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PubMed1989

Characterization, size estimation and solubilization of alpha-macroglobulin complex receptors in liver membranes.

Receptors for alpha 2-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of 125I-labelled alpha 2-macroglobulin.trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8-9.0. The half-time for association was about 5 min at 37 degrees C in contrast to about 5 h at 4 degrees C. The half-saturation constant was about 100 pM at 4 degrees C and 1 nM at 37 degrees C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 +/- 71 kDa (S.D., n = 7) for alpha 2-macroglobulin.trypsin binding activity. Affinity cross-linking to rat and human membranes of 125I-labelled rat alpha 1-inhibitor-3.chymotrypsin, a 210 kDa analogue which binds to the alpha 2-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55-60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked alpha 2-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound 125I-alpha 1-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400-500 kDa alpha 2-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.

Animals↗

PubMed1984

Circular dichroic spectroscopy of non-human alpha-macroglobulins.

Bovine, chicken and frog alpha-macroglobulins and ovomacroglobulin were studied by circular dichroic spectroscopy over the region 205-250 nm. All four spectra exhibited negative ellipticity with minima at about 215 nm similar to that reported for human alpha 2-macroglobulin. On reaction of the alpha-macroglobulins with trypsin, the spectrum of each of the four changed similarly. However, these proteins exhibited different conformational changes when treated with methylamine. These differences were exploited to determine which characteristics of alpha-macroglobulins correlate with changes in circular dichroic spectroscopy.

Animals↗

PubMed1983

Characterization of alpha 2-macroglobulin from plasma of cystic fibrosis patients and controls.

The physical, kinetic, and isoelectric focusing properties of native alpha 2-macroglobulin from cystic fibrosis and control plasmas were studied. No differences were found in the esterolytic activity levels of control, obligate heterozygote, and cystic fibrosis plasmas. Stability studies indicated that both control and cystic fibrosis alpha 2-macroglobulin retained full activity for at least 8 months at -20 degrees C, a week at 0-4 degrees C, 11 hr at 50 degrees C, and showed no differences in thermostability at several preincubation temperatures. The microheterogeneity of native alpha 2-macroglobulin was studied by column isoelectric focusing of five control and five cystic fibrosis plasmas. The number and pI values of the isoelectric forms between pH 4.5-8.0 were quite similar for both groups even though consistently less cystic fibrosis alpha 2-macroglobulin activity was recovered after isoelectric focusing.

Cystic Fibrosis↗

PubMed1977

[Production of interferon and alpha 2-macroglobulin involvement in immune response during human viral hepatitis A (author's transl)].

In viral hepatitis A, we could distinguish between monophasic and polyphasic forms. Monophasic and polyphasic hepatitis A induce the production of plasma interferon. Interferon level, elevated as early as the first days following the appearance of clinical signs, decreases and reaches a minimum value on the seventh day. A new rise of interferon is characterized by a maximum level on the twelfth day and a minimum level on the thirtieth. Beyond the first month we could still detect the presence of interferon. In the two forms of hepatitis, a complement system is activated both by classical and alternate pathways. IgM levels increase early, IgA levels remain unchanged. On the other hand, IgG levels, only slightly elevated in monophasic hepatitis A, are highly increased in polyphasic hepatitis A beyond the first month. Alpha 2-macroglobulin reaches levels above normal during convalescence in monophasic hepatitis A; on the contrary, in polyphasic hepatitis A, alpha 2-macroglobulin levles are above normal as early as the thirty first days of illness and remain above normal for several months. Elevated levels in alpha 2-macroglobulin may inhibit cellular immunity which is accountable for immunological injury of virus infected hepatocytes. We wonder whether this earlier increase in alpha 2-macroglobulin is responsible for the lasting character of viral infection observed in polyphasic hepatitis A.

Hepatitis, Viral, Human↗

PubMed1978

Human alpha2-macroglobulin and its antitrypsic and antithrombin activities in serum and plasma.

alpha2-Macroglobulin level, trypsin protein esterase and progressive antithrombin activities were measured in normal and nephrotic sera and plasma. Trypsin protein esterase activity was proportional to the alpha2-macroglobulin concentration in serum and plasma from both normal and nephrotic patients. The results were different, however, with progressive antithrombin activity: in normal plasma, antithrombin III is the main thrombin inhibitor, then alpha2-macroglobulin and alpha1-antitrypsin, whereas in nephrotic syndrome patients, alpha2-macroglobulin is the main thrombin inhibitor.

Antithrombins↗

PubMed1989

Purification of the human placental alpha 2-macroglobulin receptor.

The alpha 2-macroglobulin receptor was solubilized from human placental membranes, purified and characterized. Affinity cross-linking of labelled ligand to intact membranes showed a receptor size compatible with 400-500 kDa. The membranes were solubilized in 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonate (CHAPS) and affinity chromatography was performed using Sepharose-immobilized alpha 2-macroglobulin-methylamine with elution in buffer containing 2 mM EDTA, pH 6.0. SDS-PAGE of the resulting receptor preparation showed a predominant approx. 440 kDa band (reducing conditions) and some minor accompanying proteins of 70-90 kDa and 40 kDa. The yield was 400-800 micrograms receptor preparation per placenta. The receptor preparation immobilized on nitrocellulose bound the alpha 2-macroglobulin-trypsin complex with a dissociation constant of about 400 pM. 125I-iodinated receptor preparation bound almost quantitatively to Sepharose-immobilized alpha 2-macroglobulin-methylamine in the presence of CHAPS alone, and bound 70-80% in the presence of 0.2% SDS. The labelled proteins were separated in the presence of 0.2% SDS by gel filtration or SDS-PAGE (unboiled samples). The 440 kDa protein accounted for the major part of the binding, although some approx. 80 kDa proteins, perhaps proteolytic degradation products, also showed binding activity.

Cell Membrane↗

PubMed1993

The synthesis of alpha 2-macroglobulin by rat mammary myoepithelial cells is regulated by synergism between glucocorticoids and cytokines.

Addition of hydrocortisone to the rat mammary gland myoepithelial cell line, G4.2.3, induces the synthesis and secretion of alpha 2-macroglobulin. Interleukin-1 beta, interleukin-6 and transforming growth factor-beta synergize with hydrocortisone, increasing the synthesis of alpha 2-macroglobulin 2- to 4-fold, although they have no effect in the absence of hydrocortisone. Interleukin-6 is the most potent inducer having an optimum concentration of 1 ng/ml. Interleukin-6, unlike interleukin-1 beta or transforming growth factor-beta, decreases the lag phase from 10 h to 4 h before alpha 2-macroglobulin synthesis is induced by hydrocortisone. These results suggest that the mechanism of activation of transcription of the alpha 2-macroglobulin gene in mammary myoepithelial cells might differ from that operating in hepatic cells.

Animals↗

PubMed1982

Isolation and purification of rat acute-phase alpha 2-macroglobulin.

Acute-phase alpha 2-macroglobulin was highly purified from the serum of rats in which this protein had been induced 48 h previously by the injection of croton oil, an inflammatory agent. The isolation protocol involved two non-denaturing steps; first, separation according to molecular weight by gel filtration on Ultrogel AcA 22 and second, negative affinity chromatography which bound contaminating proteins to the column while allowing acute-phase alpha 2-macroglobulin to pass through. Several criteria were used to assess the purity of acute-phase alpha 2-macroglobulin, after which the protein by mass determination and by two different protein assays. Pure rat acute-phase alpha 2-macroglobulin was used to produce a monospecific antiserum and to calibrate a secondary standard of rat acute-phase serum by developing and characterizing rocket immunoelectrophoresis assay.

Animals↗

PubMed1983

Arming alpha 2-macroglobulin with ricin A chain forms a cytotoxic conjugate that inhibits protein synthesis and kills human fibroblasts.

A conjugate containing alpha 2-macroglobulin and highly purified ricin A chain was made using N-succinimidyl-3-(2-pyridyldithio)propionate. Radioimmunoassay indicated that it contained 1.2 mol A chain per mol alpha 2-macroglobulin. The conjugate inhibited polyuridylic-acid directed translation by rat liver ribosomes and protein synthesis in human fibroblasts. There was a 90 min lag period before the beginning of inhibition in fibroblasts, but complete inhibition could be achieved. By measuring protein synthesis as a function of protein concentration, it was demonstrated that 8.25 X 10(-9) M conjugate was required to inhibit 50% of protein synthesis in 6 h. To achieve the same level of inhibition, 165-times more (1.3 X 10(-6) M) unconjugated A chain was required, and 180-times less ricin (4.6 X 10(-11) M). Ricin was more than 28 000 times more inhibitory than A chain alone. The presence of alpha 2-macroglobulin did not increase the cytotoxicity of unconjugated A chain, and it even protected the cells to a slight extent. The inhibitory action of the conjugate was blocked by antibodies specific for alpha 2-macroglobulin or ricin, and it was not prevented by galactose or antibodies specific for ricin B chain. Electron microscopy of the conjugate indirectly labelled with ferritin demonstrated that it was internalized by receptor mediated endocytosis through coated pits. These data indicate that the A chain portion of the conjugate survives the conditions in the lysosomes to the extent that it retains its ability to inactivate cytoplasmic ribosomes.

Animals↗

PubMed1989

High-molecular-mass proteinases in rabbit reticulocytes: the multicatalytic proteinase is an ATP-independent enzyme and ATP-activated proteolysis is in part associated with a cysteine proteinase complexed to alpha 1-macroglobulin.

We have investigated the proteolytic degradation of [14C]methylcasein and 125I-labeled bovine serum albumin at pH 7.8 and 37 degrees C by lysates of rabbit reticulocytes purified from rabbit blood by two different procedures. (I) Lysates obtained from reticulocytes after removal of plasma and buffy coat as well as after washing of cells, degraded casein and albumin, and released from the two substrates 1.3%/h and 0.4%/h, respectively, of acid-soluble radioactivity. The activity towards both substrates was stimulated about 4-fold by ATP/Mg2+. Chromatography of whole blood on a column of cellulose prior to washing and lysis of cells had profound but differential effects on these activities in that stimulation of casein-degradation by ATP/Mg2+ was almost completely lost, whereas degradation of albumin, albeit at a low rate, was measurable in the presence of ATP/Mg2+ only. (II) Degradation of casein by these lysates is largely inhibited by a monospecific antibody against rabbit multicatalytic proteinase, whereas digestion of albumin is not affected by the antibody, either in the presence or absence of ATP/Mg2+. The latter activity is partially inhibited by a specific antibody against rabbit alpha 1-macroglobulin. (III) The immunoreactive amount of multicatalytic proteinase is about 1.2 micrograms per mg of lysate protein and almost identical in the two lysates. In contrast, the immunologically detectable levels of alpha 1-macroglobulin vary and are much lower in reticulocyte-lysates following chromatography on cellulose than in lysates from washed reticulocytes. (IV) Caseinolytic activity of multicatalytic proteinase, purified from rabbit reticulocyte lysate, is not activated by ATP/Mg2+ and the enzyme is proteolytically inactive towards albumin. On the other hand, a complex consisting of the proteinase inhibitor alpha 1-macroglobulin and the cysteine proteinase, cathepsin B, does degrade both substrates at pH 7.8, in an ATP/Mg2+-activated fashion. From these results it is concluded that the multicatalytic proteinase is an ATP-independent enzyme and a cellular constituent of rabbit reticulocytes whereas the activity stimulated by ATP/Mg2+ appears to be associated, at least in part, with a cysteine proteinase complexed to alpha 1-macroglobulin.

Adenosine Triphosphate↗

PubMed2005

Expression of Panza, an alpha2-macroglobulin, in a restricted dorsal domain of the primitive gut in Xenopus laevis.

Alpha2-macroglobulin is a major serum protein with diverse functions, including inhibition of protease activity and binding of growth factors, cytokines, and disease factors. We have cloned and characterized Panza, a new Xenopus laevis alpha2-macroglobulin. Panza has 56-60% amino acid similarity with previously identified Xenopus, mouse, rat and human alpha2-macroglobulins, indicating that Panza is a new member of the alpha2-macroglobulin family. Panza mRNA is first detected at the beginning of neurulation in the dorsal endoderm lining the primitive gut (archenteron roof). At the completion of neurulation and continuing through the late tadpole stage, Panza is restricted to a dorsal domain of the gut endoderm adjacent to the notochord and extending along the entire anterior-posterior axis. With outgrowth of the tailbud, Panza expression persists in the chordaneural hinge at the posterior end of the differentiating notochord and extends into the floor plate of the posterior neural tube. As gut coiling commences, Panza expression is initiated in the liver, and the dorsal domain of Panza expression becomes limited to the midgut and hindgut. With further gut coiling, strong Panza expression persists in the liver, but is lost from other regions of the gut. The expression of Panza in endodermal cells adjacent to the notochord points to a potential role for Panza in signal modulation and/or morphogenesis of the primitive gut.

Amino Acid Sequence↗

PubMed2000

Alpha(2)-macroglobulin levels are high in adult patients with congenital antithrombin deficiency.

Antithrombin is responsible for about 80% of the progressive inhibitory activity of thrombin in human plasma. The role of other protease inhibitors known to inhibit thrombin is not completely clarified. However, their contribution may become relevant when antithrombin is low. We elected to investigate adult patients with congenital antithrombin deficiency to assess the concentration of other naturally occurring thrombin inhibitors such as alpha(2)-macroglobulin, alpha(1)-antitrypsin, heparin cofactor II, and C(1)-inhibitor. The study included 59 patients with congenital antithrombin deficiency with and without a previous history of thrombosis, together with an equal number of control subjects matched for age and sex. Statistically significant differences (patients vs. controls) were observed only for alpha(2)-macroglobulin (i.e., 120 vs. 102%, p<0.01). Further analysis of antithrombin-deficient carriers with and without a past history of thrombosis showed that alpha(2)-macroglobulin levels were higher than the 90th percentile of control distribution more often in asymptomatic than symptomatic men (odds ratio=0.04; confidence interval=0.003-0.60), but not in women (odds ratio=2.14; confidence interval=0.35-13.1). In conclusion, results from this cross sectional study showed that alpha(2)-macroglobulin levels were high in patients with congenital antithrombin deficiency. Furthermore, the high levels were found more often in asymptomatic than symptomatic men. Whether this increase provides protection against thrombosis should be evaluated in a prospective study.

Adolescent↗

PubMed1984

Kinetics of the conformational alterations associated with nucleophilic modification of alpha 2-macroglobulin.

The effect of nucleophilic modification of alpha 2-macroglobulin (alpha 2M) with methylamine on the kinetics of sulfhydryl exposure was investigated. The generated sulfhydryl groups were detected with 4,4'-dithiodipyridine. The bimolecular rate constant for sulfhydryl exposure was determined to be 11.6 +/- 0.8 M-1 s-1 at 30 degrees C and pH 8.0. Treatment of alpha 2-macroglobulin with methylamine or proteases, such as plasmin and trypsin, results in a substantial increase in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonic acid. This probe was used to monitor the kinetics of the conformational change occurring in alpha 2-macroglobulin upon treatment with methylamine. It was found that the conformational change did not occur simultaneously with the cleavage of the thiol ester bonds by the nucleophile but, rather, the conformational alterations occurred following a lag phase. The data are consistent with a mechanism requiring the random cleavage of two thiol ester bonds of a dimeric unit in the molecule prior to the unimolecular process representing the conformational change. According to this model, the two dimeric units present in alpha 2M act as independent entities. On the basis of the best fit with the model, the bimolecular rate constant, for hydrolysis of the thiol ester bonds, was determined to be 11.9 +/- 0.7 M-1 s-1, while the rate constant for the conformational change was (9.7 +/- 2.0) X 10(-3) s-1. The measured rate of conformational change is rate limited by thiol ester cleavage at lower methylamine concentrations. The conformational change, measured with this fluorescence probe, approximately parallels the loss of trypsin binding activity of alpha 2-macroglobulin, measured by resistance of the bound trypsin to soybean trypsin inhibitor. A much slower loss of plasmin binding activity was observed than was found for trypsin, suggesting that the structural requirements on alpha 2M for the interaction with plasmin are disrupted much more slowly than the structural requirements for trypsin binding upon methylamine treatment of the molecule.

Fluorescent Dyes↗

PubMed1986

A unique pair of zinc binding sites in the human alpha 2-macroglobulin tetramer. A 35Cl and 37Cl NMR study.

35Cl NMR has been used to demonstrate that human alpha 2-macroglobulin tetramer possesses a unique pair of zinc binding sites. Zinc bound at these sites does not affect the 35Cl NMR line width of free Cl-. Additional lower affinity zinc sites exist that bind chloride weakly and cause broadening of the free chloride resonance through fast exchange with bound chloride. Using both 35Cl and 37Cl relaxation measurements it has been shown that chloride bound at these sites has an internal correlation time of 5.1 ns and a quadrupolar interaction, chi, of 4.2 MHz with zinc. Manganese binds to apo-alpha 2-macroglobulin analogously to zinc. alpha 2-macroglobulin that has been reacted with methylamine still possesses two classes of zinc sites per tetramer, but their relative affinities differ more than for unreacted alpha 2-macroglobulin. These data are discussed with respect to possible models for the subunit arrangement in the tetramer.

Binding Sites↗

PubMed1988

Structure and acute-phase regulation of the rat alpha 2-macroglobulin gene.

Seven genomic DNA clones representing the rat alpha 2-macroglobulin gene were isolated and characterized. The cloned sequence covered the entire gene (48 kilobases) plus 2 kilobases of 3'- and 13.7 kilobases of 5'-flanking sequences. A restriction cleavage map of the gene was produced, and the restriction cleavage pattern of genomic DNA suggested that the alpha 2-macroglobulin gene is a single-copy gene. A 7.7-kilobase fragment from the 5'-terminal region and a 250 base pair fragment from the 3'-terminal region of the gene were sequenced, and the 3' end of the gene was mapped. The sequenced 5'-terminal fragment contained 4.5 kilobases of 5'-flanking sequences plus the first three exons and two introns of the gene. Two transcription start sites, a minor and a major site, located 65 nucleotides apart, were defined by primer extension, S1 mapping, and RNaseH experiments. During an acute-phase response, transcription from both sites was induced in the liver, and over 90% of the transcripts originated from the major site. Very high concentrations of alpha 2-macroglobulin mRNA originating from both start sites were also found in the uterus but not in the liver of pregnant females. A glucocorticoid response element (GRE), a conserved consensus sequence for a potential glucocorticoid receptor DNA binding site, was found by computer search in the promoter-proximal 5'-flanking region of the alpha 2-macroglobulin gene.

Animals↗

PubMed1990

Sertoli cell synthesizes and secretes a protease inhibitor, alpha 2-macroglobulin.

The mechanism by which the seminiferous epithelium limits the damaging effects of proteases that are released from degenerating late spermatids does not depend upon protease inhibitors in the systemic circulation since these proteins are excluded from the seminiferous tubule by the blood-testis barrier. The purpose of this study was to identify the major protease inhibitor of the testis and determine its cellular origin. Sertoli cells, the major epithelial component of the seminiferous epithelium, release a protease inhibitor, testicular alpha 2-macroglobulin, in vitro. Immunoprecipitation using [35S]methionine and a monospecific polyclonal antibody prepared against purified testicular alpha 2-macroglobulin establishes that this protein is actively synthesized and secreted by Sertoli cells. Measurements of immunoreactive protease inhibitors in tubular and rete testis fluids collected by micropuncture suggest that alpha 2-macroglobulin rather than alpha 1-antitrypsin is the major protease inhibitor in the seminiferous tubules in vivo. The ability of alpha 2-macroglobulin to inactivate proteases and growth factors such as TGF-beta by a common mechanism suggests that this protein may have a dual function in the testis.

Amino Acid Sequence↗

PubMed1979

The identification of distinctive forms of human alpha 2-macroglobulin by using the numerical relationship between trypsin binding in alpha- and beta-modes.

Interactions between the serine proteinase trypsin and the protein proteinase inhibitors in human blood were expressed in terms of a coupled set of non-linear differential equations, which has been solved for each of 110 samples of serum obtained from colleagues and from a variety of hospital sources. Optimization of nine unknown theoretical parameters and 21 experimental rate measurements of the hydrolytic activity of trypsin in free and bound states after admixture with various amounts of a given serum was achieved by an iterative procedure using initial estimates of the parameters derived from the "four-straight-line" model described in the preceding paper [Topping & Seilman (1979) Biochem. J. 177, 493--499.] Such a procedure yielded the following information for each sample of serum examined: (a) the concentrations of alpha 1-antitrypsin and alpha 2-macroglobulin; (b) the unequivocal assignment of alpha 2-macroglobulin into one of seven categories on the basis of trypsin binding in two kinetically differentiated modes (alpha and beta); (c) the hydrolytic activities of trypsin (versus Bz-Arg-OEt) when bound to alpha 1-antitrypsin, and to alpha 2-macroglobulin in the alpha- and beta-modes. Molecular interpretations of the binding of trypsin to alpha 2-macroglobulin are discussed and the potential clinical value of recognizing the nature of such binding is reported.

Animals↗

PubMed1987

Inhibition of chicken calpain II by proteins of the cystatin superfamily and alpha 2-macroglobulin.

Inhibition of chicken calpain II by proteins of the cystatin superfamily and alpha 2-macroglobulin was investigated. Human liver cystatins A and B, human cystatin C, chicken cystatin and rat T-kininogen were found not to be inhibitory. Inhibition was, however, observed for bovine and rat kininogens, with Ki (inhibition constant) values of 0.8 nM and 30 nM respectively. alpha 2-Macroglobulin inhibits calpain with an initial rate constant of the order of 3 X 10(4) M-1.S-1. Calpain complexed with alpha 2-macroglobulin showed only limited reactivity towards azocasein, but reacted readily with the peptide substrate Suc-Leu-Tyr-4-methyl-7-coumarylamide and with L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane (E-64). The calpain in the complexes was at least partially protected from loss of activity due to autolysis. The calpain-alpha 2-macroglobulin complexes contained both the calpain subunits.

Animals↗