Specificity of serine proteases for cleavage sites on proatrial natriuretic factor.
An immunological approach was used to investigate the specificity of protease cleavage sites on proANF. Cleavage of 35S-cysteine biosynthetically-labeled proANF by whole serum, thrombin and kallikrein was examined. Reaction products were immunoprecipitated with two antibodies directed to different epitopes: a previously characterized antibody directed toward the carboxy-terminus of ANF103-126, which cross reacts with proANF, ANF99-126 and ANF103-126, and a newly prepared antisera to synthetic ANF99-105, which uniquely recognizes ANF99-126, but not proANF or ANF103-126. With increasing time of incubation with rat serum, proANF is sequentially cleaved at the C-terminus of a monobasic Pro-Arg dipeptide sequence to form ANF99-126, and then at the C-terminus of a dibasic Arg-Arg dipeptide sequence to yield ANF103-126. This cleavage activity of serum is blocked by leupeptin (40 micrograms/ml), but not by hirudin (100 nM), a specific inhibitor of thrombin, or by aprotinin (200 KIU/ml), a kallikrein inhibitor. When 100-fold purified serum cleavage enzyme was used in place of crude serum, similar results were obtained. Thrombin cleaves proANF only at the monobasic site to produce ANF99-126 while kallikrein cleaves only at the dibasic site to produce ANF103-126. As expected, the generation of these cleavage products can be inhibited by hirudin or aprotinin respectively. These data indicate that the substrate specificity of the serum cleavage activity is broader than that of thrombin or kallikrein, and that cleavage of proANF by serum proteases may be influenced by conformational restraints. The methods developed here should help in the future characterization of the physiological proANF cleaving enzyme.
Fischman
A J
AJ
Division of Nuclear Medicine, Massachusetts General Hospital, Boston 02114.
Wildey
G M
GM
Matsueda
G R
GR
Margolies
M N
MN
Zisfein
J B
JB
Homcy
C J
CJ
Graham
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D009320:Atrial Natriuretic Factor / Q000097:blood / Q000276:immunology / Q000378:metabolism*; D003429:Cross Reactions; D007610:Kallikreins / Q000378:metabolism; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000097:blood / Q000276:immunology / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism
D000906:Antibodies; D011480:Protease Inhibitors; D011498:Protein Precursors; D009320:Atrial Natriuretic Factor; D007610:Kallikreins; D012697:Serine Endopeptidases; D013917:Thrombin