This project will continue a long-standing investigation into the role of gamma-carboxyglutamic acid (Gla). This structure is involved in calcium binding to the vitamin K-dependent blood clotting proteins of the plasma and their subsequent interaction with membrane surfaces. In the membrane-bound state, the enzymes of the blood coagulation cascade are much more active in conversion of their substrates (zymogens) to active proteases. This project covers nearly all aspects of these reactions. Metal ion binding is investigated and attempts will be made to identify specific amino acids involved in ion chelation or in subsequent protein conformational changes. These studies will involve modification with chemical reagents followed by analysis of protein structure to determine if specific sites are modified, if they are protected by metal ions and if there is metal ion specificity for the protection (e. g. Ca versus Mg). For example, we have already shown that the amino terminal is required for the membrane-binding event, it is protected by calcium but not by magnesium. Another site (residues 101 or 102) is protected by either Mg or Ca. Chemically modified proteins will be studied to examine their metal ion and membrane binding properties. Chemical modification will also be used to generate proteins with spectroscopic probes at specific sites. These probes will allow investigation of membrane-binding properties. A protein with a built-in fluorescent probe is protein Z and this will be the focus of many studies including the isolation of residues 1-46 (these contain aH the Gla residues of protein Z) and thorough examination of its metal ion and membrane-binding properties. Membrane binding is essential for maximum activity of the prothrombinase complex. This enzymatic complex will be examined with special attempts to determine if it has the kinetic properties of a collisionally limited reaction. This is important to determine if the kinetic parameters obtained for pure systems in vitro can be extrapolated to physiological circumstances. The difference might consist of the number of complexes per particle. In vitro, with one or a few enzyme complexes, the reaction may conform to normal Michaelis-Menton kinetics. However, if the number of enzymes per particle is large in vivo, the type of kinetics may be altered. We will try to demonstrate this behavior by studying the kinetics of prothrombinase with various numbers of enzymes per vesicle. This kind of kinetic behavior will also be investigated with several other systems. Overall, these studies will add to our knowledge of the important blood clotting plasma proteins. Findings may reveal essential structural features of these proteins and lead to new understanding of the blood clotting process. The findings will also reveal general characteristics of membrane-binding events and provide a basis for investigation of an expanding class of peripheral membrane-binding proteins.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hematology Subcommittee 2 (HEM)
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University of Minnesota Twin Cities
Schools of Arts and Sciences
United States
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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
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
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
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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