7 Interaction of Prothrombin and Ca
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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 4, 2016 | http://pubs.acs.org Publication Date: June 1, 1980 | doi: 10.1021/ba-1980-0188.ch007
Phospholipid Monolayers Containing Phosphatidyl Serine M.-F.
LECOMPTE
1
and I. R. M I L L E R
Department of Membrane Research, The Weizmann Institute of Science, Rehovot, Israel
This work deals with the stoichiometry and the structural aspects of the interaction between prothrombin, negatively charged phospholipids, and Ca . We studied the adsorption equilibria of Ca and of prothrombin on monolayers containing phosphatidyl serine. Surface radioactivity of Ca and of H-prothrombin served as a measure for adsorption of the two compounds to the phospholipid monolayers. Synergetic attachment of Ca and prothrombin to the phospholipid water interface was observed and the number of Ca per attached prothrombin was determined. Electrochemical investigations on lipid monolayers interacting with prothrombin and Ca showed that prothrombin increases the lipid layer capacitance and produces a pseudocapacitance peak at the cystine-cysteine redox potential, indicating penetration of the lipid layer with a preferential onentation. ++
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" D l o o d coagulation is preceded by a sequence of zymogen-to-enzyme conversions brought about by some very specific proteases ( I ) . In these processes the interaction between coagulation factors and phospholipids plays a crucial role (2) by accelerating some of the reaction steps leading to clot formation. The key role played by the thrombin i n the blood coagulation process has led to its extensive study and mainly to the understanding of its complex formation from its zymogen, prothrombin. On leave of absence from the Chemistry Department, C.S.P., Université ParisNord, Avenue J. B. Clément, 93430 Villetaneuse, France. 1
0-8412-0473-X/80/33-188-117$05:00/l © 1980 American Chemical Society
In Bioelectrochemistry: Ions, Surfaces, Membranes; Blank, Martin; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.
BIOELECTROCHEMISTRY: IONS, SURFACES, M E M B R A N E S
118
The conversion of prothrombin to thrombin, as well as some other conversions of this series, require membrane-bound complexes of protease, substrate, and a conversion accelerating protein—Factor V (3). Factor Xa, obtained from Factor X, is instrumental i n the proteolytic decomposition of prothrombin to thrombin. Prothrombin and Factor X, being Vitamin K-dependent proteins, contain y-carboxyglutamic acid residues (4,5) and bind to membranes containing acidic phospholipids (e.g., Platelet Factor 3) i n the presence of C a (6). Intact protein structure, along with the y-carboxyglutamic acid and C a or other cations, is essential for the formation of protein membrane complexes (7). Yet our understanding on a molecular level is still fragmentary. Recently efforts have been made to obtain a clue to the following fundamental questions concerning the structure of the lipid-prothrombin interaction products on the membrane. Does prothrombin penetrate the phospholipid layer or is it bound only to the membrane surface? What is the ionic-hydrophobic balance i n the protein-membrane phospholipid interaction? Can we say something about the orientation of the interacting prothrombin molecule w i t h respect to the lipid layer from platelets? Nelsestuen and his co-workers (8) tried to answer some of these questions by quasielastic light scattering. They observed the increase in the radii of the negatively charged phospholipid vesicles upon adding prothrombin and C a \ They also determined the binding stoichiometry (9,10). Their conclusion was that Fragment I of prothrombin binds by C a bridges to the negative phospholipids with the long axis of the prolate elipsoid mole perpendicular to the plane of the lipid layer.
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 4, 2016 | http://pubs.acs.org Publication Date: June 1, 1980 | doi: 10.1021/ba-1980-0188.ch007
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In the present work we studied the interaction of prothrombin with lipid monolayers in the presence of C a . The stoichiometric relations of the interacting components were determined by measuring surface radioactivity of H-labelled prothrombin and C a . The penetration was i n ferred from the effect of the interaction on the capacitance of the surface layer. ++
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Materials
45
and
Methods
The phospholipid, supplied in chloroform-methanol solution, was purchased from L i p i d Products, Nutfield, England. For spreading, samples were evaporated i n a stream of nitrogen, their lipid content was determined by weight, and they were dissolved i n hexane (Spectra Grade, F l u k a ) . The lipid monolayers were maintained above the collapse pressure by spreading a two-to-fivefold excess of the lipid over an aqueous subphase. The experiments were performed in a 0.1M K N 0 solution, buffered with 10~ N frfa-HNOa to p H 7.6-7.8. The p H was checked at the beginning of the experiments. 3
3
In Bioelectrochemistry: Ions, Surfaces, Membranes; Blank, Martin; Advances in Chemistry; American Chemical Society: Washington, DC, 1980.
7.
LECOMPTE AND MILLER
119
Prothrombin and Ca'
Prothrombin ( ~ 74,000 mol wt) was a gift of R. Benarous and J. Elion, Institut de Pathologie Moleculaire, 24 rue d u Faubourg SaintJacques, 75014 Paris (France). Radioactive H-labelled prothrombin was prepared by a method described previously (11); sialic acid was oxidized with sodium metaperiodate. The obtained aldehyde was reduced by sodium ( H ) - b o r o hydride. Radioactive sodium- ( H)-borohydride and C a were purchased from the Radiochemical Center, Amersham, England. Water was doubly distilled over permanganate. The different i n organic salts and acids were of analytical grades. Purified mercury was triple distilled under vacuum before use. Surface radioactivity was determined with an end-window, gas-flow counter equipped with a 100-200 nm thick Formvar window supported by a thin, perforated aluminum sheet as described earlier (12). The counter was calibrated with a spread monolayer of H oleic acid, the radioactivity of which was determined also in bulk. The surface concentration of the prothrombin r was given by 3
3
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 4, 2016 | http://pubs.acs.org Publication Date: June 1, 1980 | doi: 10.1021/ba-1980-0188.ch007
3
4 5
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3
p t
(cpmyy N A (cpm)
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(cpm) o, (cpm)
