Fragment 1 Research Articles

Embedding of prethrombin domains of human prothrombin into phospholipid membranes

In a coagulation cascade, the conversion of the proenzyme prothrombin into the active enzyme thrombin occurs on the membrane of activated platelets. Since the kinetics of this conversion are considerably increased in the presence of negatively charged phospholipids, we investigated the modes of interaction of prothrombin(II) with such phospholipidic membranes. Since we have previously found that prothrombin penetrates into phospholipid monolayers and vesicles, in this work we studied which domains of this protein are involved in this penetration. Two complementary approaches were used with two complementary domains, fragment 1 (F1) and prethrombin 1 (P1). Penetration of the protein and its isolated and purified fragments into lipid monolayers with different phosphatidylserine contents was inferred from variations in the capacitance of this layer, measured by ac polarography. It was found that P1 and F1 penetrate in a cooperative manner and contribute in a synergic manner to the penetration of II, while prethrombin 2 (P2) has the highest tendency to penetrate alone. In the case of vesicles, the method is based on the photolabeling of the protein domains embedded in the membrane, using the apolar reagent 5- 125I-iodonaphthalene-1-azide. In the whole protein, P2 is the main penetrating domain, while P1 and F1 are weakly labeled. The results provide good evidence that the prethrombin 2 domain penetrates into both membrane models.

Immunological properties of canine prothrombin, its activation products, plasma and serum

Immunological properties of canine prothrombin(PO), prethrombin 1 (P1), fragment 1(F1), fragment 2(F2), thrombin(T1), plasma and serum were studied by the use of immunoelectrophoresis(IEP), crossed immunoelectrophoresis (CIEP) and Ouchterlony analysis. Antibodies against P0 and T1 were used. The results showed that P0, P1, F1, F2 and T1 all had a variable affinity with anti-PO antibody but that only P0, P1 and T1 had a demonstrable affinity with anti-T1 antibody. F1 and F2 did not show any detectable affinity. Quantitative affinity study by the use of I-125-labeled proteins gave the following average values for % bound I-125-proteins in 3 hr incubation with the antibody against P0 or T1: with anti-P0 antibody, 81 ± 2 (SD) % for I-125-P0, 79.1 ± 2(SD) % for I-125-P1, 23.5 ± 3(SD) % for I-125-F1, 25.5 ± 2(SD) % for I-125-F2 and 25.2 ± 3(SD) % for I-125-T1, and with anti-T1 antibody, 28 ± 2(SD) % for I-125-P0, 80.5 ± 2(SD) % for I-125-P1, 1.0 ± 0.03(SD) % for I-125-F1, 1.3 ± 0.03(SD) % for I-125-F2, and 81 ± 3(SD) % for I-125-T1. With the use of CIEP and anti-P0 antibody, serum was found to contain F1, F2 and a trace amount of P1, and plasma contained only P0, but upon activation with tissue thromboplastin, plasma was found to generate P1, F1, F2 and T1 with time. These results indicate that a simple, specific and quantitative immunoassay of T1 by the use of anti-T1 antibody or anti-P0 antibody may not be possible at least in dogs, but that CIEP by the use of anti-P0 antibody might be used as a diagnostic means of in vivo P0 activation.

Light dissociates enzymatically-cleaved rhodopsin into two different fragments

Thermolysin cleaves rhodopsin in retinal disc membranes into two large fragments, which can be solubilized in detergent as a non-covalent complex. Fragment 1 (F1) contains a binding site for concanavalin A, whereas fragment 2 (F2) contains an N-retinyl group after borohydride reduction and a reactive sulfhydryl group. The complex of F1 and F2 exhibits 500 nm absorbance and is regenerable after bleaching. Light dissociates F2 from F1, which can be separated by concanavalin A-agarose chromatography. The photodissociation of F1 and F2 may be an expression of conformational changes that are part of the process of visual excitation.