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PubMed1974

The interaction of alpha2-macroglobulin with proteinases. Binding and inhibition of mammalian collagenases and other metal proteinases.

1. Experiments were performed to determine whether the specific collagenases and other metal proteinases are bound and inhibited by alpha(2)-macroglobulin, as are endopeptidases of other classes. 2. A specific collagenase from rabbit synovial cells was inhibited by human serum. The inhibition could be attributed entirely to alpha(2)-macroglobulin; alpha(1)-trypsin inhibitor was not inhibitory. alpha(2)-Macroglobulin presaturated with trypsin or cathepsin B1 did not inhibit collagenase, and pretreatment of alpha(2)-macroglobulin with collagenase prevented subsequent reaction with trypsin. The binding of collagenase by alpha(2)-macroglobulin was not reversible in gel chromatography. 3. The collagenolytic activity of several rheumatoid synovial fluids was completely inhibited by incubation of the fluids with alpha(2)-macroglobulin. 4. The collagenase of human polymorphonuclear-leucocyte granules showed time-dependent inhibition by alpha(2)-macroglobulin. 5. The collagenolytic metal proteinase of Crotalus atrox venom was inhibited by alpha(2)-macroglobulin. 6. The collagenase of Clostridium histolyticum was bound by alpha(2)-macroglobulin, and inhibited more strongly with respect to collagen than with respect to a peptide substrate. 7. Thermolysin, the metal proteinase of Bacillus thermoproteolyticus, was bound and inhibited by alpha(2)-macroglobulin. 8. It was shown by polyacrylamidegel electrophoresis of reduced alpha(2)-macroglobulin in the presence of sodium dodecyl sulphate that synovial-cell collagenase, clostridial collagenase and thermolysin cleave the quarter subunit of alpha(2)-macroglobulin near its mid-point, as do serine proteinases. 9. The results are discussed in relation to previous work, and it is concluded that the characteristics of interaction of the metal proteinases with alpha(2)-macroglobulin are the same as those of other proteinases.

Animals↗

PubMed1976

The alpha macroglobulins of rat serum.

A three-stage method for isolation of alpha1 macroglobulin and alpha2 macroglobulin from the serum of normal and injured rats is described. The methods successively used, namely gel filtration, ultracentrifugation and chromatography on DEAE-cellulose, were chosen to minimize loss of tryptic esterase-protecting activity. The two proteins differed slightly with respect to the following properties: mol.wt., alpha1 macroglobulin 7.46 X 10(5), alpha2 macroglobulin 7.16 X 10(5); isoelectric focusing, alpha1, macroglobulin pI 4.4, alpha2 macroglobulin pI4.5. Amino acid analyses were identical, except with respect to tyrosine: alpha1 macroglobulin 3.96 +/- 0.24, alpha2 macroglobulin 3.16 +/- 0.32 mol/100 mol of total amino acids. When isolated from the serum of uninjured rats, alpha1 macroglobulin retained the capacity to bind 1.05 mol of trypsin/mol. However, if isolated from serum 2 days after injury only 0.78 mol of trypsin/mol of alpha1 macroglobulin was bound. alpha2 macroglobulin isolated from this latter serum bound on average 0.97 mol of trypsin/mol. When reduced with N-acetylcysteine, both molecules formed subunits of size corresponding to that expected for quarter molecules. When alpha2 macroglobulin was reduced with dithiothreitol, quarter molecules were again produced. alpha1 macroglobulin, however, when thus treated gave a more complex mixture, containing a component having a mol.wt. of less than 6 X 10(4). Antisera raised against the two proteins permitted estimation of the concentration of each protein in the plasmas or sera of normal and injured rats. Plasma from normal male rats contained 3.76 +/- 0.56 mg of alpha1 macroglobulin/ml (n = 33) and 0.016 +/- 0.001 mg of alpha2 macroglobulin/ml (n=33). After injury by injection of turpentine and cortisone, the concentrations in plasma were at 3 days 5.19 +/- 0.81 mg of alpha1 macroglobulin/ml (n = 12) and at 2 days 1.38 +/- 0.35 mg of alpha2 macroglobulin/ml (n = 12). Antisera to the two proteins did not cross-react with one another. The quarter molecules formed by reduction of both proteins showed increased antigenicity.

Amino Acids↗

PubMed1987

Cultured Ito cells of rat liver express the alpha 2-macroglobulin gene.

Ito cells were isolated from rat liver and kept in culture for up to 13 days. The capability of the Ito cells to synthesize alpha 2-macroglobulin was analyzed at different times after isolation and by pulse-chase experiments. Newly synthesized alpha 2-macroglobulin was determined by immunoprecipitation followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. alpha 2-Macroglobulin synthesis was hardly detectable in Ito cells and their media 3 days after plating. However, 5-11 days after the isolation of the cells, increasing amounts of alpha 2-macroglobulin were synthesized. The results of pulse-chase experiments performed on day 7 showed that radioactively labeled alpha 2-macroglobulin decreased in the intracellular compartment and increased in the culture medium. alpha 2-Macroglobulin was identified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions. Furthermore, when unlabeled alpha 2-macroglobulin was added during the immunoprecipitation, a competition was observed. Incubation of pancreatic elastase with culture medium of rat Ito cells or rat hepatocytes led to the same cleavage products as found with alpha 2-macroglobulin. alpha 2-Macroglobulin-specific mRNA could be demonstrated by Northern blot analysis of total RNA extracted from rat Ito cells. Under the conditions where alpha 2-macroglobulin was synthesized in Ito cells, no synthesis of alpha 1-macroglobulin, alpha 1-inhibitor 3, alpha 1-proteinase inhibitor, alpha 1-acid glycoprotein, alpha 1-acute-phase globulin (T-kininogen) and albumin could be demonstrated. It is concluded that alpha 2-macroglobulin is a true secretory protein of rat Ito cells in culture. This could be of importance for collagen metabolism in liver diseases.

Animals↗

PubMed1993

A comparison of the roles of the low density lipoprotein (LDL) receptor and the LDL receptor-related protein/alpha 2-macroglobulin receptor in chylomicron remnant removal in the mouse in vivo.

Two cell surface molecules, the low density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP)/alpha 2-macroglobulin receptor, have been found to have a role in the removal of apoE-rich lipoproteins. To further study this process and to assess the relative contributions of these proteins to the removal of chylomicron remnants, we used an anti-LDL receptor antibody, activated alpha 2-macroglobulin and the 39-kDa receptor-associated protein (RAP) as inhibitors of chylomicron remnant removal in intact mice. The advantage of this study is that we were able to use the same litters of animals and batches of lipoprotein for the experiment. In cultured Chinese hamster ovary cells, the anti-LDL receptor antibody inhibited remnant uptake and degradation about 80% as well as did unlabeled remnants. It did not affect activated alpha 2-macroglobulin uptake or degradation. Activated alpha 2-macroglobulin did not inhibit remnant uptake in normal cells but is reported to block uptake in cells that lack LDL receptors. Chylomicron remnants blocked activated alpha 2-macroglobulin uptake as effectively as unlabeled activated alpha 2-macroglobulin. This confirmed that the two ligands are cross-competitors but suggests that the LDL receptor is the primary mediator of remnant uptake in cultured cells that express the LDL receptor. In vivo, pretreatment with the anti-LDL receptor antibody decreased remnant uptake 5 min after injection by one-third. The antibody did not affect the removal of activated alpha 2-macroglobulin. Activated alpha 2-macroglobulin had a small, but reproducible effect on remnant removal, decreasing it by about 7% at 5 min. Injection of chylomicron remnants affected activated alpha 2-macroglobulin removal slightly. A similar pattern, but somewhat greater effect was seen on the hepatic uptake of the ligands. Anti-LDL receptor antibody reduced chylomicron remnant uptake by about half, and activated alpha 2-macroglobulin reduced it by about 15%. Studies with RAP provided results generally similar to those with activated alpha 2-macroglobulin, although RAP appears to bind to a site not recognized by either remnants or activated alpha 2-macroglobulin in addition to sharing a site with these ligands. Together, these results add support for the hypothesis that, although both receptors can play a role in chylomicron remnant removal, in the normal mouse in vivo the LDL receptor plays a substantially greater role, making the role of the LRP difficult to appreciate. The same is true in cells that express LDL receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1981

Purification and immunological determination of alpha 2-macroglobulin in serum from injured rats.

Rat alpha 2-macroglobulin was isolated and purified from the pooled sera of turpentine-injected rats by sequential use of dextran sulphate, DEAE-cellulose and gel filtration chromatography. The final protein product obtained by this procedure proved to be alpha 2-macroglobulin of a high degree of purity based on electrophoretic, immunologic and centrifugal analysis. The alpha 2-macroglobulin preparation also binds stoichiometrically to trypsin preventing subsequent inhibition by protein trypsin inhibitors. SDS-polyacrylamide gel electrophoresis of rat alpha 2-macroglobulin after incubation with trypsin suggested that there are at least two susceptible peptide bonds in the 170,000-dalton alpha 2-macroglobulin subunit. The concentration of alpha 2-macroglobulin in the sera of rats was measured by electroimmuno assay a monospecific antiserum against alpha 2-macroglobulin. Purified alpha 2-macroglobulin was used as a standard. Sera from normal male rats contained 32 +/- 4 micrograms of alpha 2-macroglobulin per ml. To determine the time course of response of alpha 2-macroglobulin to inflammation, rats were subjected to either laparotomy or subcutaneous injection of turpentine. After either type of injury, the concentration of alpha 2-macroglobulin increased rapidly, reaching a maximum value of 110-140 times that of the control value by 24 h. Little difference was noted in responsiveness between the two sexes.

Animals↗

PubMed1985

Altered isoelectric focusing of alpha 2-macroglobulin from plasma of patients with diabetes mellitus.

Plasma from both type I and type II patients with diabetes mellitus displayed elevated alpha 2-macroglobulin trypsin-binding activity relative to normals. Column isoelectric focusing indicated that the average isoelectric point (pI) of the major form of crude alpha 2-macroglobulin was 6.6, 5.9, and 6.2 for normal, type I and type II subjects, respectively. Focused crude diabetic alpha 2-macroglobulin displayed significantly less recovered trypsin-binding activity relative to controls, particularly above a pI value of 6.0. Following preincubation with trypsin, the crude diabetic alpha 2-macroglobulin displayed a single isoelectric form (pI value of 5.3-5.5) and a substantial increase in recovered activity which were both comparable to normal alpha 2-macroglobulin. Purified alpha 2-macroglobulin from both normal and diabetic subjects displayed similar focusing profiles having a distribution of three forms with a pI value of 5.3-5.4 for the principal form. Trypsin-preincubated samples of purified alpha 2-macroglobulin displayed a single form with a pI value of 5.5-6.0. Preincubation of purified normal or diabetic alpha 2-macroglobulin with a plasma preparation devoid of alpha 2-macroglobulin from normal or diabetic plasma resulted in significantly lowered recovery of activity. All of the above studies suggest that the altered focusing properties observed for crude diabetic alpha 2-macroglobulin are due to components of the plasma rather than to alpha 2-macroglobulin itself.

Adult↗

PubMed1999

Incorporation of non-proteolytic proteins by murine alpha2-macroglobulin.

Human alpha2-macroglobulin is a tetrameric glycoprotein with a molecular weight of 718 kDa that is present in human plasma at high concentrations. Murine alpha2-macroglobulin is homologous to human alpha2-macroglobulin but it undergoes post-translational cleavage in the subunits. Each subunit of alpha2-macroglobulin contains a thiolester which can be cleaved by small nucleophiles. In human alpha2-macroglobulin this results in a conformational change to a receptor-recognized form and a change in the electrophoretic mobility. Recent work has demonstrated that this process is reversible and during this reversal non-proteolytic proteins can become covalently trapped within the human alpha2-macroglobulin molecule. The present study further investigates this observation and examines the question whether reversal of thiolester cleavage occurs in mouse alpha2-macroglobulin. Previous studies suggest that small nucleophiles only partially convert mouse alpha2-macroglobulin to a receptor-recognized form. We demonstrate here that under appropriate conditions, mouse alpha2-macroglobulin is fully converted by NH3. We also demonstrate that despite structural and kinetic differences between human and mouse alpha2-macroglobulin, both molecules are able to incorporate non-proteolytic ligands in a similar manner. This leads us to propose a general model of ligand incorporation via nucleophilic exchange in multimeric alpha-macroglobulins.

Ammonia↗

PubMed1997

Transforming growth factor-alpha's effects on astroglial-cholinergic cell interactions in the medial septal area in vitro are mediated by alpha 2-macroglobulin.

We reported previously that two epidermal growth factor receptor ligands, epidermal growth factor and transforming growth factor-alpha, inhibit medial septal cholinergic cell phenotypic expression (choline acetyltransferase and acetylcholinesterase activities) in vitro indirectly via (a) soluble molecule(s) released from astrocytes [Kenigsberg R. L. et al. (1992) Neuroscience 50, 85-97; Kenigsberg R. L. and Mazzoni I. E. (1995) J. Neurosci. Res. 41, 734-744; Mazzoni I. E. and Kenigsberg R. L. (1996) Brain Res. 707, 88-99]. In the present study, we found that this response to transforming growth factor-alpha is mediated, for the most part, by alpha 2-macroglobulin, a potent protease inhibitor with a wide spectrum of biological activities. In this regard, the effects of transforming growth factor-alpha on cholinergic cells can be blocked with immunoneutralizing antibodies raised against alpha 2-macroglobulin. Furthermore, western blot analysis reveals that although alpha 2-macroglobulin is present in conditioned media from control septal cultures, it is more abundant in those treated with transforming growth factor-alpha. In addition, exogenous alpha 2-macroglobulin, both in its native and trypsin-activated forms, can mimic transforming growth factor-alpha's effects on septal cholinergic cell expression. However, while the native antiprotease can slightly but significantly decrease choline acetyltransferase activity, trypsin-activated alpha 2-macroglobulin, in the nanomolar range, induces as marked a decrease in this enzyme activity as that noted with transforming growth factor-alpha. Furthermore, trypsin-activated alpha 2-macroglobulin, like epidermal growth factor/transforming growth factor-alpha, decreases choline acetyltransferase activity by arresting its spontaneous increase that occurs with time in culture, does so in a reversible manner and is not neurotoxic. In addition, trypsin-activated alpha 2-macroglobulin, in the nanomolar range, can affect choline acetyltransferase in a dual manner, up-regulating it at low concentrations while down-regulating it at higher ones. These responses are identical in mixed neuronal-glial and pure neuronal septal cultures. Furthermore, when concentrations of trypsin-activated alpha 2-macroglobulin, which alone decrease choline acetyltransferase, are added simultaneously with nerve growth factor, they serve to potentiate the nerve growth factor-induced increase in enzymatic activity. As GABAergic cell expression is not affected by alpha 2-macroglobulin, it appears that the effects of this protease inhibitor on medial septal neuronal expression are neurotransmitter-specific. Finally, trypsin-activated but not native alpha 2-macroglobulin promotes a dose-dependent aggregation of the septal neurons. This change in morphology, however, is not related to those noted in choline acetyltransferase activity. In summary, these data suggest that the expression of alpha 2-macroglobulin in astroglia from the medial septal nucleus can be controlled by epidermal growth factor receptor ligands to impact the functioning of basal forebrain cholinergic neurons.

Acetylcholinesterase↗

PubMed1990

Interaction of hemorrhagic metalloproteinases with human alpha 2-macroglobulin.

The interaction between four Crotalus atrox hemorrhagic metalloproteinases and human alpha 2-macroglobulin was investigated. The proteolytic activity of the hemorrhagic toxins Ht-c, -d, and -e against the large molecular weight protein substrates, gelatin type I and collagen type IV, was completely inhibited by alpha 2-macroglobulin. The proteolytic activity of Ht-a against the same substrates was not significantly inhibited. Each mole of alpha 2-macroglobulin bound maximally 2 mol of Ht-e and 1.1 mol of Ht-c and Ht-d. These proteinases interacted with alpha 2-macroglobulin rapidly at 22 degrees C. Rate constants based on intrinsic fluorescence measurements were 0.62 X 10(5) M-1 s-1 for interaction of alpha 2-macroglobulin with Ht-c and -d and 2.3 X 10(5) M-1 s-1 for the interaction of alpha 2-macroglobulin with Ht-e. Ht-a interacted with alpha 2-macroglobulin very slowly at 22 degrees C. Increasing the temperature to 37 degrees C and prolonging the time of interaction with alpha 2-macroglobulin resulted in the formation of Mr 90,000 fragments and high molecular weight complexes (Mr greater than 180,000), in which Ht-a is covalently bound to the carboxy-terminal fragment of alpha 2-M. The identification of the sites of specific proteolysis of alpha 2-macroglobulin shows that the cleavage sites for the four metalloproteinases are within the bait region of alpha 2-macroglobulin. Ht-c and -d cleave only at one site, the Arg696-Leu697 peptide bond, which is also the site of cleavage for plasmin, thrombin, trypsin, and thermolysin. Ht-a cleaves alpha 2-macroglobulin primarily at the same site, but a secondary cleavage site at the His694-Ala695 peptide bond was also identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1973

The interaction of alpha 2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism.

1. alpha(2)-Macroglobulin is known to bind and inhibit a number of serine proteinases. We show that it binds thiol and carboxyl proteinases, and there is now reason to believe that alpha(2)-macroglobulin can bind essentially all proteinases. 2. Radiochemically labelled trypsin, chymotrypsin, cathepsin B1 and papain are bound by alpha(2)-macroglobulin in an approximately equimolar ratio. Equimolar binding was confirmed for trypsin by activesite titration. 3. Pretreatment of alpha(2)-macroglobulin with a saturating amount of one proteinase prevented the subsequent binding of another. We conclude that each molecule of alpha(2)-macroglobulin is able to react with one molecule of proteinase only. 4. alpha(2)-Macroglobulin did not react with exopeptidases, non-proteolytic hydrolases or inactive forms of endopeptidases. 5. The literature on binding and inhibition of proteinases by alpha(2)-macroglobulin is reviewed, and from consideration of this and our own work several general characteristics of the interaction can be discerned. 6. A model is proposed for the molecular mechanism of the interaction of alpha(2)-macroglobulin with proteinases. It is suggested that the enzyme cleaves a peptide bond in a sensitive region of the macroglobulin, and that this results in a conformational change in the alpha(2)-macroglobulin molecule that traps the enzyme irreversibly. Access of substrates to the active site of the enzyme becomes sterically hindered, causing inhibition that is most pronounced with large substrate molecules. 7. The possible physiological importance of the unique binding characteristics of alpha(2)-macroglobulin is discussed.

Binding Sites↗

PubMed1983

Cell-free synthesis of rat alpha 2-macroglobulin and induction of its mRNA during experimental inflammation.

Poly(A)-rich RNA was isolated from the livers of acutely inflamed rats by extraction with guanidinium HCl and oligo(dT)-cellulose chromatography. After translation in a recticulocyte lysate and immunoprecipitation with a specific antiserum to alpha 2-macroglobulin a polypeptide with an apparent molecular weight of 162000 could be detected. The cell-free synthesis of alpha 2-macroglobulin was stimulated 8-fold by the addition of RNase inhibitor. Full-length alpha 2-macroglobulin polypeptide chains appeared after 35 min in the presence of 1.85 mM Mg2+ and 100 mM K+. A nucleotide number of about 5100 was estimated for alpha 2-macroglobulin by means of sucrose gradient centrifugation of poly(A)-rich RNA followed by translation in vitro and immunoprecipitation of alpha 2-macroglobulin. In normal liver alpha 2-macroglobulin mRNA represented about 0.0007% of total translatable RNA. Acute inflammation generated by intramuscular injection of turpentine led to a 66-fold increase in translatable alpha 2-macroglobulin mRNA after 18 h, followed by a rapid decrease. In accordance to the induction of alpha 2-macroglobulin mRNA serum concentrations of alpha 2-macroglobulin increased to about 2 mg/ml. Unlike alpha 2-macroglobulin mRNA serum alpha 2-macroglobulin levels remained unchanged up to 60 h.

Animals↗

PubMed2004

Bacterial alpha2-macroglobulins: colonization factors acquired by horizontal gene transfer from the metazoan genome?

BACKGROUND: Invasive bacteria are known to have captured and adapted eukaryotic host genes. They also readily acquire colonizing genes from other bacteria by horizontal gene transfer. Closely related species such as Helicobacter pylori and Helicobacter hepaticus, which exploit different host tissues, share almost none of their colonization genes. The protease inhibitor alpha2-macroglobulin provides a major metazoan defense against invasive bacteria, trapping attacking proteases required by parasites for successful invasion. RESULTS: Database searches with metazoan alpha2-macroglobulin sequences revealed homologous sequences in bacterial proteomes. The bacterial alpha2-macroglobulin phylogenetic distribution is patchy and violates the vertical descent model. Bacterial alpha2-macroglobulin genes are found in diverse clades, including purple bacteria (proteobacteria), fusobacteria, spirochetes, bacteroidetes, deinococcids, cyanobacteria, planctomycetes and thermotogae. Most bacterial species with bacterial alpha2-macroglobulin genes exploit higher eukaryotes (multicellular plants and animals) as hosts. Both pathogenically invasive and saprophytically colonizing species possess bacterial alpha2-macroglobulins, indicating that bacterial alpha2-macroglobulin is a colonization rather than a virulence factor. CONCLUSIONS: Metazoan alpha2-macroglobulins inhibit proteases of pathogens. The bacterial homologs may function in reverse to block host antimicrobial defenses. Alpha2-macroglobulin was probably acquired one or more times from metazoan hosts and has then spread widely through other colonizing bacterial species by more than 10 independent horizontal gene transfers. yfhM-like bacterial alpha2-macroglobulin genes are often found tightly linked with pbpC, encoding an atypical peptidoglycan transglycosylase, PBP1C, that does not function in vegetative peptidoglycan synthesis. We suggest that YfhM and PBP1C are coupled together as a periplasmic defense and repair system. Bacterial alpha2-macroglobulins might provide useful targets for enhancing vaccine efficacy in combating infections.

Alphaproteobacteria↗

PubMed1999

[Alpha 2-macroglobulin in the blood of patients with diabetes mellitus].

Human alpha 2-macroglobulin, a plasma glycoprotein, traps and inhibits proteolytic enzymes which participate in inflammation and homeostasis. There is no doubt about the significant role of alpha 2-macroglobulin in immunoregulatory processes. The aim of the present study was to investigate the levels of alpha 2-macroglobulin in patients with diabetes mellitus and to investigate whether an association exists between alpha 2-macroglobulin level and the type of diabetes (type I or type II) or alpha 2-macroglobulin and diabetic complications. The patient population consisted of 48 patients with diabetes mellitus type I (average age 36.5 years) and 50 patients with diabetes type II (average age 55.3 years). Patients were divided into three groups according to the presence of complications (group I--patients without complications, group II--patients with diabetic retinopathy, group III--patients with several complications). alpha 2-macroglobulin was determined by electroimmunoassay according to Laurell. alpha 2-macroglobulin levels were significantly raised in group of diabetics type I (3190 mg/l vs. 1880 mg/l). In the group of diabetics type II alpha 2-macroglobulin levels are within the normal range (2030 mg/l vs. 1880 mg/l). After dividing of diabetics according to the presence of diabetic complications, alpha 2-macroglobulin levels in patients with diabetic complications were significantly higher than in the group of diabetics without complications (3180 mg/l vs. 2040 mg/l). The possible explanations of elevated alpha 2-macroglobulin levels in diabetics and possible participation of elevated a2-macroglobulin in the development of diabetic angiopathy are discussed. (Fig. 2, Ref. 14.)

Adult↗

PubMed1998

Binding of soluble myelin basic protein to various conformational forms of alpha2-macroglobulin.

Myelin basic protein is known to be released into the circulation following traumatic injuries or demyelination within the central nervous system, resulting in the generation of potentially immunogenic myelin basic protein material. In this investigation we have studied the binding of bovine and human myelin basic protein to human alpha2-macroglobulin, which was found to be the only major myelin basic protein-binding protein in human plasma. Myelin basic protein bound to all three conformational forms of alpha2-macroglobulin studied, i.e., native alpha2-macroglobulin, methylamine-treated alpha2-macroglobulin, and chymotrypsin-treated alpha2-macroglobulin. Zinc chloride (1 mM) or 1 mM iodoacetamide partly blocked the complex formation between myelin basic protein and alpha2-macroglobulin, while 1 mM magnesium chloride, 1 mM calcium chloride, or 1 mM EDTA had no effect on binding. Chymotrypsin and trypsin can degrade myelin basic protein to fragments which do not bind to alpha2-macroglobulin. However, when myelin basic protein was complexed with any of the conformational forms of alpha2-macroglobulin, no significant release of Na[125I]-labeled myelin basic protein occurred after proteinase treatment. The results suggest that binding of myelin basic protein to alpha2-macroglobulin may protect extracellular compartments in vivo from immunogenic myelin basic protein fragments and alpha2-macroglobulin may participate in the specific clearance of myelin basic protein from the circulation.

Blood Proteins↗

PubMed1984

The effect of zinc and other divalent cations on the structure and function of human alpha 2-macroglobulin.

Zinc binding to human alpha 2-macroglobulin was studied to assess its involvement in the structure and function alpha 2-macroglobulin. Equilibrium dialysis experiments indicated multiple classes of zinc-binding sites, the one of highest affinity having a site number of 20 and a Kd value of 8 X 10(-7) M. Native alpha 2-macroglobulin and alpha 2-macroglobulin-trypsin complexes bound comparable amount of zinc. The proteinase inhibitory activity of alpha 2-macroglobulin was not affected by zinc binding at physiological concentrations nor by the removal of zinc by EDTA. Above 25 microM zinc, alpha 2-macroglobulin activity decreased, although binding of [125I]trypsin was not affected. When nondenaturing gel electrophoresis was performed, the preparation of alpha 2-macroglobulin migrated as half-molecules at increasing zinc concentration. Experiments with other divalent cations correlated decreases in alpha 2-macroglobulin activity with apparent dissociation of the alpha 2-macroglobulin tetramer in the presence of copper and mercury, but not barium, cadmium or nickel. While zinc binding to alpha 2-macroglobulin does not function in proteinase inhibition, it might be involved in zinc transport in vivo. At nonphysiological concentrations, zinc and other divalent cations are useful as probes of protein quaternary structure.

Binding Sites↗

PubMed1994

Steroidogenic adrenocortical cells synthesize alpha 2-macroglobulin in vitro, not in vivo.

We previously identified alpha 2-macroglobulin as the major protein secreted by primary cultures of adrenocortical cells. We report here that in the adrenal gland, the distribution of alpha 2-macroglobulin in the adrenocortical tissue is restricted to the endothelium of blood vessels and that no immunoreactivity is found in steroidogenic cells. A time course study revealed that freshly dissociated bovine adrenocortical cells were void of alpha 2-macroglobulin immunoreactivity whereas the proportion of alpha 2-macroglobulin-positive cells reached more than two-thirds of the population between day 4 and day 7 of culture. Double immunoenzymatic labeling of 6-day-old cultures revealed a co-localization of alpha 2-macroglobulin and the steroidogenic enzyme P-450SCC. Treatment of 5-day-old cultures (expressing alpha 2-macroglobulin) for 24 h by either ACTH (10(-9)-10(-6) M) or alpha 2-macroglobulin (2.5 mg/ml) resulted in a marked decrease of the expression of alpha 2-macroglobulin. These data indicate that ACTH and plasmatic alpha 2-macroglobulin could physiologically repress alpha 2-macroglobulin expression in the adrenal cortex in vivo.

Adrenal Cortex↗

PubMed2000

Aberrant forms of alpha(2)-macroglobulin purified from patients with multiple sclerosis.

The biochemical properties of alpha(2)-macroglobulin were investigated in four patients with multiple sclerosis and compared to alpha(2)-macroglobulin from healthy controls. An impaired stability of alpha(2)-macroglobulin from the multiple sclerosis patients was demonstrated as a spontaneous conversion to an electrophoretic"fast" form of alpha(2)-macroglobulin upon purification and storage, with a concomitant decrease in functional capacity to inhibit proteinases. The ability to form complexes with proteinases was significantly reduced in alpha(2)-macroglobulin purified from the multiple sclerosis patients. The aberrant molecular arrangements of the protein were not due to proteinase cleavages in the bait regions of alpha(2)-macroglobulin, as demonstrated by gel electrophoresis and protein sequencing. The number of functional thiol esters, however, was reduced in alpha(2)-macroglobulin purified from the multiple sclerosis patients, an observation compatible with the impaired proteinase binding property. Furthermore, differences in isoelectric points were observed between alpha(2)-macroglobulin from the multiple sclerosis patients and alpha(2)-macroglobulin from healthy controls. The results suggest that aberrant forms of alpha(2)-macroglobulin may be present in patients with multiple sclerosis.

Adult↗

PubMed1983

Proteolytic activity of alpha 2-macroglobulin-enzyme complexes toward human factor VIII/von Willebrand factor.

The low level of enzymatic activity of certain alpha 2-macroglobulin-proteinase complexes could be important to the function of factor VIII/von Willebrand glycoprotein since it is especially sensitive to proteolytic cleavage. To test this possibility, complexes of alpha 2-macroglobulin with plasmin, trypsin, and thrombin were formed in at least a 2:1 molar ratio of alpha 2-macroglobulin:proteinase and tested for effects on the factor VIII procoagulant activity of the factor VIII/von Willebrand glycoprotein. Neither the alpha 2-macroglobulin-trypsin complex nor the alpha 2-macroglobulin-plasmin complex affected factor VIII procoagulant activity. The behavior of the alpha 2-macroglobulin-thrombin complex was different. When alpha 2-macroglobulin and thrombin were incubated in a mole ratio of 3:1 or less, factor VIII procoagulant activity was enhanced to about the same extent as with free thrombin. Even at a 24:1 mole ratio, the mixture could produce 45% of the increase in factor VIII activity obtained with free thrombin. The isolated alpha 2-macroglobulin-thrombin complex could also activate the factor VIII procoagulant function to about 45% of the level obtained with an identical amount of uncomplexed thrombin. Analysis of the alpha 2-macroglobulin-125I-labeled thrombin complexes by rechromatography or by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that this activation was not due to free thrombin. We conclude that the alpha 2-macroglobulin-thrombin complex retains sufficient proteolytic activity to activate the procoagulant function of factor VIII/von Willebrand glycoprotein despite the latter being a very large substrate, having an estimated molecular weight of 1-20 million.

Antigens↗