1991 Fiscal Year Final Research Report Summary
Determination of the reactive site (s) of kininogens for cysteine proteases using technique of site-directed mutagenesis
| Project/Area Number |
02670114
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Shiga university of Medical Science |
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Principal Investigator |
OHKUBO Iwao Shiga University of Medical Science, Professor, 医学部, 教授 (80152073)
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| Project Period (FY) |
1990 – 1991
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| Keywords | Mutagenesis / kininogens / cysteine protease inhibitor / reactive site |
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| Research Abstract |
High and low molecular weight kininogens strongly inactivate cysteine proteases such as cathepsin B, H and L, calpains I and II, papain, and ficin in noncovalent and noncompetitive manners. It is thought that both kininogens play important physiological roles in the regulation of cysteine proteases. There are two inhibitory domains on the common heavy chain of both kininogens. It is speculated that domains 2 and 3 contain two regions as potential candidates for the reactive site of cysteine proteases : Gln-Val-Val-Ala-Gly (QVVAG) located at approximately two-thirds of the distance from the N-terminus of the domains and Gly located at 44 amino acid residues upstream from the QVVAG region.
In this project, using a cDNA coding human low molecular weight kininogen, we have attemed to express the wild type protein and proteins with deletions of domains 2 and 3, and with deletions or substitutions of the QVVAG and Gly regions, in eukaryotic cells such as BHK, COS and CHO.
Although the amounts of the wild type protein and two proteins with deletions of domains 2 or 3 (<50ng/ml) expressed by the induction with dexamethasone in BHK cells were very low, we collected these mediums from the cells (totally about 5 L each), partially purified these three proteins by using Red-Sepharose and Mono Q column chromatographies, and finally obtained about 3 ug of each protein. Now, we are attempting to analyze their kinetic parameters, and determine the real reactive site (s) of kininogens for cysteine proteases.
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