Human cathepsin B1. Inhibition by α2-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.     

Contribution of the allelicMtz 3 andMtz 4 allotype genes to the formation of individual rabbit serum α2-macroglobulin molecules

The contribution of the allelicMtz ³ andMtz ⁴ genes to the formation of individual rabbit serum α₂-macroglobulin (α₂M) molecules was examined by precipitation of α₂M from rabbits of known genotype with antiallotype antisera. The α₂M was isolated fromz ³z³ andz ⁴z⁴ homozygous andz ³z⁴ heterozygous rabbits, iodinated with I¹²⁵ and precipitated by sequential reactions with antiallotype antiserum and goat anti-rabbit IgG. Purified unlabeled α₂M or α₂M in serum was used to inhibit competitively the reaction of antiallotype antiserum and labeled α₂M. Nearly all α₂M molecules have z3 or z4 antigenic determinants; approximately 50% of α₂M molecules in heterozygotes have both. Altogether, the z3, z3,4, and z4 molecules in heterozygotes have approximately 60% of the number of z3 and 40% of the number of z4 determinants as compared to the respective homozygotes. Unlike all other known allelic blood protein systems of rabbits, allelic exclusion does not occur in α₂M molecules of heterozygotes; rather, hybrid molecules are formed. Presented in part at the Fifty-fourth and Fifty-fifth Annual Meetings of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April 12–17, 1970, and Chicago, Illinois, April 12–17, 1971. This investigation was supported in part by U.S. Public Health Service Grants AI-09241 and AI-07043. B.H.B. performed this investigation in partial fulfillment of the requirements for the Doctor of Philosophy Degree in the Graduate College; he is supported by a postdoctoral fellowship from the Schweppe Foundation. K.L.K. is the recipient of U.S. Public Health Service Research Career Development Award AI-28687.     

The interaction between α2-macroglobulin and cationic aspartate aminotransferase

On starch-gel or polyacrylamide-gel electrophoresis of human serum, a supernumerary zone of aspartate aminotransferase activity may be demonstrated, migrating with the slow alpha(2) protein zone. This appearance is due only to cationic aspartate aminotransferase, bound by alpha(2)-macroglobulin. The binding is strongly potentiated by dilute borate buffers.     

Interaction of α2-macroglobulin with L-asparaginase

Summary Obvious protection of the catalytic activity of Esch. coli L-asparaginase by ₂-macroglobulin (₂M) was observed under conditions otherwise propitious to the dissociation of the tetrameric molecule into inactive subunits, i.e. very diluted enzyme solutions or the presence of either SDS or urea. The degree of protection depended on enzyme and ₂M concentrations respectively, and on the preincubation time of the ₂M-enzyme mixture prior to substrate addition. The formation of a catalytically active complex between ₂M and L-asparaginase was confirmed by gel filtration on a Sephadex-G column and by polyacrylamide gel electrophoresis. The fact that the migration distance of the active complex corresponded to the migration of ₂M and the absence in that case of a migration band corresponding to the intact molecule suggest that complexing of the enzyme with ₂M prevented its dissociation into subunits and thus its inactivation. Addition of ₂M to the already dissociated enzyme molecule did not restore its catalytic activity.Alpha₂-macroglobulin was shown to have an inhibiting effect on the proteolytic activity of almost all proteases and no effect on their esterolytic activity. Furthermore, it prevents the inhibition of esterolytic activity by some natural compounds^1–5. The effect of ₂M on other types of catalytic activity has not been investigated enough to afford a generalization of the possible role of this macroglobulin in the control of enzyme activity in the body.This paper reports the results of an in vitro study of the effect of ₂M on the catalytic activity of an important amidase, i.e. L-asparaginase (L-asparagine amidohydrolase 3.5.1.1), which in recent years has been used in the treatment of acute lymphocytic leukemia in children^6,7.Abbreviations ₂M ₂-macroglobulin - E enzyme - SDS sodium dodecylsulfate Part of the results were reported at the 10th International Congress of Biochemistry, Hamburg 1976, Abst. p. 377.     

The isolation and partial characterization of α2-macroglobulin from the serum of the ostrich (Struthio camelus)

• 1.1. α₂-Macroglobulin (α₂M) activity is present in the serum of the ostrich, Struthio camelus. The chromogenic synthetic peptide substrates BAPNA and ATNA were hydrolysed by trypsin and chymotrypsin, respectively, in the presence of ostrich serum and the α₂M in ostrich serum protected trypsin from being inhibited by soybean trypsin inhibitor. Ostrich α₂M proved to be a potent inhibitor of bovine pancreatic trypsin and chymotrypsin.
• 2.2. α₂M was purified to apparent homogeneity by PEG precipitation, DEAE-Toyopearl 650M, Bio-Gel A-5m and Zn²⁺-affinity chromatography.
• 3.3. Ostrich α₂M migrated as a single band (M_r 779,000) during non-denaturing gradient gel electrophoresis and showed increased mobility after reaction with trypsin. Denaturation dissociated ostrich α₂ M into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits.
• 4.4. Isoelectric focusing revealed a pI of 5.3.
• 5.5. The amino acid composition of ostrich α₂M is typical of an α₂M, comparing favourably with those of other animal species. The carbohydrate composition of the purified protein, in percentage dry weight of the molecule, was galactose: mannose (1:1), 4.55; N-acetylglucosamine, 2.35; N-acetylneuraminic acid, 0.58; and fucose, 0.77.
• 6.6. α₂M was assessed immunologically by Ouchterlony double-diffusion and Western blot analysis with polyvalent antisera directed against ostrich α₂M.
• 7.7. Ostrich α₂M seems to show many physical, chemical and kinetic properties similar to those of other known α₂Ms, but is expected to differ from other αMs when considering the primary structure of the bait region, the area differing among α Ms from different species and determining its specificity.

     

Lack of association between α2-macroglobulin polymorphisms and Alzheimer's disease

This study was undertaken to investigate the role of two alpha2-macroglobulin (A2M) polymorphisms, an intronic 5-bp deletion and Ile1000Val, in the development of Alzheimer's disease (AD) and to evaluate the interaction between the apolipoprotein E (APOE) and A2M polymorphisms. The A2M polymorphisms were screened by using polymerase-chain-reaction-based assays in 555 white late-onset AD cases and 446 controls. The gentoype distributions of the 5-bp deletion and Ile1000Val polymorphisms were comparable between cases and controls (P = 0.158 and P = 0.148, respectively). Likewise, there was no significant difference in allele frequencies of each polymorphism among cases and controls (P = 0.361 and P = 0.062, respectively). The stratification of data by APOE*4 status also did not yield any significant association. In conclusion, we observed no association between either the intronic deletion polymorphism or the Ile1000Val polymorphism of A2M and AD in our case-control cohort.     

Nickel is a specific antagonist for the catabolism of activated α2-macroglobulin

The multifunctional low density lipoprotein receptor-related protein/α₂-macroglobulin receptor (LRP) binds and degrades several ligands involved in protease and lipoprotein metabolism. We previously reported that nickel (Ni²⁺) specifically inhibits the binding of activated α₂-macroglobulin (α₂M^*) at 4°C to LRP and had no effect on the binding of other ligands to the receptor (Hussain et al. (1995) Biochem. 34, 16074–16081). In the current investigation, we have examined the effect of Ni²⁺ on the catabolism of ¹²⁵I-labeled α₂M^*, receptor-associated protein (RAP) and lactoferrin at physiologic temperatures by fibroblasts. Nickel completely inhibited the degradation of α₂M^* over a wide range of concentrations (0.3–2.4 nM); 50% inhibition for the degradation of 1.2 nM α₂M^* was observed at 0.5 mM Ni²⁺. Furthermore, nickel inhibited the binding, internalization and degradation of ¹²⁵I-α₂M^* in a dose- and time- dependent manner. In contrast, the degradation of several concentrations of ¹²⁵I-RAP by fibroblasts was not affected by different amounts of Ni²⁺ for various times. Similarly, Ni²⁺ did not inhibit the degradation of lactoferrin either before or after treating the cells with heparitinase to remove cell-surface proteoglycans. The degradation of lactoferrin was, however, inhibited by the RAP indicating that lactoferrin degradation was mediated by the LRP. These data suggest that Ni²⁺ is a specific inhibitor for the degradation of α₂M^*.     

Immunogenetic study on the polymorphism of serum α2-lipoproteins in mink. I. Identification and genetic control of five Lpm allotypes

Five allotypes, Lpm 1, Lpm 2, Lpm 3, Lpm 4, and Lpm 5, were detected by isoimmunization in mink sera. Immunoelectrophoresis, preparative ultracentrifugation, and histochemical tests for lipids and esterase permitted reference of these alloantigenic markers to a very high density ₂-lipoprotein. Based on population analysis and breeding tests, five genetic units are postulated: Lpm ¹, Lpm⁴, Lpm^3,4, Lpm^1,2, and Lpm ^2,4,5. These units determine the polymorphism of the Lpm system and behave as Mendelian alleles.     

Functional modifications of α2-macroglobulin by primary amines. Kinetics of inactivation of α2-macroglobulin by methylamine, and formation of anomalous complexes with trypsin

The unique steric inhibition of endopeptidases by human alpha(2)M (alpha(2)-macroglobulin) and the inactivation of the latter by methylamine were examined in relation to each other. Progressive binding of trypsin by alpha(2)M was closely correlated with the loss of the methylamine-reactive sites in alpha(2)M: for each trypsin molecule bound, two such sites were inactivated. The results further showed that, even at low proteinase/alpha(2)M ratios, no unaccounted loss of trypsin-binding capacity occurred. As alpha(2)M is bivalent for trypsin binding and no trypsin bound to electrophoretic slow-form alpha(2)M was observed, this indicates that the two sites must react (bind trypsin) in rapid succession. Reaction of [(14)C]methylamine with alpha(2)M was biphasic in time; in the initial rapid phase complex-formation with trypsin caused a largely increased incorporation of methylamine. In the subsequent slow phase trypsin had no such effect. These results prompted further studies on the kinetics of methylamine inactivation of alpha(2)M with time of methylamine treatment. It was found that conformational change of alpha(2)M and decrease in trypsin binding (activity resistant to soya-bean trypsin inhibitor) showed different kinetics. The latter decreased rapidly, following pseudo-first-order kinetics. Conformational change was much slower and followed complex kinetics. On the other hand, binding of (125)I-labelled trypsin to alpha(2)M did follow the same kinetics as the conformational change. This discrepancy between total binding ((125)I radioactivity) and trypsin-inhibitor-resistant binding of trypsin indicated formation of anomalous complexes, in which trypsin could still be inhibited by soya-bean trypsin inhibitor. Further examination confirmed that these complexes were proteolytically active towards haemoglobin and bound (125)I-labelled soya-bean trypsin inhibitor to the active site of trypsin. The inhibition by soya-bean trypsin inhibitor was slowed down as compared with reaction with free trypsin. The results are discussed in relation to the subunit structure of alpha(2)M and to the mechanism of formation of the complex.     

Electrophoretic and chromatographic evidence for allelic polymorphisms in the river buffalo α-globin gene complex

Isoelectric focusing in the ultranarrow immobilized (7.1–7.5) pH gradient (IPG) of hemoglobin and high-performances liquid chromatography (HPLC) of globin chains were used to investigate Hb polymorphism in Italian river buffalo. Six different phenotypes, each characterized by two or four different Hbs, were detected by IPG, whereas two different^IIα-globin chains were separated from two different^Iα-chains by HPLC. Two α-chains (^Iα¹ and^IIα³), and Hbs with similar mobilities (Hb1 andHb3), were associated with the AA Hb phenotype: two α-chains (^Iα² and^IIα⁴), and Hbs with different mobilities (Hb2 andHb4), were associated with the BB phenotype: two sets of doublet Hbs were associated with the AB phenotype, thus suggesting allelic polymorphisms at the two α loci. An allele at the β locus is responsible for increasing to as many as eight the number of different Hbs, thus further complicating the notable Hb polymorphism of the river buffalo.     

Affinity of different α1-agonists and antagonists for the α1-adrenoceptors of rabbit and rat liver membranes

In membranes prepared from rabbit liver, competition with [³H] prazosin by different α₁-agonists and antagonists revealed different affinities in comparison to the results obtained on rat liver membranes, and showed a good correlation with the affinity of the same compounds for the cloned α_1c-adrenoceptor subtype. The potencies observed on rat liver membranes were well correlated with the affinity observed for the cloned α_1b-adrenoceptors. These results confirm that rabbit and rat liver membranes preparations can be utilized to evaluate the affinity of compounds for these α₁-adrenergic subtypes.     

Kinetics of the inhibition of free and elastin-bound human pancreatic elastase by α1-proteinase inhibitor and α2-macroglobulin

At pH 8.0 and 25°C α₁-proteinase inhibitor and α₂-macroglobulin bind human pancreatic elastase with rate constants of 4.7·10⁵ M⁻¹·s⁻¹ and 6.4·10⁶ 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 α₁-proteinase inhibitor and α₂-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, α₂-macroglobulin is a fast-binding inhibitor of elastin-bound elastase, but only one of its two sites is functioning in presence of elastin.     

Localisation and synthesis of α-fetoprotein in the rabbit

Summary The cellular location and sites of synthesis of -fetoprotein (AFP) in the foetal, neonatal and maternal rabbit, were studied by the fluorescent antibody technique and by culturing tissuesin vitro with labelled amino acids. AFP was found to be localised intracellularly within liver hepatocytes and yolk sac endoderm of the foetus, and within the maternal uterine epithelium. Analysis of extracts of the cultured tissues for incorporation of radioactivity into serum proteins separated by polyacrylamide gel electrophoresis or analysed by autoradiography of immuno-precipitation lines, confirmed that the foetal liver and yolk sac splanchnopleur were the principal sites of primary synthesis of AFP. Localisation of AFP in the uterine epithelium and other foetal organs was consistent with a secondary derivation from the uterine fluid or from the blood circulation. These findings are discussed in relationship to findings in man and other mammals.Supported by an award from the Medical Research Council to whom grateful acknowledgement is made.     

Tissue and serum α2-3- and α2-6-linkage specific sialylation changes in oral carcinogenesis

Increased sialylation of cell surface glycoconjugates is among the key molecular changes associated with malignant transformation and cancer progression. We investigated significance of linkage-specific sialylation changes in oral carcinogenesis. Tissue and serum levels of total sialic acid (TSA), linkage-specific sialyltransferases (ST) and sialoproteins were analyzed from patients with oral precancerous conditions (OPC) and oral cancer as well as the post-treatment follow-up blood samples of oral cancer patients. TSA levels were measured using a spectrophotometric method. The linkage-specific lectins, Sambusus nigra (SNA) and Maackia amurensis (MAM) detects α2-6- and α2-3-linked sialic acid, respectively, were used to analyze ST activity and sialoproteins. Malignant tissues showed significantly higher levels of TSA, reactivity of SNA and MAM, and α2,3-ST activity compared to the adjacent normal tissues. α2,6-ST was also higher in malignant tissues. Similarly, the marker levels were higher in precancerous tissues than their adjacent normal tissues. Serum levels of TSA, TSA/ total proteins, α2-6-sialoproteins and α2,6-ST were markedly increased in untreated oral cancer patients compared to the controls and OPC as well as responder (CR) patients. Serum levels of the markers were higher or comparable between untreated oral cancer patients and non-responders (NR). Serum levels of α2-3-sialylation were elevated in non-responders compared with the responders. Further, the observed sialylation changes in tissue and serum were found to be associated with various clinicopathological features and disease progression. Thus, the data suggest potential utility of sialylation markers in early detection, prognostication and treatment monitoring of oral cancer.     

Localization of the α2-macroglobulin gene and Lpm gene family on mink chromosome 9

Summary Using cloned cDNA for human ₂-macroglobulin (A2M) as a probe, mink-Chinese hamster hybrid cells were analysed. The results allowed us to assign a gene for A2M to mink chromosome 9. Breeding tests demonstrated that the Lpm-locus coding for other related -macroglobulin protein and the gene for peptidase B (PEPB) are linked 11±3 cm apart. The PEPB gene is located on mink chromosome 9, and hence, the Lpw-locus is on the same mink chromosome. The relationship of the genetic systems controlling the isotypically different -macroglobulins in mink serum are discussed.     

Interaction between human α2-macroglobulin and duodenase, a serine proteinase with dual specificity

Interaction between a serine proteinase from bovine duodenum and human serum alpha(2)-macroglobulin (alpha(2)-MG) was studied. alpha(2)-MG is established to be one of the most effective duodenase inhibitors. The enzyme is completely inhibited in less than 30 sec at equimolar ratio of the inhibitor and enzyme (concentration 2 x 10(-8) M). Under identical conditions, the rate of duodenase association with alpha(2)-MG is at least 2.5-fold higher than the rate of chymotrypsin association with this inhibitor. The interaction with duodenase results in proteolysis of the inhibitor subunit in the "bait region". Similarly to other proteases, duodenase in the complex with alpha(2)-MG retains the intact catalytic apparatus and ability to hydrolyze some small substrates. But the duodenase-inhibitor complex is fully inactive to proteins (bovine serum albumin). The stoichiometry of the enzyme interaction with the inhibitor is 2 : 1 (mol/mol). Based on the association rate constant and the termination time of the duodenase and alpha(2)-MG in vivo association, alpha(2)-MG is suggested to be a physiological regulator of the enzyme.     

Thiol reduction of human α2-macroglobulin. The subunit structure

1. Human alpha(2)-macroglobulin was prepared from a fraction obtained during the large-scale separation of normal human plasma proteins for clinical use. 2. Sedimentation-equilibrium measurements indicated a molecular weight of 725000. A value of 18.1S was obtained for s(0) (20,w). 3. The dissociation that occurs in the pH range 4.5-2.5 and in the region of neutrality in urea-containing solutions is consistent with a dimeric structure of the molecule. 4. The effects of the thiol reagents mercaptoethanol, mercaptoethylamine and N-acetylcysteine were investigated over a range of experimental conditions. Distinct components having sedimentation coefficients of 15, 12 and 8.5S were identified. 5. Conditions were found under which limited reduction with thiol liberated a subunit with a molecular weight approximately one-quarter of that of the intact molecule. This subunit retains the serological specificity of the whole molecule.