Interaction of murine intestinal mast cell proteinase with inhibitors (serpins) in blood; analysis by SDS-PAGE and western blotting.
The interaction of mouse intestinal mast cell proteinase (IMCP) with serine proteinase inhibitors (serpins) in blood was analysed: (i) by examining the capacity of the inhibitors in blood to block the binding of the irreversible serine esterase inhibitor [3H]diisopropyl fluorophosphate (DFP); (ii) by Western blotting. The binding of [3H]DFP to IMCP was blocked very rapidly by inhibitors in mouse serum and, by Western blotting, this inhibition was associated with the appearance of a 73,000 MW proteinase/inhibitor complex together with a series of higher (greater than 100,000) MW complexes. IMCP was not dissociated from these complexes when electrophoresed under reducing conditions, although prior heat treatment of mouse serum (60 for 30-160 min) abolished the formation of all proteinase/inhibitor complexes. Similarly, the activity of a 48,000 MW inhibitor of chymotrypsin was abolished by heat treatment. A titration experiment established that between 0.5 and 5 mg IMCP were inhibited per ml of serum. The properties and MW of the IMCP inhibitor complexes are typical of serpins and suggest that IMCP secreted during intestinal immunological reactions would be rapidly and irreversibly inactivated by plasma-derived inhibitors.
Irvine
J
J
Moredun Research Institute, Edinburgh, U.K.
Newlands
G F
GF
Huntley
J F
JF
Miller
H R
HR
D000818:Animals; D015153:Blotting, Western; D053818:Chymases; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007700:Kinetics; D051379:Mice; D008815:Mice, Inbred Strains; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors; D015843:Serpins / Q000097:blood*; D014235:Trichinellosis / Q000097:blood / Q000201:enzymology*
D015842:Serine Proteinase Inhibitors; D015843:Serpins; D012697:Serine Endopeptidases; D053818:Chymases