Sheep mast-cell proteinase-1 (sMCP-1) is a serine proteinase expressed predominantly by mucosal mast cells, with specificity for cleavage C-terminal to basic and hydrophobic amino acid residues. A cDNA encoding sMCP-1 has been cloned using reverse transcriptase (RT)-PCR. It appears to be translated as a pre-proenzyme with a 17-amino-acid signal peptide, a basic 2-amino-acid propeptide and a 226-amino-acid catalytic domain. A second cDNA, encoding a serine proteinase 90% identical with sMCP-1, was also cloned and named sMCP-3. Molecular models were constructed for both enzymes using coordinates for the refined X-ray structures of human cathepsin G, chymase and rat mast-cell proteinase-2. The model for sMCP-1 suggests that the acidic Asp-226 side chain extends into the substrate-binding pocket, hydrogen-bonding with Ser-190 on the opposite side and bisecting the pocket. The location of an acidic moiety in this position would favour interaction with basic substrate residues and binding of aromatic residues is rationalized by interaction of the positively charged equatorial plane with Asp-226. The balance between chymotryptic and tryptic activities of sMCP-1 was found to be sensitive to salt concentration, with increasing univalent cation concentration favouring chymotryptic activity relative to the tryptic. Using a peptide substrate representing residues 36–59 of the human thrombin receptor, increasing salt concentration favoured cleavage at Phe-43 rather than at Arg-41.
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August 1998
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Research Article|
August 01 1998
Sheep mast-cell proteinases-1 and -3: cDNA cloning, primary structure and molecular modelling of the enzymes and further studies on substrate specificity
Sybil M. McALEESE;
Sybil M. McALEESE
*Department of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian, EH25 9RG, Scotland, U.K.
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Alan D. PEMBERTON;
Alan D. PEMBERTON
1
*Department of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian, EH25 9RG, Scotland, U.K.
1To whom correspondence should be addressed (e-mail: alan.pemberton@ed.ac.uk).
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Mary E. McGRATH;
Mary E. McGRATH
†Axys Pharmaceuticals, Inc., 180 Kimball Way, South San Francisco, California 94080, U.S.A.
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John F. HUNTLEY;
John F. HUNTLEY
‡Moredun Research Institute, International Research Centre, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, Scotland, U.K.
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Hugh R. P. MILLER
Hugh R. P. MILLER
*Department of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian, EH25 9RG, Scotland, U.K.
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Publisher: Portland Press Ltd
Received:
February 13 1998
Revision Received:
April 29 1998
Accepted:
May 14 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 333 (3): 801–809.
Article history
Received:
February 13 1998
Revision Received:
April 29 1998
Accepted:
May 14 1998
Citation
Sybil M. McALEESE, Alan D. PEMBERTON, Mary E. McGRATH, John F. HUNTLEY, Hugh R. P. MILLER; Sheep mast-cell proteinases-1 and -3: cDNA cloning, primary structure and molecular modelling of the enzymes and further studies on substrate specificity. Biochem J 1 August 1998; 333 (3): 801–809. doi: https://doi.org/10.1042/bj3330801
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