This research has three general objectives that are related to understanding the physical and kinetic properties of assembling the blood coagulation cascade. The first is to understand the function of the vitamin K-dependent amino acid, Gamma-carboxyglutamic acid in calcium binding and protein-membrane association. The protein structures that are involved in, and change as a result of, calcium ion binding will be investigated by use of nmr and lanthanide substitution experiments utilizing the vitamin K-dependent, membrane-binding portion of prothrombin. The lanthanide experiments will also make extensive use of fluorescence techniques. The structure at the protein-membrane interface will be studied by measuring dynamic and equilibrium properties of protein-membrane association and by substitution of lanthanides for calcium and determining various distances and changes in the complex detected by fluorescence techniques. The second major area is the structure and function of membrane complexes containing more than one protein. The initial hypothesis is that the membrane-binding site of the vitamin K-dependent proteins functions as a portion of an overall binding interaction. That is, these proteins normally bind simultaneously to a membrane plus at least one other membrane-bound protein. Specific protein-protein complexes on the membrane surface to be studied include: protein S with protein C(a) and with complement C4-binding protein. Other examples include protein C(a) with factor Va, factor VIIIa or thrombomodulin. Equilibrium, dynamic and hydrodynamic aspects of these interactions will be measured to determine if the proteins associate with each other while they are membrane bound. Light scattering, quasielastic light scattering and various fluorescence techniques will be used. Protein binding to phospholipid monolayers will also be used to document the properties of the protein-lipid associations. The third area of study involves proteins of the contract phase of coagulation. At least some of these proteins bind to lipid membranes and undergo activation. The various properties of the protein-lipid associations describe above will be measured to help understand the physical forces involved in binding. Analysis of the kinetics of factor activation will be correlated with protein-lipid binding to try to understand how the surface influences this kinetic event. These studies should increase our knowledge of protein-membrane interactions in general and how such interactions are specifically responsible for enhancing the rates of the blood coagulation process.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL015728-14
Application #
3335041
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1976-05-01
Project End
1990-04-30
Budget Start
1986-05-01
Budget End
1987-04-30
Support Year
14
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Arts and Sciences
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Martinez, M B; Flickinger, M C; Nelsestuen, G L (1999) Steady-state enzyme kinetics in the Escherichia coli periplasm: a model of a whole cell biocatalyst. J Biotechnol 71:59-66
McDonald, J F; Nelsestuen, G L (1997) Potent inhibition of terminal complement assembly by clusterin: characterization of its impact on C9 polymerization. Biochemistry 36:7464-73
McDonald, J F; Shah, A M; Schwalbe, R A et al. (1997) Comparison of naturally occurring vitamin K-dependent proteins: correlation of amino acid sequences and membrane binding properties suggests a membrane contact site. Biochemistry 36:5120-7
McDonald, J F; Evans Jr, T C; Emeagwali, D B et al. (1997) Ionic properties of membrane association by vitamin K-dependent proteins: the case for univalency. Biochemistry 36:15589-98
Nelsestuen, G L; Martinez, M B (1997) Steady state enzyme velocities that are independent of [enzyme]: an important behavior in many membrane and particle-bound states. Biochemistry 36:9081-6
Lu, Y; Nelsestuen, G L (1996) Dynamic features of prothrombin interaction with phospholipid vesicles of different size and composition: implications for protein--membrane contact. Biochemistry 35:8193-200
Lu, Y; Nelsestuen, G L (1996) The prothrombinase reaction: ""mechanism switching"" between Michaelis-Menten and non-Michaelis-Menten behaviors. Biochemistry 35:8201-9
Evans Jr, T C; Nelsestuen, G L (1996) Importance of cis-proline 22 in the membrane-binding conformation of bovine prothrombin. Biochemistry 35:8210-5
Martinez, M B; Flickinger, M C; Nelsestuen, G L (1996) Accurate kinetic modeling of alkaline phosphatase in the Escherichia coli periplasm: implications for enzyme properties and substrate diffusion. Biochemistry 35:1179-86
Schwalbe, R A; Coe, J E; Nelsestuen, G L (1995) Association of rat C-reactive protein and other pentraxins with rat lipoproteins containing apolipoproteins E and A1. Biochemistry 34:10432-9

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