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PubMed1998

Prevention of the influence of fibrin and alpha2-macroglobulin in the continuous measurement of the thrombin potential: implications for an endpoint determination of the optical density.

We proposed the endogenous thrombin potential (ETP) as an overall function test of the coagulation system. We recently introduced a routine test which requires defibrinated plasma. In order to develop an assay in which the ETP-value can be directly obtained by measuring the optical density, we investigated two methods to inhibit fibrinogen clottability and to inactivate alpha2-macroglobulin. The first method makes use of hydroxylamine to inactivate alpha2-macroglobulin and H-Gly-Pro-Arg-Pro-OH to inhibit fibrin polymerization. At pH 7.35, plasma incubated with 25 mM hydroxylamine and 1.5 mg/mL H-Gly-Pro-Arg-Pro-OH for 5 minutes at 37 degrees C resulted in a reduced endlevel of the amidolytic activity on small chromogenic substrates. The second method uses a metalloprotease purified from Crotalus basiliscus to remove alpha2-macroglobulin from plasma in combination with H-Gly-Pro-Arg-Pro-OH. Herein plasma is incubated with 3.5 LM protease during 15 minutes at 37 degrees C in the presence of 1 mg/mL polymerization inhibitor. The enzymatic method results in a zero endlevel of the amidolytic activity and this would imply that measurement of the ETP is reduced to an endpoint determination of the optical density. We show that the endpoint determination of the optical density correlates well with the calculated ETP in plasmas with different degrees of anticoagulation.

Biomarkers↗

PubMed2000

Polymorphisms in the alpha-2 macroglobulin gene in psychogeriatric patients.

Recent reports have suggested that genetic polymorphisms in the alpha-2 macroglobulin (A2M) gene are associated with an increased risk for Alzheimer's disease. In the present study we tested two polymorphisms in the alpha-2 macroglobulin gene, a 5 bp deletion at the 5' splice site of exon 18 and a G/A point mutation (V1000I) in exon 24, in a sample of 118 healthy, non demented controls and 238 consecutively recruited gerontopsychiatric patients, diagnosed as: Alzheimer's disease (N=88), mild cognitive impairment (N=32), subjective cognitive complaints (N=54) and depression/other psychiatric disorders (N=64). The aim of this study was to test whether the investigated polymorphisms has a high enough selectivity and specificity to distinguish between the different gerontopsychiatric disorders or to differentiate genetically AD from other forms of dementia, respectively. Also a possible relation to the APOE varepsilon4 polymorphism was examined. Our study failed to show an association between the two investigated polymorphisms in the alpha-2 macroglobulin gene and any of the four different psychogeriatric patient subgroups, either alone or in combination with the APOE varepsilon4 genotype.

Aged↗

PubMed1998

Relationships of liver weight, cholesterol, albumin and alpha2-macroglobulin concentrations with ovarian function in swine.

In two genetic swine models selected for diversity in ovulation rates (White composite controls and ovulation rate selection line, n = 131; 1/2 White composite: 1/2 Meishan crossbreds, n = 387), a positive relationship was established with liver weight and ovulation rate (P < 0.01). Serum changes of cholesterol, albumin and alpha2-macroglobulin were monitored during various stages of the luteal phase and follicular phase (days 17 and 19 of the estrous cycle; 1/2 White composite: 1/2 Meishan gilts). Serum cholesterol concentrations increased with liver weights (r = 0.19; P < 0.01) and corpora lutea numbers (r = 0.14; P < 0.01). Albumin concentrations were negatively correlated with corpora luteal numbers (r = -0.3; P < 0.01) but had no relationship with liver weight. Serum concentrations of alpha2-macroglobulin were not related to liver weight or corpora lutea numbers. Circulating concentrations of cholesterol and alpha2-macroglobulin increased with day of the estrous cycle (P < 0.01). Testosterone concentrations were inversely related to circulating cholesterol concentrations during the estrous cycle, but testosterone concentrations on day 17 or 19 of the cycle were unrelated to corpora lutea numbers. Concentrations of estrone on day 17 or 19 (as an index of follicles destined to ovulate) were also not related to numbers of corpora lutea. Many interactions between liver and ovarian function involving metabolic and endocrine systems are plausible, but defined mechanisms resulting in coordinate increases in liver weight and ovulation rates are presently unelucidated.

Animals↗

PubMed2000

Specific binding of alpha-macroglobulin to complement-type repeat CR4 of the low-density lipoprotein receptor-related protein.

The low-density lipoprotein receptor-related protein (LRP) is a large surface receptor that mediates binding and internalization of a large number of structurally and functionally unrelated ligands. The ligand binding sites are located in clusters of complement-type repeats (CR), where the general absence of mutual binding competition suggests that different ligands map to distinct sites. Binding of alpha(2)-macroglobulin-protease complexes to the LRP is mediated by the receptor binding domain (RBD) of alpha(2)-macroglobulin (alpha(2)M). To determine the major binding epitope(s) in the LRP, we generated a complete set of tandem CR proteins spanning the second cluster of CR domains, and identified a binding site for alpha(2)M in the N-terminal part of the cluster comprising CR3-CR6, using ligand blotting and surface plasmon resonance (SPR) analysis. The specific site involved in alpha(2)M recognition resides in the fourth CR domain, CR4, whereas another site is identified in CR5. An acidic epitope in CR4 is identified as important for binding alpha(2)M by mutagenesis and SPR analysis. The formation of the complex between the rat alpha(1)-macroglobulin RBD and CR domain pairs is characterized by analytical size-exclusion chromatography, which demonstrates a sufficiently strong interaction between the alpha(1)M RBD and CR34 or CR45 for the isolation of a complex.

Amino Acid Sequence↗

PubMed1991

X-ray scattering study of hagfish protease inhibitor, a protein structurally related to complement and alpha 2-macroglobulin.

A protease inhibitor from hagfish blood plasma, homologous to human alpha 2-macroglobulin, has been studied in solution using small-angle X-ray scattering; the radius of gyration, R, was found to be 7.0 nm, the molecular weight 340,000 +/- 20,000, and the largest distance within the molecule, Dmax, 22 nm. When the inhibitor reacts with chymotrypsin, its 1:1 chymotrypsin complex is found to be more compact than the native molecule, R = 6.1 nm. A very similar conformational change is observed after the protein is reacted with methylamine. The data are consistent with models consisting of two equal elliptic cylinders with the same size as the one used as a model for the complement proteins C3 and C4 [cf. Osterberg et al. (1989) Eur. J. Biochem. 183, 507-511]. In the model for the native protein, these cylinders are arranged in an extended form, and in the one for the methylamine derivative (or chymotrypsin complex), they are closer together so that the projection of their elliptic surfaces forms an angle of about 70 degrees. These models for the hagfish protease inhibitor were expanded to models for the twice as large human alpha 2-macroglobulin using symmetry operations, and the resulting alpha 2-macroglobulin models were found to agree with those emerged from earlier studies involving electron microscopy and X-ray scattering methods.

Animals↗

PubMed1984

Kinetics of appearance of sulfhydryl groups in alpha 2-macroglobulin on reaction of the inhibitor with amines.

The mechanism of the appearance of sulfhydryl groups in alpha 2-macroglobulin in the reaction with amines was characterized by analyses of the kinetics with ammonia and methylamine. All reactions occurred under pseudo-first-order conditions in the range of pH (7.0-8.6) and amine concentration (10-600 mM) investigated. The logarithm of the pseudo-first-order rate constant increased linearly as a function of pH with a slope of unity, indicating that the unprotonated amine is the active species in the reaction. Plots of the observed pseudo-first-order rate constants vs. concentration of unprotonated amine at constant pH were also linear and gave second-order-rate constants of 0.32 and 13.8 M-1 s-1 for ammonia and methylamine, respectively, at pH 8.0; similar values were obtained at pH 8.6. Activation energies of 85 and 100 kJ mol-1 and activation entropies of 10 and 95 J K-1 mol-1 for ammonia and methylamine, respectively, were estimated from Arrhenius plots, suggesting that the higher reaction rate for methylamine is due primarily to a higher activation entropy. These results are consistent with the release of sulfhydryl groups being caused by a nucleophilic attack of the uncharged amine on a thio ester bond of alpha 2-macroglobulin in a bimolecular reaction occurring under pseudo-first-order conditions. The characteristics of the reaction suggest that the thio ester in each alpha 2-macroglobulin subunit reacts independently and equivalently with the amine and also that the thio ester bond cleavage initiates the reaction sequence leading to inactivation of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Amines↗

PubMed1985

Purification and characterization of frog alpha-macroglobulin: receptor recognition of an amphibian glycoprotein.

Frog alpha-macroglobulin was purified to apparent homogeneity by Ni2+ chelate affinity chromatography. Frog alpha-macroglobulin migrated as an alpha 1-globulin in cellulose acetate electrophoresis. A molecular weight of 730 000 was obtained by equilibrium sedimentation, and in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), the protein migrated as a single band of Mr approximately 360 000 before reduction and Mr approximately 180 000 after reduction. Treatment with trypsin resulted in subunit cleavage to yield a fragment of Mr approximately 90 000. After being heated, the protein fragmented, migrating in SDS-PAGE as two bands of Mr approximately 120 000 and 60 000. This fragmentation was inhibited by prior reaction of the protein with methylamine. In native pore-limit electrophoresis the protein exhibited the characteristic "slow" to "fast" conformational change of protease-treated alpha-macroglobulins. In contrast, typical "slow" to "fast" conformational change was not observed in native PAGE with this preparation. Moreover, the protein incorporated approximately 2 mol of [14C]methylamine/mol of inhibitor without demonstrating a change in mobility in native PAGE. In circular dichroism studies, the protein exhibited a spectrum similar to that of human alpha 2M. Reaction with trypsin resulted in a broadening and decrease in the magnitude of the spectrum. Reaction with methylamine resulted in similar changes, but of smaller magnitude. The inhibitor bound approximately 0.7 mol of trypsin in both radiolabeled protease binding and amidolytic titration studies. 125I-Labeled native frog alpha 1M was removed slowly from the circulation of mice with a t1/2 greater than 2h.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1986

Identification of 1H resonances from the bait region of human alpha 2-macroglobulin and effects of proteases and methylamine.

The 1H NMR spectrum of human alpha 2-macroglobulin, Mr 716,000, consists of predominantly extremely broad unresolved resonances but also has nine relatively sharp (delta nu 1/2 less than 25 Hz) resonances from aromatic residues. By treatment of alpha 2-macroglobulin with methylamine, chymotrypsin, and subtilisin, it has been shown that eight of these resonances arise from bait region residues. More specifically, assignment has been made of resonances at 6.80 and 7.11 ppm to the ortho and meta protons, respectively, of tyrosine-685 and tentative assignment of a resonance at 7.29 ppm to the aromatic protons of phenylalanine-684. C2 proton resonances from five histidine residues are also visible. Four of these are attributed to residues in the bait region or immediately adjacent to this, at positions 675, 694, 699, and 704. The sharpness of resonances from bait region residues demonstrates the great flexibility of this region of the polypeptide. It is proposed that the flexible region extends from residue 675 to residue 710. These resonances are all affected by proteolytic cleavage in the bait region but are not influenced by the subsequent conformational rearrangement of the whole protein tetramer. The significance of these findings is discussed in relation to the current structural models of alpha 2-macroglobulin.

Humans↗

PubMed1987

In support of the trap hypothesis. Chymotrypsin is not rigidly held in its complex with human alpha 2-macroglobulin.

Complexes (2:1) of chymotrypsin with human alpha 2-macroglobulin have been prepared in the presence of 200 mM methylamine such that 90% of the chymotrypsin remains noncovalently bound to the alpha 2-macroglobulin. Reaction of this complex with the active-site-directed spin-labeling reagent 4-[(ethoxyfluorophosphinyl)oxy]-2,2,6,6-tetramethylpiperidinyl+ ++-1-oxy results in nitroxide labeling of the active-site serine residue of the complexed chymotrypsin. Electron spin resonance (ESR) spectra of this complex were recorded at 275 K in buffer and at 263 K in 50% glycerol. At 263 K in 50% glycerol the spectrum is that expected for a rigid glass, whereas at room temperature the ESR spectrum shows that the chymotrypsin is only slightly immobilized compared with free spin-labeled chymotrypsin. These findings are discussed in relation to possible models of inhibition of protease activity by alpha 2-macroglobulin. It is concluded that the trap mechanism of Barrett and Starkey [Barrett, A. J., & Starkey, P. M. (1973) Biochem. J. 133, 709-724] is the only model currently considered that can account for the present findings.

Chymotrypsin↗

PubMed1989

Preparation and initial characterization of an intermediate, half-cleaved form of human alpha 2-macroglobulin.

A form of human alpha 2-macroglobulin (alpha 2M) has been prepared that has properties intermediate to those of native alpha 2-macroglobulin and 2:1 protease-alpha 2 M ternary complex by using Sepharose-linked chymotrypsin. The intermediate form has mobility on native polyacrylamide gels between the fast and slow forms of alpha 2M and migrates as a diffuse band. Two bait regions and two thiol esters per alpha 2M tetramer are cleaved, although no chymotrypsin is detectable in the modified alpha 2-macroglobulin species. The remaining bait regions and thiol esters can be cleaved by further reaction with other proteases. Intermediate-form alpha 2M can trap 1.18 mol of chymotrypsin, 0.85 mol of trypsin, and 0.65 mol of thrombin. Although both thrombin and methylamine react with intermediate-form alpha 2M at rates not distinguishable within experimental error from those of their reactions with native alpha 2M, chymotrypsin-Sepharose reacts much more slowly with the intermediate form than with native alpha 2 M, indicating a nonequivalence of the two reactive sites on alpha 2M. This nonequivalence may be present initially or be induced by reaction at the first site. Comparison of ESR results obtained from spin-labeling methylamine-treated or protease-reacted alpha 2M with those from spin-labeling of the free SH groups in intermediate-form alpha 2M shows that trapped protease influences the mobility of the attached nitroxide either through direct contact or by producing a different conformation from that present in methylamine-treated or intermediate-form alpha 2M.

Binding Sites↗

PubMed1973

Inhibition by alpha-macroglobulin and other serum proteins.

1. Normal human serum was found to inhibit human cathepsin B1. 2. The major inhibitor present in serum was purified and identified as alpha(2)-macroglobulin. 3. alpha(2)-Macroglobulin was found to bind cathepsin B1 in an approximately 1:1 molar ratio. When bound, the enzyme retained about 50% of its proteolytic activity, and up to 80% of its activity against alpha-N-benzoyl-dl-arginine 2-naphthylamide. 4. Pretreatment of alpha(2)-macroglobulin with cathepsin B1 inactivated by exposure to pH8.5 or iodoacetic acid, in large molar excess, did not prevent the subsequent binding of active enzyme. Active enzyme, once bound, was not protected from inhibition by 1-chloro-4-phenyl-3-tosylamido-l-butan-2-one. 5. Cathepsin B1 was also inhibited by human immunoglobulin G, at high concentration. 6. Because it had been suggested that haptoglobin is responsible for the inhibition of ;cathepsin B' by serum, a method was devised for the selective removal of haptoglobin from mixtures of serum proteins by adsorption on haemoglobin covalently linked to Sepharose. No evidence was obtained that haptoglobin has any inhibitory activity against the enzyme.

Animals↗

PubMed1986

Structural analysis of acute-phase alpha 2-macroglobulin.

High resolution images of rat acute-phase alpha 2-macroglobulin (AP alpha 2M) have been obtained by using dark-field electron microscopy. No staining or artifact-inducing procedures were used. Analysis of unfiltered electron microscope plates, exposed to minimal electron beam radiation, revealed highly contrasted particles of variable morphology with dimensions of approx. 19 nm X 14 nm. An electron-dense core with four to six projections could be seen. Two-fold symmetry was evident in selected images, supporting the four-subunit composition of the protein. Image processing and filtering confirmed the presence and configuration of the projections by demonstrating exact molecular dimensions of 16 nm X 9.5 nm and a shape with six projections like that of the Russian letter zh. SDS/polyacrylamide-gel electrophoresis revealed that this molecule was in the proteinase-bound form. C.d. data revealed a surprisingly low content of alpha-helical secondary structure (12%) and an atypically large content of beta-form structure (33%). Comparison of the amino acid compositions of AP alpha 2M and human alpha 2-macroglobulin indicated a high degree of homology between the two molecules. It is concluded that the conformation of rat AP alpha 2M, both at the molecular and secondary structural levels, is strikingly similar to that of human alpha 2-macroglobulin.

Amino Acids↗

PubMed1996

Structural and functional analysis of the spontaneous re-formation of the thiol ester bond in human alpha 2-macroglobulin, rat alpha 1-inhibitor-3 and chemically modified derivatives.

The alpha-macroglobulins are proteinase inhibitors that form part of a superfamily along with components of the complement system. Internal beta-cysteinyl-gamma-glutamyl thiol ester bonds are an important structural feature of most alpha-macroglobulins and several complement components. We have studied the reversibility of thiol ester cleavage caused by NH3 or CH3NH2 in tetrameric human alpha 2-macroglobulin (alpha 2M) and monomeric rat alpha 1-inhibitor-3 (alpha 1I3). When employing NH3 as the nucleophile, the thiol ester in alpha 1I3 re-formed spontaneously at room temperature after gel filtration to remove excess nucleophile, and an active proteinase inhibitor was regained. When CH3NH2 was employed as the nucleophile, thiol ester reversibility was more energy-demanding. With either nucleophile, alpha 2M once inactivated did not regain proteinase-inhibitory capacity at room temperature. At elevated temperatures, however, the reaction between alpha 2M and NH3 or CH3NH2 was reversible and the inhibitory capacity could be recovered. Modification of the cysteinyl groups from the thiol ester prevented its re-formation but did not prevent the heat-induced retrieval of inhibitory capacity, suggesting that conformational features rather than the thiol ester are essential for alpha 2M to function as an inhibitor. As demonstrated by non-denaturing PAGE, the conformation of native alpha 2M is restored when the proteinase-inhibitory capacity is recovered.

Acute-Phase Proteins↗

PubMed1997

Modification of the erythrocyte surface in rats bearing Yoshida ascites sarcoma is brought about by a tumour variant of alpha2-macroglobulin.

Erythrocytes from the circulation of rats bearing Yoshida ascites sarcoma exhibit higher concanavalin A (ConA)-mediated agglutinability than those from normal animals. A tetrameric glycoprotein of subunit molecular mass 170 kDa, purified from the cell-free ascites fluid, was found to confer higher ConA-mediated agglutinability on erythrocytes in vitro. An antiserum to this tumour-derived protein failed to detect any cross-reactive component in normal rat plasma or in any of the normal tissues examined. An immunoreactive protein was, however, detected in blood plasma when the acute-phase reaction was stimulated by injection of turpentine. The cross-reactive acute-phase protein was purified by ConA-affinity, gel-filtration and ion-exchange chromatography, and identified as alpha2-macroglobulin. The acute-phase protein and the protein obtained from the ascites fluid have identical or very similar native and subunit molecular masses, subunit arrangement and pI. They both are able to inhibit trypsin and, as a consequence, acquire greater mobility in native PAGE. In addition, the two proteins bind to rat erythrocytes non-specifically, and in similar amounts. However, despite these similarities, the acute-phase protein is unable to enhance the agglutinability of erythrocytes. The two proteins differ in their carbohydrate content, but this differential glycosylation is not the cause of the difference in their surface modification activity. The chemically deglycosylated proteins show a small but consistent difference in the size of their polypeptides. Their tryptic peptide maps, although largely similar, show some differences, as do their amino acid compositions. It is probable that the proteins are independent members of the same (alpha-macroglobulin) family. The rat embryo is also found to express a soluble protein consisting of a 170 kDa polypeptide that cross-reacts with the antibody to the tumour-derived protein. The purified embryo protein is able to alter the ConA-mediated agglutinability of erythrocytes in vitro, and also yields a tryptic peptide map that is identical to that of the tumour-derived protein. The modification of the host cell surface in the tumour-bearing rats is thus caused by what appears to be a tumour (oncofetal?) variant of alpha2-macroglobulin.

Agglutination↗

PubMed2000

Conformational variants of human alpha2-macroglobulin are reflected in a C-terminal 'switch region'.

Human alpha2-macroglobulin displays extensive conformational changes when induced to transform into new quaternary structures, which are eliminated from the systemic circulation by receptor-mediated endocytosis. One major region involved in these conformational changes is located in a segment of 30 amino acids from Glu1314 to Ser1343 (-Glu-Glu-Phe-Pro-Phe-Ala-Leu-Gly-Val-Gln-Thr-Leu-Pro-Gln-Thr-Cys-Asp -Glu-Pro-Lys-Ala-His-Thr-Ser-Phe-Gln-Ile-Ser-Leu-Ser-), which we term the 'switch region' of alpha2-macroglobulin, as deduced by immunochemical techniques. Monoclonal antibodies were generated using either native, methylamine-treated or the 18-kDa C-terminal receptor-binding fragment as the immunogen. From an extensive number of obtained hybridomas, 11 mAbs were selected because of their capacity to bind to the C-terminal fragment. Irrespective of the original configuration of the antigen used for immunization, seven of the antibodies were shown to be reactive with a set of overlapping epitopes, closely positioned within the 'switch region', as confirmed by the use of synthetic peptides covering the entire C-terminal fragment. The specificities of the seven individual antibodies, as determined by ELISA and BIAcore technologies, revealed a pronounced conformational pleomorphism in the 'switch region'. The results indicate that the 'switch region' may be involved in the exposure of the receptor recognition site and can be used as an indicator region for different conformational states of alpha2-macroglobulin.

Amino Acid Sequence↗

PubMed2000

Rat alpha1-macroglobulin enhances nerve growth factor-promoted neurite outgrowth, TrkA phosphorylation, and gene expression of pheochromocytoma PC12 cells.

Monoamine-activated human alpha2-macroglobulin (alpha2M) has been previously demonstrated to inhibit TrkA-, TrkB-, and TrkC-mediated signal transduction. Rat alpha1-macroglobulin (alpha1M) and alpha2M are structural homologues of human alpha2M, but rat alpha1M is distinctly different from rat alpha2M in many ways and its role in the mammalian nervous system is unknown. In this report, monoamine-activated rat alpha1M was demonstrated to enhance in a dose-dependent manner nerve growth factor (NGF)-promoted neurite outgrowth in pheochromocytoma PC12 cells. Monoamine-activated alpha1M by itself, however, was neither neurotrophic nor mitogenic to PC12 cells. To investigate further its possible mode of action, the ability of monoamine-activated alpha1M and normal alpha1M to bind and to activate the NGF receptor (TrkA) was investigated. Monoamine-activated alpha1M formed a more stable complex with TrkA than normal alpha1 M, but the binding of monoamine-activated alpha1M to TrkA was adversely affected by prior stimulation of TrkA with NGF. In addition, monoamine-activated alpha1M enhanced the NGF-promoted TrkA phosphorylation and up-regulated the expression of NGF-inducible immediate-early genes (c-jun and NGFI-A) and delayed-response genes (SCG10 and transin) in PC12 cells; normal alpha1M, in contrast, produced little or no effect. This study demonstrates that alpha1M, the constitutive form of alpha-macroglobulin in the rat, possesses the ability to promote NGF-mediated differentiation in PC12 cells, possibly via its direct action on TrkA receptors and TrkA-mediated signal transduction and gene expression.

Animals↗

PubMed1998

Collagenase-1 complexes with alpha2-macroglobulin in the acute and chronic wound environments.

The purpose of this study was to examine the appearance and activation of collagenase-1 (MMP-1) in the wound environment. We found that MMP-1 accumulates in the fluid phase of the burn wound environment within 2 d of injury and reaches maximal levels by day 4. Two forms of the enzyme were evident; one that corresponded to proMMP-1 and another that corresponded to a group of high molecular mass (approximately 200 kDa and >200 kDa doublet) MMP-1 containing complexes. ProMMP-1 and MMP-1 containing complexes also occurred in wound fluid from venous stasis ulcers, but neither was detected in mastectomy fluid or in plasma. Levels of the proteinase inhibitor alpha2-macroglobulin in burn fluid and chronic ulcer wound fluid were almost as high as in plasma, and the high molecular mass MMP-1 containing complexes in burn fluid appeared to result from binding between alpha2-macroglobulin and activated MMP-1. These observations provide direct evidence that active MMP-1 in the fluid phase of the wound environment becomes complexed to alpha2-macroglobulin.

Acute Disease↗

PubMed2002

A slow form of alpha-2-macroglobulin in diseased and healthy dogs.

Alpha-macroglobulins (AMs) function as non-specific protease inhibitors by using a so-called trapping mechanism, which is a compaction of the molecule that can be seen as a "fast" form in native polyacrylamide gel electrophoresis (PAGE). AMs also play a role in the transport and clearance of cytokines and growth factors from the circulation. In the dog, two AMs are known, alpha-1-macroglobulin (A1M) and alpha-2-macroglobulin (A2M). Using agarose and polyacrylamide gel electrophoresis of canine serum or plasma, we detected a cathodal, slow form of A2M. Upon activation with elastase, slow A2M resembled normal A2M in agarose gel electrophoresis, showing decreased negative charge at semi-saturation but not at full saturation with enzyme. In PAGE, however, slow A2M, unlike normal A2M, did not exhibit a "fast" form after short-term incubation with elastase. After incubation overnight, the "fast" form was seen, indicating a retarded reaction. Incubation of slow A2M with ammonium sulphate, a known activator of AMs, resulted in decreased negative charge in agarose gel electrophoresis and no reaction or partial reaction in PAGE. Slow A2M is present in fresh blood samples or may develop from partial alterations within a few days of storage. Moreover, it is sometimes reversible. Our findings may indicate that slow A2M is a result of an instability of the molecule, leading to a conformational change, which affects electrical charge and impairs the ability to develop into the "fast" form upon activation. This may lead to a delayed clearance of protease and inflammatory mediators from the circulation. Slow A2M was predominantly found in diseased dogs, especially in the Labrador retriever.

Animals↗