Mapping the heparin-binding site of mucus proteinase inhibitor.
Heparin accelerates the inhibition of neutrophil elastase by mucus proteinase inhibitor (MPI), the physiological antielastase of airways as a result of its binding with the inhibitor [Faller, B., Mély, Y., Gérard, D., & Bieth, J. G. (1992) Biochemistry 31, 8285-8290]. To explore the heparin-binding site of the inhibitor, we have modified the lysine and arginine residues of MPI and its isolated C-terminal domain by using 4-N,N-(dimethylamino)azobenzene-4'-isothiocyano-2'-sulfonic acid (S-DABITC) [Chang, J. Y. (1989) J. Biol. Chem. 264, 3111-3115] and (p-hydroxyphenyl)glyoxal (HPG) (Yamasaki, R. B., Vega, A., & Feeney, R. E. (1980) Anal. Biochem. 109, 32-40], respectively. The derivatizations were done in the absence and presence of a 4.5 kDa heparin fraction with a low degree of polydispersity. The effect of chemical modification of the inhibitors on their affinity for heparin was tested using two complementary procedures, one based on the ability of heparin to accelerate the inhibition of chymotrypsin by the inhibitors and the other exploiting the affinity of the inhibitors for immobilized heparin. Modification of a limited number of lysine and arginine residues in full-length MPI led to a 6-fold decrease in affinity for heparin. The presence of the polymer during the modification reactions significantly prevented this effect. Amino acid sequencing unambiguously identified the heparin-protected lysines as Lys 13 and Lys 87, located on the N-terminal and C-terminal domains of MPI, respectively. Heparin apparently protects mainly two arginine residues from modification by HPG.(ABSTRACT TRUNCATED AT 250 WORDS)
Mellet
P
P
Laboratoire d'Enzymologie, INSERM Unité 392, Université Louis Pasteur de Strasbourg, Illkirch, France.
Ermolieff
J
J
Bieth
J G
JG
D000595:Amino Acid Sequence; D001665:Binding Sites; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D017879:Isothiocyanates; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010449:Peptide Mapping; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
D017879:Isothiocyanates; D009842:Oligopeptides; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C043307:succinyl-alanyl-alanyl-prolyl-phenylalanine-4-nitroanilide; C012926:4-(N,N-dimethylaminoazobenzene)-4'-isothiocyanate; D006493:Heparin; D004124:p-Dimethylaminoazobenzene; D002918:Chymotrypsin