Differences in the binding of transforming growth factor beta 1 to the acute-phase reactant and constitutively synthesized alpha-macroglobulins of rat.

Human alpha 2-macroglobulin (alpha 2M) is a proteinase inhibitor and carrier of certain growth factors, including transforming growth factor beta 1 (TGF-beta 1). The constitutively synthesized homologue of human alpha 2M in the adult rat is alpha 1M. Rat alpha 2M is an acute-phase reactant, expressed at high levels in experimental trauma, pregnancy and in certain pathological conditions. The physiological role of rat alpha 2M is not known. In this investigation, we demonstrated that rat alpha 1M and rat alpha 2M bind TGF-beta 1. The equilibrium dissociation constants (KD) for the binding of TGF-beta 1 to the native forms of alpha 1M and alpha 2M were 257 and 109 nM respectively. alpha 1M underwent conformational change when it reacted with methylamine. The resulting product bound TGF-beta 1 with higher affinity (32 nM). Methylamine-treated rat alpha 2M did not undergo conformational change and did not bind TGF-beta 1 with increased affinity. Previous studies suggest that the native conformation may be the principal form responsible for the cytokine-carrier activity of alpha 2M in plasma and serum-supplemented cell culture medium. To confirm that native rat alpha 2M is a more efficient TGF-beta 1 carrier than native alpha 1M, fetal bovine heart endothelial cell (FBHE) proliferation assays were performed. TGF-beta 1 (5 pM) inhibited FBHE proliferation, and native alpha 2M (0.3 microM) counteracted this activity whereas alpha 1M (0.3 microM) had almost no effect. Rat alpha 2M underwent conformational change when it reacted with plasmin incorporating 1.1 mol of plasmin/mol. alpha 2M-plasmin bound TGF-beta 1; the KD (61 nM) was lower (P < 0.01) than that determined for the native alpha 2M-TGF-beta 1 interaction. These studies demonstrate that both rat alpha-macroglobulins are carriers of TGF-beta 1. The native form of rat alpha 2M probably has a predominant role, compared with native alpha 1M, as a TGF-beta 1 carrier in the plasma during the acute-phase response.

Electron microscopy studies of alpha 2-macroglobulin subunit association after limited reduction with dithiothreitol.

The subunits of human alpha 2-macroglobulin (alpha 2M) were dissociated by treatment with reductant (0.5 mM dithiothreitol) under mild conditions. Intact tetramers, half-molecules (subunit dimers), and monomers were identified by chromatography on Superose-6. These products were not in rapidly reversible equilibrium since purified half-molecules were completely stable for up to 18 h. Monomers slowly associated to form some half-molecules in the same time period. Negatively stained preparations of monomers and half-molecules demonstrated significant heterogeneity by electron microscopy. This heterogeneity probably reflected structural differences as well as variation in projection and staining. After reaction with methylamine or proteinase, half-molecules and purified monomers reassociated. The principal product was an intact tetramer displaying an "H-like" image which was visually indistinguishable from the structure of intact tetrameric alpha 2M after conformational change. Incompletely reassociated alpha 2M species were also identified after performing chromatography to increase the fraction of these products. Images resembling one-half of the intact tetrameric alpha 2M structure (epsilon-image) or three-quarters of the intact tetramer (chair-image) were observed. These studies demonstrate that reassociation of reductant-dissociated alpha 2M subunits occurs by the formation of appropriate interactions, so that the resulting tetramers are equivalent to nontreated alpha 2M-trypsin. Unlike native alpha 2M, the structure of conformationally transformed alpha 2M is relatively insensitive to the loss of interchain disulfide bonds. We propose that reassembly of alpha 2M subunits occurs by the binding of two half-molecules or by the addition of individual subunits to half-molecules or trimers.

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.

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)

Alpha-macroglobulin from Limulus polyphemus exhibits proteinase inhibitory activity and participates in a hemolytic system.

Significant primary sequence homology between the alpha-macroglobulin family of proteinase inhibitors and the complement components C3, C4, and C5 implies that these proteins arose from a common ancestor. Hemolymph from the ancient invertebrate Limulus polyphemus contains both complement-like and proteinase inhibitory activity. In this report, we present evidence that L. polyphemus alpha-macroglobulin not only possesses proteinase inhibitory activity, but it also participates in the lytic system of the horseshoe crab. The protein is a disulfide-linked dimer of subunits of molecular mass 185 kDa. Upon reaction with proteinase or methylamine, L. polyphemus alpha-macroglobulin underwent a major conformational change and no proteinase-associated multimerization was detected. L. polyphemus alpha-macroglobulin is the only detectable inhibitor of a number of proteinases in L. polyphemus hemolymph. Proteinase inhibition follows the general "trapping" mechanism shared by most alpha-macroglobulins; however, no covalent linking of proteinases to the inhibitor was detected despite the presence of a functional thiolester. Moreover, the inhibitor demonstrated thiolester-mediated binding to sheep erythrocytes, a property also observed with complement components such as C3. Depletion of functional protein by treatment of hemolymph with methylamine destroyed the proteinase inhibitory capacity and the lytic activity of the hemolymph. Both activities were restored by adding purified protein to depleted hemolymph. Studies with purified L. polyphemus alpha-macroglobulin demonstrated that the thiolester incorporates glycerol as well as methylamine, a property shared by human C3. The data support the hypothesis that L. polyphemus alpha-macroglobulin is both a proteinase inhibitor and part of a lytic system, providing a link between the two distinct sides of the alpha-macroglobulin family. Because both properties are contained in one molecule, we propose the name "limac" to describe this Limulus alpha-macroglobulin complement-like protein.

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.

Electron microscopy studies of alpha 2-macroglobulin conformational intermediates obtained by derivatization with cis-dichlorodiammineplatinum (II).

The structure of human alpha 2-macroglobulin (alpha 2M) after reaction with cis-dichlorodiammineplatinum (II) (cis-DDP) was studied by electron microscopy. The cis-DDP stabilized a novel conformation of the native inhibitor resembling a doughnut surrounded by two, three, of four well defined spherules. When only two spherules were present, these structures were usually oriented on opposite sides of the doughnut. The protein region joining a spherule to the central structure did not include sufficient mass to exclude stain and was, therefore, invisible. Other images showed spherules that were partially superimposed on the doughnut. A comparison of many molecules suggested great flexibility of the peripheral spherules relative to the central structure. The cis-DDP prevented complete conformational change when the alpha 2M was reacted with trypsin. The products of this reaction included apparent conformational intermediates. These intermediates most closely resembled either native alpha 2M or the well established "H" structure of alpha 2M-proteinase, depending on the initial conditions used to modify the alpha 2 M with cis-DDP. When cis-DDP-treated alpha 2M was reacted with trypsin, purified by chromatography and subsequently treated with diethyldithiocarbamate, complete conformational change was observed. Based on an analysis of the alpha 2M structural intermediates obtained using the chemical modification procedures described here, a new model of alpha 2M conformational change was developed. We postulate that conformational change initially involves contraction of the peripheral spherules towards the central doughnut. These spherules then unfold and elongate in the perpendicular direction to form the lateral walls of the proteinase transformed alpha 2M H structure.

Regulation of streptokinase-human plasmin complex by the plasma proteinase inhibitors alpha 2-antiplasmin and alpha 2-macroglobulin is species specific and temperature dependent.

Streptokinase-plasmin complex (SkPl) was prepared with human plasminogen. Regulation of SkPl and plasmin by the plasma proteinase inhibitors, alpha 2-antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M), was studied as a function of temperature in plasminogen-depleted human plasma, mouse plasma, and solutions of purified proteins. The reaction of plasmin with proteinase inhibitors in human plasma was complete. alpha 2AP was the predominant inhibitor. The fraction of alpha 2M-plasmin recovered was not affected significantly by incubation temperature. In contrast, the reaction of SkPl with human proteinase inhibitors was markedly temperature dependent. The apparent second-order rate constant for the reaction of SkPl with purified alpha 2AP at 37 degrees C (1.5 x 10(2) mol/L-1 s-1) was greater than 150-fold higher than the constant derived at 4 degrees C. In human plasma and in solutions containing mixtures of purified human proteins, alpha 2AP was the principal inhibitor of SkPl. Elevating the temperature enhanced the reaction of SkPl with alpha 2AP and alpha 2M comparably. Equivalent results were obtained when incubations were performed in platelet-rich plasma (PRP) or whole blood. In murine plasma, SkPl reacted readily with the proteinase inhibitors. The principal inhibitor of SkPl was alpha 2M. Maximum reaction between SkPl and murine alpha 2M was observed at 37 degrees C; however, significant reaction also occurred at 4 degrees C. alpha 2 AP was the predominant inhibitor of plasmin in mouse plasma. Reaction of alpha 2AP with SkPl in murine plasma was significant only after the alpha 2M was inactivated with methylamine. These results were not affected by platelets or whole blood cells. We conclude that the thrombolytic efficacy of streptokinase reflects not only the nature of the plasminogen activator complex but also the function of the proteinase inhibitors.

Alpha 2-macroglobulin is the primary inhibitor of miniplasmin in vitro and in vivo in the mouse. Comparison with alpha 2-antiplasmin in simultaneous reaction experiments.

Miniplasmin reacted rapidly with purified human alpha 2-macroglobulin (alpha 2M). More than 98% of the complexes were stabilized by at least one covalent bond. The second-order rate constant for the reaction of alpha 2M with miniplasmin at 4 degrees C was 5.1 x 10(5) M-1.s-1. This value was determined by measuring the formation of covalent alpha 2M-125I-miniplasmin complex; however, the rate constant most likely reflects the bait-region cleavage step in the reaction mechanism. Miniplasmin bound primarily to alpha 2M when incubated at 37 degrees C with various mixtures of alpha 2-antiplasmin (alpha 2AP) and alpha 2M. A 2.4-fold molar excess of alpha 2AP was required to yield an equal distribution of proteinase between the two inhibitors. alpha 2M was the primary miniplasmin inhibitor in human and murine plasma (4 degrees C and 37 degrees C). The extent of covalent-bond formation with murine alpha 2M was approx. 96%. Intravenously injected miniplasmin cleared rapidly from the circulation of mice and was recovered principally in the liver. The catabolic pathway was distinctly different from that of miniplasminogen, which was sequestered mainly in the kidneys. The rate of miniplasmin clearance was much faster than that of purified alpha 2AP-miniplasmin complex, suggesting reaction with alpha 2M in vivo. This was confirmed in clearance competition experiments with alpha 2M-methylamine.