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PubMed1996

Localisation of the major reactive lysine residue involved in the self-crosslinking of proteinase-activated Limulus alpha 2-macroglobulin.

When alpha 2-macroglobulin (alpha 2M) from the American horseshoe crab, Limulus polyphemus, reacts with proteinases, its thiol esters, like those of other alpha-macroglobulins, become activated, leading to the formation of covalently crosslinked species that can be detected as high molecular weight bands in reducing SDS-PAGE. While other alpha-macroglobulins extensively form crosslinks to the reacting proteinase, Limulus alpha 2M does not. It rather becomes internally crosslinked. It was found from N-terminal sequence analysis of purified [14C]carboxymethylated peptides from Limulus alpha 2M-trypsin complexes that an isopeptide bond formed in approx. 60% yield from the thiol esterified Gln-1002 specifically to Lys-254 in the opposing monomer of the disulphide bridged dimer is the main cause of the internal crosslinking.

Animals↗
PubMed1995

Studies of complexes between proteases, substrates and the protease inhibitor alpha 2-macroglobulin using capillary electrophoresis with laser-induced fluorescence detection.

Capillary zone electrophoresis (CZE) with laser-induced fluorescence (LIF) detection is shown to constitute a unique technique for the investigation of the interaction between proteases, protease inhibitors and substrates. Under optimized analysis conditions, the formation of a complex between FITC-labelled proteases such as trypsin, plasmin, alpha-chymotrypsin and the (unlabelled) protease inhibitor alpha 2-macroglobulin was studied. This is not possible with UV detection, since under such conditions the complex cannot be distinguished from the unreacted protease inhibitor. Low ratios of FITC bonded to the proteases further complex formation, while high ratios often prevent the reaction. Complex formation shows a strong dependence on the incubation conditions (pH, salt concentration, temperature, incubation time). Once formed, however, the complexes are stable under CZE conditions (e.g., a pH of the electrophoresis buffer of 10.5) for at least 30 min. Treatment with sodium dodecyl sulfate (5 min at 90 degrees C or 30 min at 75 degrees C) does not destroy the complexes, whereas treatment with mercaptoethanol (reduction of disulfide bonds) eliminates the peak from the electropherogram. Both findings argue for the formation of a covalent bond between the protease and the inhibitor during complex formation. Since the reaction of the proteases with alpha 2-macroglobulin does not involve the binding site of the former, a residual proteolytic activity is still observed in the ensuing complex. The extent of the inhibition of the remaining trypsin activity in a trypsin--alpha 2-macroglobulin complex was established to depend on the molecular mass of the second trypsin inhibitor.

Electrophoresis, Capillary↗
PubMed1990

Measurement of the 'fast' or complexed form of alpha 2 macroglobulin in biological fluids using a sandwich enzyme immunoassay.

A sandwich enzyme immunoassay has been developed for measuring the 'fast' or complexed form of alpha 2 macroglobulin using a complex-specific monoclonal antibody. The working range of this assay is 1.5-15 micrograms/l and is suitable for use with various biological fluids. Using this assay the normal plasma levels were found to range from 4.2 mg/l to 14.4 mg/l (0.17%-0.70% of total alpha 2 macroglobulin) with a mean value of 7.6 mg/l +/- 2.6 (0.37% +/- 0.12% of total alpha 2 macroglobulin). Elevated levels were seen in plasma samples taken on the day of admission from patients with acute pancreatitis and in some synovial fluid samples from patients with various arthritides.

Antibodies, Monoclonal↗
PubMed1992

Temperature dependence of the kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin into half-molecules. A minimum rate at 15 degrees C indicates hydrophobic interaction between the subunits.

The kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin to half-molecules has been studied as a function of temperature by using small-angle scattering of X-rays and neutrons. The most striking result of the present investigation is that there is a minimum in reaction rate at about 15 degrees C, and that the rate increases when the temperature is lowered, or raised, from that value. By analyzing the first-order rate constants in terms of transition-state theory it was found that the dissociation is associated with a large and positive change in heat capacity between the activated complex and native alpha 2-macroglobulin (delta CP is in the range 5 to 6 kJ mol-1 K-1). In analogy with pure thermodynamic investigations, where a large change in heat capacity normally is interpreted as a melting of hydrophobic interaction, we therefore propose that hydrophobic interaction is involved in the so-called non-covalent interactions between the subunits of alpha 2-macroglobulin. As a result of the present investigation, it also follows that the free energy of activation delta G has a maximum at about 32 degrees C, whereas the enthalpy of activation delta H and the entropy of activation delta S are zero at about 15 degrees C and 32 degrees C, respectively. These temperatures are slightly dependent upon the concentration of urea and upon whether the reaction is run in a 1H or a 2H medium. Furthermore, from the kinetic point of view, at low temperature the reaction can be characterized as enthalpy driven, whereas at high temperature, it can be characterized as entropy driven.

Humans↗
PubMed1978

Collection of insulin, EGF and alpha2-macroglobulin in the same patches on the surface of cultured fibroblasts and common internalization.

We have used video intensification microscopy to observe fluorescent derivatives of insulin, epidermal growth factor and alpha2-macroglobulin added to Swiss 3T3-4 cells. At 4 degrees C, each of these polypeptides binds diffusely to specific receptors on the cell surface. When the cells are warmed to 23 or 37 degrees C, the bound insulin epidermal growth factor or alpha2-macroglobulin rapidly forms patches on the cell surface and is internalized. Using fluorescein-labeled alpha2-macroglobulin and rhodamine-labeled derivatives of insulin and epidermal growth factor, we show that all three polypeptides are internalized within the same vesicles by a common pathway. The mechanism for the internalization of these molecules is discussed.

Cell Line↗
PubMed1984

The two alpha 2-macroglobulin-bound trypsin molecules have different affinities for the basic pancreatic trypsin inhibitor.

We have investigated the enzymatic properties of alpha 2-macroglobulin-bound porcine trypsin using a substrate: Z-Gly-Gly-Arg-p-nitroanilide and two inhibitors: p-aminobenzamidine and basic pancreatic trypsin inhibitor. The ternary alpha 2-macroglobulin-(trypsin)2 complex behaves like a mixture of two enzymes which bind basic pancreatic trypsin inhibitor with widely different affinities (Ki = 0.11 microM and 23 microM). About one-half of the trypsin molecules of the ternary complex are covalently bound to alpha 2-macroglobulin. Preparation of the complex in the presence of hydroxylamine prevents covalent bond formation, but the two trypsins of this artificial complex still exhibit large differences in affinity for basic pancreatic trypsin inhibitor. The trypsin molecules of the ternary complex also exhibit small differences in their affinity for Z-Gly-Gly-Arg-p-nitroanilide and p-aminobenzamidine.

Benzamidines↗
PubMed1987

Some consequences of the covalent and non-covalent binding modes of plasmin with alpha 2-macroglobulin.

Analysis of plasmin-alpha 2-macroglobulin interactions by polyacrylamide gel electrophoresis showed that both the light and heavy chains of the proteinase have covalent links with the inhibitor. This covalent binding occurs with a 95 +/- 5% yield and can be abolished in the presence of hydroxylamine without modification of the plasmin-alpha 2-macroglobulin stoichiometry, the extent of the 180-kDa peptide chain cleavage and the generation of the -SH groups. However, these two different binding modes greatly influence the enzymatic properties of the proteinase as well as the occupancy by an other proteinase molecule of the free binding site of the (1:1) plasmin-alpha 2-macroglobulin complex. Non-covalently bound plasmin is more active on synthetic substrates and interacts more tightly with the basic pancreatic trypsin inhibitor than the covalently bound enzyme. Furthermore, the former complex incorporates significantly more chymotrypsin than the latter. The incorporation of chymotrypsin influences the catalytic properties of plasmin within the ternary complex.

Chymotrypsin↗
PubMed1989

Kinetics of the inhibition of free and elastin-bound human pancreatic elastase by alpha 1-proteinase inhibitor and alpha 2-macroglobulin.

At pH 8.0 and 25 degrees C alpha 1-proteinase inhibitor and alpha 2-macroglobulin bind human pancreatic elastase with rate constants of 4.7.10(5) M-1.s-1 and 6.4.10(6) M-1.s-1, respectively. The corresponding delay times of elastase inhibition in plasma are 0.4 s and 0.2 s, respectively, indicating that both inhibitors may act as physiological antielastases. Elastin impairs the elastase inhibitory capacity of alpha 1-proteinase inhibitor and alpha 2-macroglobulin. In presence of human elastin, the former behaves like a slow-binding elastase inhibitor, with a rate constant of about 260 M-1.s-1. In contrast, alpha 2-macroglobulin is a fast-binding inhibitor of elastin-bound elastase, but only one of its two sites is functioning in presence of elastin.

Animals↗
PubMed1991

The exposure of murine macrophages to alpha 2-macroglobulin 'fast' forms results in the rapid secretion of eicosanoids.

The exposure of [3H]arachidonate-radiolabelled murine peritoneal macrophages to alpha 2-macroglobulin-methylamine or alpha 2-macroglobulin-trypsin but not native alpha 2-macroglobulin (alpha 2M) results in the rapid secretion of [3H]eicosanoids. Resident peritoneal macrophages stimulated with 0.1 microM alpha 2M-methylamine exhibited an enhanced secretion within 10 min. The ability of alpha 2M 'fast' forms to stimulate secretion of [3H]eicosanoids was similar to that observed in the presence of the murine macrophage chemoattractant platelet-activating factor. As observed for total [3H]eicosanoid secretion, alpha 2M 'fast' forms also rapidly enhanced the secretion of the cAMP-elevating prostanoid, prostaglandin E2, from resident peritoneal macrophages. Stimulated secretion of prostaglandin E2 in response to 0.1 microM alpha 2M-methylamine was less rapid than that observed using 0.1 microM platelet-activating factor. Similar amounts of secreted prostaglandin E2 were present in media of macrophage cultures after 1 h exposure to the two stimuli. In the presence of 0.1 microM alpha 2M-methylamine, secreted prostaglandin E2 remained elevated, compared to the appropriate buffer control, for at least 24 h. The present results indicate that receptor recognition of alpha 2M 'fast' forms by macrophages results in the rapid stimulation of eicosanoid secretion and suggest that secretion of prostaglandin E2 and other eicosanoids may be involved in the ability of alpha 2 M 'fast' forms to regulate various macrophage functional responses.

Animals↗
PubMed1990

Interactions between cytokines and alpha 2-macroglobulin.

Cytokines are often thought of as short range, short half-life molecules. This view may now be challenged by recent findings that indicate that alpha 2-macroglobulin and antibodies to cytokines may alter cytokine kinetics in vivo. alpha 2-Macroglobulin, one of the major proteins in serum, can bind a wide range of physiologically important molecules. As Keith James reviews here, the interaction between alpha 2-macroglobulin and a number of cytokines has recently come under scrutiny, revealing a new and potentially important mechanism for the modulation of cytokine activity, while in the following paper a modulatory role for anti-cytokine antibodies is considered.

Animals↗
PubMed1988

Image processing of electron micrographs of human alpha 2-macroglobulin half-molecules induced by Cd2+.

Human alpha 2-macroglobulin is a tetrameric plasma inhibitor of proteinases. Its dissociation by Cd2+ gives functional dimers. Electron microscopy of negatively stained dimers shows their round-ended cylindrical shape with furrows delimiting 3 main stain-excluding domains. Image processing of electron micrographs shows the existence of 2 main orientations of the dimers on the carbon support film. The dimer is composed of 2 curved monomers linked in a central domain, and related by a 90 degree rotation. Taking into account the known primary structure of alpha 2-macroglobulin and the linkage of the 2 constitutive monomers by 2 disulfide bonds, the molecular organization of the dimer is discussed, extended to the tetrameric molecule and compared to the published models of human alpha 2-macroglobulin.

Cadmium↗
PubMed1984

alpha 2-Macroglobulin-proteinase complexes in cystic fibrosis serum. Analysis by monoclonal antibodies and receptor-mediated endocytosis by normal human fibroblasts.

alpha 2-Macroglobulin complexed to proteinases activated during clotting of cystic fibrosis and control sera was quantitated with the complex-specific monoclonal antibody F2B2 . Similar amounts of alpha 2-macroglobulin complexes (between 40 and 90 micrograms/ml) were generated in cystic fibrosis and control sera. Endocytosis of the complexes by normal human fibroblasts was compared to the amount of complexes detected by the F2B2 -radioimmunoassay. Normal uptake was observed with 13 out of 14 cystic fibrosis sera. One cystic fibrosis serum showed strongly reduced endocytosis of the complexes. Complexes isolated from this serum on immobilized F2B2 failed to inhibit binding of purified alpha 2-macroglobulin-trypsin to its receptor, demonstrating deficient receptor-binding of these complexes. The low uptake complexes could not be distinguished from complexes isolated from control or other cystic fibrosis sera by isoelectric focusing, rate electrophoresis or SDS-polyacrylamide gel electrophoresis.

Animals↗
PubMed1986

Alpha-2-macroglobulin, albumin, and chymotrypsin inhibitory capacity in cerebrospinal fluid as indices of blood-cerebrospinal fluid barrier.

Concentration of alpha-2-macroglobulin, albumin, and chymotrypsin inhibitory capacity representing mainly alpha-1-proteinase inhibitor were estimated in cerebrospinal fluid in disorders of the central nervous system. While chymotrypsin inhibitory capacity was elevated in all cases with derangement of the blood-cerebrospinal fluid barrier, in 30% of the cases alpha-2-macroglobulin levels were in the normal range. The difference can be attributed to the much larger size of the latter. Better correlation between albumin concentration and chymotrypsin inhibitory capacity (r = 0.84) than between albumin and alpha-2-macroglobulin (r = 0.62) supports the view that the rate of entry of proteins from blood into cerebrospinal fluid is inversely related to their size.

Albumins↗
PubMed1991

Structural studies of human alpha 2-macroglobulin: concordance between projected views obtained by negative-stain and cryoelectron microscopy.

Two views of native alpha 2-macroglobulin are revealed by electron microscopy of negatively stained samples; in one view the molecule resembles a padlock and in the other, a pair of lips. Interconversion of the two views upon tilting establishes that these are two different projected views of the same structure. Furthermore, the two views are related by a 45 degrees rotation about their major axis because they interconvert when the specimens are titled +/- 22.5 degrees. Negatively stained molecules on Butvar films present a nearly equal distribution of the two views, whereas in frozen-hydrated samples the molecules almost exclusively are oriented in the lip view. Measurements from both views indicate that the alpha 2-macroglobulin molecule is approximately 200 A long and approximately 140 A wide. Our results suggest that alpha 2-macroglobulin is composed of two protomeric units, each in the shape of a twisted letter S. These units are joined together at their ends to form a complex with point group symmetry 222. The 45 degrees interconversion angle between the lip and padlock views support this arrangement. Average images of unstained and stained lips are quite similar, indicating that the native structure is consistently preserved by the two electron microscopy procedures used in this investigation. This is substantiated by the interconversion between the lip and padlock views that occurs when the molecule is rotated 45 degrees [corrected] about its major twofold axis.

Cryopreservation↗
PubMedDate not supplied

Three-dimensional structures of the human alpha 2-macroglobulin-methylamine and chymotrypsin complexes.

The three-dimensional structures of chymotrypsin- and methylamine-treated negatively stained human alpha 2-macroglobulin have been determined by weighted back projection from electron microscope data. Projections of the reconstructions show good concordance with two-dimensional averages of both stained and frozen-hydrated molecules. The reconstructions reveal that the H-shaped front projection of the molecule is related to the smaller ellipsoidal end view by a rotation of 90 degrees about the crossbar (minor axis) of the H. This finding is in agreement with tilt studies. The reconstruction of the alpha 2-macroglobulin-methylamine reveals an hour-glass shaped void which is filled by the two proteinase molecules in the reconstruction of alpha 2-macroglobulin-chymotrypsin. Protein plugs which appear to block the exterior entrances to the cavity may function to prevent access of proteins to the encapsulated proteinase and serve to block its escape. Extensive thresholding of each reconstruction leaves a "backbone" consisting of two side-by-side rod-like structures, suggesting that this is the arrangement of the two protomeric units which form the molecule. Both structures show some departure from the expected symmetry. The asymmetries are robust features of the reconstructions and may reflect structurally asymmetric features of the transformation from the native to the chymotrypsin-treated form of the molecule.

Binding Sites↗
PubMed2004

A novel serine protease complexed with alpha2-macroglobulin from skeletal muscle of lizard fish (Saurida undosquamis).

A novel fish muscle serine protease named muscle soluble serine protease (MSSP) was purified from the soluble fraction of lizard fish (Saurida undosquamis: Synodontidae) muscle by ammonium sulfate fractionation followed by four steps of column chromatographies. In native-PAGE, the purified enzyme appeared as a single band with an estimated mol. mass of approximately 380 kDa by gel filtration. In SDS-PAGE under reducing conditions, the purified enzyme migrated as two protein bands at 110 and 100 kDa, named subunits A and B, respectively. The 20 residues of N-terminal amino acid sequence of subunit B showed 70% of homology to beta-chain of carp alpha(2)-macroglobulin-1. Moreover, both subunits A and B showed immunoreactivity with anti carp alpha(2)-macroglobulin antibody. Purified MSSP was inactivated by Pefabloc SC, aprotinin, benzamidine and TLCK, but not by alpha(1)-antitrypsin. After acid treatment (pH 2, 24 h), however, the enzyme activity eluted at 14 kDa from Sephacryl S-200 carried out under acidic conditions was inhibited by alpha(1)-antitrypsin. Lizard fish MSSP most rapidly hydrolyzed Boc-Val-Pro-Arg-MCA and Boc-Gln-Arg-Arg-MCA, but did not hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA and Suc-Ala-Ala-Pro-Phe-MCA, and was not suppressed either by E-64, pepstatin A and ethylenediaminetetraacetic acid (EDTA). These results indicate that the purified MSSP is a serine protease complexed with alpha(2)-macroglobulin, and the entrapped protease was dissociated by the acid treatment. Purified and free MSSPs were most active at pH 10.0 and 9.0, respectively. Purified MSSP degraded myofibrillar proteins and casein but time courses of degradation of these substrates by the enzyme differed.

Amino Acid Sequence↗
PubMed1999

alpha2-macroglobulin- and murinoglobulin-1- deficient mice. A mouse model for acute pancreatitis.

Mice deficient in either or both mouse alpha2-macroglobulin (MAM) and murinoglobulin-1 (MUG1) were generated and proved phenotypically normal under standard conditions. Acute pancreatitis was induced with a diet deficient in choline and methionine, supplemented with ethionine. The mortality was less than 25% in wild-type mice, as opposed to at least 56% in knockout mice, and was highest (70%) in MAM-/- mice, with earliest onset at 2 days. Plasma amylase and lipase levels were increased, but pancreatic tissue appeared histologically variable in individual mice. The clinical symptoms were most severe in MAM-/- mice and, surprisingly, were not aggravated in the double knockout mice, suggesting that the lack of proteinase inhibition capacity was not the major problem. Therefore, we analyzed the expression of 21 different cytokines and polypeptide factors in the pancreas of all experimental groups of mice. Interleukin-1-receptor antagonist mRNA was consistently induced by the diet in the pancreas of MAM-/- mice, and transforming growth factor-beta, tumor necrosis factor-alpha, tumor necrosis factor-beta, beta-lymphotoxin, and interferon-gamma mRNA levels were also increased. The data demonstrate the important role of alpha2-macroglobulin (A2M) in acute pancreatitis as both a proteinase inhibitor and a cytokine carrier. Mice deficient in MAM and/or MUG thus offer new experimental models for defining in vivo the role of the macroglobulins in pancreatitis and in other normal and pathological processes.

Acute Disease↗