Affordable Access

Publisher Website

Domains of human prothrombin embedded into vesicles: relation with biological activity

Bioelectrochemistry and Bioenergetics
Publication Date
DOI: 10.1016/s0302-4598(98)00185-8
  • Prothrombin-Membranes Interactions
  • 5[125 I]-Iodonaphthalene-1-Azide
  • Photolabeling
  • Alternative Current Polarography
  • Hmde
  • Biology
  • Chemistry


Abstract In our previous works, ac polarography experiments gave evidence of penetration of prothrombin into phosphatidylserine (PS) containing monolayers and labeling of prothrombin by the highly apolar azide 5[ 125 I ]-iodonaphthalene-1-azide (INA) could be used to monitor penetration of the zymogen into phospholipid vesicles. In the present study, the photochemical labeling method was used to detect which domains in the whole prothrombin molecule are embedded in 75% PS–25% PC containing vesicles; the results are compared to those obtained on monolayers by ac polarography with isolated purified fragments of the protein. The kinetics of cleavage of prothrombin following INA labeling, in the presence of phospholipids, by factor Xa or thrombin, as well as the nature of the products formed, are reported here. The cleavages of radiolabeled INA-II and non-radiolabeled II could be independently followed by the formation of radiolabeled and non-radiolabeled products. It was found that prethrombin 2 is the main penetrating domain, as compared with prethombin 1 and fragment 1, while fragment 2 does not penetrate. Both INA-II and II were activated by factor Xa to give the expected products but INA-II was cleaved faster at Arg 271. This finding suggests that the interaction with vesicles which leads to INA-labeling of prothrombin, results in a conformational change of the protein that enhances its susceptibility to activation. In gel electrophoresis under reducing conditions, subsequent cleavage at Arg 320 resulted in the formation of radiolabeled thrombin B-chain (TB). Since thrombin is formed from radiolabeled prothrombin, this shows that the prothrombin molecules which are embedded in the membrane are biologically active.

There are no comments yet on this publication. Be the first to share your thoughts.