Separation of free and chymotrypsin-bound alpha 2-macroglobulin by affinity chromatography. Its use to demonstrate that the two chymotrypsin-binding sites of alpha 2-macroglobulin are equivalent and independent.

A monoclonal antibody was obtained from the fusion of spleen cells of mice, immunized with methylamine-treated alpha 2-macroglobulin (alpha 2M), with the myeloma cell line P3-X63-Ag8.653. A competitive binding assay demonstrated that the antibody was specific for a neoantigen expressed on alpha 2M when the inhibitor reacts with proteinases or with methylamine. When immobilized, the monoclonal antibody retained its ability to specifically bind alpha 2M-proteinase complexes or methylamine-treated alpha 2M, both of which could be quantitatively recovered from the immunoaffinity column by lowering the pH to 5.0. Binary alpha 2M-proteinase complexes of trypsin, plasmin, and thrombin, prepared by incubating large amounts of alpha 2M with a small amount of enzyme, were isolated by immunoaffinity chromatography. Each purified complex was characterized with regard to proteinase content, extent of alpha 2M subunit cleavage, extent of thiol ester hydrolysis, and extent of conformational change. Each complex contained 0.8-0.9 mol of proteinase/mol of inhibitor. In the alpha 2M-thrombin, alpha 2M-plasmin, and alpha 2M-trypsin complexes, approximately 50%, 60%, and 75% of the subunits are cleaved, respectively. Titration of sulfhydryl groups revealed that all purified binary complexes contained 2 +/- 0.5 mol of thiol/mol of complex, suggesting that each complex retains two intact thiol ester bonds. When the purified complexes were incubated with excess trypsin or with methylamine, an additional 1-2 mol of sulfhydryl/mol of complex could be titrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Specificity Of Monoclonal Antibody 7h11d6 For A2mmentioning

confidence: 99%

Identification of a monoclonal antibody specific for a neoantigenic determinant on .alpha.2-macroglobulin: use for the purification and characterization of binary proteinase-inhibitor complexes

Strickland¹,

Steiner²,

Migliorini³

et al. 1988

“…Each mole of a2M is capable of inhibiting 2 mol of trypsin or chymotrypsin (Pochon et al, 1978;Barrett et al, 1979;Swenson & Floward, 1979) but only 1 mol of plasmin (Ganrot, , This work was supported by National Heart, Lung, and Blood Institute Grants HL-24066 and HL-31932 and by National Cancer Institute Grant CA-29589. 0006-2960/87/0426-0486S01.50/0 1967; Pochon et al, 1978;Gonias et al, 1982a) or a synthetic chymotrypsin dimer (Pochon et al, 1981). This difference in binding can be explained by a model of a2M structure that contains two adjacent and equivalent binding sites (Pochon et al, 1981;Pochon & Bieth, 1982;Feldman et al, 1985). A large proteinase such as plasmin can bind to one site and sterically inhibit the binding of a second proteinase (Pochon et al, 1981;Gonias & Pizzo, 1983a;Feldman et al, 1985).…”

mentioning

confidence: 99%

Characterization of .alpha.2-macroglobulin-plasmin complexes: complete subunit cleavage alters receptor recognition in vivo and in vitro

Roche¹,

Pizzo²

1987

When human alpha 2-macroglobulin (alpha 2M) binds proteinases, it undergoes subunit cleavage. Binding of small proteinases such as trypsin results in proteolysis of each of the four subunits of the inhibitor. By contrast, previous studies suggest that reaction of plasmin with alpha 2M results in cleavage of only two or three of the inhibitor subunits. In this paper, we demonstrate that the extent of subunit cleavage of alpha 2M is a function of plasmin concentration. When alpha 2M was incubated with a 2.5-fold excess of plasmin, half of the subunits were cleaved; however, at a 20-fold enzyme to inhibitor ratio, greater than 90% of the subunits were cleaved with no additional plasmin binding. This increased cleavage was catalyzed by free rather than bound plasmin. It is concluded that this "nonproductive" subunit cleavage is dependent upon the molar ratio of proteinase to inhibitor. The consequence of complete subunit cleavage on receptor recognition of alpha 2M-plasmin (alpha 2M-Pm) complexes was studied. Preparations of alpha 2M-Pm with only two cleaved subunits bound to the murine macrophage receptor with a Kd of 0.4 nM and 60 fmol of bound complex/mg of cell protein. When preparations of alpha 2-M-Pm with four cleaved subunits were studied, the Kd was unaltered but ligand binding increased to 140 fmol/mg of cell protein. The receptor binding behavior of the latter preparation is equivalent to that observed when alpha 2M is treated with small proteinases such as trypsin. This study suggests that receptor recognition site exposure is not complete in the alpha 2M-Pm complex with half of the subunits cleaved. Proteolytic cleavage of the remaining subunits of the inhibitor results in a further conformational change exposing the remaining receptor recognition sites.

“…Effect of Chymotrypsin. Chymotrypsin cleaves the bait region of a2M at a unique site between residues Tyr-685 and Glu-686 (Mortensen et al, 1981) and is trapped by a2M at a stoichiometry between 1 and 2 mol of chymotrypsin per mole of tetrameric a2M (Pochon et al, 1978;Pochon & Bieth, 1982;Howell et al, 1983). The aromatic region spectra of a computer summation of 1 equiv of a2M and 2 equiv of bovine chymotrypsin and of a 1:2 mixture of these proteins are shown in Figure 4, together with their difference.…”

Section: Resultsmentioning

confidence: 99%

Identification of proton resonances from the bait region of human .alpha.2-macroglobulin and effects of proteases and methylamine

Gettins¹,

Cunningham²

1986

The 1H NMR spectrum of human alpha 2-macroglobulin, Mr 716,000, consists of predominantly extremely broad unresolved resonances but also has nine relatively sharp (delta nu 1/2 less than 25 Hz) resonances from aromatic residues. By treatment of alpha 2-macroglobulin with methylamine, chymotrypsin, and subtilisin, it has been shown that eight of these resonances arise from bait region residues. More specifically, assignment has been made of resonances at 6.80 and 7.11 ppm to the ortho and meta protons, respectively, of tyrosine-685 and tentative assignment of a resonance at 7.29 ppm to the aromatic protons of phenylalanine-684. C2 proton resonances from five histidine residues are also visible. Four of these are attributed to residues in the bait region or immediately adjacent to this, at positions 675, 694, 699, and 704. The sharpness of resonances from bait region residues demonstrates the great flexibility of this region of the polypeptide. It is proposed that the flexible region extends from residue 675 to residue 710. These resonances are all affected by proteolytic cleavage in the bait region but are not influenced by the subsequent conformational rearrangement of the whole protein tetramer. The significance of these findings is discussed in relation to the current structural models of alpha 2-macroglobulin.