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Biomedical subjects

G Sanyal

Publications and source records attributed to G Sanyal.

At least 37 records · Page 2Linked to original sources
PubMed1992

Purification and characterization of a functionally homogeneous 60-kDa species of the retinoblastoma gene product.

The retinoblastoma susceptibility gene (RB) encodes a 928-amino acid protein (pRB) that is hypothesized to function in a pathway that restricts cell proliferation. The immortalizing proteins from three distinct DNA tumor viruses (SV40 large T antigen, adenovirus E1a, and human papilloma virus Type 16 E7) have been shown to interact with RB protein through two noncontiguous regions comprised of amino acids 393-572 (domain A) and 646-772 (domain B). We constructed a truncated form of RB (RB p60) that retains these two domains but eliminates the N-terminal 386 amino acids of RB. RB p60 was expressed in Escherichia coli in inclusion bodies. After solubilization, it was refolded in the presence of magnesium chloride, and the active protein was isolated with an E7 peptide affinity column. The protein that elutes from this column is functionally homogenous in its ability to bind immobilized E7 protein. Thermal denaturation studies provide additional evidence for the conformational homogeneity of the isolated protein. This purification scheme allows the isolation of significant amounts of RB p60 protein that is suitable for structural and functional studies.

Chromatography, Affinity↗
PubMed1991

Proton NMR assignments and secondary structure of the snake venom protein echistatin.

The snake venom protein echistatin is a potent inhibitor of platelet aggregation. The inhibitory properties of echistatin have been attributed to the Arg-Gly-Asp sequence at residues 24-26. In this paper, sequence-specific nuclear magnetic resonance assignments are presented for the proton resonances of echistatin in water. The single-chain protein contains 49 amino acids and 4 cystine bridges. All of the backbone amide, C alpha H, and side-chain resonances, except for the eta-NH of the arginines, have been assigned. The secondary structure of the protein was characterized from the pattern of nuclear Overhauser enhancements, from the identification of slowly exchanging amide protons, from 3JC alpha H-NH coupling constants, and from circular dichroism studies. The data suggest that the secondary structure consists of a type I beta-turn, a short beta-hairpin, and a short, irregular, antiparallel beta-sheet and that the Arg-Gly-Asp sequence is in a flexible loop connecting two strands of the distorted antiparallel beta-sheet.

Amino Acid Sequence↗
PubMed1991

Effect of polyanions on the refolding of human acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) is unstable at physiological temperatures in the absence of polyanions such as heparin. Therefore, the effect of temperature on the kinetics of refolding of aFGF has been examined in the presence and absence of several polyanions. The protein folds into its native state at temperatures up to 30 degrees C without polyanions with an activation energy of approximately 14 kcal/mol, but does not acquire native structure above this temperature. When heparin, inositol hexasulfate, or sulfate ion are present, aFGF refolds below 30 degrees C with a slightly reduced activation energy (10-11 kcal/mol). In addition, the protein now also renatures between 30 and 50 degrees C with activation energies of 1-2 (heparin), 16 (inositol hexasulfate), and 7 (sulfate) kcal/mol. Trace heavy metals appear to inhibit the refolding process, but a molecular chaperone (bovine 70-kDa heat shock cognate protein) and a peptidylprolyl isomerase (the FK506-binding protein) have no effect. It is concluded that the rate of refolding of aFGF at physiological temperatures is probably controlled by the interaction of a native-like state of the protein with an unknown polyanionic species.

Anions↗
PubMed1991

Stabilization of the FK506 binding protein by ligand binding.

Although the rotamase activity of the FK506 binding protein is inhibited by ligand binding, it is hypothesized that the ligand/protein complex itself may be responsible for the immunosuppressive effects of FK506. We have therefore examined the structure of the FK506 binding protein in the presence of an analog of FK506 (FK520) by a combination of fluorescence, CD, FTIR and calorimetry. While only small changes in the overall structure of the protein may be induced by ligand, a large change in thermal stability of the binding protein is observed.

Anti-Bacterial Agents↗
PubMed1989

Static quenching of tryptophan fluorescence by oxidized dithiothreitol.

The quenching of fluorescence of 5-methoxyindole, N-acetyl-L-tryptophanamide and two single tryptophan containing peptides, melittin and mastoparan X, by oxidized dithiothreitol was studied. The slopes of the Stern-Volmer plots for steady-state fluorescence quenching were 133 M-1, 71.2 M-1, 75.5 M-1 and 35.0 M-1 at 21 degrees C and pH 7.0 for 5-methyoxyindole, N-acetyl-L-tryptophanamide, melittin and mastoparan X respectively. Fluorescence lifetimes of indole or tryptophan in these compounds, as determined by multifrequency phase fluorometry, were decreased by 15% or less at concentrations that produced 50% or more quenching of steady-state fluorescence. Thus, quenching of fluorescence by oxidized dithiothreitol for these derivatives of indole appears to be largely static in nature, suggesting a ground-state interaction.

Dithiothreitol↗
PubMed1988

Binding of amphiphilic peptides to a carboxy-terminal tryptic fragment of calmodulin.

Calmodulin (CaM) fragments 1-77 (CaM 1-77) and 78-148 (CaM 78-148) were prepared by tryptic cleavage of CaM. CaM 78-148 exhibited Ca2+-dependent binding to mastoparan X, Polistes mastoparan, and melittin with apparent dissociation constants less than 0.2 microM as judged from changes in the fluorescence spectrum and anisotropy of the single tryptophan residue of each of these cationic, amphiphilic peptides. This interaction was accompanied by a large spectral blue shift of the peptide fluorescence spectrum. These findings are consistent with earlier results [Malencik, D.A., & Anderson, S.R. (1984) Biochemistry 23, 2420-2428] on the binding of mastoparan X to CaM fragment 72-148. The binding of the peptide to CaM 78-148 also caused a significant loss of the accessibility of the peptide tryptophan to the fluorescence quencher acrylamide. The CaM 78-148 induced effects on the fluorescence spectra and tryptophan accessibility of the peptides were most pronounced for mastoparan X, a peptide with tryptophan on the apolar face of the putative amphiphilic helix. The data were comparable with results from parallel experiments on the Ca2+-dependent interaction of these peptides with intact CaM. Difference circular dichroic spectra suggested that binding to CaM 78-148 was associated with the induction of considerable degrees of helicity in the amphiphilic peptides, which by themselves have predominantly random coil structures in aqueous solution. This finding is also reminiscent of the interaction of these peptides with intact CaM.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1987

Tryptophan fluorescence studies of subunit interaction and rotational dynamics of human luteinizing hormone.

Human luteinizing hormone (hLH) has a single tryptophan residue occurring in the beta-subunit (beta hLH). This provides an intrinsic fluorescent probe, in native hLH and beta hLH, that is unambiguously assigned. The fluorescence intensities of hLH and beta hLH are, however, significantly different. This difference has been utilized in studying the interaction of fluorescent beta hLH with the nonfluorescent alpha-subunit. The accessibility of the tryptophan residue in native hLH and beta hLH has been assessed by measuring the rate of collisional fluorescence quenching and by solvent perturbation (D2O/H2O) of fluorescence. Fluorescence anisotropy measurements have been used in studying the intramolecular dynamics and segmental tryptophan mobility in hLH and beta hLH. Lifetime-resolved anisotropy, measured by the technique of oxygen quenching of fluorescence, has revealed the presence of segmental tryptophan motion. These data can be satisfactorily explained in terms of fast segmental tryptophan motion and rotational diffusion of the whole protein and do not require that intersubunit motion be invoked for intact hLH as it was suggested earlier on the basis of fluorescence depolarization of fluorescein-labeled hLH [Bishop, W. H., & Ryan, R. J. (1975) Biochem. Biophys. Res. Commun. 65, 1184-1190].

Fluorescence Polarization↗
PubMed1985

Probable role of amphiphilicity in the binding of mastoparan to calmodulin.

Two-dimensional helical wheel diagrams and calculations of mean hydrophobic moments show mastoparan, mastoparan X, and Polistes mastoparan to have all the properties expected for amphiphilic helices. Circular dichroic properties are consistent with a random form for these peptides in dilute aqueous solution, but greater than 50% helix is apparent when the peptides are dissolved in 70% trifluoroethanol/water mixtures (v/v) or when the peptides are bound to calmodulin. Changes in the fluorescence spectra, anisotropy, and accessibility of tryptophan whose indole side chain is on the apolar surface of the amphiphilic helix imply a significant role for the apolar surface in the binding of the mastoparans and another amphiphilic peptide, melittin, to calmodulin. These data provide a useful model for designing high-affinity synthetic peptide inhibitors of calmodulin.

Animals↗
PubMed1984

The carbon dioxide hydration activity of the sulfonamide-resistant carbonic anhydrase from the liver of male rat: pH independence of the steady-state kinetics.

The steady-state kinetic constants for the catalysis of CO2 hydration by the sulfonamide-resistant and testosterone-induced carbonic anhydrase from the liver of the male rat has been determined by stopped-flow spectrophotometry. The turnover number was 2.6 +/- 0.6 X 10(3) s-1 at 25 degrees C, and was invariant with pH ranging from 6.2 to 8.2 within experimental error. The Km at 25 degrees C was 5 +/- 1 mM, and was also pH independent. These data are in quantitative agreement with earlier findings of pH-independent CO2 hydration activity for the mammalian skeletal muscle carbonic anhydrase isozyme III. The turnover numbers for higher-activity isozymes I and II are strongly pH dependent in this pH range. Thus, the kinetic status of the male rat liver enzyme is that of carbonic anhydrase III. This finding is consistent with preliminary structural and immunologic data from other laboratories.

Animals↗
PubMed1983

The pH dependence of the hydration of CO2 catalyzed by carbonic anhydrase III from skeletal muscle of the cat. Steady state and equilibrium studies.

We have measured the pH dependence of the kinetics of CO2 hydration catalyzed by carbonic anhydrase III from the skeletal muscle of the cat. Two methods were used: an initial velocity study in which the change in absorbance of a pH indicator was measured in a stopped flow spectrophotometer, and an equilibrium study in which the rate of exchange of 18O between CO2 and H2O was measured with a mass spectrometer. We have found that the steady state constants kCO2 cat and KCO2 m are independent of pH within experimental error in the range of pH 5.0 to 8.5; the rate of release from the enzyme of the oxygen abstracted from substrate HCO-3 in the dehydration is also independent of pH in this range. This behavior is very different from that observed for carbonic anhydrase II for which kCO2 cat and the rate of release of substrate oxygen are very pH-dependent. The rate of interconversion of CO2 and HCO-3 at equilibrium catalyzed by carbonic anhydrase III is not altered when the solvent is changed from H2O to 98% D2O and 2% H2O. Thus, the interconversion probably proceeds without proton transfer in its rate-limiting steps, similar to isozymes I and II.

Animals↗
PubMed1983

Kinetics and inhibition of carbonic anhydrase-catalyzed hydrolysis of 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone. Comparison with reactions of other substrates.

The catalytic activities of human red cell carbonic anhydrase (EC 4.2.1.1) isozymes B and C for the hydrolysis of 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone have been compared with their activities towards three other substrates. The substrate specificity (measured as kcat/Km) for either isozyme decreases in this order: CO2 greater than 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone greater than acetaldehyde greater than p-nitrophenyl acetate. Unlike CO2 hydration, enzyme B is slightly more active towards sultone hydrolysis than C. Despite these widely differing activities of both isozymes with regard to different substrates, the inhibition constants for anion and sulfonamide inhibitors are nearly independent of the substrate used. This suggests that the binding sites of these substrates in the enzyme are the same or nearly the same. Earlier studies on 2-hydroxy-5-nitro-alpha-toluenesulfonic acid sultone from this and other laboratories had underestimated both the intrinsic activity and the susceptibility to anion inhibition of human carbonic anhydrase B. We now find that this was due to the use of acetonitrile as the substrate solvent, which is often contaminated with cyanide, a powerful inhibitor of carbonic anhydrase. The inhibition of human carbonic anhydrase B by several industrial batches of acetonitrile agrees completely with the spectrophotometrically determined cyanide content of these batches.

Acetaldehyde↗
PubMed1983

The transcorneal permeability of sulfonamide carbonic anhydrase inhibitors and their effect on aqueous humor secretion.

Eleven sulfonamide carbonic anhydrase inhibitors of varied chemical and physical types were studied with respect to transcorneal permeability and reduction of intraocular flow and pressure. Using the isolated rabbit cornea, a constant drug concentration on the epithelial side and 6 ml solution in the endothelial chamber, first order rate constants (kin) ranged from 0 . 1-40 X 10(-3)/hr, roughly proportional to their lipid solubility. Drugs on the high side of this range were generally water insoluble and had pKa's too high to yield sodium salts at useful pH; therefore, the actual amount of drug delivered was small. We sought compounds which combined low pKa, good lipid solubility, and high activity against the enzyme. Trifluormethazolamide (TFM) has a pKa of 6 . 6, ether partition coefficient of 6, and a K1 of 2 X 10(-8)M. kin is 3 X 10(-3)/hr. TFM and five other compounds were also studied in vivo for their ability to penetrate the eye into the anterior and posterior chambers. These rate constants were roughly proportional to those measured in vitro; however, significant differences in accession to the two chambers were observed, as a function of varying physico-chemical properties of the drugs. A 3% solution of TFM (100 mM) applied to the rabbit eye for 25 min generated 0 . 7 mM in the anterior chamber and 0 . 07 mM in the posterior. Tissue distribution of TFM (and its metabolite) showed a relatively high concentration in the ciliary body 6 hr after dose. Intraocular pressure was reduced by 4 mmHg. With 10 min exposure this concentration of TFM reduced pressure by about 1 . 7 mmHg. Although the use of this drug is limited by its chemical instability and the length of exposure needed, the principle of treating glaucoma by the topical use of carbonic anhydrase inhibitors appears feasible.

Animals↗
PubMed1981

Thermodynamics of carbonic anhydrase catalysis. A comparison between human isoenzymes B and C.

The CO2 hydration and HCO3- dehydration activities of human red cell carbonic anhydrase isozymes B and C (HCAB and HCAC) have been studied as a function of temperature from 0 degrees to 37 degrees C. The Arrhenius plots of ln kcat versus 1/T are linear for both isozymes in both hydration and dehydration reactions, indicating that the rate-determining steps remain unchanged over this temperature range. The 37 degrees C hydration kcat, at pH 7.5, is 13 X 10(5) s-1 for isozyme C and 0.71 X 10(5) s-1 for isozyme B. Km, for hydration, is 10 mM for C and 5 mM for B, and invariant with temperature. The uncatalyzed reactions are significantly affected by temperature, 30- to 40-fold rate enhancements being observed from 0 degrees to 37 degrees C. The enzyme-catalyzed processes are much less sensitive to temperature, the rate enhancements being 2- to 3-fold for HCAB and 5- to 6-fold for HCAC in this temperature range. These observations are consistent with a significant lowering of the free energy of activation by both isozymes. This effect is greater for C accounting for its higher catalytic power. The enthalpy of activation, at pH 7.5 and 8.2, in the rate-limiting step is considerably less for the B enzyme compared to C. This is, however, more than offset by a large negative entropy of activation in the case of HCAB. This observation indicates either a mechanistic difference in the rate-limiting events or a difference in the structural organizations of the active sites of the two isozymes, or both.

Carbonic Anhydrases↗
PubMed1981

Kinetics and inhibition of membrane-bound carbonic anhydrase from canine renal cortex.

The membrane-bound carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) in the canine renal cortex has been characterized in terms of its CO2 hydration kinetics and inhibition by sulfonamides and inorganic anions. Comparing these properties with those of the renal cytoplasmic and the human red cell B and C isozymes, it appears that the membrane enzyme is quite different from the soluble carbonic anhydrases. The turnover number of the particulate enzyme is about 3-times lower than that of the cytoplasmic enzyme. The membrane-bound enzyme is also different from its cytoplasmic counterpart in being more resistant against inhibition, particularly against Cl-. Microsomes from the renal cortex were purified to yield luminal and anti-luminal fractions. Carbonic anhydrase activity was found in both. The luminal and anti-luminal carbonic anhydrases appeared similar in terms of their kinetic properties and susceptibility to inhibition.

Animals↗