Abstract
Digestion of human alpha 2-macroglobulin-methylamine (alpha 2M-CH3NH2) with papain prior to gel filtration resulted in the resolution of three distinct peaks. The material in peak I (Mr approximately 600,000) and peak II (Mr approximately 55,000) did not have any receptor binding ability as determined by in vivo clearance studies and in vitro competitive binding studies using mouse peritoneal macrophages. In contrast, the material in peak III (Mr approximately 20,000) bound to macrophage alpha 2-macroglobulin (alpha 2M) receptors with a Kd of 250 nM. This represents a 500-fold decrease in affinity relative to undigested alpha 2M-CH3NH2. Sequence analysis demonstrated that this material constituted the carboxyl-terminal fragment (COOH-terminal fragment) of alpha 2M. alpha 2M is known to possess a methionyl residue which is susceptible to modification by cis-dichlorodiammineplatinum (II) (cis-DDP) with the result being a loss of receptor binding ability by alpha 2M. For this reason, experiments were performed to determine if the platinum-reactive methionyl residue is located in the COOH-terminal receptor binding fragment of alpha 2M. The results of this investigation demonstrate that cis-DDP is not reactive with either the isolated COOH-terminal fragment or the COOH-terminal fragment isolated from alpha 2M-CH3NH2 which had been pretreated with cis-DDP. In addition, the COOH-terminal fragment did not bind to monoclonal antibody 7H11D6, a monoclonal antibody which binds to the platinum-reactive epitope of the alpha 2M-CH3NH2 receptor recognition site. In contrast, the 55-kDa fragment of alpha 2M bound approximately 1 mol platinum/mol of 55-kDa fragment and also bound to monoclonal antibody 7H11D6. Since the COOH-terminal fragment retains some receptor binding ability, the results of this investigation demonstrate that this fragment is not the complete receptor recognition site and suggest that a platinum-reactive methionyl residue located in the 55-kDa fragment of alpha 2M is another component of this site.
Highlights
On many different cell types, with the result being endocytosis and degradation of azM.Digestion of a,M-CH3NHzwith small amounts of a novel bacterial proteinase or papain results in cleavage of a COOH-terminal 138-residue fragment of a2M which retains binding ability to both fibroblast and hepatocyte a2M receptors as well as monoclonal antibodies which bind to the a,M-receptor recognition site [33, 34]
COOH-terminal fragment of a2M binds to the macrophage a,M receptor
The Kdfor the binding to themacrophage might not react with the COOH-terminal fragment of azM azMreceptor was 250 nM; this isin excellent agreement with because of a conformational change in the polypeptide in- the Kd determined for the binding of the COOH-terminal curred during the isolation procedure
Summary
CHsNH2 with papain or a novel, as yet uncharac~rized, rophages were plated at a density of 250,000 cells/cm2in RPMI-1640 bacterial proteinase [33,34]. Incubation media were receptor recognition of a2M-CH3NH2I.n this report, we show prepared by adding various concentrations of unlabeled competing that the isolated COOH-terminal fragment binds to the ligand to 0.3 nM '251-a2M-CH3NHi2n binding buffer. The final volume macrophage a2Mreceptor but does not react with cis-DDP or bind to our receptor recognition site-specific monoclonal antibody described in the accompanying paper [30]. Nonspecific binding was determined by Determination of Platinum Content of cis-DDP-treated Proteins- incubation in the presence of a 100-fold molar excess of unlabeled. DDP-treated a2M-CH,NH2 (3.5 p ~w)as reacted with papain (201, excess of H202over Na"% After incubation for 30 min at room w/w) in a buffer containing 50 mM CH3COONa,100mM NaC1,l mM temperature, NaN, was added to inactivate the enzyme, excess KI. Mac- performed in 12% gels using the Tris/sulfate system of Neville [42]
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