Activated Hageman factor (Factor XII-a) is a serine proteinase that is involved in blood coagulation: it activates Factor XI to bring about formation of thrombin, converts plasma prekallikrein to kallikrein, and plasminogen to plasmin, and increases the clot-promoting properties of Factor VII. Trypsin-inhibitors isolated from pumpkin (Cucurbita maxima) seeds, viz., CMTI-I, CMTI-III, CMTI-IV, each of which has a molecular weight of 3 Kd, and CMTI-V (Mr 7 Kd), are highly specific inhibitors of Factor XII-a; other serine proteases which bear moderate sequence homology (32 to 37%) to Factor XII-a, such as human plasmin, bovine chymotrypsin, and human thrombin are either weakly or not at all inhibited. CMTI-V has recently been isolated and characterized: it belongs to the Potato 1 inhibitor family with one disulfide bridge, and it does not bear any sequence homology to the smaller 3 Kd proteins. While virgin CMTI-I, CMTI-III, and CMTI-V all inhibit Factor XII-a, their reactive-site hydrolyzed (modified) forms do not. Determination of the solution structures of the intact and modified inhibitors by two- dimensional Nuclear Magnetic Resonance (2D NMR) spectroscopy will lead to a better understanding of the mechanism of specific inhibition of Factor XII-a and may lead to development of new therapeutic strategies for blood coagulation disorders. The objectives of the proposed work are: 1) to continue the sequence-specific hydrogen-1 NMR assignments of intact and modified CMTI-V and identify their secondary structural elements; 2) to deduce the three-dimensional structures of virgin and modified CMTI-V by the use of nuclear Overhauser effect-derived distances and a distance-geometry algorithm (DSPACE) in conjection with an energy minimization program (X-PLOR); and 3) to continue the comparative study of intact and reactive-site hydrolyzed CMTI-I and CMTI-III by multiple- quantum proton-detected heteronuclear 2D NMR spectroscopy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL040789-01A3
Application #
3358020
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1993-08-01
Project End
1996-07-31
Budget Start
1993-08-01
Budget End
1994-07-31
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Kansas State University
Department
Type
Schools of Arts and Sciences
DUNS #
City
Manhattan
State
KS
Country
United States
Zip Code
66506