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At least 109 records · Page 6Linked to original sources
PubMed1989

Identification of alpha 2-macroglobulin as a cytokine binding plasma protein. Binding of interleukin-1 beta to "F" alpha 2-macroglobulin.

Treatment of normal human plasma with methylamine resulted in the discovery of an interleukin-1 beta(IL-1 beta) binding protein. The protein was labeled with 125I-IL-1 beta and the relative molecular mass (Mr) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein-IL-1 beta complex had a Mr of approximately 400,000 in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis but became dissociated when exposed to beta-mercaptoethanol. The 125I-IL-1 beta labeled protein complex could be immunoprecipitated from plasma by using an anti-alpha 2-macroglobulin (alpha 2M) antiserum. Similarly, a monoclonal antibody (mAb) specific for electrophoretically fast ("F")alpha 2M was able to adsorb the 125I-IL-1 beta labeled complex from plasma. The mAb was also capable of adsorbing "F" alpha 2M-125I-IL-1 beta complexes from binary reaction mixtures, but failed to adsorb free 125I-IL-1 beta. Experiments carried out with purified plasma alpha 2M established that IL-1 beta became bound to alpha 2M only upon reaction with trypsin or methylamine, which results in the appearance of free thiol groups in alpha 2M ("F" alpha 2M). There was no binding of IL-1 beta to the native form of alpha 2M (electrophoretically slow or "S" alpha 2M), which lacks free thiol groups. Pretreatment of "F" alpha 2M with N-ethylmaleimide or [ethylenebis(oxyethylenenitrilo)] tetraacetic acid prevented complex formation between "F" alpha 2M and IL-1 beta. In contrast, the yield of "F" alpha 2M IL-1 beta complex formation was increased severalfold in the presence of 2.5 mM Zn2+. These findings indicate that "F" alpha 2M interacts with IL-1 beta through a thiol-disulfide exchange reaction. Zn2+ may play a major role in bringing together the reactive domains of the adjoining peptide backbones into proper orientation. The ready complex formation between "F" alpha 2M and the pleiotropic cytokine IL-1 beta suggests a novel biological role for "F" alpha 2M, since "F" alpha 2M-IL-1 beta complexes, but not "F" alpha 2M alone, retained IL-1-like activity in the thymocyte costimulator bioassay.

Biological Factors↗
PubMed1994

Transforming growth factor beta isoform 2-specific high affinity binding to native alpha 2-macroglobulin. Chimeras identify a sequence that determines affinity for native but not activated alpha 2-macroglobulin.

Transforming growth factor beta 2 (TGF-beta 2) is less potent than TGF-beta 1 in some endothelial cell proliferation assays due to the greater tendency of TGF-beta 2 to bind alpha 2-macroglobulin (alpha 2M). Substitution of TGF-beta 1 residues 40-47 into the TGF-beta 2 sequence yields a chimeric molecule that, like TGF-beta 1, expresses activity that is not substantially affected by serum alpha 2M (Burmester, J. K., Qian, S. W., Roberts, A. B., Huang, A., Amatayakul-Chantler, S., Suardet, L., Odartchenko, N., Madri, J. A., and Sporn, M. B. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8628-8632). In this investigation, we studied the binding of TGF-beta chimeras, which contain TGF-beta 1 residues 40-47, to both major conformations of human alpha 2M under apparent equilibrium conditions. Native alpha 2M, the primary form of this protein in serum, bound TGF-beta 2/beta 1 (40-82) and TGF-beta 2/beta 1 (40-47) with low affinity. The apparent KD values for the two chimeras and native alpha 2M were 310 and 330 nM, respectively. These values were much higher than the KD determined for TGF-beta 2 and native alpha 2M (11 nM) and equivalent to the KD determined for TGF-beta 1 and native alpha 2M. By contrast, both TGF-beta chimeras bound alpha 2M-methylamine, an altered conformation of alpha 2M, with high affinity (16 and 19 nM), which is characteristic of TGF-beta 2 and not TGF-beta 1. Fetal bovine heart endothelial cell DNA synthesis was inhibited to a similar degree by TGF-beta 1, TGF-beta 2, TGF-beta 2/beta 1 (40-82), and TGF-beta 2/beta 1 (40-47) in the presence of dilute (0.2%) fetal bovine serum. When 0.07 microM alpha 2M-methylamine was added, the activities of TGF-beta 2, TGF-beta 2/beta 1 (40-82), and TGF-beta 2/beta 1 (40-47) were significantly counteracted while the activity of TGF-beta 1 was unchanged, as would be predicted by the equilibrium binding analyses. These studies indicate that the TGF-beta structural elements, which mediate binding to native alpha 2M and conformationally transformed alpha 2M, are not equivalent. Residues 40-47 are critical in determining affinity for native alpha 2M but are less important in determining affinity for alpha 2M-methylamine.

Animals↗
PubMed1993

Alpha 2-macroglobulin and pregnancy zone protein. Serum levels, alpha 2-macroglobulin receptors, cellular synthesis and aspects of function in relation to immunology.

alpha 2-Macroglobulin, a 720 kDa tetramer, and Pregnancy Zone Protein, a 360 kDa dimer, are strongly homologous glycoprotein proteinase inhibitors of human plasma. alpha 2M and PZP monomers contain two regions of decisive functional importance i.e. the bait- and the thiol ester regions. The bait region serves as a substrate to a variety of proteinases, and cleavage in this domain causes a conformational change of the molecule. In addition, PZP forms tetramers when reacted with proteinase. As a result of the conformational change the thiol ester, which is hidden in the native structure, becomes readily accessible, and rapidly reacts with available nucleophiles preferably from the surface of the 'attacking' proteinase. The proteinase is thus covalently linked to alpha 2M/PZP but its active site is not affected. However, due to the structural changes in activated alpha 2M/PZP, bound proteinase is entrapped and its activity towards other than small substrates is sterically hindered. Presentation of receptor recognition sites (maximum four per tetramer) is also a consequence of reaction with proteinase. alpha 2M constitutes a large part of the plasma proteins and in early childhood serum contains as much as 4-5 g/l. Nearly all alpha 2M in the blood is native, since alpha 2M-proteinase complex is subject to rapid receptor-mediated clearance. The serum concentration in adults is about 2 g/l and shows little variation with age. The level is slightly higher in women than in men, and a modest (approximately 20%), possibly oestrogen induced, increase is observed during pregnancy. Sexual hormones, in particular androgens, might also account for the difference between childhood and adult alpha 2M levels. Yet, the reason for the downregulation during adolescence is not firmly established. In contrast to several of its animal homologues, human alpha 2M is not an acute phase protein. The serum level may change during acute and chronic disease, but the change is generally moderate and is not associated with any particular disease. Decreased alpha 2M concentration typically results from enhanced clearance of alpha 2M-proteinase complex and occurs in states of increased proteolytic activity, e.g. pancreatitis. Increased serum alpha 2M is frequently seen in nephrotic conditions and may mirror a change in plasma volume as well as in alpha 2M metabolism. Normal PZP serum levels are > 100 fold lower than those of alpha 2M. Male and female levels are similar in childhood, but unlike alpha 2M, PZP is strongly influenced by hormones.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1997

Studies on the interaction of IL-8 with human plasma alpha 2-macroglobulin: evidence for the presence of IL-8 complexed to alpha 2-macroglobulin in lung fluids of patients with adult respiratory distress syndrome.

alpha 2-Macroglobulin (alpha 2m) is a major plasma proteinase inhibitor, as well as a carrier and regulator of the function of many cytokines. IL-8 is a potent neutrophil attractant and activator, and it plays an important role in the pathogenesis of adult respiratory distress syndrome (ARDS). The concentration of both IL-8 and alpha 2m is increased in lung fluids from patients with ARDS. Therefore, interaction of IL-8 with human alpha 2m was studied. Mixtures of native and methylamine-treated alpha 2m (fast alpha 2m) with 125I-labeled IL-8 were analyzed using nonreducing gel electrophoresis. 125I-labeled IL-8 exclusively bound to fast alpha 2m, and the binding could be inhibited by unlabeled IL-8. Analysis of the IL-8-alpha 2m interaction using SDS-PAGE gels indicated that the binding was mainly noncovalent. The affinity of the binding of alpha 2m to IL-8 was measured using an equilibrium dialysis technique, and Kd was 30 nM. Bioassays revealed that fast alpha 2m did not affect IL-8-induced neutrophil degranulation or chemotaxis. However, it protected IL-8 from proteolytic degradation. In addition, IL-8 complexed to alpha 2m was detected in lung fluids from patients with ARDS. alpha 2m may therefore modulate IL-8 function in the lung.

Antigens, CD↗
PubMed1991

Reaction of proteinases with alpha 2-macroglobulin: rapid-kinetic evidence for a conformational rearrangement of the initial alpha 2-macroglobulin-trypsin complex.

The kinetics of the reaction of trypsin with alpha 2M were examined under pseudo-first-order conditions with excess inhibitor. Initial studies indicated that the fluorescent dye TNS is a suitable probe for monitoring the reaction over a wide concentration range of reactants. Titration experiments showed that the conformational changes associated with the binding of trypsin to alpha 2M result in an increased affinity of the inhibitor for TNS. Two distinct phases were observed when this dye was used to monitor the progress of the reaction. Approximately half of the fluorescence signal was generated during a rapid phase, with the remainder generated during a second, slower phase. The observed pseudo-first-order rate constant of the first phase varied linearly with the concentration of alpha 2M up to the highest concentration of inhibitor used, whereas the rate constant of the second phase was independent of alpha 2M concentration. The data fit a mechanism in which the association of trypsin with alpha 2M occurs in two consecutive, essentially irreversible steps, both leading to alterations in TNS fluorescence. The initial association occurs with a second-order rate constant of (1.0 +/- 0.1) X 10(7) M-1 s-1 and is followed by a slower, intramolecular conformational rearrangement of the initial complex with a rate constant of 1.4 +/- 0.2 s-1. The data are consistent with a previously proposed model for the reaction of proteinases with alpha 2M [Larsson et al. (1989) Biochemistry 28, 7636-7643].2+ this model, once an initial 1:1 alpha 2M-proteinase

Endopeptidases↗
PubMed1983

Receptor-mediated endocytosis of alpha 2-macroglobulin: solubilization and partial purification of the fibroblast alpha 2-macroglobulin receptor.

Recent studies in our laboratory have been aimed at biochemically characterizing the alpha 2M receptor present on fibroblast membranes. The approach we have taken is to develop a means of assessing binding to solubilized alpha 2M binding sites. The binding activity was not removed by treatment with high salt concentration or treatment with chaotropic agents. Removal of the binding activity from membranes did occur using a variety of detergents which suggests that the receptor molecules may be "intrinsic" membrane proteins. The most useful detergent for solubilizing the alpha 2M receptor was octyl-beta-D-glucoside. The alpha 2M binding activity could be removed from NRK fibroblasts and Vero cells using this detergent and was found to remain in solution at 100,000 X g. Removal of the octyl-beta-D-glucoside by extensive dialysis resulted in formation of protein-lipid aggregates that bind to 125I-alpha 2M specifically and with high affinity. Such binding sites were not generated when KB cells (which lack receptors) were extracted with the detergent. Significantly, the observed affinities detected for both high- and low-affinity binding sites were similar to those reported with intact cells or membranes. In addition, binding to the solubilized sites could be inhibited using compounds known to block the receptor-mediated endocytosis of alpha 2M (bacitracin, IBHNA). Other compounds (monensin, dansylcadaverine), which did not directly inhibit the high-affinity binding sites, may exert their effects at different stages in receptor-mediated endocytosis (i.e., receptor recycling). alpha 2M binding sites from NIH-3T3 (spontaneous) tumors have been purified approximately 95-fold by a combination of ion exchange and gel permeation chromatography. The receptor appears to be an acidic protein that approximately coelutes with aldolase (45 A, 158,000 daltons) on gel filtration. Ion exchange chromatography appears to remove an endogenous inhibitor of alpha 2M binding and may also remove binding sites having lower affinity for 125I-alpha 2M. Recent studies using immobilized alpha 2M as an affinity resin suggest that the high-affinity alpha 2M receptor may have a subunit molecular weight of approximately 85,000. Studies are now in progress aimed at further characterizing these high-affinity binding sites. Once bound to the alpha 2M receptor, alpha 2M enters cells via coated pits and is rapidly transferred to receptosomes. These organelles carry the ligand into the Golgi region, from which it is transferred to lysosomes where it is slowly degraded.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗