeong-Im (Department of Orthopedic surgery, Ajou University) ;
  • Bahk, Yun-Kyoung (Department of Biochemistry, College of Medicine, Inha University) ;
  • Paik, Seung R. (Department of Biochemistry, College of Medicine, Inha University) ;
  • Kim, Jung-Gyu (Department of Pediatrics, College of Medicine, Inha University) ;
  • Chang, Chung-Soon (Department of Biochemistry, College of Medicine, Inha University)
  • Received : 0
  • Accepted : 0
  • Published : 0

Abstract

We have previously shown that a DNA fragment is responsible for the anticoagulatory effect of an earthworm, Lumbricus rubellus. The anticoagluant increased the activated partial thromboplastin time (APTT) and also inhibited the thrombin activity observed with either N-${\alpha}$-p-tosyl-L-arginine methyl ester (TAME) or H-D-phenyl-alanyl-L-pipecoil-L-arginine-p-nitroanilide (S-2238). Since trypsin digestion of the anticoagulant further increased the APTT, the possible presence of a regulatory protein for the anticoagulatory DNA was investigated by digesting the anticoagulant with trypsin and isolating the DNA fragment with C4-reversed phase HPLC. The DNA fragment lacking a regulatory protein was eluted in the flow-through fraction, and analyzed with thrombin and activated factor X. Activated factor X activity was more strongly inhibited than thrombin activity. For DNA digestion, we treated the anticoagulant with DNase and purified the DNA-binding protein with a FPLC Resource-S cation exchange column. The regulatory protein, with an $M_r$ of 55.0 kDa, reduced the anticoagulatory effect of the DNA fragment.

Keywords

Activated factor X;Anticoagulatory DNA;DNA-binding protein;Earthworm;Thrombin