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S Krishnaswamy

Publications and source records attributed to S Krishnaswamy.

At least 73 records · Page 4Linked to original sources
PubMed1990

Structural refinement and analysis of Mengo virus.

The structure of Mengo encephalomyelitis virus was refined at 3 A resolution with a final R-factor of 0.221 and a root-mean-square deviation from idealized bond lengths of 0.019 A for 10 A to 3 A data with F greater than or equal to 3 sigma(F). The Hendrickson-Konnert refinement was restrained by the phases derived from the molecular replacement averaging procedure and constrained by the icosahedral symmetry of the virus. The virus consists of 60 protomers each having three major subunits, VP1, VP2 and VP3, along with one smaller internal protein, VP4. The three major subunits form similar eight-stranded beta-barrel structures. Alterations in the original sequence were found at position 45 in VP1 (Arg to Ala) and at position 58 in VP3 (Met to Val). The residues in loops I and II of VP1 (82 to 102), the "FMDV loop" in VP1 (205 to 213), the flexible loop of VP3 in the putative receptor attachment site (175 to 185) as well as the terminal regions 260 to 268 in VP1, 253 to 256 in VP2 and 13 to 15 in VP4 were built or modified in regions of weak density. The variation in temperature factors at the end of the refinement is over a wide range (from 2 to 80 A2), with the disordered outer and inner regions showing high mobility. Four cis proline residues, 105 in VP1, 85 and 152 in VP2 and 59 in VP3, have been identified. The disulfide bridge Cys86 to Cys88 in VP3 has been characterized. One phosphate ion and 233 water positions were included in the refinement. It is suggested that this phosphate is associated with the receptor attachment site. There are two major hydrogen-bonding networks involving solvent atoms; one involving only the subunits of a protomer, and the other connecting the protomers in a pentamer. The distribution of atom types around the icosahedral symmetry axes shows that the 5-fold channel is more hydrophobic than that along the 3-fold axis and that there are more charged residues around the 2-fold axis. The analysis of contacts between the different subunits supports the assignment of the protomeric unit. The five protomers that form the pentameric unit are held together by interactions involving the smaller VP4 protein and the amino termini of VP1 and VP3. The pentamers are associated by means of the amino-terminal region of the VP2 subunits, the beta F strand of the VP3 subunits, the C terminus of the VP4 subunits and the electrostatic helical (alpha A) interactions of VP2 subunits across the icosahedral 2-fold axes. The superposition of the corresponding subunits of Mengo virus, human rhinovirus 14 and southern bean mosaic virus has provided an improved sequence alignment. The largest structural similarity is between the VP3 subunits of Mengo virus and rhinovirus, while the least similarity is between the VP1 subunits. The various specialized insertions in the different subunits can be associated with specific functional requirements.

Amino Acid Sequence↗
PubMed1990

Conformational variability of a picornavirus capsid: pH-dependent structural changes of Mengo virus related to its host receptor attachment site and disassembly.

The structure of Mengo virus had been determined from crystals grown in the presence of 100 mM phosphate buffer at pH 7.4. It is shown that Mengo virus is poorly infectious at the phosphate concentration similar to that in which it was crystallized. Maximal infectivity is achieved at 10 mM phosphate or less in physiological saline. The phosphate effect is ameliorated when the pH is lowered to 4.6. Although it has not been possible to study the crystal structure of the virus at low phosphate concentrations, it is shown that increasing the Cl- concentration at pH 6.2 or decreasing the pH to 4.6 causes substantial conformational changes confined to the "pit," a deep surface depression. These structural changes involve a movement of the "FMDV loop" (GH loop) in VP1, an ordering of the "VP3 loop" (GH loop in VP3) between 3176 and 3182, the displacement of a bound phosphate near the "FMDV loop" (GH loop in VP1), and movement of the carboxy terminus of VP2. The changes in conformation are correlated with the dissociation of the virion into pentamers at pH 6.2 and 150 mM Cl-. The localization of the conformational changes and the correlated role of the phosphate in controlling infectivity support the hypothesis that the "pit" is the receptor attachment site.

Amino Acid Sequence↗
PubMed1989

Three-dimensional structures of drug-resistant mutants of human rhinovirus 14.

Mutants of human rhinovirus 14 were isolated and characterized by searching for resistance to compounds that inhibit viral uncoating. The portions of the RNA that code for amino acids that surround the antiviral compound binding site were sequenced. X-ray analysis of two of these mutants, 1188 Val----Leu and 1199 Cys----Tyr, shows that these were single-site substitutions which would sterically hinder drug binding. Differences in the resistance of mutant viruses to various antiviral compounds may be rationalized in terms of the three-dimensional structures of these mutants. Predictions of the structures of mutant rhinovirus 14 with the substitutions 1188 Val----Leu, 1199 Cys----Tyr and 1199 Cys----Trp in VP1 were made using a molecular dynamics technique. The predicted structure of the 1199 Cys----Tyr mutant was consistent with the electron density map, while the 1188 Val----Leu prediction was not. Large (up to 1.4 A) conformational differences between native rhinovirus 14 and the 1199 Cys----Tyr mutant occurred in main-chain atoms near the mutation site. These changes, as well as the orientation of the 1199 tyrosine side-chain, were correctly predicted by the molecular dynamics calculation. The structure of the predicted 1199 Cys----Trp mutation is consistent with the drug-resistant properties of this virus.

Chromosome Aberrations↗
PubMed1989

Zymogen/enzyme discrimination using peptide chloromethyl ketones.

Glutamylglycinylarginyl chloromethyl ketone, tyrosylglycinylarginyl chloromethyl ketone, and phenylalanylprolylarginyl chloromethyl ketone have been labeled at their amino termini using fluorescein, rhodamine-X, lissamine-rhodamine, pyrene, and the 1,5-, 2,5-, and 2,6-dimethylaminonaphthalene-1-sulfonyl moieties. These peptidyl chloromethyl ketones have also been modified by incorporation of biotin and epsilon-amino caproyl biotin. The ability of these various chloromethyl ketones to be incorporated into a collection of zymogen-enzyme pairs has been evaluated using a variety of coagulation and fibrinolytic proteins. All labeled chloromethyl ketones were efficiently incorporated into the proteases tested, with the exception of urokinase which was refractory to inhibition by phenylalanylprolylarginyl chloromethyl ketone derivatives. No modification of any zymogen species was observed even under conditions designed to detect minimal reactivity. When enzymes were modified using chloromethyl ketones labeled with epsilon-amino caproyl biotin, the modified proteins readily reacted with avidin under a variety of different conditions. The observed reactivity with avidin was used in enzyme "blotting" following electrophoretic resolution of polypeptide chains and to remove active enzyme present in enzyme-zymogen mixtures. These reagents have been used to evaluate the potential for active site expression by the single-chain human factor VII molecule. Studies conducted with tissue factor, phospholipids, and calcium using factor X as substrate demonstrate that no activity can be obtained without initial activation of either factor X to factor Xa or factor VII to factor VIIa by an external source. We thus conclude that factor VII is a true zymogen, inert in the blood clotting process prior to its cleavage to factor VIIa.

Amino Acid Chloromethyl Ketones↗
PubMed1989

The interaction of bone Gla protein (osteocalcin) with phospholipid vesicles.

The binding interaction of bone Gla protein (BGP), or osteocalcin, to phospholipid vesicles in the presence of calcium has been investigated. Two separate indirect methodologies involving displacement of pyrene-modified Factor Va bound to phospholipid vesicles, and competition with several coagulation proteins in a prothrombin activation assay were performed. Titration of BGP into a cuvette containing phospholipid vesicles (75:25, L-alpha-phosphatidylcholine/L-alpha-phosphatidylserine (PCPS] saturated with pyrene-modified Factor Va resulted in a systematic decrease in steady-state anisotropy, suggesting competition for membrane binding sites with pyrene-modified Factor Va. BGP was also found to inhibit thrombin generation in the prothrombin activation assay. Approximately 50% inhibition was observed at 3 microM BGP under phospholipid-limiting (0.5 microM PCPS) concentrations. No inhibition was observed under phospholipid excess (30 microM PCPS) concentrations. Direct measurement of phospholipid binding was measured using equilibrium gel filtration. Elution profiles using fixed lipid (3.4 mumol of PCPS) and varying BGP concentrations (1-17 microM) in the presence of 3 mM CaCl2 showed a BGP-phospholipid association. Quantitation of determined isotherm yielded a dissociation constant of 6 +/- 1 microM with a stoichiometry of 102 +/- 9 BGP molecules/vesicle at saturation (35 PCPS lipids/BGP) in the presence of 3 mM CaCl2. These results support the hypothesis that protein gamma-carboxylation events are coincident with membrane binding potential.

Binding, Competitive↗
PubMed1989

The reassociation of factor Va from its isolated subunits.

Factor Va is an essential cofactor for the activation of prothrombin catalyzed by factor Xa. The cofactor is a heterodimer composed of a light chain and a heavy chain that are associated noncovalently in the presence of divalent metal ions. The kinetics of the formation of factor Va from the isolated and separated subunits was examined by the time-dependent regain in cofactor activity using direct assays of prothrombin activation catalyzed by prothrombinase. The rate of reassociation at saturating concentrations of calcium ions was slow with a strong temperature dependence. The product of the association reaction was indistinguishable from native factor Va on the basis of activity. The second order rate constant for the process at 37 degrees C in the presence of 2 mM CaCl2 was 1.58 X 10(5) M-1.min-1. Manganese ion increased the rate of regain of activity without influencing the extent of the reaction. The previous identification of a single reactive sulfhydryl in each subunit of factor Va permitted the modification of the separated subunits with sulfhydryl-directed fluorophores. Subunit reassociation was directly measured by fluorescence energy transfer using light chain modified with 6-acryloyl-2-dimethylaminonaphthalene (fluorescence donor) and heavy chain modified with fluorescein 5-maleimide (fluorescence acceptor). Fluorescence measurements indicate that the heavy and light chains associate tightly (Kd = 5.9 x 10(-9) M) and reversibly with a stoichiometry of 1:1. The dissociation of the subunits from the cofactor is first order with a rate constant of 1.03 X 10(-3) min-1. These interpretations were confirmed by physical measurements of subunit reassociation by sedimentation velocity studies.

2-Naphthylamine↗
PubMed1988

The binding of factor Va to phospholipid vesicles.

The analysis of free sulfhydryl groups in factor Va using dithiobis-(nitrobenzoic acid) (DTNB) indicated the presence of one accessible thiol in each of the two subunits of the cofactor. Intact factor Va contained one readily accessible sulfhydryl group under native conditions and approximately two such groups after denaturation. A comparison of the rate of modification of the accessible thiol in factor Va under native conditions to those observed with the isolated subunits indicated that the thiol present in component D of the cofactor was readily accessible to reaction with DTNB. Factor Va was reacted with the sulfhydryl-directed fluorophore N-(1-pyrene)maleimide, resulting in the concomitant loss of the accessible thiol with no detectable alteration in the activity of the cofactor. This fluorescent derivative of factor Va (Pyr-Va) was used to examine the binding of factor Va to phospholipid vesicles by fluorescence polarization. Fluorescence polarization of the pyrene moiety increased saturably when Pyr-Va was titrated with increasing concentrations of vesicles composed of phosphatidylcholine and phosphatidylserine (PS). Systematic analysis of the binding of Pyr-Va to PCPS (75% phosphatidylcholine, 25% PS) indicated that the binding interaction was characterized by a dissociation constant of 2.7 x 10(-9) M with 42 mol of PCPS bound per mol of Va at saturation. The data obtained by varying the PS content of the vesicles are consistent with the interpretation that the Va-combining site on the vesicle surface is composed of a discrete number of PS molecules. The binding of Pyr-Va to PCPS was independent of added calcium ion and could be reversed by the addition of unlabeled Va or isolated component E but not by component D. Analysis of the displacement curves indicated that native factor Va or isolated component E and Pyr-Va mutually excluded each other on the vesicle surface with identical affinities. Competition experiments conducted using component E digested by factor Xa or the isolated derivative peptides indicated that the cleavage of component E by factor Xa had no effect on the PCPS binding properties of this subunit. Further, the data obtained with the isolated peptides suggest that the lipid-binding domain of component E is present in the amino-terminal region of this subunit.

Animals↗
PubMed1988

Prothrombinase complex assembly. Kinetic mechanism of enzyme assembly on phospholipid vesicles.

The kinetics of the assembly of the prothrombinase complex was studied using factor Xa modified with the active site-directed fluorophore dansylglutamylglycylarginyl chloromethyl ester (DEGR.Xa) as a reporter for the process. Stopped-flow kinetic studies of prothrombinase assembly were undertaken at saturating concentrations of calcium ion, using vesicles composed of phosphatidylcholine and phosphatidylserine (PCPS), factor Va, and DEGR.Xa. The rate of complex formation, studied under pseudo first-order conditions, depended on the premixing protocol used to initiate complex assembly and was most rapid when prothrombinase assembly was initiated by reacting Va with the preformed DEGR.Xa.PCPS complex. Under these conditions, reaction rate was linearly dependent on the concentration of PCPS with no evidence for saturation and was independent of the concentration of VA. Essentially identical reaction rates were observed when the association of Va with PCPS was independently assessed by stopped-flow light scattering. The data indicate that prothrombinase assembly initiated by mixing Va with the DEGR.Xa.PCPS complex is rate limited by the initial reaction between Va and available combining sites on the vesicle surface (k = 5.7 x 10(7) M-1.s-1) followed by extremely rapid reactions (k greater than 1 x 10(9) M-1.s-1) between PCPS-bound DEGR.Xa and PCPS-bound Va. The formation of a separate Va.PCPS complex is therefore required for prothrombinase assembly to proceed. Reaction rates obtained when prothrombinase assembly was initiated by reacting DEGR.Xa with the preformed Va.PCPS complex or by reacting PCPS with a mixture of DEGR.Xa and Va were lower. Under these initiation conditions, reaction rates were inhibited by increasing concentrations of factor Va. These data indicate that the formation of the DEGR.Xa.PCPS binary complex is also a prerequisite for prothrombinase assembly. The second-order rate constant for the reaction between factor Xa and PCPS (k = 2.9 x 10(7) M-1.s-1) and the first-order rates of dissociation of the Va.PCPS complex (koff = 0.17 s-1) and the Xa.PCPS complex (koff = 3.3 s-1) were independently determined by stopped-flow light scattering. Collectively, the data are consistent with the interpretation that prothrombinase assembly proceeds via the initial formation of separate Xa.PCPS and Va. PCPS binary complexes at near diffusion-limited rates on the same vesicle. The lipid-bound constituents then react rapidly via rearrangements on the vesicle surface to form prothrombinase.

Amino Acid Chloromethyl Ketones↗
PubMed1988

Control of ciliary beat by calcium: the effects of lindane, a potent insecticide.

The ciliary beat and cell motility of Dunaliella, a biflagellate unicellular green alga, have been studied by means of computer analysis of high-speed microcinematography and laser doppler velocimetry. Lindane was found rapidly to inhibit cell velocity in less than 5 min. and in a dose-related manner. After the initial 5-min. period, inhibition remained nearly stable for at least 3 hr. The waveform of the ciliary beat was not uniformly affected by lindane and only the effective stroke was greatly slowed down. The recovery stroke was not significantly modified, and the general form of the wave did not seem to be altered. Bending parameters automatically measured from high-speed movies showed that lindane induced a considerable lengthening of the initiation phase of wave propagation. Since lindane interacts specifically with calcium transport and can induce an increase in cytoplasmic calcium, the strong effect of lindane on ciliary beat is probably correlated with a modification of the calcium balance of the cell. These results support the hypothesis of a control of bend initiation by calcium.

Calcium↗
PubMed1988

Structural analysis of a series of antiviral agents complexed with human rhinovirus 14.

The binding to human rhinovirus 14 of a series of eight antiviral agents that inhibit picornaviral uncoating after entry into host cells has been characterized crystallographically. All of these bind into the same hydrophobic pocket within the viral protein VP1 beta-barrel structure, although the orientation and position of each compound within the pocket was found to differ. The compounds cause the protein shell to be less flexible, thereby inhibiting disassembly. Although the antiviral potency of these compounds varies by 120-fold, they all induce the same conformational changes on the virion. The interactions of these compounds with the viral capsid are consistent with their observed antiviral activities against human rhinovirus 14 drug-resistant mutants and other rhinovirus serotypes. Crystallographic studies of one of these mutants confirm the partial sequencing data and support the finding that this is a single mutation that occurs within the binding pocket.

Antiviral Agents↗
PubMed1987

Activation of human prothrombin by human prothrombinase. Influence of factor Va on the reaction mechanism.

The kinetics of the activation of human prothrombin catalyzed by human prothrombinase was studied using the fluorescent alpha-thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA). Prothrombinase proteolytically activates prothrombin to alpha-thrombin by cleavages at Arg273-Thr274 (bond A) and Arg322-Ile323 (bond B). The differential fluorescence properties of DAPA complexed with the intermediates and products of human prothrombin activation were exploited to study the kinetics of the individual bond cleavages in the zymogen. When the catalyst was composed of prothrombinase (human factor Xa, human factor Va, synthetic phospholipid vesicles, and calcium ion), initial velocity studies of alpha-thrombin formation indicated that the kinetic constants for the cleavage of bonds A or B were similar to the constants that were obtained for the overall reaction (bonds A + B). The progress of the reaction was also monitored by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results indicated that the activation of human prothrombin catalyzed by prothrombinase proceeded exclusively via the formation of meizothrombin (bond B-cleaved) as an intermediate. Kinetic studies of the cofactor dependence of the rates of cleavage of the individual bonds indicated that, in the absence of the cofactor, cleavage at bond B would constitute the rate-limiting step in prothrombin activation. Progress curves for prothrombin activation catalyzed by prothrombinase and monitored using the fluorophore DAPA were typified by the appearance of a transient maximum, indicating the formation of meizothrombin as an intermediate. When factor Xa alone was the catalyst, progress curves were characterized by an initial burst phase, suggesting the rapid production of prethrombin 2 (bond A-cleaved) followed by its slow conversion to alpha-thrombin. Gel electrophoresis followed by autoradiography was used to confirm these results. Collectively, the results indicate that the activation of human prothrombin via the formation of meizothrombin as an intermediate is a consequence of the association of the cofactor, human factor Va, with the enzyme, human factor Xa, on the phospholipid surface.

Arginine↗
PubMed1987

Membrane penetration of bovine factor V and Va detected by labeling with 5-iodonaphthalene-1-azide.

The membrane-binding properties of Factor V and Factor Va were investigated using the lipophyllic, photoactivable probe 5-[125I]iodonaphthalene-1-azide. In the presence of vesicles composed of 75% phosphatidylcholine and 25% phosphatidylserine, both Factor V and Va were found to be labeled by the probe. The label was almost exclusively localized to the carboxyl-terminal-derived component E of Factor Va. The results are consistent with the interpretation that component E is the membrane binding subunit of Factor Va and that the interaction between Factor V or Factor Va and the membrane involves the penetration of the protein into the lipid bilayer.

Affinity Labels↗
PubMed1987

Effect of fluoride ingestion on dental fluorosis in sheep.

The effects of fluoride ingestion (3.5 mg/kg of body weight) were evaluated in 9 ewes at 6 through 9, 10 through 13, or 14 through 17 months of age (3 ewes/age group). In the 3 groups, the plasma fluoride concentration rapidly increased to 0.45, 0.46, and 0.50 microgram/ml, respectively, and decreased rapidly to 0.1 microgram/ml after fluoride was removed from the ration. In 5 of the 9 ewes, this short-term exposure caused symmetrical, moderate damage to the molars, which is characteristic of fluorosis in sheep. In 7 of the 9 ewes, abnormal gaps were found between incisors, which are not characteristic of fluorosis in sheep. Fluoride accumulation was higher in the roots of the incisors and in the dentine of the molars and was particularly evident in teeth that were developing when fluoride was administered; however, the fully developed teeth and teeth that developed several months after administration of fluoride also were affected. The increase in fluoride concentration in the enamel was confined to a maximum of 2 molars. The abnormal wear of the molars was not directly linked to fluoride accumulation.

Animals↗
PubMed1986

The binding of activated protein C to factors V and Va.

Activated protein C has been derivatized with the active site-directed fluorophore 2-(dimethylamino)-6-naphthalenesulfonylglutamylglycylarginyl chloromethyl ketone (2,6-DEGR-APC). Covalently modified activated protein C has been used to investigate the binding interactions of the protein to factors V and Va in the presence of phospholipid vesicles. The fluorescence polarization of the 6-dimethylaminonaphthalene-2-sulfonyl moiety increased saturably with increasing phospholipid concentrations in the presence or absence of factor V or Va. Differences in the limiting polarization values indicated distinguishable differences in the interactions between 2,6-DEGR-APC and phospholipid in the presence of factor V or Va. The dissociation constant calculated for the 2,6-DEGR-APC/phospholipid interaction (7.3 X 10(-8) M) was not significantly altered by factor V but was decreased to 7 X 10(-9) M in the presence of factor Va. The interaction between 2,6-DEGR-APC and factor V or Va was characterized by a 1:1 stoichiometry. The binding of 2,6-DEGR-APC to factor V or Va in the presence of phospholipid could be reduced in a competitive manner by diisopropylphosphofluoridate-treated activated protein C. An analysis of the displacement curves indicated that the binding of 2,6-DEGR-APC was indistinguishable from the binding of diisopropylphosphofluoridate-treated activated protein C. The interaction between 2,6-DEGR-APC and phospholipid-bound factor Va was further examined using the isolated subunits of factor Va. Fluorescence polarization changes observed with component E of Va (light chain) closely corresponded with the changes observed with factor Va, whereas isolated component D (heavy chain) had little influence on the binding of 2,6-DEGR-APC to phospholipid vesicles. The data presented are consistent with the interpretation that component E of factor Va contains a binding site for activated protein C.

Amino Acid Chloromethyl Ketones↗
PubMed1986

The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction.

The activation of bovine prothrombin by prothrombinase (Factor Xa, Factor Va, synthetic phospholipid vesicles, and calcium ion) was studied in the presence of the fluorescent, reversible thrombin inhibitor dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide (DAPA). Recordings of fluorescence intensity during prothrombin activation exhibited maxima that decreased to stable limiting values. These data suggested the transient appearance of the meizothrombin-DAPA complex, which exhibits fluorescence with 1.5-fold greater intensity than the thrombin-DAPA complex. At substrate concentrations well below Km, progress curves could be fitted by equations describing an ordered, sequential conversion of prothrombin to thrombin through the intermediate meizothrombin via two pseudo-first order steps. The pseudo-first order rate constants for both steps varied linearly with enzyme concentration, indicating that both steps are catalyzed by prothrombinase. The progress of the reaction was also monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry analyses of aliquots removed at intervals spanning the reaction. These analyses confirmed both the existence of meizothrombin and its time course as predicted from the equations used to analyze fluorescence intensity profiles. Meizothrombin levels peaked at about 0.3 mol/mol initial prothrombin under the conditions typically studied. In addition, prethrombin 2, which is the intermediate expected from cleavages occurring in the order opposite that required to form meizothrombin, was not observed under any of the conditions examined. These data indicate that prothrombin activation catalyzed by the fully assembled prothrombinase complex proceeds via an ordered, sequential reaction with meizothrombin as the sole intermediate.

Animals↗