Purification and characterization of dog mast cell protease-3, an oligomeric relative of tryptases.
The existence of a protein approximately 48% identical with mast cell tryptases was predicted previously from a dog mastocytoma cDNA. Antibodies raised against a peptide based on the deduced sequence suggested that the protein (dog mast cell protease-3, dMCP-3) is expressed in mast cells. In this report, characterization of the protein purified from mastocytomas reveals an N-glycosylated, high molecular weight, tryptic serine protease, which appears to be a tetramer of catalytic subunits, approximately half of which are linked by disulfide bonds. The oligomeric complex yields a single NH2-terminal sequence, which is identical with that predicted by dMCP-3 cDNA. This finding, and the lack of closely related genes on blots of genomic DNA, predict that each subunit is the product of one gene. Although dMCP-3 binds to heparin, it is active and stable at low ionic strength in heparin's absence. It resists inactivation by inhibitors in plasma but is sensitive to small inhibitors, e.g. leupeptin and bis(5-amidino-2-benzimidazolyl)methane (BABIM). dMCP-3 hydrolyzes extended peptidyl p-nitroanilides ending in basic residues, with P1 arginine preferred to lysine; it hydrolyzes the Arg18-Ser19 bond of calcitonin gene-related peptide but cleaves neither vasoactive intestinal peptide nor casein. These data suggest that dMCP-3 is a unique serine protease whose stability, formation of intersubunit disulfide bonds, inhibitor susceptibilities and substrate preferences differ from those of its closest relatives, the mast cell tryptases.
Raymond
W W
WW
Cardiovascular Research Institute, University of California, San Francisco 94143-0911, USA.
Tam
E K
EK
Blount
J L
JL
Caughey
G H
GH
D000595:Amino Acid Sequence; D000818:Animals; D001704:Biopolymers; D002364:Caseins / Q000378:metabolism; D053818:Chymases; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D053802:Tryptases; D014407:Tumor Cells, Cultured
D001704:Biopolymers; D002364:Caseins; D010455:Peptides; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; C083294:chymase 2; C092818:mast cell protease 3; D053818:Chymases; D053802:Tryptases