Resistance of tropoelastin and elastin peptides to degradation by alpha 2-macroglobulin-protease complexes

When α-ketoglutarate is the substrate, malate is a considerably more effective inhibitor of glutamate dehydrogenase than glutamate, oxalacetate, aspartate, or glutarate. Malate is a considerably poorer inhibitor when glutamate is the substrate. Malate is competitive with α-ketoglutarate, uncompetitive with TPNH, and noncompetitive with glutamate. The above, plus the fact that malate is a considerably more potent inhibitor when TPNH rather than TPN is the coenzyme, indicates that malate is predominantly bound to the α-ketoglutarate site of the enzyme-TPNH complex and has a considerably lower affinity for the enzyme-TPN complex. Ligands which decrease binding of TPNH to the enzyme such as ADP and leucine markedly decrease inhibition by malate. Conversely, GTP, which increases binding of TPNH to the enzyme also enhances inhibition by malate. Malate also decreases interaction between mitochondrial aspartate aminotransferase and glutamate dehydrogenase. This effect of malate on enzyme-enzyme interaction is enhanced by DPNH and GTP which also increase inhibition of glutamate dehydrogenase by malate and is decreased by TPN, ADP, ATP, α-ketoglutarate, and leucine which decrease inhibition of glutamate dehydrogenase by malate. These results indicate that malate could decrease α-ketoglutarate utilization by inhibiting glutamate dehydrogenase and retarding transfer of α-ketoglutarate from the aminotransferase to glutamate dehydrogenase. These effects of malate would be most pronounced when the mitochondrial level of α-ketoglutarate is low and the level of malate and reduced pyridine nucleotide is high.     

Decreased formation of alpha 2-macroglobulin-protease complexes in plasma of patients with cystic fibrosis.

Alpha₂-macroglobulin from patients with cystic fibrosis is shown to have reduced binding with papain, trypsin, and thrombin. The obligate heterozygotes for cystic fibrosis revealed intermediate values between the controls and the patients. Since papain and trypsin are not plasma endopeptidases, it becomes evident that the absence of α₂-macroglobulin-protease complex in cystic fibrosis is due to a molecular defect within the macroglobulin.     

Structure of alpha 2-macroglobulin-protease complexes. Methylamine competition shows that proteases bridge two disulfide-bonded half-molecules.

alpha 2-Macroglobulin (alpha 2M) forms several different covalent complexes with proteases. These include unusual forms in which more than one of the four identical subunits of alpha 2M are cross-linked by amide bonds to more than one lysyl amino group of the bound protease. The structure of these complexes and the question of how the identical subunits are arranged to form two protease binding sites are matters of current controversy. The 185-kDa subunits are arranged into two disulfide-bonded half-molecules which are, in turn, noncovalently associated. We have provided evidence that, in the major multivalent cross-linked form, proteases can span the two half-molecules, forming a covalently bonded tetramer [Wang, D., Yuan, A. I., & Feinman, R. D. (1984) Biochemistry 23, 2807-2811]. An alternative theory has recently been proposed in which the major high molecular weight form has two bonds to protease that are within half-molecules--a multivalent cross-linked dimer [Sottrup-Jensen, L., Hansen, H. F., Pedersen, H. S., & Kristensen, L. (1990) J. Biol. Chem. 265, 17727-17737]. To resolve this conflict, experiments were carried out to determine the structure of one of the high molecular weight bands (band 3) seen on SDS-PAGE. Band 3 has anomalous migration, corresponding to markers of apparent molecular mass of 550 kDa (between the tetramer and dimer). In the experiments described here, reactions of thrombin with alpha 2M were run in the presence of methylamine, which competes for one of the two thrombin-alpha 2M covalent bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the parameters affecting covalent binding stoichiometry in binary and ternary complexes of thymidylate synthase

Covalent binding stoichiometries for both the enzyme:5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) binary complex and the enzyme:FdUMP:5,10-methylenetetrahydrofolate (inhibitory ternary) complex at equilibrium were measured by the trichloroacetic acid precipitation assay and shown to be a function of temperature, time, pH, salt concentration, buffer composition and thiol concentration. Incubation at 37 degrees C yielded the maximum covalent binding ratio (mol FdUMP/mol enzyme) for the latter binary (0.7) and ternary (1.7) complexes. In most buffers studied, the maximum covalent binding ratio (1.5-1.7) for the inhibitory ternary complex occurred over a broad pH range (4.5-8.0), while the optimum covalent binding ratio for binary complex was observed at a much narrower region centered between pH 5.5-6.5. In the presence of increasing concentrations of phosphate buffer, the maximum binding ratio for the covalent binary complex decreased from 0.63 in the absence of phosphate to 0.1 in the presence of 225 mM phosphate, while that for the inhibitory ternary complex was unchanged. When a ternary complex was formed with enzyme, FdUMP and (+/-)-tetrahydrofolate in the absence of phosphate, the FdUMP:enzyme covalent binding ratio was 1.8, while in the presence of 75 mM phosphate, the binding ratio was only 1.0. When exogenous thiol was removed by centrifugal column chromatography, the maximum binding stoichiometry of the resulting inhibitory ternary complex was 1.7 and was independent of added thiol over a 2 h incubation period at 37 degrees C. When extensive dialysis at 5 degrees C was used to remove the thiol, the maximum binding stoichiometry of the resulting inhibitory ternary complex was found to be dependent on both the concentration of added thiol and the time of incubation at 37 degrees C and did not exceed a value of 1.0.     

Formation of vitamin D-binding protein-actin and binary and ternary plasma gelsolin-actin complexes in human serum

After the addition of actin to serum, the binding of actin to serum actin-binding proteins was analyzed by the method of immunoblotting using monospecific antibodies against vitamin D-binding protein (DBP) (group-specific component, Gc), human skeletal actin and human plasma gelsolin. When increasing amounts of globular actin were added to serum, actin bound to DBP preferentially. After exhausting DBP, actin began to bind to plasma gelsolin. When equally increasing amounts of filamentous actin were added to serum, actin was bound to both plasma gelsolin and DBP, and then uncomplexed DBP removed one actin molecule from gelsolin-actin 1:2 complex, resulting in a gelsolin-actin 1:1 complex. These results support the theory that the actin-depolymerizing activity of serum is due to the concerted role of plasma gelsolin and DBP.     

Calcium-dependent distance changes in binary and ternary complexes of troponin

Calcium-dependent distance changes have been determined by resonance energy transfer in binary and ternary troponin complexes in order to collect evidence for the structural rearrangements which are part of the hypothetical trigger mechanism of skeletal muscle contraction. Donor and acceptor fluorophores were either intrinsic tryptophans in subunits with a favourable sequence from different species, quasi-intrinsic Tb3+ ions bound to troponin C or extrinsic labels attached to specific cysteine or methionine residues. All chemically modified subunits proved fully active in conferring calcium sensitivity onto myosin ATPase. Nine distances were determined between five sites which allowed construction of a three-dimensional lattice representing the spatial distribution of four sites in the ternary complex of troponin C, I and T. Distances in binary complexes were nearly unaltered upon addition of the third subunit. Regulatory calcium binding caused distance changes of the order of 0.7-1.1 nm. In view of the large displacements of the hypothetical mechanism, they turned out to be smaller than anticipated. The fluorophoric sites selected may be localized in a zone of the troponin complex which happens to be relatively little affected by the mechanism. Alternatively, amplification of the moderate changes seen here would require the complete set of thin filament proteins.     

Reaction of human alpha 2-macroglobulin half-molecules with plasmin as a probe of protease binding site structure

Human alpha 2-macroglobulin (alpha 2M) half-molecules were prepared by limited reduction and alkylation of the native protein. Reaction with plasmin resulted in nearly quantitative cleavage of the half-molecule Mr approximately 180000 subunits into Mr approximately 90000 fragments. Subunit cleavage was significantly less complete when plasmin was reacted with alpha 2M whole molecules. The plasmin and trypsin binding capacities of the two forms of alpha 2M were compared by using radioiodinated proteases. alpha 2M half-molecules bound an equivalent number of moles of plasmin or trypsin. Native unreduced alpha 2M bound only half as much plasmin as trypsin. These data are consistent with the hypothesis that the two protease binding sites are adjacent in native alpha 2M. alpha 2M half-molecule-plasmin complexes reassociated less readily than half-molecule-trypsin complexes, supporting this interpretation. The frequency of covalent bond formation between plasmin and alpha 2M was considerably higher than that previously observed with other proteases. Approximately 80-90% of the plasmin that reacted with alpha 2M whole molecules or half-molecules became covalently bound. The reactivities of purified alpha 2M-plasmin complexes were compared with small and large substrates. Equivalent kcat/Km values were determined at 22 degrees C for the hydrolysis of H-D-Val-Leu-Lys-p-nitroanilide dihydrochloride by whole molecule-plasmin complex and half-molecule-plasmin complex (40 mM-1 s-1 and 39 mM-1 s-1, respectively, compared with 66 mM-1 s-1 determined for free plasmin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of troponin subunits: free energy of binary and ternary complexes

We have determined the free energy of formation of the binary complexes formed between skeletal troponin C and troponin T (TnC.TnT) and between troponin T and troponin I (TnT.TnI). This was accomplished by using TnC fluorescently modified at Cys-98 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine for the first complex and TnI labeled at Cys-133 with the same probe for the other complex. The free energy of the ternary complex formed between troponin C and the binary complex TnT.TnI [TnC.(TnT.TnI)] was also measured by monitoring the emission of 5-(iodoacetamido)eosin attached to Cys-133 of the troponin I in TnT.TnI. The free energies were -9.0 kcal.mol-1 for TnC.TnT, -9.2 kcal.mol-1 for TnT.TnI, and -8.7 kcal.mol-1 for TnC.(TnT.TnI). In the presence of Mg2+ the free energies of TnC.TnT and TnC.(TnT.TnI) were -10.3 and -10.9 kcal.mol-1, respectively; in the presence of Ca2+ the corresponding free energies were -10.6 and -13.5 kcal.mol-1. Mg2+ and Ca2+ had negligible effect on the free energy of TnT.TnI. From these results the free energies of the formation of troponin from the three subunits were found to be -16.8 kcal.mol-1, -18.9 kcal.mol-1, and -21.6 kcal.mol-1 in the presence of EGTA, Mg2+, and Ca2+, respectively. Most of the free energy decrease caused by Ca2+ binding to the Ca2+-specific sites is derived from stabilization of the TnI-TnC linkage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cadmium binding to human alpha 2-macroglobulin

alpha 2-Macroglobulin (alpha 2M) is one of the major cadmium-binding proteins of human plasma. As determined with equilibrium dialysis, alpha 2M bound 4.6 (+/- 0.7) mol Cd2+ per mol protein with an apparent dissociation constant of (9.6 (+/- 5.0] X 10(-7) M. Methylamine-modified alpha 2M (alpha 2M-Me) had a similar affinity for Cd2+ (Kd,app = 5.3 X 10(-7) M), but fewer binding sites. Cadmium produced a small increase in the amidolytic activity of trypsin in the presence of alpha 2M and soybean trypsin inhibitor. Using the binding parameters determined from the equilibrium dialysis studies, the Cd2+ concentration which produced a half-maximal increase in amidolytic activity corresponded to saturation of all Cd2+-binding sites in one-half of the alpha 2M molecules. From these results, a model is proposed in which one Cd2+-binding site is present in each of the four polypeptide chains which compose alpha 2M.     

Crystal structure of Pseudomonas fluorescens mannitol 2-dehydrogenase binary and ternary complexes. Specificity and catalytic mechanism

Long-chain mannitol dehydrogenases are secondary alcohol dehydrogenases that are of wide interest because of their involvement in metabolism and potential applications in agriculture, medicine, and industry. They differ from other alcohol and polyol dehydrogenases because they do not contain a conserved tyrosine and are not dependent on Zn(2+) or other metal cofactors. The structures of the long-chain mannitol 2-dehydrogenase (54 kDa) from Pseudomonas fluorescens in a binary complex with NAD(+) and ternary complex with NAD(+) and d-mannitol have been determined to resolutions of 1.7 and 1.8 A and R-factors of 0.171 and 0.176, respectively. These results show an N-terminal domain that includes a typical Rossmann fold. The C-terminal domain is primarily alpha-helical and mediates mannitol binding. The electron lone pair of Lys-295 is steered by hydrogen-bonding interactions with the amide oxygen of Asn-300 and the main-chain carbonyl oxygen of Val-229 to act as the general base. Asn-191 and Asn-300 are involved in a web of hydrogen bonding, which precisely orients the mannitol O2 proton for abstraction. These residues also aid in stabilizing a negative charge in the intermediate state and in preventing the formation of nonproductive complexes with the substrate. The catalytic lysine may be returned to its unprotonated state using a rectifying proton tunnel driven by Glu-292 oscillating among different environments. Despite low sequence homology, the closest structural neighbors are glycerol-3-phosphate dehydrogenase, N-(1-d-carboxylethyl)-l-norvaline dehydrogenase, UDP-glucose dehydrogenase, and 6-phosphogluconate dehydrogenase, indicating a possible evolutionary relationship among these enzymes.     

Binding of alpha 2-macroglobulin-thrombin complexes and methylamine-treated alpha 2-macroglobulin to human blood monocytes

The binding of alpha 2-macroglobulin (alpha 2M) to human peripheral blood monocytes was investigated. Monocytes, the precursors of tissue macrophages, were isolated from fresh blood by centrifugal elutriation or density gradient centrifugation. Binding studies were performed using 125I-labeled alpha 2M. Cells and bound ligand were separated from free ligand by rapid vacuum filtration. Nonlinear least-squares analysis of data obtained in direct binding studies at 0 degrees C showed that monocytes bound the alpha 2M-thrombin complex with a Kd of 3.0 +/- 0.9 nM and the monocyte had 1545 +/- 153 sites/cell. Thrombin alone did not compete for the site. Binding was divalent cation dependent. Direct binding studies also demonstrated that monocytes bound methylamine-treated alpha 2M in a manner similar to alpha 2M-thrombin. Competitive binding studies showed that alpha 2M-thrombin and methylamine-treated alpha 2M bound to the same sites on the monocyte. In contrast, native alpha 2M did not compete with alpha 2M-thrombin for the site. Studies done at 37 degrees C suggested that after binding, the monocyte internalized and degraded alpha 2M-thrombin and excreted the degradation products. Receptor turnover and degradation of alpha 2M-thrombin complexes were blocked in monocytes treated with chloroquine, an inhibitor of lysosomal function. Our results indicate that human monocytes have a divalent cation dependent, high-affinity binding site for alpha 2M-thrombin and methylamine-treated alpha 2M which may function to clear alpha 2M-proteinase complexes from the circulation.     

Substrate binding to chloramphenicol acetyltransferase: evidence for negative cooperativity from equilibrium and kinetic constants for binary and ternary complexes

Chloramphenicol acetyltransferase (CAT) catalyzes the acetyl-CoA-dependent acetylation of chloramphenicol by a ternary complex mechanism with a rapid equilibrium and essentially random order of addition of substrates. Such a kinetic mechanism for a two-substrate reaction provides an opportunity to compare the affinity of enzyme for each substrate in the binary complexes (1/Kd) with corresponding values (1/Km) for affinities in the ternary complex where any effect of the other substrate should be manifest. The pursuit of such information for CAT involved the use of four independent methods to determine the dissociation constant (Kd) for chloramphenicol in the binary complex, techniques which included stopped-flow measurements of on and off rates, and a novel fluorometric titration method. The binary complex dissociation constant (Kd) for acetyl-CoA was measured by fluorescence enhancement and steady-state kinetic analysis. The ternary complex dissociation constant (Km) for each substrate (in the presence of the other) was determined by kinetic and fluorometric methods, using CoA or ethyl-CoA to form nonproductive ternary complexes. The results demonstrate an unequivocal decrease in affinity of CAT for each of its substrates on progression from the binary to the ternary complex, a phenomenon most economically described as negative cooperativity. The binary complex dissociation constants (Kd) for chloramphenicol and acetyl-CoA are 4 microM and 30 microM whereas the corresponding dissociation constants in the ternary complex (Km) are 12 microM and 90 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of thiolester bond cleavage-dependent conformational changes in binary alpha 2-macroglobulin-proteinase complexes

The structures of the two proteinase-binding sites in human alpha 2-macroglobulin (alpha 2M) were probed by treatment of alpha 2M with the serine proteinases thrombin and plasmin. Each proteinase forms an equimolar complex with alpha 2M (a binary alpha 2M-proteinase complex) which results in the activation and cleavage of two internal thiolester bonds in alpha 2M. Binary alpha 2M-proteinase complexes demonstrated an incomplete conformational change as determined by nondenaturing polyacrylamide gel electrophoresis and incomplete receptor recognition site exposure as determined by in vivo plasma elimination studies. Treatment of binary alpha 2M-proteinase complexes with CH3NH2, trypsin, or elastase resulted in cleavage of an additional one or two thiolester bonds in alpha 2M and complete receptor recognition site exposure, demonstrating that a limited conformational change had occurred. Treatment of the alpha 2M-thrombin complex with elastase resulted in the incorporation of approximately 0.5 mol proteinase/mol alpha 2M and completion of the conformational change in the complex. Similar treatment of the alpha 2M-plasmin complex resulted in the incorporation of less than 0.1 mol proteinase/mol alpha 2M. Unlike the alpha 2M-thrombin complex, the alpha 2M-plasmin complex did not undergo a complete conformational change following treatment with CH3NH2 or trypsin. Incubation of this complex with elastase resulted in proteolysis of the kringle 1-4 region of the alpha 2M-bound plasmin heavy chain, and following this treatment the alpha 2M-plasmin complex underwent a complete conformational change. The results of this investigation demonstrate that binary alpha 2M-proteinase complexes retain a relatively intact proteinase-binding site. In the case of the alpha 2M-plasmin complex, however, the heavy chain of alpha 2M-bound plasmin protrudes from the proteinase-binding site and prevents a complete conformational change in the complex despite additional thiolester bond cleavage.     

A transfer nuclear Overhauser effect study of coenzyme binding to distinct sites in binary and ternary complexes in glutamate dehydrogenase

The oxidized coenzyme NAD binds to two sites per subunit of bovine liver glutamate dehydrogenase with equal affinity in the absence of dicarboxylic acid coligands. In the presence of glutarate or 2-oxoglutarate, the affinity to one site is unchanged, but the affinity to the other (presumed to be the active site) is considerably increased and now requires two dissociation constants to describe its saturation. A combination of transfer nuclear Overhauser effects (TRNOE) together with an examination of the slopes of TRNOE time dependence indicates that while NAD is bound in a syn conformation at both binding sites, NADP (which binds only to the active site) is bound in a syn-anti mixture. The existence of N6 to N3' and N6 and N2' and N1' to N3' NOE's with NAD suggests that the two coenzyme binding sites are located near enough to allow intermolecular NOE's. In the presence of 2-oxoglutarate where only binding to the active site is effectively observed, the conformation of either coenzyme is syn. Modeling studies using the distance estimates from the TRNOE results suggest that the nicotinamide ribose approximates a 3'-endo conformation. The absence of evidence for intermolecular NOE's under these conditions indicates that while the active and regulatory NAD sites per subunit are in close proximity, the six active sites per hexamer are located greater than 5 A apart.     

Proteolytic activity of alpha 2-macroglobulin-enzyme complexes toward human factor VIII/von Willebrand factor

The low level of enzymatic activity of certain alpha 2-macroglobulin-proteinase complexes could be important to the function of factor VIII/von Willebrand glycoprotein since it is especially sensitive to proteolytic cleavage. To test this possibility, complexes of alpha 2-macroglobulin with plasmin, trypsin, and thrombin were formed in at least a 2:1 molar ratio of alpha 2-macroglobulin:proteinase and tested for effects on the factor VIII procoagulant activity of the factor VIII/von Willebrand glycoprotein. Neither the alpha 2-macroglobulin-trypsin complex nor the alpha 2-macroglobulin-plasmin complex affected factor VIII procoagulant activity. The behavior of the alpha 2-macroglobulin-thrombin complex was different. When alpha 2-macroglobulin and thrombin were incubated in a mole ratio of 3:1 or less, factor VIII procoagulant activity was enhanced to about the same extent as with free thrombin. Even at a 24:1 mole ratio, the mixture could produce 45% of the increase in factor VIII activity obtained with free thrombin. The isolated alpha 2-macroglobulin-thrombin complex could also activate the factor VIII procoagulant function to about 45% of the level obtained with an identical amount of uncomplexed thrombin. Analysis of the alpha 2-macroglobulin-125I-labeled thrombin complexes by rechromatography or by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that this activation was not due to free thrombin. We conclude that the alpha 2-macroglobulin-thrombin complex retains sufficient proteolytic activity to activate the procoagulant function of factor VIII/von Willebrand glycoprotein despite the latter being a very large substrate, having an estimated molecular weight of 1-20 million.     

Thermodynamic studies of binary and ternary complexes of pig heart lactate dehydrogenase.

The thermodynamics of the reaction catalyzed by pig heart muscle lactate dehydrogenase (LDH; EC1.1.1,27) have been studied in 0,2 M potassium phosphate buffer, pH 7, over the temperature range of 10 to 35 degrees C by using oxamate and oxalate to simulate the corresponding reactions of the substrates pyruvate and lactate, respectively. The various complexes formed are characterized by Gibbs free energies, enthalpies, and entropies. The Gibbs free energies were determined by equilibrium dialysis investigations, fluorescence titrations, and ultraviolet difference spectroscopy, while the reaction enthalpies stem from direct calorimetric measurements, Formulas are given for both the temperature dependence of the equilibrium constants and the variation with temperature of the enthalpies involved in the four reactions between LDH and NADH or NAD, LDH-NADH and oxamate, and LDH-NAD and oxalate. All reactions show a marked negative temperature coefficient, deltacp, of the binding enthalpies indicating partial refolding to be associated with binary and ternary complex formation. This interpretation appears very probable in view of recent x-ray crystallographic studies on lactate dehydrogenase from dogfish, which demonstrate a volume decrease to occur on binding of oxamate to the LDH-NADH complex. The validity of the thermodynamic parameters, as derived with substrate analogues, for the actual catalytic reaction, gains strong support from the agreement between the sum of the heats involved in the four intermediary reactions reported in this study and direct determinations of the overall enthalpy associated with the catalytic process published in the literature.