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

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)     

Immunoelectron microscopy studies with a monoclonal antibody directed against a receptor recognition site epitope in human alpha 2-macroglobulin.

Alpha 2-Macroglobulin (alpha 2M) is a plasma proteinase inhibitor that binds up to 2 mole of proteinase per mole of inhibitor. Proteinase binding or reaction with small primary amines causes a major conformational change in alpha 2M. As a result of this conformational change, a new epitope recognized by monoclonal antibody 7H11D6 is exposed. The association of alpha 2M-proteinase or alpha 2M-methylamine with alpha 2M cellular receptors is prevented by 7H11D6. In this investigation, the binding of 7H11D6 to alpha 2M was studied by electron microscopy. 7H11D6 bound to alpha 2M-methylamine and alpha 2M-trypsin but not to native alpha 2M. The structure of alpha 2M after conformational change resembled the letter "H." 7H11D6 epitopes were identified near the apices of the four arms in the alpha 2M "H" structure. 7H11D6 that was adducted to colloidal gold (7HAu) retained the specificity of the free antibody (binding to alpha 2M-trypsin but not to native alpha 2M). alpha 2M conformational change intermediates prepared by sequential reaction with a protein crosslinker and trypsin also bound 7HAu. These results suggest that a complete alpha 2M conformational change is not necessary for 7H11D6 epitope exposure and may not be required for receptor recognition. 7HAu was used to isolate a preparation consisting primarily of binary alpha 2M-trypsin (1 mole trypsin per mole alpha 2M instead of 2). Structures resembling the letter "H" were most common; however, each field showed some atypical molecules with arms that were compacted instead of thin and elongated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of interferon-gamma and human alpha 2-macroglobulin on peritoneal macrophage morphology and Ia antigen expression.

While the primary role of the plasma protein alpha 2-macroglobulin (alpha 2M) appears to be related to its proteinase inhibitory activity, alpha 2M has been reported to regulate the immune response in vitro. Previous studies have demonstrated that, although native alpha 2M has no effect on macrophage function, proteinase- or CH3NH2-treated alpha 2M antagonize the IFN-gamma-induced expression of class II major histocompatibility complex (Ia) antigens on mouse peritoneal macrophages. In this investigation, we examined the effects of alpha 2M-CH3NH2 on the IFN-gamma-induced expression of macrophage Ia antigens by indirect immunofluorescence microscopy, radioimmunoassay, and immunoprecipitation of biosynthetically-labelled Ia. While alpha 2M-CH3NH2 suppressed the IFN-gamma induced increase in the percentage of Ia-positive macrophages detected by immunofluorescence microscopy, alpha 2M-CH3NH2 had no effect on the average of number of Ia molecules expressed per cell as detected by radioimmunoassay. In addition, alpha 2M-CH3NH2 had no effect on the ability of IFN-gamma to induce biosynthesis of Ia. Microscopic examination of IFN-gamma-treated macrophages revealed that treatment with alpha 2M-CH3NH2 prevented IFN-gamma-induced changes in macrophage morphology. IFN-gamma-treatment of elongated inflammatory macrophages was associated with the generation of round cells which possessed few cytoplasmic projections. By contrast, addition of alpha 2M-CH3NH2 to the incubation prevented the IFN-gamma-induced morphological changes, and the cells remained elongated with irregular cytoplasmic borders. We postulate that alpha 2M-CH3NH2 decreases the IFN-gamma-induced expression of Ia by preventing morphological changes in macrophages, resulting in the distribution of existing Ia over a larger surface area. As a consequence of this, the perceived fluorescence intensity of the bound antibody is lowered and the cells appear to be Ia-negative.     

Analysis of thiolester bond cleavage-dependent conformational changes in binary alpha 2-macroglobulin-proteinase complexes

The structures of the two proteinase-binding sites in human alpha 2-macroglobulin (alpha 2M) were probed by treatment of alpha 2M with the serine proteinases thrombin and plasmin. Each proteinase forms an equimolar complex with alpha 2M (a binary alpha 2M-proteinase complex) which results in the activation and cleavage of two internal thiolester bonds in alpha 2M. Binary alpha 2M-proteinase complexes demonstrated an incomplete conformational change as determined by nondenaturing polyacrylamide gel electrophoresis and incomplete receptor recognition site exposure as determined by in vivo plasma elimination studies. Treatment of binary alpha 2M-proteinase complexes with CH3NH2, trypsin, or elastase resulted in cleavage of an additional one or two thiolester bonds in alpha 2M and complete receptor recognition site exposure, demonstrating that a limited conformational change had occurred. Treatment of the alpha 2M-thrombin complex with elastase resulted in the incorporation of approximately 0.5 mol proteinase/mol alpha 2M and completion of the conformational change in the complex. Similar treatment of the alpha 2M-plasmin complex resulted in the incorporation of less than 0.1 mol proteinase/mol alpha 2M. Unlike the alpha 2M-thrombin complex, the alpha 2M-plasmin complex did not undergo a complete conformational change following treatment with CH3NH2 or trypsin. Incubation of this complex with elastase resulted in proteolysis of the kringle 1-4 region of the alpha 2M-bound plasmin heavy chain, and following this treatment the alpha 2M-plasmin complex underwent a complete conformational change. The results of this investigation demonstrate that binary alpha 2M-proteinase complexes retain a relatively intact proteinase-binding site. In the case of the alpha 2M-plasmin complex, however, the heavy chain of alpha 2M-bound plasmin protrudes from the proteinase-binding site and prevents a complete conformational change in the complex despite additional thiolester bond cleavage.     

Analysis of ligand recognition by the purified alpha 2-macroglobulin receptor (low density lipoprotein receptor-related protein). Evidence that high affinity of alpha 2-macroglobulin-proteinase complex is achieved by binding to adjacent receptors.

The molecular basis for binding of alpha-macroglobulin-proteinase complexes to the human two-chain 500/85-kDa (alpha/beta) alpha 2-macroglobulin (alpha 2M) receptor (alpha 2MR)/low density lipoprotein receptor-related protein was analyzed. Ligand blotting experiments showed that a 40-kDa protein, present in the affinity-purified alpha 2MR preparation, is bound to the alpha 2MR alpha-chain and released by heparin. Removal of the 40-kDa protein resulted in a 3-5-fold increase in binding of alpha 2M-trypsin. Nitrocellulose-immobilized pure two-chain alpha 2MR was incubated with human alpha 2M-trypsin, containing four identical subunits, and two monovalent ligands: rat alpha 1-inhibitor-3-chymotrypsin and the 18-kDa receptor binding fragment of the alpha 2M subunit. Binding of alpha 2M-trypsin to the alpha-chain of immobilized alpha 2MR was composed of a high (Kd = 40 pM at 4 degrees C) and a low (Kd = 2 nM) affinity component. alpha 1-Inhibitor-3-chymotrypsin bound to the same sites but with one component (Kd = 0.4 nM). Competition-inhibition experiments and dissociation experiments, using ligands with different valences, as well as experiments with alpha 2MR immobilized at different densities, led to the following model. The low (Kd = 2 nM) affinity of alpha 2M-proteinase is prevalent when only one of the four domains binds to alpha 2MR, i.e. when the receptor density is low or when neighboring receptors are occupied. The high (Kd = 40 pM) affinity is achieved by binding of at least two domains to adjacent receptors.     

Binding and endocytosis of alpha 2-macroglobulin-plasmin complexes

The clearance of 125I-labeled alpha 2-macroglobulin-plasmin complexes (125I-alpha 2M-PM) from mouse circulation is slower than that of 125I-labeled alpha 2M-methylamine complexes (125I-alpha 2M-CH3NH2). In addition, clearance of 125I-alpha 2M-PM is biphasic, but that of 125I-alpha 2M-CH3NH2 follows simple first-order kinetics. Treatment of alpha 2M-PM with trypsin yields a complex that clears like alpha 2M-CH3NH2. Complexes of alpha 2M with Val442-plasmin (alpha 2M-Val442-PM) were prepared; alpha 2M-Val442-PM has a stoichiometry of 2 mol of Val442-PM to 1 mol of alpha 2M and also clears like alpha 2M-CH3NH2. In vitro 4 degrees C binding inhibition studies with mouse peritoneal macrophages show that alpha 2M-CH3NH2, alpha 2M-PM, trypsin-treated alpha 2M-PM, and alpha 2M-Val442-PM bind with the same affinity, apparent Kd = 0.4 nM. The binding isotherms at 4 degrees C are the same for 125I-alpha 2M-CH3NH2, 125I-alpha 2M-PM, and 125I-trypsin-treated alpha 2M-PM in both mouse peritoneal macrophages and 3T3-L1 fibroblasts. The Scatchard plots for the binding isotherms in macrophages were curved; those in 3T3-L1 fibroblasts were linear with an apparent Kd of 0.48 nM and a receptor activity of 140 fmol/mg of cell protein for alpha 2M-CH3NH2, an apparent Kd of 0.29 nM and a receptor activity of 110 fmol/mg of cell protein for alpha 2M-PM, and an apparent Kd of 0.35 nM and a receptor activity of 210 fmol/mg of cell protein for trypsin-treated alpha 2M-PM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the binding of chicken alpha-macroglobulin and ovomacroglobulin to the mammalian alpha 2-macroglobulin receptor

Chicken alpha-macroglobulin (alpha M) and ovomacroglobulin were purified by Ni+2 chelate chromatography. These proteins had similar subunit structure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chicken alpha M bound 1.0 mol and ovomacroglobulin bound 0.8 mol 125I-trypsin per mol inhibitor, respectively. Ovomacroglobulin cleared rapidly from the circulation of mice, and the clearance was inhibited by asialoorosomucoid, but native chicken alpha M cleared slowly (t 1/2 greater than 1 h). After reaction with trypsin, this alpha-macroglobulin cleared rapidly (t 1/2 = 3 min), and this clearance was inhibited by a 1000-fold molar excess of human alpha 2M-methylamine. Ovomacroglobulin-trypsin did not inhibit the binding of 0.2 nM 125I-labeled human alpha 2M-methylamine to mouse peritoneal macrophages in vitro, but chicken alpha M reacted with trypsin inhibited the binding by 50% at 1.9 nM. A kappa I of 1.1 nM was calculated for the binding of chicken alpha M-trypsin to the mammalian alpha-macroglobulin receptor. This affinity is comparable to that obtained with human and bovine alpha 2M.     

Changes in the binding of "fast"-form alpha 2-macroglobulin to 3T3-L1 cells after differentiation to adipocytes

Human alpha 2-macroglobulin (alpha 2M)-CH3NH2 specifically binds to 3T3-L1 fibroblasts and adipocytes with an apparent Kd of 0.3 nM at 4 degrees C. Binding to fibroblasts follows first-order kinetics only for the first 20-30 min of reaction, k1 = 160 microM-1 h-1, and then proceeds in a non-first-order reaction that takes 28 h to reach steady state. Receptor activity is 120 fmol of alpha 2M-CH3NH2/mg of cell protein or 60 000 molecules/cell. Binding is nondissociable. In contrast, binding to adipocytes follows first-order kinetics, k1 = 720 microM-1 h-1, and reaches steady state in 6-8 h. Receptor activity is 35 fmol of alpha 2M-CH3NH2/mg of cell protein or 60 000 molecules/cell. Binding is reversible with a k2 of 0.4 h-1. Control studies with 3T3-C2 cells, which do not differentiate after hormone treatment, indicate that these differences are not due to hormone treatment alone. Binding to both fibroblasts and adipocytes is specific for "fast"-form alpha 2M but not for native alpha 2M. Inhibition studies with neoglycoproteins demonstrate that binding does not occur via any of the known carbohydrate receptors. Some cross-reactivity with antithrombin III-trypsin complexes is demonstrated. Both fibroblasts and adipocytes take up and degrade alpha 2M-CH3NH2 at 37 degrees C. For both cell types, the concentration of alpha 2M-CH3NH2 needed for half-maximal uptake is 65 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active site histidine in pig liver aminolevulic acid dehydratase modified by diethylpyrocarbonate and protected by Zn2+ ions

1. The effect of diethylpyrocarbonate (DEP) (0.1-0.35 mM) on the purified pig liver amino-levulic acid dehydratase (ALA-D) containing 0.3 g-atoms Zn/subunit, under different pHs (6.0-7.5), temperature (0-18 degrees C) and time (0-60 min) was studied. 2. Three histidyl residues/subunit were modified by DEP (0.2 mM, pH 6.8), but activity was completely lost after the first one had reacted, indicating the presence of one histidine residue essential for ALA-D catalysis. Reactivation by treatment with hydroxylamine (0.7 mM, pH 7.0) confirmed that only histidine and no other nucleophile amino acids were directly involved in DEP inhibition. 3. Zn ions (0.5 mM) and the substrate ALA (5-10 mM) protected against DEP inactivation, protection was dependent on pH. 4. Sn, Se, Hg, Cd, Mn, Co and Pb (0.01-0.1 mM) did not significantly protect ALA-D against inactivation. 5. It is concluded that the substrate and Zn binding sites and the essential histidyl residues are in close proximity in the active center. It is proposed that in the catalytic synthesis of porphobilinogen from ALA, histidine groups have the specific role of transporting protons from the aqueous media to a hydrophobic active site.     

Evidence that the platinum-reactive methionyl residue of the alpha 2-macroglobulin receptor recognition site is not in the carboxyl-terminal receptor binding domain

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.     

The structure of alpha 2-macroglobulin-methylamine after papain digestion as determined by electron microscopy.

alpha 2-Macroglobulin-methylamine (alpha 2M-CH3NH2) was digested with papain at pH 5.0. The major 600 kDa fragment was purified by molecular-exclusion chromatography. In a non-denaturing gel-electrophoresis system, the 600 kDa fragment migrated in a single band at a rate that was comparable with that for the untreated alpha 2M-CH3NH2. The elution volume of the 600 kDa fragment on Superose-6 was slightly increased. In primary cultures of rat hepatocytes, cellular uptake of 125I-alpha 2M-CH3NH2 was not affected by the 600 kDa fragment, confirming the results of other investigators. The 600 kDa fragment was negatively stained with uranyl formate and analysed by transmission electron microscopy. The major structural characteristics of the parent protein (alpha 2M-CH3NH2) remained intact. The most common image included prominent lateral walls and two centrally located regions of stain exclusion termed 'paddle structures'. The distance between the paddle structures was equivalent in alpha 2M-CH3NH2 and the 600 kDa fragment [approximately 13.5 nm (135 A)]. By contrast, the lateral walls in the 600 kDa fragment were decreased in length by approximately 0.37 nm (37 A) (19%). It is proposed that the 600 kDa structure retains the 'hollow cylinder' shape of alpha 2M-CH3NH2. The structure of the cylinder is formed by the lateral walls and four paddle structures (only two are imaged, owing to overlapping). The paddle structures in the 600 kDa fragment are intact and relatively closer to the apices of the molecule, owing to the decrease in lateral wall length. Since the alpha 2M receptor-binding sites are removed by papain digestion, the studies presented here support the location of the receptor-binding sites near the apices of the lateral walls.     

Photoinactivation and carbethoxylation of leucine aminopeptidase.

In the present paper the reactivity of histidyl residues of leucine aminopeptidase from bovine eye lens was studied by dye-sensitized photooxidation and by carbethoxylation of the enzyme protein using diethylpyrocarbonate. Of all the different amino acids modified by photooxidation only histidine is connected with the enzymic acticity, whereas tyrosine seems to be involved in structure stabilization. By changing the pH and varying the effectors (Mg2+ and/or dodecylsulfate) of the reaction mixture a different number of histidyl residues of the enzyme protein is caused to react with diethylpyrocarbonate. No secondary reactions with tyrosyl or tryptophyl residues could be observed by spectrophotometric investigations. The enzyme modified by one of the above-mentioned methods shows changes in the capacity of Mn2+ binding measured by autoradiography as well as in the degree of enhancement of enzymic activity by Mn2+ or Mg2+ ions. Of the 48 histidyl residues of the enzyme (Mr = 326000) up to 2 histidyl residues per subunit (Mr = 54000) may be involved in Mn2+ or Mg2+ binding and up to 4 histidyl residues have a strong influence on Zn2+ binding.     

Identification of alpha 2-macroglobulin conformational intermediates by electron microscopy and image analysis. Comparison of alpha 2-macroglobulin-thrombin and alpha 2-macroglobulin reacted with cis-dichlorodiammineplatinum(II) and trypsin.

Human alpha 2-macroglobulin (alpha 2M) exists in two well defined, highly distinct conformations and in less well described intermediate conformations. In this study, previously characterized reactions were used to partially or completely transform the conformation of alpha 2M. Electron micrographs of each preparation were subjected to image analysis. Ternary alpha 2M-trypsin (2 mol of trypsin/mol of alpha 2M) was analyzed as a control for the fully transformed state. Correspondence analysis (CORAN) and hierarchical ascendant classification (HAC) generated five image clusters from 330 aligned alpha 2M-trypsin complexes. Average images of each cluster resembled the letter "H" with four nearly equivalent lateral arms. Abnormally shaped lateral arms were not demonstrated by HAC, using a variety of factor sets. In a native polyacrylamide gel electrophoresis system, alpha 2M-thrombin migrated in a diffuse band partially behind alpha 2M-trypsin, suggesting conformational heterogeneity. CORAN and HAC of 733 alpha 2M-thrombin complexes identified two neighboring clusters, the average images of which showed an H-like structure in which one arm was replaced by a globular stain-excluding body. The two alpha 2M-thrombin clusters included 125 images (17.1% of image population). The complete absence of atypical lateral arm structure in the alpha 2M-trypsin clusters suggests that this variation is not the result of orientation or staining artifact. Native alpha 2M was reacted with cis-dichlorodiammineplatinum(II) and then with trypsin to form alpha 2M-Pt-trypsin, a preparation that includes partially transformed alpha 2M structures. CORAN and HAC of 580 alpha 2M-Pt-trypsin complexes generated five clusters, the average images of which showed atypical lateral arm structure equivalent to that demonstrated with alpha 2M-thrombin. The five alpha 2M-Pt-trypsin clusters accounted for 15.2% of the image population. These studies suggest that alpha 2M conformational change intermediates demonstrate common structural characteristics, permitting an elucidation of the steps involved in this complex transformation.     

Purification and characterization of human alpha 2-macroglobulin conformational variants by non-ideal high performance size-exclusion chromatography.

Human alpha 2-macroglobulin (alpha 2M) was eluted as a single nondispersed peak from a TSK-G4000SW size exclusion chromatography column equilibrated in 20 mM-sodium phosphate/100 mM-NaCl, pH 7.2 (PBS). The void volume and total accessible volume of the column were 6.08 ml and 14.42 ml. The elution volume (Ve) of native alpha 2M was 9.20 +/- 0.04 ml. The Ve was altered minimally by changing the ionic strength or adding ethanol to the equilibration buffer. Ternary alpha 2M-trypsin, containing 2 mol of proteinase/mol of inhibitor, and alpha 2M-methylamine failed to be eluted in well-defined peaks when the column was equilibrated in PBS. The majority of either preparation was recovered slowly at Ve values greater than 14.5 ml, reflecting significant nonideal interactions with the support structure. Addition of 10% ethanol or increased ionic strength in the equilibration buffer independently caused either form of reacted alpha 2M to be eluted in a distinct peak at decreased Ve, suggesting that the nonideal interactions included hydrophobic and electrostatic adsorption. When the equilibration buffer was 80 mM-sodium phosphate/320 mM-NaCl, pH 7.2, partial resolution of ternary alpha 2M-trypsin and alpha 2M-methylamine was obtained with a single column run. The Ve values of ternary alpha 2M-trypsin and alpha 2M-methylamine in this buffer were 13.15 +/- 0.08 ml and 11.94 +/- 0.14 ml, respectively. The Ve of native alpha 2M was 8.84 +/- 0.03 ml. The resolving capacity of TSK-G4000SW was exploited to purify native alpha 2M rapidly and efficiently from solutions that contained significant amounts of either ternary alpha 2M-trypsin or binary alpha 2M-trypsin (1 mol of proteinase/mol of inhibitor). This purification was complete within the limits of sensitivity of denaturing and nondenaturing polyacrylamide-gel electrophoresis. alpha 2M-plasmin was well resolved from native alpha 2M. The Ve of alpha 2M-plasmin was 12.88 +/- 0.32 ml in 80 mM-sodium phosphate/320 mM-NaCl, pH 7.2. A number of procedures were used to prepare solutions with up to 90% binary alpha 2M-trypsin. The Ve of binary alpha 2M-trypsin in these various solutions was intermediate between the values of native alpha 2M and ternary alpha 2M-trypsin. The conformations of binary and ternary complex, as reflected by mobility in nondenaturing electrophoresis, were identical, confirming previous results. Finally, in the binary alpha 2M-trypsin complex, the single trypsin cleaved more than two, and as many as all four alpha 2M subunits.     

Physical and chemical properties of human plasma alpha2-macroglobulin.

Alpha2-M (alpha2-macroglobulin) was purified from human plasma by two different procedures. As well as having no detectable impurities by the usual criteria for testing the homogeneity of protein preparations, these alpha2M preparations showed a single component, after reduction in urea, of 185000 daltons by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weight of the alpha2M was found to be 718000 by sedimentation equilibrium experiments using the gravimetrically determined -v of 0.731 ml/g. The interaction of several proteinases with alpha2M was studied by using a novel discontinuous polyacrylamide-gel system, which showed clear separation of the enzyme-complexed alpha2M from the free alpha2M. These studies indicated that urokinase, as well as trypsin, chymotrypsin, plasmin and thrombin forms complexes with alphaM. The cleavage of the 185000-dalton subunit to a 85000-dalton species on interaction of trypsin with alpha2M was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after reduction of the alpha2M-trypsin complex in urea. The amino acid composition, carbohydrate content, absorption coefficient at 280 nm, the specific refractive increment and the sedimentation coefficient for these alpha2M preparations were measured. The stability of the trypsin-binding activity of the alpha2M preparations was also studied under several storage situations.     

Yeast phenylalanyl-tRNA synthetase. Properties of the histidyl residues.

Reactivity of the histidyl groups of yeast phenylalanyl-tRNA synthetase was studied in the absence or presence of substrates. In the absence of substrates about 10 histidine residues were found to react with similar kinetic constants. Phenylalanine at 10(-3) M was found to protect two histidyl residues; increasing the amino acid concentration to 5 . 10(-3) M resulted in the protection of two more histidyl groups. tRNAPhe did not afford any protection to histidine residues, but acylated phenylalanyl-tRNA (Phe-tRNAPhe) protected two of the four histidyl groups already protected by phenylalanine. These results suggest the existence of two different sets of accepting sites for phenylalanine: one specific for the free amino acid, the other one specific for the amino acid linked to the tRNA, but being accessible to free phenylalanine, with a somewhat lower binding constant, ATP was found to mask around four histidyl residues against diethylpyrocarbonate modification. By photoirradiation of enzyme-phenylalanine complex in the presence of rose bengale, a significant amount of amino acid was bound to the alpha subunit (Mr = 73 000) of phenylalanyl-tRNA synthetase, confirming that the amino acid binding site is located on this subunit, as previously suggested by modification of thiol groups. Upon irradiation of an enzyme-tRNA complex, almost no covalent binding of tRNA occurred during enzyme inactivation, suggesting that the histidyl residues involved in the enzymic activity are not required for tRNA binding.     

Immunosuppressive properties of electrophoretically "slow" and "fast" form alpha 2-macroglobulin. Effects on cell-mediated cytotoxicity and (allo-) antigen-induced T cell proliferation

Lymphokine activated killer cell lysis of K562 cells was inhibited by alpha 2-macroglobulin (alpha 2M), soybean trypsin inhibitor, and alpha 1-proteinase inhibitor. In serum free medium 2 mg/ml alpha 2M suppressed target cell lysis in a 4-h cytotoxic assay with about 40%. Suppression was dose and time dependent. Cytotoxicity was unaffected by alpha 2M concentrations less than 0.25 mg/ml, and by alpha 2M added later than 1.5 h from start of assay. Pre-treatment of effector (but not of target) cells with alpha 2M was even more suppressive than the presence of alpha 2M during assay. Cell-mediated cytotoxicity was not inhibited by alpha 2M treated with methylamine or by various alpha 2M-proteinase complexes. In contrast, alpha 2M-proteinase complex as well as native alpha 2M suppressed the proliferation of Ag-activated T cells. However, methylamine-treated alpha 2M did not inhibit T cell proliferation, and suppression by alpha 2M-proteinase complex was significantly reduced after inhibition of the alpha 2M-bound proteinase. On incubation at 4 degrees C with lymphokine-activated killer cells, alpha 2M reacted with cell associated proteinases and changed from electrophoretically "slow" to "fast" form. Cell associated proteinases bound by alpha 2M showed chymotrypsin- and trypsin-like specificities and their activity surpassed activity caused by cellular leakage and secretion. The present results strongly indicate that alpha 2M mediates immunosuppression in its capacity as a proteinase inhibitor and suggest inhibition of (T)cell surface-associated proteinases as a possible mode of suppression.     

Reaction of proteinases with alpha 2-macroglobulin: evidence for alternate reaction pathways in the inhibition of trypsin

Titration experiments were employed to measure the binding stoichiometry of alpha 2M for trypsin at high and low concentrations of reactants. These titration experiments were performed by measuring the SBTI-resistant trypsin activity and by direct binding measurements using 125I-labeled trypsin. The binding stoichiometry displayed a marked dependence upon protein concentration. At high alpha 2M concentrations (micromolar), 2 mol of trypsin are bound/mol of inhibitor. However, at low alpha 2M concentrations (e.g., 0.5 nM), only 1.3 mol of trypsin were bound/mol of inhibitor. Sequential additions of subsaturating amounts of trypsin to a single aliquot of alpha 2M also resulted in a reduction in the final binding ratio. A model has been formulated to account for these observations. A key element of this model is the observation that purified 1:1 alpha 2M-proteinase complexes are not capable of binding a full mole of additional proteinase [Strickland et al. (1988) Biochemistry 27, 1458-1466]. The model predicts that once the 1:1 alpha 2M-proteinase complex forms, this species undergoes a time-dependent conformational rearrangement to yield a complex with greatly reduced proteinase binding ability. According to this model, the ability of alpha 2M to bind 2 mol of proteinase depends upon the association rate of the second enzyme molecule with the binary (1:1) complex, the enzyme concentration, and the rate of the conformational alteration that occurs once the initial complex forms. Modeling experiments suggest that the magnitude of the rate constant for this conformational change is in the order of 1-2 s-1.     

Ethoxyformylation of tubulin with [3H]diethyl pyrocarbonate: a reexamination of the mechanism of assembly inhibition

In this study we reexamined the basis for the profound inhibitory effects of low concentrations of diethyl pyrocarbonate (DEP) on tubulin's ability to assemble into microtubules [cf. Lee, Y. C., Houston, L. I., & Himes, R. H. (1976) Biochem. Biophys. Res. Commun. 70, 50-56]. Assembly inhibition at low DEP concentrations can be resolved into two components: a component reversible with hydroxylamine (attributed to monoethoxyformylation of histidyl residues) that contributes approximately 40% of the inhibition and a hydroxylamine-resistant component (attributed to ethoxyformylation of non-histidyl residues) that contributes approximately 60% of the inhibition. Comparisons between the extent of assembly inhibition associated with each component and the degree of residue modification argue for the involvement of a small number of highly reactive residues in the inhibition process. To identify these residues, tubulin was reacted with limiting concentrations of [3H]DEP and subjected to tryptic digestion and HPLC analysis. Only one moderately reactive histidyl residue was detected. This residue (approximately 2-3-fold more reactive than the bulk histidyl residues) eluted in an apparently large, hydrophobic fragment. We failed to detect any non-histidyl residues that were exceptionally reactive to [3H]DEP. However, we did observe that the N-terminal methionyl residues in native protein were ethoxyformylated at rates comparable to that of the bulk histidyl residues. In denatured protein these methionyl residues were ethoxyformylated to a much larger extent (approximately 3-4-fold) than the bulk histidyl residues. We suggest that the N-terminal methionyl residues in tubulin are partly buried or are in a salt-bridge interaction in native protein and that ethoxyformylation of these residues disrupts tubulin structure and interferes with microtubule assembly.     

Localization of epsilon-lysyl-gamma-glutamyl cross-links in five human alpha 2-macroglobulin-proteinase complexes. Nature of the high molecular weight cross-linked products.

Covalent binding of proteinases by human alpha 2-macroglobulin (alpha 2M) results primarily from the formation of stable epsilon-Lys-gamma-Glu isopeptide bonds. Cross-linking engages 12, 13, and 10 of the 14, 14, and 11 Lys residues in chymotrypsin, trypsin, and subtilisin, respectively, and reaction with the alpha-amino group of the C-chain of chymotrypsin and the B-chain of beta-trypsin is also seen. In contrast, cross-linking engages only 6 of the 11 Lys residues in thermolysin. In each of these proteinases, a few residues react to the greatest extent: Lys36, Lys79, Lys87, and Lys93 in chymotrypsin; Lys87, Lys109, Lys222, and Lys239 in trypsin; Lys12, Lys43, and Lys141 in subtilisin; and Lys210 and Lys219 in thermolysin. In elastase, 1 of the 3 Lys residues (Lys87) is tentatively identified as being cross-linked. Formation of unstable bonds judged to be mainly p-tyrosyl-gamma-glutamyl esters can also be significant for some proteinases. In each of the proteinases, several of the strongly reacting Lys residues are located relatively close to each other, presumably reflecting steric constraints within the alpha 2M-proteinase complexes as they form. Proteinases are covalently bound to alpha 2M to one or two of its COOH-terminal bait region-cleaved half-subunits. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern of the high molecular weight cross-linked species indicates that binding of a proteinase through two cross-links occurs not only within the 360-kDa disulfide-bridged alpha 2M dimer but also between the two dimers in the alpha 2M tetramer.