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.     

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.     

Evolution of α2-macroglobulin. The structure of a protein homologous with human α2-macroglobulin from plaice (Pleuronectes platessa L.) plasma

The plaice (Pleuronectes platessa L.) papain-binding protein previously demonstrated to be homologous with human alpha(2)-macroglobulin, and designated plaice alpha(2)-macroglobulin homologue or alphaMh, was shown to be a glycoprotein of s(20,w) 11.86S. In polyacrylamide-gel pore-limit electrophoresis under non-denaturing conditions plaice alphaMh migrated to the same position as half-molecules of human alpha(2)-macroglobulin, and treatment with methylamine or a proteinase caused no change in its electrophoretic properties. Either denaturation in urea (4m) or mild reduction by dithiothreitol (1mm) partially dissociated plaice alphaMh into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions plaice alphaMh dissociated into subunits of M(r) 105000 (I) and 90000 (II). Approximately equal amounts of each subunit were formed, and peptide ;mapping' showed subunits I and II to be distinct polypeptide chains. Under alkaline denaturing conditions, a proportion of the I chains of alphaMh were cleaved into fragments of M(r) about 60000 and 40000. This cleavage was favoured by reducing conditions and prevented by prior inactivation of the alphaMh with methylamine. [(14)C]Methylamine allowed to react with alphaMh became covalently linked to subunit I. These properties suggested the existence of an autolytic site on subunit I analogous to the autolytic site of human alpha(2)-macroglobulin. Reaction of alphaMh with a proteinase resulted in cleavage of a fragment of M(r) 10000-15000 from subunit I. A proportion of the proteinase molecules trapped by alphaMh became covalently linked to the inhibitor. A scheme is proposed for the evolution of human alpha(2)-macroglobulin and plaice alphaMh from a common ancestral protein, which may also have been an ancestor of complement components C3 and C4.     

Pregnancy zone protein,a proteinase binding α-macroglobulin. Stopped-flow kinetic studies of its interaction with chymotrypsin

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein, strongly related to α₂-macroglobulin (α₂M). The proteinase binding reaction of PZP is investigated using chymotrypsin as a model enzyme. The time-course of the interaction is studied by measuring the change in intrinsic protein fluorescence of PZP-chymotrypsin reaction mixtures as a function of time after rapid mixing in a stopped-flow apparatus. Titrations show the changes of fluorescence at equilibrium to correspond with the formation of a chymotrypsin-PZP(tetramer) species. The kinetic results show the formation of the species to take place in an overall second-order process dependent on the concentrations of chymotrypsin and of PZP(dimers), k = 5 · 10⁵M⁻¹ ·s⁻¹. Reactions of PZP-thiol groups do not give rise to fluorescence changes. The fluorescence changes most likely reflect the formation of an intermediate with intact thiol esters. Further analysis of the kinetic results suggests that the chymotrypsin-PZP(tetramer) intermediate is formed in two reaction steps: (1) initially native PZP(dimers) are cleaved at bait regions by enzyme molecules, and that is the rate determining reaction of the fluorescence changes; (2) association with another PZP(dimer) or PZP(dimer)-chymotrypsin complex in a very fast reaction that leads to the formation of 1:1-chymotrypsin-PZP(tetramer) intermediate, probably with intact thiol esters. The interactions studied apparently are established early in the path of the reaction and the fluorescence changes probably reflect noncovalent enzyme-PZP contacts, which are not changed when covalent binding occurs. Further, fluorescence changes are seen only in reactions of PZP with enzymes, not with methylamine.     

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.     

Properties of an α-1,3-Glucanase from Streptomyces sp. KI-8

Srome properties were examined of purified α-l,3-glucanase isolated from the culture supernatant of the soil microorganism Streptomyces KI-8.

The optimum pH and temperature were pH 5.4 and 60°C, respectively. The α-1,3-glucanase was stable up to 50°C on heating for 10 min. This enzyme hydrolyzed the substrate α-l,3-glucan into glucose and nigerose by an endo-type of action. Nigerotriose, nigerotetraose and nigeropentaose were hydrolyzed into glucose and nigerose, whereas nigerose was not attacked. The degree of hydrolysis of pseudonigeran, Lentinus α-1,3-glucan, mutan IG-1 (less soluble fraction) and IG-2 (more soluble fraction) by the α-1,3-glucanase were 28.5%, 14.3%, 8.8% and 10.0%, respectively. Km values (mg/ml) for pseudonigeran, Lentinus α-l,3-glucan, mutan IG-1 and IG-2 were 1.12, 1.98, 8.00 and 5.00. The enzyme solubilized 50 to 80% of mutan by concerted action with dextranase.     

Characterization of an α-agarase from Thalassomonas sp. LD5 and its hydrolysate

Applied Microbiology and Biotechnology - It has been a long time since the first α-agarase was discovered. However, only two α-agarases have been cloned and partially characterized so far...     

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.     

Interaction of α-thalassemia genes with each other and with HbC in an American black family

The relative rates of in vitro synthesis of hemoglobin chains have been studied in an American black family in which the mother is doubly heterozygous for alpha-thalassemia and HbC and the father is heterozygous for alpha-thalassemia. The alpha/non-alpha synthetic ratio was equally unbalanced in both the bone marrow and the peripheral blood of the mother. Although HbC comprised 35% of her hemoglobin (compared to 42.2 +/- 2.2 in individuals with HbC trait and balanced globin synthesis), synthetic data showed that the newly synthesized beta C chain was 44% of the total newly synthesized beta chains. Isolated membranes contained more newly synthesized beta C than beta A chains. Three of the offspring were within the normal range, and the remaining three had alpha-thalassemia. There were two spontaneous abortions during the second trimester of pregnancy. Hydrops fetalis did not occur, and none of the children had HbH disease or HbC trait.     

Characterization of hyperthermostable α-amylase from Geobacillus sp. IIPTN

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum enzyme production was in exponential phase with activity 135 U ml⁻¹ at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml⁻¹ at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg⁻¹ protein. The molecular mass of the purified enzyme was 97 KDa. The values of K _m and V _max were 36 mg ml⁻¹ and 222 μmol mg⁻¹ protein min⁻¹, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The enzyme was stimulated with Mn²⁺, whereas it was inhibited by Hg²⁺, Cu²⁺, Zn²⁺, Mg²⁺, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.     

Interaction of Insulin-Like Growth Factor-Binding Protein 2 with α2-Macroglobulin in the Circulation

Insulin-like growth factors (IGFs) play active role in mitogenic and metabolic processes. In the peripheral circulation, they are mostly bound to specific IGF-binding proteins (IGFBPs). Proteolysis of IGFBPs releases free, active IGFs. IGFBP-2 is the second most abundant of the six binding proteins and its concentration increases in catabolic states. The possible interaction between IGFBP-2 and other proteins in the circulation was investigated in this study. Our results showed that IGFBP-2 associates with α2-macroglobulin (α2M), a protease inhibitor. Formation of IGFBP-2/α2M complexes most likely contributes to the regulation of IGFBP-2 proteolysis and, thus, the activity of IGFs.     

Thermostable α-1,4- and α-1,6-glucosidase enzymes from Bacillus sp. Isolated from a marine environment

Penaeus vannamei (the shrimp) is an omnivorous species and it can be assumed that a high level of carbohydrates is necessary for its growth. -1,4- and 1,6-glucosidases are important enzymes necessary for the ultimate liberation of glucose residues from various carbohydrates, principally starch. However, the shrimp's hepatopancreas produces only -1,4-glucosidases, which limits the growth rate in different sources of starch. In order to identify strains with -1,4- and 1,6-glucosidase enzymes with potential uses in shrimp feed production, Bacillus strains were isolated from marine environments. One strain produced large amounts of an extracellular thermostable -glucosidase that permitted good growth on starch. The organism was identified by polymorphism (restriction-fragment-length polymorphism, RFLP), sequenced, and named B. subtilis LMM-12.     

Purification and Characterization of α-Hydroxyglutarate Dehydrogenase from Alcaligenes sp.

NADH-dependent soluble l-α-hydroxyglutarate dehydrogenase (l-2-hydroxyglutarate: NAD⁺ 2-oxidoreductase) was found in a bacterium belonging to the genus Alcaligenes obtained from soil by citrate enrichment culture. A mutant with about 2.5-fold higher activity of the enzyme was derived from the bacterium and used as the enzyme source. High level of the enzyme was produced at the late stage of cultivation in the presence of citrate and with limited aeration. The enzyme was purified from the cells to homogeneity to give crystals, and its enzymatic properties were studied. The enzyme strongly reduced α-ketoglutarate to stereochemically pure l-α-hydroxyglutarate with NADH as a coenzyme, but it oxidized d-α-hydroxyglutarate with about 1/10 of the rate for l-form oxidation.     

The interaction of α2-macroglobulin with proteinases. Binding and inhibition of mammalian collagenases and other metal proteinases

1. Experiments were performed to determine whether the specific collagenases and other metal proteinases are bound and inhibited by alpha(2)-macroglobulin, as are endopeptidases of other classes. 2. A specific collagenase from rabbit synovial cells was inhibited by human serum. The inhibition could be attributed entirely to alpha(2)-macroglobulin; alpha(1)-trypsin inhibitor was not inhibitory. alpha(2)-Macroglobulin presaturated with trypsin or cathepsin B1 did not inhibit collagenase, and pretreatment of alpha(2)-macroglobulin with collagenase prevented subsequent reaction with trypsin. The binding of collagenase by alpha(2)-macroglobulin was not reversible in gel chromatography. 3. The collagenolytic activity of several rheumatoid synovial fluids was completely inhibited by incubation of the fluids with alpha(2)-macroglobulin. 4. The collagenase of human polymorphonuclear-leucocyte granules showed time-dependent inhibition by alpha(2)-macroglobulin. 5. The collagenolytic metal proteinase of Crotalus atrox venom was inhibited by alpha(2)-macroglobulin. 6. The collagenase of Clostridium histolyticum was bound by alpha(2)-macroglobulin, and inhibited more strongly with respect to collagen than with respect to a peptide substrate. 7. Thermolysin, the metal proteinase of Bacillus thermoproteolyticus, was bound and inhibited by alpha(2)-macroglobulin. 8. It was shown by polyacrylamidegel electrophoresis of reduced alpha(2)-macroglobulin in the presence of sodium dodecyl sulphate that synovial-cell collagenase, clostridial collagenase and thermolysin cleave the quarter subunit of alpha(2)-macroglobulin near its mid-point, as do serine proteinases. 9. The results are discussed in relation to previous work, and it is concluded that the characteristics of interaction of the metal proteinases with alpha(2)-macroglobulin are the same as those of other proteinases.     

Characterization of a half-molecular fragment obtained by reduction of human α2-macroglobulin with dithiothreitol

A half-molecular fragment of ₂-macroglobulin has been prepared by reducing and alkylating the inter-subunit disulfide bonds in the tetrameric ₂-macroglobulin molecule with 1 mM dithiothreitol (40 min) and 3 mM iodoacetamide (40 min). Further purification was made by gel chromatography and the homogeneous population of halfmolecules has been characterized by the techniques of small-angle X-ray and neutron scattering. The radii of gyration found by the two methods are 57.0 and 58.0 Å, respectively. The match point, obtained by neutron scattering from solutions with different H₂O/D₂O rations, is at 43% D₂O; the data are consistent with a particle having a higher scattering density at large distances from the particle centre. From the X-ray and neutron intensities scattered at zero angle, the specific volume was determined to be 0.73 cm³/g at+5°C and the molecular weight to be 390,000; the latter value is associated with a relatively large error due to the uncertainty in the concentration determination. Shape analysis indicates that the best-fitting scattering-equivalent threeaxial bodies are oblate shaped, with two of their axial dimensions about three to four times larger than the third one. From the volume of the best-fitting scattering-equivalent three-axial bodies, 0.72×10⁶ ų, we obtain a water content equal to 0.38 g H₂O/g protein (dry weight).Abbreviations SANS small-angle neutron scattering - SAXS small-angle X-ray scattering - ₂ M ₂-macroglobulin - DTT dithiothreitol - Tris tris(hydroxymethyl)aminomethane     

Purification and properties of a low-molecular-weight α-mannosidase from Cellulomonas sp.

Cellulomonas sp. isolated from soil produces a high level of α-mannosidase (α-mannanase) inductively in culture fluid. The enzyme had two different molecular weight forms, and the properties of the high-molecular-weight form were reported previously (Takegawa, K. et al.: Biochim. Biophys. Acta, 991, 431–437, 1989). The low-molecular-weight α-mannosidase was purified to homogeneity by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was over 150,000 by gel filtration. Unlike the high-molecular-weight form, the low-molecular-weight enzyme readily hydrolyzed α-1,2- and α-1,3-linked mannose chains.     

Functional Changes of Polyethylene Glycol-modified Serine Proteinase from Aspergillus sojae and Interaction with α2-Macroglobulin

The covalent attachment of activated polyethylene glycol₂ (PEG₂) of 10,000 daltons to non- essential groups on a serine proteinase II (SepII) from Aspergillus sojae produced two modified preparations (PEG₂-SepII-S and PEG₂-SepII-L). The molecular weights of PEG₂-SepII-S and PEG₂-SepII-L were about 170,000 and 280,000, respectively. The PEG₂-SepII-S lost about 80 % of its antigenicity, while the PEG₂-SepU-L completely lost its antigenicity. In comparison of kinetic parameters with SepII there was less than 40 % variation in Km, but the values of k_cat towards succinyl-l-leucy 1-l-leucy 1-l-valy 1-l-tyrosine 4-methylcoumaryl-7-amide (Suc-LLVY-MCA) or succinyl-l- alanyl-l-alanyl-l-valyl-l-alanine β-nitroanilide (Suc-AAVA-/>NA) decreased to about 70% less than that of SepII. The modified preparations have about 20 % activity towards fibrin hydrolysis and a low affinity for a protein proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), with a molecular weight of 23,000, while the preparations have high affinity for a low molecular weight microbial inhibitor, chymostatin. The stoichiometry of the reaction of a₂-macroglobulin (α₂M) with PEG₂-SepII-S showed that PEG₂-SepII-L bound to α₂M in a molar ratio of 1:1. No appreciable differences were observed in the pH stabilities of the modified enzymes and the native one at pH 3.6, while the modified enzymes were more stable than that of the native one at pH 11.5. The two modified preparations were labile at 50°C, but the native enzyme was completely stable at 50°C.     

A recombinant bait region mutant of human α2-macroglobulin exhibiting an altered proteinase-inhibiting spectrum

Alpha 2-macroglobulin (α2M), a plasma glycoprotein produced in the liver, inhibits a variety of proteinases and thus considered to play important homeostatic roles in the body. This broad inhibitory spectrum has been explained by the trapping theory by which a proteinase recognizes a region of 25–30 amino acid peptide in α2M called bait region and cleaves it, leading to the conformational change of α2M, and to the subsequent entrapment and inhibition of the proteinase. We constructed α2M cDNAs with mutated DNA sequences in the bait region, and obtained recombinant CHO cell lines producing either wild type α2M, or mutant α2Ms, i.e., α2M/K692 and α2M/K696, each with substitution of Arg with Lys at codons 692 and 696, respectively. We tested if lysyl endopeptidase is not inhibited by wild type α2M, but could be inhibited by these engineered mutant α2Ms. Thus, recombinant α2M/K696 protein successfully inhibited lysyl endopeptidase activity, while recombinant α2M/K692 protein was not sensitive to lysyl endopeptidase, suggesting that not all bait region peptide bonds can equally be accessible and susceptible to proteinases. The present results not only provided the trapping theory with additional supportive evidence, but the first experimental evidence for the value of engineered α2M-derived proteinase inhibitor with an artificial proteinase inhibitory spectrum of potential industrial and/or therapeutic usefulness. This revised version was published online in August 2006 with corrections to the Cover Date.     

Interaction of α-synuclein with biomembranes in Parkinson's disease —role of cardiolipin

One of the key molecular events underlying the pathogenesis of Parkinson's disease (PD) is the aberrant misfolding and aggregation of the α-synuclein (αS) protein into higher-order oligomers that play a key role in neuronal dysfunction and degeneration. A wealth of experimental data supports the hypothesis that the neurotoxicity of αS oligomers is intrinsically linked with their ability to interact with, and disrupt, biological membranes; especially those membranes having negatively-charged surfaces and/or lipid packing defects. Consequences of αS–lipid interaction include increased membrane tension, permeation by pore formation, membrane lysis and/or leakage due to the extraction of lipids from the bilayer. Moreover, we assert that the interaction of αS with a liquid-disordering phospholipid uniquely enriched in mitochondrial membranes, namely cardiolipin (1,3-diphosphatidyl-sn-glycerol, CL), helps target the αS oligomeric complexes intracellularly to mitochondria. Binding mediated by CL may thus represent an important pathomechanism by which cytosolic αS could physically associate with mitochondrial membranes and disrupt their integrity. Impaired mitochondrial function culminates in a cellular bioenergetic crisis and apoptotic death. To conclude, we advocate the accelerated discovery of new drugs targeting this pathway in order to restore mitochondrial function in PD.