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PubMed1997

An increase in the carbohydrate moiety of alpha 2-macroglobulin is associated with systemic lupus erythematosus (SLE).

Using lectin blots in conjunction with peptide mapping, alpha 2-macroglobulin micropurified from systemic lupus erythematosus (SLE) patients was shown to become abnormally glycosylated suggesting the occurrence of complex glycosylation in this pathological condition. To confirm there is indeed a quantitative increase in specific monosaccharides in this protein; alpha 2-macroglobulin was micropurified from a battery of 37 serum samples which included 6 normal donors (3 male and 3 female), 23 SLE patients, 6 rheumatoid arthritis patients, 1 mixed connective tissue disease patient, and 1 Sjogren's syndrome patient; for carbohydrate analysis. It was noted that the concentration of total monosaccharides in alpha 2-macroglobulin micropurified from serum samples of SLE patients is significantly higher than normal donors with a mean +/- SD of 188 +/- 410 micrograms/mg protein (SLE, n = 23) versus 14.5 +/- 4 micrograms/mg protein (normal, n = 6) even though there was a high variation in the level of monosaccharides among the SLE patients. An increase in oligosaccharides in alpha 2-macroglobulin from SLE patients compared to normal subjects was confirmed by concanavalin A (Con A) blots using peptide fragments derived from the micropurified protein. Since the interaction of peptide fragments derived from alpha 2-macroglobulin with Con A requires the presence of mannose and/or glucose residues, we have also examined if there are any correlations between the levels of mannose and glucose in alpha 2-macroglobulin and SLE. The concentration of mannose (38 +/- 60 micrograms/mg protein) in alpha 2-macroglobulin derived from SLE patients was significantly higher than normal donors (mannose, 4.8 +/- 1 micrograms/mg protein) however, the concentration of glucose in alpha 2-macroglobulin derived from SLE patients when compared to normal donors was not statistically significant, 18 +/- 20 micrograms/mg protein in SLE versus 2 +/- 0.5 micrograms/mg protein in normal donors due to high variation between samples. Also, the concentration of galactose in alpha 2-macroglobulin from SLE patients was significantly higher than normal donors (45.7 +/- 173 micrograms/mg protein versus 0.13 +/- 0.03 microgram/mg protein). These results illustrate quantification of carbohydrate in selected glycoproteins such as alpha 2-macroglobulin may be a novel and alternative clinical marker for SLE.

Antibodies↗

PubMed1993

Regulation of Sertoli cell alpha 2-macroglobulin and clusterin (SGP-2) secretion by peritubular myoid cells.

alpha 2-Macroglobulin and clusterin are two putative Sertoli cell secretory products; however, the regulator(s) modulating their secretion by Sertoli cells is not known. Recent studies from this laboratory have shown that the testicular alpha 2-macroglobulin, unlike its liver homologue, is not an acute-phase reactant and its concentration is not affected by acute inflammation. We sought to determine whether FSH, testosterone, and other biomolecules would affect the secretion of alpha 2-macroglobulin and clusterin by Sertoli cells as well as whether peritubular myoid cells would affect the secretion of these proteins by Sertoli cells. It was noted that Sertoli cells cultured in vitro secreted increasing amounts of alpha 2-macroglobulin and clusterin as a function of time. FSH (50-1000 ng/ml) and testosterone (10(-11)-10(-5) M) had no apparent effect on the secretion of alpha 2-macroglobulin and clusterin by Sertoli cells. Addition of interleukin-6 to Sertoli cell-enriched cultures, in doses known to stimulate alpha 2-macroglobulin secretion by hepatocytes, did not affect the alpha 2-macroglobulin secretion. However, dexamethasone at 10(-7)-10(-5) M stimulated alpha 2-macroglobulin secretion by Sertoli cells dose-dependently while the addition of interleukin-6 had no synergistic effect on dexamethasone-stimulated alpha 2-macroglobulin secretion. These findings suggest that the synthesis and/or secretion of alpha 2-macroglobulin by Sertoli cells is regulated by a mechanism distinct from that of the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2004

The capacity of alpha2-macroglobulin to inhibit an exogenous protease is significantly increased in critically ill and septic patients.

The image of alpha2-macroglobulin is based on a paradigm evolved in the 1970s. During this decade alpha2-macroglobulin was shown to irreversibly entrap and thereby inhibit a maximum of two proteases. Additional binding of nonproteolytic proteins, i.e., inflammatory mediators and growth factors, is dependent on the conformational status of alpha2-macroglobulin. It was our aim to clarify whether the interaction of nonproteolytic proteins with alpha2-macroglobulin during inflammatory conditions might also modulate the capacity of alpha2-macroglobulin to inhibit proteases. To explore this possibility, bromelain, an exogenous protease, was titrated against plasma of critically ill or septic patients, whose pathophysiological conditions are defined by a massive release of inflammatory mediators. The stoichiometry of bromelain inhibition by alpha2-macroglobulin was quantified by caseolytic activity assays. The maximal alpha2-macroglobulin/bromelain inhibition ratios were significantly increased (1:6 and 1:8 in the two patient groups, P < 0.01) as compared with control groups (1:2 with purified alpha2-macroglobulin and 1:4 in healthy volunteers). The increase of alpha2-macroglobulin inhibition capacity in patients was paralleled by the appearance of a large signal on Western blots, suggesting the formation of additional complexes. Our results demonstrate alpha2-macroglobulin to have more flexibility than had been thought, and it may thereby contribute to a shift in a 30-year-old paradigm.

Adult↗

PubMed1977

Synthesis and secretion of alpha-2-macroglobulin by cultured adherent lung cells. Comparison with cell strains derived from other tissues.

We studied the synthesis and secretion of alpha-2-macroglobulin by cultures of human adherent cells. Much more alpha-2-macroglobulin (measured by radioimmunoassay) accumulated in media of established strains of adherent cells derived from embryonic lung than in media of established strains derived from adult skin or rheumatoid synovium. Alpha-2-macroglobulin accumulated in media of primary cultures of adherent cells from a variety of embryonic tissues. However, the amount of alpha-2-macroglobulin accumulating in media of subsequent passage of these cells declined for all strains except those derived from lung. Immunodiffusion and double-antibody immunoprecipitation studies of cell extracts and media after incubation of cells with l-[(35)S]methionine supported the radioimmunoassay finding that adherent cells from lung synthesized and secreted more alpha-2-macroglobulin than adherent cells from skin. Intracellular alpha-2-macroglobulin could not be detected by radio-immunoassay or visualized by immunofluorescent microscopy, suggesting that synthesized alpha-2-macroglobulin is rapidly secreted. Plasminogen-rich fibrin clots were lysed in culture media of adherent cells from embryonic lung and, to a lesser extent, heart. Adherent cells from other tissues, which produced less alpha-2-macroglobulin, did not lyse fibrin clots. However, all cultures of adherent cells contained pericellular fibronectin, a large, external, transformation-sensitive glycoprotein known to be cleaved by plasmin. We speculate that production of alpha-2-macroglobulin may be a means for protease-secreting normal cells to preserve cell surface integrity and that alpha-2-macroglobulin synthesized locally in lung may protect lung tissues from a variety of proteases.

Cell Adhesion↗

PubMed1992

Localization of cleavage sites for human fibroblast collagenase in the bait region of five mammalian alpha-macroglobulins.

The interaction between human fibroblast collagenase and five mammalian alpha-macroglobulins (human alpha 2-macroglobulin and pregnancy zone protein, rat alpha 1- and alpha 2-macroglobulin and rat alpha 1-inhibitor 3) is discussed. Complex formation involves specific limited proteolysis in the alpha-macroglobulin bait regions, activation of the internal beta-cysteinyl-gamma-glutamyl thiol esters, conformational changes and covalent complex formation. For human alpha 2-macroglobulin, and rat alpha 1-macroglobulin and alpha 2-macroglobulin it is estimated that the overall rate constant of complex formation is greater than 1.10(6) M-1s-1, while it is much lower for human pregnancy zone protein and rat alpha 1-inhibitor 3. More than 95% of the complexed collagenase is covalently bound. The identification of the sites of specific limited proteolysis in the bait regions of the five alpha-macroglobulins shows that cleavage may take place in sequences that are not related to those identified earlier in the collagens. These results greatly expand the repertoire of sequences known to be cleaved by fibroblast collagenase, and suggest that this proteinase has a primary substrate specificity resembling that of the microbial proteinase thermolysin as it preferentially cleaves at the NH2-terminal side of large hydrophobic residues. In addition, the results highlight the unique structure of the flexible alpha-macroglobulin bait region in that it can accommodate a conformation required by the highly restrictive fibroblasts collagenase. It is suggested that alpha-macroglobulins may play an important role in locally controlling the activity of collagenases and perhaps other proteinases of the extracellular matrix.

Amino Acid Sequence↗

PubMed1991

Reaction of proteinases with alpha 2-macroglobulin from the American horseshoe crab, Limulus.

The products generated by the reaction of Limulus alpha 2-macroglobulin with trypsin were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Unreacted Limulus alpha 2-macroglobulin had a subunit molecular mass of 185 kDa. Trypsin-reacted samples contained two prominent peptides smaller (85 and 100 kDa) and three peptides larger (200, 250, and 300-350 kDa) than the unreacted subunit. Reaction of methylamine-treated Limulus alpha 2-macroglobulin with trypsin resulted in the same two prominent reaction products smaller than 185 kDa, but all of the reaction products larger than 185 kDa were absent. The covalent binding of biotinylated trypsin with Limulus alpha 2-macroglobulin was detected by probing Western blots with horseradish peroxidase-avidin. Surprisingly, the only reaction products that contained trypsin were bands at 100 and 120 kDa. The staining of these bands with horseradish peroxidase-avidin was weak: most of the biotinylated trypsin that remained associated with alpha 2-macroglobulin during gel filtration chromatography was located at the dye front following reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The reaction products larger than 185 kDa did not contain trypsin. Methylamine-reacted Limulus alpha 2-macroglobulin failed to bind any biotinylated trypsin. In contrast to the reaction of trypsin with Limulus alpha 2-macroglobulin, all high molecular mass bands generated by the reaction of human alpha 2-macroglobulin with biotinylated trypsin stained intensely with horseradish peroxidase-avidin. Thus, Limulus alpha 2-macroglobulin forms thiol ester-dependent, high molecular mass products involving isopeptide bonding between trypsin-generated fragments, without the incorporation of trypsin into the complexes. Most of the alpha 2-macroglobulin-associated trypsin is non-covalently trapped rather than covalently cross-linked.

Animals↗

PubMed1988

Conformational changes of alpha-macroglobulin and ovomacroglobulin from the green turtle (Chelonia mydas japonica).

Green turtle plasma alpha-macroglobulin and ovomacroglobulin underwent conformational changes when they were treated with proteinases or methylamine. Their conformational changes were studied by HPLC gel chromatography, circular dichroism, and electron microscopy. The Stokes radii of native green turtle alpha-macroglobulin and ovomacroglobulin were estimated to be 84.3 +/- 0.5 A, and 93.0 +/- 0.5 A, respectively, by means of an HPLC experiment. After reaction with methylamine or proteinases, the Stokes radius of alpha-macroglobulin changed to 83.0 +/- 0.5 A or 85.4 +/- 0.5 A, respectively, and that of ovomacroglobulin to 93.0 +/- 0.5 A or 87.1 +/- 0.5 A. The circular dichroic spectra of native alpha-macroglobulin and ovomacroglobulin exhibited a negative band at around 215 nm, indicating the presence of beta-structure. Reaction of the two macroglobulins with methylamine resulted in a slight decrease in the ellipticity and reaction with proteinases led to a slight increase. The electron micrographic images of native alpha-macroglobulin and ovomacroglobulin can be described as deformed rings for the former and rugby balls for the latter. A common characteristic feature of the two molecules was that the central parts of the molecules were only thinly occupied by subunit. After reaction of macroglobulins with proteinases, the void spaces became partially filled and their overall shape more rectangular. Methylamine treatment caused a structural change only in alpha-macroglobulin but not in ovomacroglobulin. The difference in the susceptibility of the macroglobulins to methylamine was taken as an indication of evolutional divergence of the two homologous proteins within the last 300 million years.

Animals↗

PubMed1986

Alpha 2-macroglobulin gene expression during rat development studied by in situ hybridization.

The sites of alpha 2-macroglobulin mRNA synthesis during rat development have been localized by in situ hybridization using a rat alpha 2-macroglobulin cDNA probe. Fetal liver was found to be the major site of alpha 2-macroglobulin mRNA synthesis. In addition, alpha 2-macroglobulin mRNA was detected in brain, spinal cord and eye. Alpha 2-Macroglobulin mRNA was quantitated by use of a sensitive RNAse protection assay. Maximal levels of alpha 2-macroglobulin mRNA were found in fetal livers shortly before birth. A rapid decline of alpha 2-macroglobulin mRNA occurred within 1 day after parturition. A similar time course, although at an approximately 20-fold lower level, was observed for alpha 2-macroglobulin mRNA in livers of pregnant rats. Alpha 2-Macroglobulin mRNA could also be detected in placenta. The levels were comparable to those found in maternal livers.

Aging↗

PubMed1977

Binding of zinc to alpha-2-macroglobulin and its role in enzyme binding activity.

Physicochemical studies performed on alpha-2-macroglobulin were correlated with the biological activities of this protein. Equilibrium dialysis of the binding of 65Zn by alpha-2-macroglobulin at pH 7.9 showed heterogeneous binding which could be attributed to two classes of binding sites. The site of greatest affinity for zinc had an apparent stoichiometry (n1 in gatoms/mol of alpha-2-macroglobulin monomer) of 12 and an apparent association constant (K1) of 3.06.10(7). The second binding site had an n2 of 60 and K2 of 1.32.10(5). The trypsin binding activity of alpha-2-macroglobulin did not depend on the presence of zinc in this protein since all but traces of this metal could be removed by EDTA without loss of trypsin binding activity. Saturation of site 1 with zinc did not affect the trypsin binding activity of alpha-2-macroglobulin, but binding of the metal by site 2 progressively decreased the trypsin binding activity by causing an irreversable association of the alpha-2-macroglobulin molecules. Removal of excess zinc from alpha-2-macroglobulin did not restore its trypsin binding activity. Our results also indicate that the high zinc content of alpha-2-macroglobulin (320--770 microgram/g protein) reported in the literature is an artifact and that native alpha-2-macroglobulin contains approximately 150--180 microgram Zn/g protein.

Dialysis↗

PubMed1983

Enzymatic properties of alpha 2-macroglobulin-proteinase complexes. Apparent discrimination between covalently and noncovalently bound trypsin by reaction with soybean trypsin inhibitor.

The binding of trypsin to alpha 2-macroglobulin, the appearance of free beta-cysteinyl thiol groups of the formed complexes, the steady-state kinetics of their enzymic hydrolysis of carbobenzoxy-L-valyl-glycyl-L-arginyl-4-nitroanilide and finally their reactions with soybean trypsin inhibitor leading to the formation of ternary alpha 2-macroglobulin-trypsin-soybean trypsin inhibitor complexes were investigated. Each alpha 2-macroglobulin molecule binds two trypsin tightly; the dissociation constants were found to be unmeasureably small, but the extent of formation of 1:1 and 1:2 complexes at different molar ratios of alpha 2-macroglobulin to trypsin as determined from the appearance of thiol groups clearly indicated that binding of trypsin to alpha 2-macroglobulin shows negative cooperativity. Binding of the first trypsin makes the access of the second less easy. The kinetic results showed a decrease of the kc/Km value of hydrolysis of the tripeptide substrate by approx. 4-fold compared to that of free trypsin for each alpha 2-macroglobulin-bound trypsin. Here no differences were seen between the bound trypsins. The analysis of the reactions between the alpha 2-macroglobulin-trypsin complexes and soybean trypsin inhibitor shows that ternary complexes do form, although slowly, and that two processes occur, not only when 1:2 complexes but also when 1:1 complexes react with soybean trypsin inhibitor. Soybean trypsin inhibitor apparently discriminates between two distinct binding modes of trypsin to alpha 2-macroglobulin, the covalently and the noncovalently alpha 2-macroglobulin-bound trypsins.

Animals↗

PubMed1980

Binding and degradation of alpha 2-macroglobulin by cultured fibroblasts.

We studied the interactions of alpha 2-macroglobulin, a major protease inhibitor of plasma and of serum-containing culture medium, with cultured fibroblasts. Iodinated human alpha 2-macroglobulin bound specifically to washed cell layers of cultured human fibroblasts. At 0--4 degrees C, binding was saturated at a concentration of 10--20 micrograms/ml. At 37 degrees C, radiolabel appeared in the medium in a form soluble in 10% trichloroacetic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that ingested iodinated alpha 2-macroglobulin transiently forms a complex with a trypsin-like protease. Indirect immunofluorescence demonstrated alpha 2-macroglobulin in vacuoles of fibroblasts grown in 10% human serum or incubated with purified alpha 2-macroglobulin. Fibroblasts transformed by SV-40 (VA-13 cells) bound and degraded less 125I-labeled alpha 2-macroglobulin than non-transformed fibroblasts and had fewer vacuoles containing alpha 2-macroglobulin. These observations indicate that cultured fibroblasts bind, take up by endocytosis, and degrade alpha 2-macroglobulin. Binding and endocytosis of alpha 2-macroglobulin by a cell may be a means of modulating proteases in the microenvironment of the cell and during endocytosis.

Cell Transformation, Viral↗

PubMed1998

Inhibition of a snake venom hemorrhagic metalloproteinase by human and rat alpha-macroglobulins.

Jararafibrase I is a hemorrhagic metalloproteinase purified from Bothrops jararaca venom, which induces local hemorrhage by degrading the basement membrane components. The present study was undertaken to investigate the inhibition of jararafibrase I by human and rat serum proteinase inhibitors. The proteolytic activity of jararafibrase I was completely inhibited by human and rat sera. In particular, rat serum displayed a greater inhibitory capacity. The inhibitory capacities of both sera were dependent on alpha-macroglobulins. SDS-PAGE analysis revealed that jararafibrase I formed complexes with alpha-macroglobulins that were present in normal sera. The proteolytic activity of jararafibrase I was completely inhibited by alpha1-macroglobulin and murinoglobulin in rat serum, and by human alpha2-macroglobulin. The inhibition molar ratios of alpha-macroglobulin/jararafibrase I were 1.5 for rat alpha1-macroglobulin and human alpha2-macroglobulin, and 2.4 for rat murinoglobulin. SDS-PAGE under reducing conditions demonstrated that the bait region of human alpha2-macroglobulin and rat murinoglobulin was cleaved by jararafibrase I. The bait region cleavage sites were identified as being situated at the 696Arg-697Leu peptide bond in human alpha2-macroglobulin, and at the 686Ala-687Val peptide bond in rat murinoglobulin.

Amino Acid Sequence↗

PubMed1990

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.

Amino Acid Sequence↗

PubMed1987

The elastase inhibitors of swine serum: an improved method for the isolation of alpha 2-macroglobulin.

A previous communication from this laboratory as well as one from another described the separation of alpha 2-macroglobulin from swine serum. The products from both laboratories contained, in addition to alpha 2-macroglobulin, an additional macroglobulin contaminant with alpha 2-globulin mobility. Due to their physicochemical similarity these macroglobulins are not resolved using conventional column procedures such as ion exchange chromatography and gel filtration. Subsequent experiments have shown that immunoelectrophoretically pure swine alpha 2-macroglobulin is present, in good yield (65%) in the breakthrough effluent of columns of Bio-Gel A-1.5m-Reactive Blue 2 while the contaminating macroglobulin is tightly bound. The production of highly purified swine alpha 2-macroglobulin utilizing this observation is the subject of the present report. The product of the separation was found to be homogeneous when subjected to immunoelectrophoresis, at a concentration of 14-16 mg/ml, and diffused against antiswine whole serum antibody. The production of monospecific antibody, a more stringent test for homogeneity, resulted when the purified alpha 2-macroglobulin was injected into rabbits. Physicochemical analyses on the purified product showed that swine and human alpha 2-macroglobulins are true homologs.

Animals↗

PubMed1970

Human plasma alpha 2-macroglobulin. An inhibitor of plasma kallikrein.

Activation of plasma kallikrein arginine esterase activity by kaolin resulted in peak activity at 1 min of incubation and a 50% reduction in activity at 5 min in normal plasma, and 30% reduction in the plasma of patients with hereditary angioneurotic edema who lacked the C1 inactivator. The peak esterolytic activity was inhibited by soybean trypsin inhibitor whereas the 5 min activity was resistant to this inhibitor. Acid treatment of normal and hereditary angioneurotic edema plasma destroyed the factor responsible for the fall in esterase activity at 5 min and the factor which rendered the esterase resistant to soybean trypsin inhibitor. Purified alpha(2)-macroglobulin inhibited approximately 50% of the TAMe esterase activity of purified plasma kallikrein without changing its activity toward basic amino acid esters. The interaction between the alpha(2)-macroglobulin and kallikrein resulted in alterations in the gel filtration chromatographic pattern of the TAMe esterase and biologic activity of kallikrein, indicating that kallikrein was bound to the alpha(2)-macroglobulin. The TAMe esterase activity of this complex, isolated by column chromatography, was resistant to C1 inactivator and SBTI. Studies of incubation mixtures of kallikrein, alpha(2)-macroglobulin and C1 inactivator suggested that these inhibitors compete for the enzyme, and that the alpha(2)-macroglobulin partially protects the esterase activity of kallikrein from C1 inactivator. The alpha(2)-macroglobulin isolated from kaolin-activated plasma possessed 240 times the esterolytic activity of the alpha(2)-macroglobulin purified from plasma treated with inhibitors of kallikrein and of its activation. The alpha(2)-macroglobulin blocked the uterine-containing activity and vascular permeability-inducing effects of plasma kallikrein. These studies suggest that the alpha(2)-macroglobulin is a major plasma inhibitor of kallikrein and provide a new example of an interrelationship between the coagulation, fibrinolytic, and kallikrein enzyme systems.

Alpha-Globulins↗

PubMed1986

Isolation and characterization of alpha-macroglobulin from guinea pig plasma.

Guinea pig alpha-macroglobulin was purified to apparent homogeneity by sequential chromatography on Sephacryl S-300, DEAE-cellulose, and hydroxyapatite. A molecular weight of 780,000 was obtained by equilibrium sedimentation. The preparation migrated as a single band of Mr = 180,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Rabbit antiserum raised against the final preparation partially cross-reacted with human and rat alpha-2-macroglobulins but not with rat alpha-1-macroglobulin. Guinea pig alpha-macroglobulin stimulated the amidolytic activity of trypsin towards a small substrate, but inhibited the proteolytic activity of trypsin towards remazol brilliant blue hide powder. When treated with trypsin or methylamine, four thiol groups per molecule were newly generated. The reaction with trypsin proceeded with at least at two different rates: half of the thiol groups were generated in a fast reaction and the remaining half in a slower reaction. On the other hand, such a two-step reaction was not detected in the reaction with methylamine. The methylamine-treated alpha-macroglobulin retained half the capacity to bind trypsin and its mobility in polyacrylamide gel under nondenaturing conditions remained virtually unchanged. These properties are in marked contrast to those reported for human alpha-2-macroglobulin, but resemble those of rat alpha-2- and mouse alpha-macroglobulins. The amidase activity of trypsin bound to guinea pig alpha-macroglobulin was impaired by soybean trypsin inhibitor to a much greater degree than that of trypsin bound to human or rat alpha-2-macroglobulin.

Animals↗

PubMed1984

Receptor-mediated endocytosis of alpha 2-macroglobulin and transferrin in rat caput epididymal epithelial cells in vitro.

The endocytic activity of epithelial cells from the rat epididymis in vitro has been examined by following the uptake of tracer compounds conjugated to proteins. Transferrin-gold and alpha 2-macroglobulin-gold were taken up initially in coated pits, internalized and sequestered into tubular-vesicular structures, multivesicular bodies and, in the case of alpha 2-macroglobulin, into lysosomes. Uptake could be prevented by an excess of unlabeled protein. Studies using 125I-alpha 2-macroglobulin and 125I-transferrin also showed that the uptake of these proteins was specific and could be displaced with increasing amounts of unlabeled protein. In addition, binding of 125I-transferrin to cells was saturable at 4 degrees C. These studies indicate that transferrin and alpha 2-macroglobulin are taken up by receptor-mediated endocytosis. In contrast, a fluid phase marker, bovine serum albumin-gold (BSA-gold), was initially taken up predominantly in uncoated caveolae rather than coated pits, and could not be displaced with excess BSA. By virtue of their charge, polycationized ferritin and unlabeled colloidal gold were taken up and internalized by adsorptive endocytosis, a pathway which is similar to fluid phase endocytosis. The uptake and internalization of alpha 2-macroglobulin and transferrin differed in a number of respects. Uptake and internalization of alpha 2-macroglobulin but not of transferrin was dependent on extracellular calcium. Only alpha 2-macroglobulin was transferred into lysosomes, whereas transferrin was recycled to the cell surface. Although the proton ionophore, monensin, and the transglutaminase inhibitor, dansylcadaverine, did not stop uptake and internalization of either alpha 2-macroglobulin or transferrin, they did prevent the transfer of alpha 2-macroglobulin to lysosomes.

Adsorption↗

PubMed1988

The development of rat alpha 2-macroglobulin. Studies in vivo and in cultured fetal rat hepatocytes.

During inflammation and tissue injury, there is an increase in the plasma concentration of several proteins, the acute-phase proteins. The levels of some acute-phase proteins have been reported to increase in pregnant and tumour-bearing animals. Rat alpha 2-macroglobulin is classified as an acute-phase protein. In this study we report the expression of alpha 2-macroglobulin in various tissues during development of the rat embryo by analysis of mRNA. The tissues studied are liver, visceral yolk sac, placental labyrinth, decidua and trophoblast. In addition, the sites of alpha 2-macroglobulin expression are localized by in situ hybridization of cDNA for alpha 2-macroglobulin to mid-sagittal cryosections of rat embryos. The level of mRNA coding for alpha 2-macroglobulin is determined in the liver of rats aged between 12 days gestation and 2 days postnatal. alpha 2-Macroglobulin mRNA is first observed in fetal liver from 12 days of gestation and increases after day 17, reaching a maximum on day 20. At this time the level is greater than that found in the liver of an adult rat suffering from acute inflammation. alpha 2-Macroglobulin mRNA is detectable in the yolk sac, placental labyrinth, trophoblast tissue and decidua. In the decidua the alpha 2-macroglobulin message is first detected at 8 days of gestation, with high levels observed from 10 to 21 days of gestation. These observations are supported by in situ hybridization studies. Experiments using cultured hepatocytes show that cells derived from rats at 15 days and 19 days of gestation are capable of synthesizing and secreting alpha 2-macroglobulin. Both synthesis and secretion can be induced by the addition of dexamethasone to the culture medium.

Animals↗