Biomedical subjects

G B Villanueva

Publications and source records attributed to G B Villanueva.

At least 19 recordsLinked to original sources

PubMed1994

The low pH stability of human coagulation factor XII (Hageman factor) is due to reversible conformational transitions.

Factor XII undergoes autoactivation when bound to negatively charged surfaces. To gain insight into the mechanism of factor XII autoactivation and stability at low pH, structural studies in the presence and absence of a soluble surface, dextran sulfate, at pH 5.3 and pH 8.3 were carried out. The circular dichroism data indicate that the secondary structure at pH 5.3 is only modestly different from that at pH 8.3. However, large changes in the secondary structure are found to occur when factor XII is exposed to pH 5.3 in the presence of surface. Changes in tertiary structure at low pH are also evident from the difference in tryptophan fluorescence and chemical reactivity of the histidine residues. Factor XII binds to the surface even at pH 5.3 though it is inactive at this pH. It is concluded that factor XII adopts a different conformation at pH 5.3 and causes it to interact differently with dextran sulfate. This results in an obstructed cleavage site that accounts for its stability at low pH.

Binding Sites↗

PubMed1994

A model demonstrating different interactions of human coagulation factor XII (Hageman factor) with the surface at physiological and lower pH.

The surface binding activity of human coagulation factor XII is independent of pH while its proteolytic breakdown decreases considerably on lowering the pH (Samuel, M. and Villanueva, G.B. (1992) Biophysical J. 61, 1895). In the present study we showed that the amidolytic activity of factor XII in the presence of a soluble surface, dextran sulfate (DS500; Mr 500,000) decreases on lowering the pH. Electrophoretic and ultraviolet difference spectral studies indicate that factor XII binding stoichiometry to DS500 is reduced to half at pH 5.3 when compared to that at pH 7.4. A model is presented to show the different interactions of factor XII with DS500 which explains its well known stability at low pH.

Chromogenic Compounds↗

PubMed1993

Histidine residues are essential for the surface binding and autoactivation of human coagulation factor XII.

The role of histidine residue in the surface binding and autoactivation of human factor XII has been investigated by chemical modification with diethyl pyrocarbonate. It is found that low concentrations of diethyl pyrocarbonate have profound inhibitory effects on the surface binding activity of factor XII. At 2.5-fold molar excess of the reagent, six histidines are modified and 80% of the amidolytic activity is lost. Electrophoretic studies show that the modified protein has lost the capacity to bind to the surface, resulting in diminished proteolytic autoactivation. When modification is performed in the presence of the surface, dextran sulfate, two of the six histidines are protected from modification and the amidolytic activity is completely preserved. It is concluded that histidine residues in factor XII play key role in its surface binding activity.

Binding Sites↗

PubMed1993

Effect of zinc removal on the conformation of Escherichia coli DNA topoisomerase I.

Escherichia coli DNA topoisomerase I contains three Zn(II) in each enzyme molecule required for relaxation of negatively supercoiled DNA. Apoenzymes were prepared from both the intact topoisomerase (M(r) 97,000) and the truncated active form top85 (M(r) 85,000) that lacks the carboxyl terminal domain but still contains the three Zn(II). Fluorescence and circular dichroism spectroscopy were used to compare the apoenzymes with topoisomerase and top85 reconstituted with Zn2+. The results indicated structural changes affecting the environment of the tryptophan residues and increasing the alpha-helical and beta-sheets content of the protein occurred upon zinc removal. These structural changes probably account for the loss of enzyme activity.

Apoenzymes↗

PubMed1992

A simple and rapid method to study the association of the contact proteins of blood coagulation.

Native and reduced SDS polyacrylamide gel electrophoresis on the automated PhastSystem (Pharmacia) were used to demonstrate protein-protein binding interactions and structural changes during proteolytic activations of the proteins involved in contact activation. The "mobility shift" assay in native gels has been used to visualize the kinetics of activation of factor XII by dextran sulfate as well as the formation of kallikrein-cleaved high molecular weight kininogen. It shows the formation of prekallikrein-high molecular weight kininogen complexes and factor XII-dextran sulfate complex for the first time in gels. The use of automation makes this procedure fast and reproducible using nanogram amounts of protein in relatively short time.

Amino Acid Sequence↗

PubMed1992

Human factor XII (Hageman factor) autoactivation by dextran sulfate. Circular dichroism, fluorescence, and ultraviolet difference spectroscopic studies.

The first event leading to the activation of the plasma kallikrein-kinin system is the surface-dependent conversion of factor XII to an active enzyme. Factor XII autoactivation was investigated using dextran sulfate as a soluble activating surface, and the significance of aggregation and the nature of the conformational change were examined by ultraviolet difference spectroscopy, fluorescence and circular dichroism. Results indicate that DS500 (500-kDa dextran sulfate) induces aggregation of factor XII. Analysis of the binding data suggests that 165-192 factor XII molecules can bind to one DS500 chain, while a 1:1 stoichiometry is observed with 5-kDa dextran sulfate. The interaction of factor XII and dextran sulfate is a biphasic process. It is initiated by a fast contraction of the molecule upon binding, as revealed by an apparent increase in organized secondary structures, and then followed by a slow relaxation process during cleavage and subsequent activation. Overall, the results are consistent with a model in which factor XII undergoes conformational changes upon binding to the activating surface. The rapidity of autoactivation in the presence of DS500, as opposed to 5-kDa dextran sulfate, implies that aggregation provides a special mechanism whereby proteolytic cleavage is accomplished efficiently when factor XII molecules are bound side by side on the DS500 molecule.

Circular Dichroism↗

PubMed1991

Thrombin inhibition by hirudin: how hirudin inhibits thrombin.

In addition to its classical active-site regions (catalytic site and adjacent regions), alpha-thrombin has a unique anion-binding exosite, which is functionally independent of the catalytic site and is involved in fibrin(ogen) recognition. This exosite also accounts for adhesion to negatively charged surfaces (e.g., glass), binding to cell surfaces, and interactions with the anionic tail of hirudin. Hirudin (as an apolar, tridisulfide-linked core structure followed by its anionic tail) interacts with alpha-thrombin by apolar (e.g., catalytic-site and adjacent regions of thrombin), as well as by ionic binding (e.g., anion-binding exosite). Circular dichroism measurements reveal a sigmoidal nonadditivity for the hirudin tail fragments, which block fibrinogen-clotting activity without interfering with tripeptide chromogenic substrate activities. Such fragments, however, inhibit factor V activation to much lesser extents than hirudin, where factor V activation is the key step in regulating thrombin generation by hirudin or heparin/antithrombin III. Hirudin-derived antithrombotics may thus have differential modes of action in hemostasis and wound healing processes.

Amino Acid Sequence↗

PubMed1989

Conformation of high molecular weight kininogen: effects of kallikrein and factor XIa cleavage.

The effect of kallikrein and factor XIa proteolysis of high molecular weight kininogen (HK) was investigated. Circular dichroism (CD) spectroscopy showed that cleavage of HK by plasma kallikrein or urinary kallikrein, both of which result in an active cofactor (HKa), results in conformational change that is characterized by increase in CD ellipticity at 222 nm. This suggests an increase in organized secondary structures. By contrast, cleavage of HK by factor XIa which results in an inactive cofactor (HKi) is characterized by a dramatic decrease in CD ellipticity at 222 nm suggesting an entirely different type of conformational change. The intrinsic fluorescence of HK is enhanced after cleavage by all three proteases. These conformational changes may play a role in determining the structure and function of HKa and HKi.

Circular Dichroism↗

PubMed1988

Analysis of the secondary structure of hirudin and the mechanism of its interaction with thrombin.

Highly purified hirudin with a specific activity of 13,950 antithrombin units/mg was isolated from a commercial preparation by reversed-phase chromatography. The circular dichroism (CD) spectrum of hirudin was investigated and it was found that the spectrum cannot be accounted for solely in terms of the traditional three components of peptide backbone. It was also found that the CD spectrum of the thrombin-hirudin complex was not additive with respect to the individual spectra of thrombin and hirudin. This deviation from additivity was significant between 210 and 225 nm, indicating alterations in the secondary structures of the proteins during complex formation. When thrombin was titrated with hirudin, the spectral deviation from additivity was sigmoidal, suggesting the cooperative nature of the binding process. Gel filtration of the thrombin-hirudin mixture showed no molecular species greater than a 1:1 complex (Mr 45,500), but gel filtration of free hirudin showed a multimeric form (Mr 51,300) under the same experimental conditions. It is concluded that the cooperative nature of the binding process is due to the binding of thrombin molecules to the multimeric form of hirudin. This initial binding occurs with little or no change in the CD spectrum. In the second step, the multiple complex dissociates to form 1:1 complexes, resulting in larger conformational changes and a considerable increase in binding affinity.

Binding Sites↗

PubMed1988

Circular dichroism of platelet factor 4.

The circular dichroism of platelet factor 4 was investigated and it was found to contain 15% alpha-helix, 25% beta-structure, and the rest of the molecule in unordered conformation. In the presence of heparin, no change in the circular dichroism was observed, suggesting no significant changes in the secondary structure of platelet factor 4 when heparin binds. The CD spectrum of platelet factor 4 was also investigated in the presence of increasing concentrations of guanidine hydrochloride. A two-state transition was observed with midpoints at 0.125 and 2.0 M guanidine hydrochloride. Based on gel filtration studies, the first unfolding transition was correlated with the dissociation of the tetrameric structure. This first unfolding domain was not observed in the presence of heparin, suggesting that heparin stabilizes the tetrameric structure. The second unfolding transition corresponds to the disruption of the overall secondary structure which is generally observed with most proteins. It is concluded that a relatively weak force of attraction holds the tetrameric structure of platelet factor 4 and the dissociation of the subunits is accompanied by loss of some helical secondary structure.

Chromatography, Gel↗

PubMed1988

Demonstration of altered antithrombin III activity due to nonenzymatic glycosylation at glucose concentration expected to be encountered in severely diabetic patients.

The effect of nonenzymatic glycosylation on the kinetics and structure-function relationships of antithrombin III were investigated at normal physiologic concentrations of antithrombin III and glucose, which are 5.2 microM and 5 mM, respectively. The results were compared with antithrombin III incubated at the glucose concentration expected to be found in severely diabetic patients (15 mM). Antithrombin III incubated at 5 mM lost 33% of the heparin cofactor activity after 7 days, whereas antithrombin III incubated at 15 mM lost 50% for the same period. Under both conditions, half of the heparin cofactor activity was lost after 15 days. When D-[U-14C]glucose was used as tracer, approximately 0.6 mol glucose/mol protein was incorporated after 10 days at both concentrations of glucose. A detailed evaluation of the kinetics of inhibition of thrombin by glycosylated antithrombin III revealed that the second-order rate constant is three times smaller than that of normal antithrombin III. On the basis of these data, it is concluded that glycosylated antithrombin III with 50% depressed heparin cofactor activity is three times weaker than normal antithrombin III as an inhibitor of thrombin. The implications of these observations with respect to the possible pathogenesis of thrombosis in diabetes are discussed.

Antithrombin III↗

PubMed1986

Light-scattering investigation of the subunit structure and dissociation of octopoda hemocyanins.

The molecular weights, subunit dissociation, and conformation in solution of the hemocyanins of three species of octopi were investigated by light-scattering, ultracentrifugation, absorbance, and circular dichroism methods. The molecular weights of the hemocyanins of Octopus bimaculoides, Octopus bimaculatus, and Octopus rubescens obtained by light scattering were 3.3 X 10(6), 3.4 X 10(6), and 3.5 (+/- 0.3) X 10(6), respectively. The average molecular weights of the fully dissociated hemocyanins of the same octopi, investigated at alkaline pH and in the presence of 8 M urea and 6 M guanidinium chloride (GdmCl), were found to be close to one-tenth of those of the parent proteins, with average molecular masses of 3.4 X 10(5), 3.3 X 10(5), and 3.3 (+/- 0.3) X 10(5). These findings confirm the earlier observations of van Holde and co-workers with other cephalopod hemocyanins that the basic cylindrical assembly of molluscan hemocyanins consists of 10 subunits. Circular dichroism and absorbance measurements suggest that the dissociated subunits at alkaline pH and in concentrated urea solutions retain their native, multidomain folding. Fairly concentrated GdmCl above 3-4 M is necessary to unfold fully the dissociated hemocyanin chains. Molecular weight measurements studied as a function of reagent concentration with the urea and Hofmeister salt series as dissociating agents show that the ureas are very effective dissociating agents, while the salts are ineffective to moderately effective reagents for octopus hemocyanin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Structure-function relationships in heparin cofactor II: chemical modification of arginine and tryptophan and demonstration of a two-domain structure.

Heparin cofactor II and antithrombin III are plasma proteins functionally similar in their ability to inhibit thrombin at accelerated rates in the presence of heparin. To further characterize the structural and functional properties of human heparin cofactor II as compared to antithrombin III, we studied the possible significance of arginyl and tryptophanyl residues and the changes in protein structure and activity during guanidinium chloride (GdmCl) denaturation. Both antithrombin and heparin cofactor activities of heparin cofactor II are inactivated by the arginine-specific reagent, 2,3-butanedione. Saturation kinetics are observed during modification and suggest formation of a reversible protease inhibitor-butanedione complex. Quantitation of arginyl residues following butanedione modification shows a loss of about four residues for total inactivation, one of which is essential for antithrombin activity. Arginine-modified heparin cofactor II did not bind to heparin-agarose and implies a role for the other modified arginyl residues during heparin cofactor activity. N-Bromosuccinimide oxidation (20 mol of reagent/mol of protein) of heparin cofactor II results in modification of approximately two tryptophanyl residues with no concomitant loss of heparin cofactor activity. Moreover, there is no enhancement of intrinsic protein fluorescence during heparin binding to the native inhibitor. Circular dichroism measurements show that the structural transition of heparin cofactor II during denaturation is distinctly biphasic, yielding midpoints at 0.6 and 2.6 M GdmCl. Functional protease inhibitory activities are affected to the same extent following denaturation-renaturation at various GdmCl concentrations. The results indicate that arginyl residues are critical for both antithrombin and heparin binding activities. In contrast, tryptophanyl residues are apparently not essential for heparin-dependent interactions. The results also suggest that heparin cofactor II contains two structural domains which unfold at different GdmCl concentrations.

Amino Acids↗

PubMed1986

Acetylation of antithrombin III by aspirin.

Although the effect of aspirin in blood coagulation has been believed to be due to its ability to interfere with platelet function, very few studies have reported its effect on various blood coagulation proteins. Since aspirin (acetylsalicylic acid) is known to acetylate numerous biologic macromolecules, the effect of aspirin on antithrombin III was investigated. It was found that antithrombin III is irreversibly inactivated by treatment with aspirin. The inactivation follows pseudo first-order kinetics and incorporation of one molecule of aspirin per molecule of the protein is necessary for complete inactivation. Reaction with acetyl-[14C]-salicylic acid incorporated 1.4 mol of acetyl group per mole of protein but reaction with carboxyl-[14C]-acetyl salicylic acid incorporated only 0.03 mol of radioactive label per mole of the protein. Furthermore, sodium salicylate does not inactivate the protein. This suggests that the reaction occurs through the acetylation of antithrombin III. Amino group analysis of aspirin-treated antithrombin III using trinitrobenzenesulfonic acid revealed that one to two primary amino groups are lost relative to the untreated antithrombin III. It is concluded that the reaction of aspirin with antithrombin III results in specific acetylation of lysine residues.

Acetylation↗

PubMed1985

Light-scattering investigation of the dissociation behavior of Lunatia heros and Littorina littorea hemocyanins.

The subunit structure and dissociation of the hemocyanins of two marine snails, Lunatia heros and Littorina littorea, were investigated by light-scattering molecular weight methods. The hemocyanins of both species of snails are readily dissociated to fragments of one-tenth and one-twentieth of the parent proteins of close to 9 X 10(6) daltons by either increasing the pH or using dissociating reagents of the hydrophobic urea series or some of the Hofmeister salts. The lower members of the latter group of reagents, NaCl, and to some extent also NaBr were found to have only marginal effects on the observed molecular weight transitions, suggesting that the two hemocyanins investigated possess beta-type subunits, which are known to be resistant to NaCl dissociation. The molecular weight profiles obtained with the various dissociating reagents were single inverted sigmoidal-shaped curves for both Lunatia and Littorina hemocyanins, suggesting overlapping transitions. The ultracentrifugation patterns and the species-distribution plots based on the urea dissociation data of Littorina hemocyanin suggest the presence of whole, half, and one-tenth molecular weight species in the dissociation transition region. Fitting of the urea dissociation data of Littorina hemocyanin obtained at both pH 5.7 and pH 8.0, assuming a sequential two-step dissociation scheme used in our previous studies [Herskovits, T. T., & Russell, M. W. (1984) Biochemistry 23, 2812-2819], was found to be consistent with a model of a few hydrophobic binding sites at the contact areas of the half-molecules and a much larger apparent number of binding sites (Napp) at the side to side contacts of the one-tenth molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Subunit dissociation of Busycon canaliculatum hemocyanin.

The hemocyanin of the channeled whelk, Busycon canaliculatum, is a multisubunit protein with a molecular weight close to 9 X 10(6). The increase in pH above neutrality and the addition of 0-5 M urea and 0-2 M GdnHCl is found to dissociate the whole molecules to half-molecules and smaller dimeric and monomeric fragments of one-tenth and one-twentieth mass of the parent hemocyanin. The molecular weight transitions investigated at constant protein concentration of 5 X 10(-2) g X l-1 show no clearly discernible plateau regions, where essentially only half-molecules and one-tenth molecules are present. The ultracentrifugation patterns in much of the dissociation region produced by urea at pH 6.9 suggests the presence of three distinct components consisting of whole molecules, half-molecules and largely one-tenth molecular weight fragments. At pH 8.2 and higher, where whole molecules are largely absent, the effects of urea on the dissociation of half-molecules to tenths and tenth-molecules to twentieth molecule was investigated by means of light scattering. Analysis of the urea data based on a decamer to dimer and dimer to monomer scheme of dissociation used in our earlier studies gave apparent estimates of about 90 amino acid groups at the contact areas of the dimers in the half-molecules and 110 groups at the monomer contacts forming the dimers. The latter relatively large estimate of groups suggests that the dissociation of the tenth molecules or dimers must occur by longitudinal splitting of the contact areas along both the folded domains and the connecting chain segments of the twentieth molecules. Circular dichroism, absorbance and viscosity data suggest that the secondary structure and conformation of the folded domains of the hemocyanin subunits are largely retained at both high pH and in 3-8 M urea solutions. The molecular weights at pH 9.0-10.6 and in 3-8 M urea are found to be (4.2-4.7) X 10(5), close to one-twentieth of the mass of the parent hemocyanin. Denaturation and unfolding of the subunit domains is observed between 3 and 6 M GdnHCl solutions, as evidenced by the abolition of the characteristic copper absorbance in the neighborhood of 346 nm and the relatively pronounced changes in circular dichroism at 222 nm and intrinsic viscosity. The further decrease in molecular weights to about (2.6-3.2) X 10(5), below one-twentieth of the mass of hemocyanin suggests the presence of hidden breaks or scissions in the polypeptide chains suffered during isolation, which become exposed as a result of complete unfolding in GdnHCl solutions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1984

Conformational integrity of human alpha-thrombin.

It is known that storage at pH 6 stabilizes thrombin against inactivation. In order to determine whether structural changes accompany this stabilization, the conformation of human alpha-thrombin at pH 6.0 and 7.5 was investigated by chemical modification, difference spectroscopy, circular dichrosim, and thermal stability. It was shown that the CD spectra at the 230-200 nm peptide transition were indistinguishable at the two pH values, indicating no differences in the secondary structure as also indicated by the thermal stability of the enzyme at pH 6.0, 7.4 and 8.3. However, differences were observed in the 300-250 nm aromatic transition suggesting some changes in the microenvironment of the aromatic chromophores. Solvent perturbation in 20% ethylene glycol and 20% dimethylsulfoxide showed that at pH 7.5, 4.3 +/- 0.3 tryptophan and 8.6 +/- 0.4 tyrosine residues were exposed and accessible to the solvent whereas at pH 6.0 these values were 3.6 +/- 0.1 tryptophan and 7.8 +/- 0.4 tyrosine residues. At pH 7.5, 6.0 +/- 0.5 tryptophan residues were found reactive toward dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide while 2.5 +/- 0.3 were found reactive at pH 6.0. Accompanying these structural changes were ultraviolet absorption and CD spectral changes with transition midpoints at pH 6.45 characteristic of histidine ionization. These spectral changes were lost when alpha-thrombin was modified by diethylpyrocarbonate but not by N-alpha-tosyl-L-Lysinechloro-methyl ketone. It is concluded that a second histidine residue, not the active site His-43, is associated with the pH dependent conformational changes at pH 6.0. The ionization of this histidine residue and the accompanying conformational changes could explain the reduced catalytic efficiency and stability of alpha-thrombin at pH 6.

Circular Dichroism↗

PubMed1984

Effect of heparin modification on its circular dichroism spectrum.

The effect of modification of the carboxyl groups of high affinity heparin was investigated. The binding affinity toward antithrombin III decreases in the following order: Heparin greater than heparin methyl ester greater than heparinylglycine greater than heparinylglycine methyl ester. This result agrees qualitatively with the previous studies using unfractionated heparin. Esterification of the carboxyl groups (i.e., HME) does not affect the CD profile of heparin at 210 nm but introduction of a bulkier glycine methyl ester (i.e., HGME) leads to formation of a very intense band at 235 nm. Based on reported CD analyses of uronic acid derivatives and our model building studies, it is concluded that the large difference in CD spectra of HGME as compared to unmodified heparin and HME is due to a change in ring conformation of the uronic acid moiety (i.e., 4C1 to 1C4 or vice versa).

Carbohydrate Conformation↗