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Biomedical subjects

V Prakash

Publications and source records attributed to V Prakash.

At least 55 records · Page 3Linked to original sources
PubMed1996

Interaction of sheep liver apo-serine hydroxymethyltransferase with pyridoxal-5'-phosphate: a physicochemical, kinetic, and thermodynamic study.

Sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) is a homotetramer of Mr 213,000 requiring pyridoxal-5'-phosphate (PLP) as cofactor. Removal of PLP from the holoenzyme converted the enzyme to the apo form which, in addition to being inactive, was devoid of the characteristic absorption spectrum. Upon the addition of PLP to the apoenzyme, complete activity was restored and the visible absorption spectrum with a maximum at 425 nm was regained. The interaction of PLP with the apoenzyme revealed two phases of reaction with pseudo-first-order rate constants of 20 +/- 5 s(-1) and 12.2 +/- 2.0 x 10(-3) s(-1), respectively. However, addition of PLP to the apoenzyme did not cause gross conformational changes as evidenced by circular dichroic and fluorescence spectroscopy. Although conformationally apoenzyme and holoenzyme were indistinguishable, they had distinct apparent melting temperatures of 51 +/- 2 and 58 +/- 2 degrees C, respectively, and the reconstituted holoenzyme was thermally as stable as the native holoenzyme. These results suggested that there was no apparent difference in the secondary structure of holoenzyme, apoenzyme, and reconstituted holoenzyme. However, sedimentation analysis of the apoenzyme revealed the presence of two peaks of S20,w values of 8.7 +/- 0.5 and 5.7 +/- 0.3 S, respectively. A similar pattern was observed when the apoenzyme was chromatographed on a calibrated Sephadex G-150 column. The first peak corresponded to the tetrameric form (Mr 200,000 +/- 15,000) while the second peak had a Mr of 130,000 +/- 10,000. Reconstitution experiments revealed that only the tetrameric form of the apoenzyme could be converted into an active holoenzyme while the dimeric form could not be reconstituted into an active enzyme. These results demonstrate that PLP plays an important role in maintaining the structural integrity of the enzyme by preventing the dissociation of the enzyme into subunits, in addition to its function in catalysis.

Animals↗
PubMed1996

Preferential interaction of denaturants with rice bran lipase.

The catalytic stability of rice bran lipase has been determined in the presence of three denaturants viz, urea, GuHCl and GuHSCN. The enzyme is completely inactive above 7 M urea, 4 M GuHCl and 2 M GuHSCN concentration. The extent of denaturant interaction has been determined by the partial specific volume measurements of the enzyme. The preferential interaction parameter (xi 3) has values of 0.042, 0.064 and 0.075 g/g, and the denaturation volume changes are -180, -240 and -270 ml/mol in presence of 8 M urea, 6 M GuHC1; and 3 M GuHSCN, respectively. The experimental values of number of denaturant molecules bound (A3) are 0.418, 0.566 and 0.320 g/g and the calculated values are 0.321, 0.511 and 0.632 g/g in presence of 8 M urea, 6 M GuHCl and 3 M GuHSCN, respectively. Fluorescence emission measurements indicated a decrease in the fluorescence emission intensity and a red shift in the emission maximum as the denaturant concentration is increased indicating the gradual exposure of aromatic chromophores. The instability of the enzyme in the presence of these denaturants has been indicated by a decreased value of apparent thermal denaturation temperature (Tm) of the enzyme from a control value of 67 degrees C. The results obtained in the present study explain the extent of inactivation/stability of rice bran lipase in presence of these denaturants at different concentrations.

Enzyme Stability↗
PubMed1996

Interaction of 5,7-dihydroxy-4'-methoxyflavone with a multisubunit protein, carmin: thermodynamics and kinetics of interaction.

Acacetin (5,7-dihydroxy-4'-methoxy flavone) is a flavone intrinsically present in the seeds of Carthamus tinctorius. Carmin is a multimeric, high molecular weight protein from the seeds of Carthamus tinctorius. The association constant of interaction of acacetin and carmin is maximum at 37.2 degrees C with a value of (3.96 +/- 0.61) x 10(4) M-1 as measured by fluorescence quenching. Acacetin has at least two binding sites on carmin. The interaction follows pseudo-first-order kinetics with a reaction rate constant of 3.4 +/- 0.4 s-1. The titration calorimetric data suggest that binding sites for acacetin and its structural analogue, biochanin A, are conserved. The interaction does not affect the association-dissociation equilibrium of the protein. Also, the binding does not induce any significant conformational changes in the protein as monitored by circular dichroic spectra. Biochanin A (5, 7-dihydroxy-4'-methoxyisoflavone), a structural analogue, interacts with carmin with an association constant of (9.33 +/- 1.44) x 10(4) M-1 at 36.9 degrees C. This indicates that the stereochemistry of the ligand plays an important role in the binding process of flavone to protein. Interaction studies of chemically modified lysyl and tryptophanyl groups separately, and lysyl and tryptophanyl groups sequentially, in the protein carmin with the ligands reveal the involvement of tryptophanyl residues in the binding process and show that it is predominantly an entropically driven hydrophobic interaction.

Flavones↗
PubMed1996

Clinicocolonoscopic profile of colonic tuberculosis.

BACKGROUND: Colonic tuberculosis is common in developing countries. However, its diagnosis is difficult. Nevertheless, colonoscopy and biopsy examination have shown promising results. METHODS: We evaluated the clinical spectrum and colonoscopic features of 62 patients with colonic tuberculosis. RESULTS: Abdominal pain, fever, anorexia, weight loss, and change in bowel habit were seen in more than 50% of the patients. Massive rectal bleeding was frequently (13%) observed. Colonoscopy revealed strictures in 17, deformed ileocecal valve in 34, ulcers in 52, nodules in 49, polypoidal lesions in three, and fibrous bands in five patients. Segmental tuberculosis, lesions mimicking carcinoma, and multiple site involvement were observed in 19%, 20%, and 50% of the patients, respectively. Histopathologically, well formed granulomas were seen in 27, collections of epithelioid cells in 18, and chronic, nonspecific inflammatory changes in 17 of the cases. Acid-fast bacilli could not be isolated from any of the patients. All of the patients responded to the anti-tubercular treatment. Follow-up colonoscopy in 22 patients demonstrated regression of lesions. CONCLUSIONS: Colonic tuberculosis is common in India. Our findings indicate that colonoscopy is useful for its diagnosis. However, histopathology many not always be helpful. Therefore, in a given clinical and colonosopic setting, a therapeutic trial may be indicated.

Adolescent↗
PubMed1995

Interaction of 3'-O-caffeoyl D-quinic acid with human serum albumin.

The interaction of chlorogenic acid (CGA) with human serum albumin (HSA) was studied from the view-point of thermodynamics and mechanism of binding at pH 6.0. The association constants (Ka) for the HSA-CGA interaction at 10, 25 and 40 degrees C were 6.0 x 10(4), 9.0 x 10(3) and 2 x 10(4) M-1, resulting in delta G of -6.21, -5.80, -6.32 kcal/mol, respectively. These high Ka-values showed that the interaction between CGA and HSA is strong, endothermic and entropically driven. Binding of chlorogenic acid induces conformational change in HSA as indicated by quenching of fluorescence emission intensity along with a red shift in the emission maxima from 338 to 350 nm. This suggested the involvement of the lone tryptophan residue in the region of binding. Far-ultraviolet circular dichroic data showed a decrease in the alpha-helical content of HSA from 56 to 50% upon binding of CGA. These data are also supported by the decrease in the apparent Tm of HSA by 4 degrees C upon binding of CGA causing destabilization of the HSA molecule. The kinetics of the interaction involves a single step in the binding, and the kinetic curve attains equilibrium within 180 +/- 5 s. Data on caffeic acid (CA) and quinic acid (QA), which are the hydrolysis products of the bidentate CGA molecule, indicate that CA interacts more strongly than CGA. CA binds with an association constant of 8 x 10(4) M-1 and with a maximum number of binding sites of four. Microcalorimetric investigation of the interaction of these ligands with HSA suggests that the strength of binding follows the order CA >> CGA >>> QA with a single class of binding sites. The effect of temperature on the binding of CGA to HSA showed that the interaction is dominated by hydrophobic forces and hydrogen bonding.

Binding Sites↗
PubMed1995

Mechanism of solvent-induced thermal stabilization of alpha-amylase from Bacillus amyloliquefaciens.

The transition temperature of irreversible thermal inactivation of alpha-amylase from Bacillus amyloliquefaciens was estimated to be 60 degrees C. At this temperature, the enzyme inactivation followed first-order kinetics, having a half-life (t 1/2) of 12 min with a rate constant (k) of 0.06 min-1. Conformational change was a prerequisite for this thermal inactivation. This is governed by stepwise temperature-dependent phenomena. Among the solvent stabilizers tested, the enzyme was thermally stable in presence of DMSO and PEG 300 and the stabilizing efficiency of these cosolvents was concentration-dependent. The enzyme was partially stabilized by 5.0 M DMSO and 1.9 M PEG 300 up to 78 degrees C. However, above 78 degrees C the enzyme was inactivated in these cosolvents also. The mechanism of stabilization has been explained by preferential hydration of the enzyme in these structure stabilizing solvents by exclusion from the protein surface and interface by measurement of partial specific volume in these cosolvents. The data suggest a high value of preferential interaction parameter, (delta g3/delta g2)tau, mu 1, mu 3 being -0.606/g/g g/g in 40% DMSO and a low value of -0.025 g/g in 5% glycerol. The preferential interaction parameters in sucrose and glycerol suggests that (delta g3/delta g2)tau, mu 1, mu 3m is highest of -0.420 g/g in 10% glycerol than any other cosolvent.

Bacillus↗
PubMed1994

Interactions of L-serine at the active site of serine hydroxymethyltransferases: induction of thermal stability.

Serine hydroxymethyltransferase (SHMT), EC 2.1.2.1, exhibits broad substrate and reaction specificity. In addition to cleaving many 3-hydroxyamino acids to glycine and an aldehyde, the enzyme also catalyzed the decarboxylation, transamination and racemization of several substrate analogues of amino acids. To elucidate the mechanism of interaction of substrates, especially L-serine with the enzyme, a comparative study of interaction of L-serine with the enzyme from sheep liver and Escherichia coli, was carried out. The heat stability of both the enzymes was enhanced in the presence of serine, although to different extents. Thermal denaturation monitored by spectral changes indicated an alteration in the apparent Tm of sheep liver and E. coli SHMTs from 55 +/- 1 degrees C to 72 +/- 3 degrees C at 40 mM serine and from 67 +/- 1 degrees C to 72 +/- 1 degrees C at 20 mM serine, respectively. Using stopped flow spectrophotometry k values of (49 +/- 5) x 10(-3) s-1 and (69 +/- 7) x 10(-3) s-1 for sheep liver and E. coli enzymes were determined at 50 mM serine. The binding of serine monitored by intrinsic fluorescence and sedimentation velocity measurements indicated that there was no generalized change in the structure of both proteins. However, visible CD measurements indicated a change in the asymmetric environment of pyridoxal 5'-phosphate at the active site upon binding of serine to both the enzymes. The formation of an external aldimine was accompanied by a change in the secondary structure of the enzymes monitored by far UV-CD spectra. Titration microcalorimetric studies in the presence of serine (8 mM) also demonstrated a single class of binding and the conformational changes accompanying the binding of serine to the enzyme resulted in a more compact structure leading to increased thermal stability of the enzyme.

Animals↗
PubMed1994

Structural stability of lipase from wheat germ.

Purified lipase from wheat germ was used for the determination of preferential interaction parameters under different stabilizing cosolvent conditions. The partial specific volume of the enzyme was measured under both isomolal and isopotential conditions in phosphate buffer at pH 7.0, 0.02 M, and the value was found to be 0.730 +/- 0.001 and 0.731 +/- 0.002 mL/g, respectively. The partial specific volume measurements with different cosolvents indicated that the enzyme has a (delta g3/delta g2)T,mu1,mu3 values of -0.119 +/- 0.012, -0.073 +/- 0.009 and -0.141 +/- 0.020 g/g, respectively, in 25% glucose, 25% sucrose and 25% DMSO. The (delta g3/delta g2)T,mu1,mu3 values in 10 and 20% glycerol were -0.054 +/- 0.012 and -0.073 +/- 0.016 g/g, respectively. Based on these values it is clear that the enzyme is stabilized in the presence of these cosolvents by increasing its hydration, of which DMSO is stabilizing to the maximum extent. The stabilization of the enzyme was also confirmed by the thermal denaturation measurements in the presence of these cosolvents which indicated a shift in the apparent thermal denaturation temperature of the enzyme towards higher temperatures. The data are supported further by the ultraviolet difference spectral as well as fluorescence measurements in the presence of these cosolvents. The stabilization has been attributed to the preferential hydration of the enzyme in the presence of these cosolvents.

Dimethyl Sulfoxide↗
PubMed1994

Interaction of guanidine hydrochloride and guanidine thiocyanate with wheat germ lipase.

Effect of two classical and potent denaturants, guanidine hydrochloride (GuHCl) and guanidine thiocyanate (GuHSCN) on purified wheat germ lipase has been studied. Lipase was found to be active only up to 5 M GuHCl and 1.5 M GuHSCN. The extent of interaction was determined by the measurement of apparent partial specific volume of the enzyme in presence of these two denaturants. While the preferential interaction parameter (zeta 3) has values of 0.08 +/- 0.02 and 0.14 +/- 0.03 g/g, the interaction parameter (delta m3/delta m2)T,mu 1, mu3 has values of 35 +/- 9 and 50 +/- 10 mole/mole for GuHCl and GuHSCN, respectively. The number of denaturant molecules bound to the enzyme, A3, obtained experimentally were 0.486 +/- 0.020 and 0.348 +/- 0.020 g/g and the calculated values were 0.459 +/- 0.023 and 0.567 +/- 0.030 g/g for 6 M GuHCl and 3 M GuHSCN, respectively. The volume change occurring upon denaturation results in -420 +/- 42 and -462 +/- 84 ml/mole in 6 M GuHCl and 3 M GuHSCN, respectively. The denaturation is accompanied by exposure of hydrophobic groups to the bulk solvent as confirmed by fluorescence emission measurements of the enzyme. The Tm measurements indicated a control value of 56 +/- 1 degree C. In presence of 6 M GuHCl/3 M GuHSCN, the value was 42 +/- 1 degree C. These results explain the retention of lipase activity even at 5 M GuHCl from a mechanistic point of view.

Guanidine↗
PubMed1993

Interaction of sodium dodecyl sulfate with multi-subunit proteins. A case study with carmin.

Sodium dodecyl sulfate (SDS) dissociates the multimeric protein, carmin, into its monomers (2 S) at low concentration. Dissociation begins at and above 1.6 mM SDS concentration and reaches 50% at 5 mM SDS concentration. Denaturation occurs above 5 mM SDS concentration. The dissociation step involves binding of 540 +/- 50 mol of SDS/mol of protein with an association constant, K alpha of 6.90 +/- 0.35 x 10(2) M-1. The interaction reflects a delta G(0) = -4.0 +/- 0.1 kcal mol-1. In the denaturation step, the K alpha has the same value, and the gamma value is nearly 2-fold higher. Dissociation of carmin thus begins only above a binding of 0.60 g of SDS/g of protein. Analysis of the binding data at 37 degrees C indicates a maximum of 1030 +/- 90 mol of SDS bound/mol of protein, which is equivalent to 1.14 +/- 0.10 g of SDS/g of protein. Upon denaturation, the alpha-helix content of the protein increases from 4 to 15%. Kinetically, the denaturation process consists of a two-step process (a fast and a slow step). The first order rate constants for these steps are 89.6 +/- 8.1 and 15.8 +/- 1.5 min-1, respectively, at 6.3 mM SDS concentration. The processes of dissociation and denaturation occur sequentially. Dissociation of the protein is reversible, whereas the process of denaturation is only partially reversible as reflected by sedimentation velocity and conformational analysis. These data are taken as a model for general understanding of the dissociation and/or denaturation processes, which could be either sequential or simultaneous in multimeric proteins.

Circular Dichroism↗
PubMed1993

Kinetics and thermodynamics of the mechanism of interaction of sodium phytate with alpha-globulin.

The precipitation mechanism of alpha-globulin in the presence of myo-inositol hexaphosphate (sodium phytate) was studied in detail. The maximum interaction was found at pH 2.3 where the protein was in a dissociated state having an 8.3S aggregate and a 1.5S monomer. This interaction was predominantly dependent upon the sodium phytate to protein ratio. Velocity sedimentation studies indicated polymer formation due to preferential progressive binding of ligand to polymer, whose size and concentration increased with an increase in sodium phytate concentration. The polymer formation was shown to be ligand mediated and exists independently in solution along with the monomer. The binding isotherm by equilibrium dialysis confirmed differential binding of sodium phytate to the polymer and the monomer as indicated by two sets of binding sites, one having 7 +/- 2 sites of a K value 1.3 x 10(-4) mol-1 and the other having 56 +/- 3 sites with a K value of 2.8 x 10(-3) mol-1. Binding resulted in perturbation of chromophores of protein due to charge effects. The kinetics of the polymer formation was shown to be a pseudo-first-order reaction having two steps. The initial fast reaction involving conformational changes has rate constants of k1 = 52.4 x 10(-3) s-1 and k' = 67.5 x 10(-3) s-1, followed by a slow reaction step of rate constants k2 = 4.3 x 10(-3) s-1 and k'2 = 2.9 x 10(-3) s-1 at sodium phytate concentrations of 1 x 10(-4) M and 5 x 10(-4) M, respectively.

Alpha-Globulins↗
PubMed1993

Interaction of myo-inositol hexaphosphate (MIHP) with beta-globulin from Sesamum indicum L. Kinetics and thermodynamics.

beta-Globulin, the low molecular weight protein fraction from Sesamum indicum L., interacts with myo-inositol hexaphosphate (MIHP) maximally at pH 3.0, with concomitant precipitation up to 85 +/- 2% at an MIHP concentration of 8 x 10(-4) M. The kinetics of interaction as followed by stopped-flow spectrophotometry suggested the reaction to be of pseudo first-order, having an initial fast step followed by a relatively slow step of rate constants 1.9 x 10(-2) s-1 and 1.2 x 10(-3) s-1, respectively at 1 x 10(-4) M MIHP concentration. The analysis of the complex indicated the presence of polymer as seen in sedimentation velocity experiment. This was accompanied by conformational change of a three-fold decrease in beta-structure and also an increase in fluorescence emission intensity accompanied with a red shift from 330 to 334 nm. Stoichiometric analysis of MIHP binding suggested four independent binding sites for MIHP, with a free energy change, delta G zero = 5.1 kcal mol-1 resulting from a binding constant of 3.6 x 10(3) M-1.

Beta-Globulins↗
PubMed1993

Structural stability of beta-globulin, the low molecular weight protein fraction from sesame seed (Sesamum indicum L.) in alkaline solution.

Beta-globulin, a single polypeptide chain of molecular weight 15,000 +/- 1,000, undergoes denaturation in alkaline pH (7.0-13.0), thereby affecting the hydrodynamic properties of the protein, viz. a decrease in sedimentation coefficient from a value of 2.0s to 1.4s at pH 11.3, an increase in reduced viscosity from 0.042 dl/g to 0.158 dl/g at pH 12.6 and a decrease in partial specific volume resulting in a volume change of 6.3 +/- 1.0 ml/mole residue at pH 11.7. The perturbation of tryptophanyl residues and ionization of tyrosyl residues are preceded by alteration in conformational status of the protein. The fluorescence emission measurements indicate initial unfolding of the protein molecule which exposes the tryptophan and tyrosyl residues to the solvent. The tyrosyl phenolic group ionization is anomalous having a pKint value of 11.2. The reduced viscosity value reaches a plateau region at pH 12.5.

Hydrogen-Ion Concentration↗
PubMed1992

Aging of tubulin at neutral pH: the destabilizing effect of vinca alkaloids.

The effect of the vinca alkaloid drugs, vincristine, vinblastine, catharanthine, and vindoline, on the aging process of tubulin has been examined. It was found that addition of vincristine or vinblastine accelerated by a factor of 3-3.5 the transformation of tubulin from the 5.8 S alpha-beta-tubulin dimer to paucidisperse polymers, with an average sedimentation coefficient of 9 S, previously observed in the absence of drugs (V. Prakash and S. N. Timasheff, 1982, J. Mol. Biol. 160, 499-515). This transformation of tubulin from 5.8 S to "9 S" followed pseudo-first-order kinetics whether the starting protein was predominantly dimeric (i.e., at low drug concentration) or self-associated into the reversible linear polymers induced by the vinca alkaloid drugs at high drug concentration (G. C. Na and S. N. Timasheff, 1980, Biochemistry 19, 1355-1365; V. Prakash and S. N. Timasheff, 1985, Biochemistry 24, 5004-5010). Identical kinetics were found in a fluorescence examination of the loss by tubulin of its ability to bind colchicine specifically, indicating that the rate determining step is a protein conformational change that induces a major change in the far uv circular dichroism spectrum of tubulin. The found lack of an effect of dithiothreitol on the aging and aggregation processes is consistent with the irreversible aggregation being due to the intermolecular coalescence of nonpolar patches on the protein. The observations that vincristine binds to aged tubulin and that the aging of tubulin is accompanied by quenching of the tryptophan fluorescence similar to that which occurs on the binding of the vinca drugs has led to the proposal that the vinca alkaloids stabilize the aged conformation of the protein by interacting with nonpolar regions that may be related to the aggregation sites.

Animals↗
PubMed1992

Aging of tubulin at neutral pH: stabilization by colchicine and its analogues.

The effect of colchicine and its analogues, allocolchicine, 2,3,4-trimethoxy-4'-carbomethoxy-1,1'biphenyl, 2,3,4,4'-tetramethoxy-1,1'-biphenyl, 2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl, and tropolone methyl ether, on the aging process of tubulin has been examined. In contrast to the vinca alkaloid drugs which accelerate the formation of the paucidisperse 9 S polymers by a factor of 3.5, the colchicine class of ligands stabilize alpha,beta-tubulin. Less than 10% of the protein is transformed into the aggregates after 50 h of incubation in the presence of 1 x 10(-3) M colchicine, as compared to nearly 70-75% transformation in its absence. These results are supported by fluorescence examination of the retention of colchicine binding ability, as well as circular dichroism spectroscopy. In the presence of colchicine, the rate determining step is a conformational change, just as in its absence. The colchicine analogues which bind to tubulin in a rapidly reversible equilibrium were almost as effective in tubulin stabilization. Addition of vincristine to the system reduced the stability of the tubulin-colchicine complex. Furthermore, vincristine was found to have the same effects on the fresh complex as it does on pure tubulin; i.e., it induced the isodesmic linear polymerization and inhibited assembly into the microtubule-mimicking large polymers. This inhibition, however, was stoichiometric, whereas it is substoichiometric in the case of microtubules.

Animals↗