The structure, physical and kinetic properties as well as the regulatory mechanisms of skeletal muscle phosphorylase kinase have been studied extensively. In contrast to our current understanding of muscle phosphorylase kinase, the... more
The structure, physical and kinetic properties as well as the regulatory mechanisms of skeletal muscle phosphorylase kinase have been studied extensively. In contrast to our current understanding of muscle phosphorylase kinase, the structure and regulatory properties of liver enzyme have not yet been fully clarified. Results obtained with crude and partially purified preparations of liver phosphorylase kinase suggest two main mechanisms of regulation: firstly, covalent modification through a phosphorylation reaction catalyzed by cyclic AMP-dependent protein kinase in response to glucagon and epinephrine (Vandenheede et al. 1979), and secondly, Ca2+ has been proposed as a possible candidate for an intracellular mediator in the hormonal control by cyclic AMP-independent hormones (e.g., vasopressin, angiotensin and α adrenergic agents) (Van de Werve et al. 1977); besides that, the control of kinase reaction can occur due to binding of effectors to the substrate, phosphorylase b, to the enzyme, phosphorylase kinase, or both.
Research Interests:
Recent studies forecast that many ectothermic animals, especially aquatic stenotherms, may not be able to thrive or even survive predicted climate change. These projections, however, generally do not call much attention to the role of... more
Recent studies forecast that many ectothermic animals, especially aquatic stenotherms, may not be able to thrive or even survive predicted climate change. These projections, however, generally do not call much attention to the role of behaviour, an essential thermoregulatory mechanism of many ectotherms. Here we characterize species-specific locomotor and respiratory responses to acute ambient warming in two highly stenothermic Antarctic Notothenioid fishes, one of which (Chaenocephalus aceratus) lacks haemoglobin and appears to be less tolerant to thermal stress as compared to the other (Notothenia coriiceps), which expresses haemoglobin. At the onset of ambient warming, both species perform distinct locomotor manoeuvres that appear to include avoidance reactions. In response to unavoidable progressive hyperthermia, fishes demonstrate a range of species-specific manoeuvres, all of which appear to provide some mitigation of the deleterious effects of obligatory thermoconformation an...
Research Interests:
Research Interests:
The structure, physical and kinetic properties as well as the regulatory mechanisms of skeletal muscle phosphorylase kinase have been studied extensively. In contrast to our current understanding of muscle phosphorylase kinase, the... more
The structure, physical and kinetic properties as well as the regulatory mechanisms of skeletal muscle phosphorylase kinase have been studied extensively. In contrast to our current understanding of muscle phosphorylase kinase, the structure and regulatory properties of liver enzyme have not yet been fully clarified. Results obtained with crude and partially purified preparations of liver phosphorylase kinase suggest two main mechanisms of regulation: firstly, covalent modification through a phosphorylation reaction catalyzed by cyclic AMP-dependent protein kinase in response to glucagon and epinephrine (Vandenheede et al. 1979), and secondly, Ca2+ has been proposed as a possible candidate for an intracellular mediator in the hormonal control by cyclic AMP-independent hormones (e.g., vasopressin, angiotensin and α adrenergic agents) (Van de Werve et al. 1977); besides that, the control of kinase reaction can occur due to binding of effectors to the substrate, phosphorylase b, to the enzyme, phosphorylase kinase, or both.
Research Interests:
Research Interests:
Recent studies forecast that many ectothermic animals, especially aquatic stenotherms, may not be able to thrive or even survive predicted climate change. These projections, however, generally do not call much attention to the role of... more
Recent studies forecast that many ectothermic animals, especially aquatic stenotherms, may not be able to thrive or even survive predicted climate change. These projections, however, generally do not call much attention to the role of behavior, an essential thermoregulatory mechanism of many ectotherms. Here we characterize species-specific locomotor and respiratory responses to acute ambient warming in two highly stenothermic Antarctic Notothenioid fishes, one of which (Chaenocephalus aceratus) lacks hemoglobin and appears to be less tolerant to thermal stress as compared to the other (Notothenia coriiceps), which expresses hemoglobin. At the onset of ambient warming, both species perform distinct locomotor maneuvers that appear to include avoidance reactions. In response to unavoidable progressive hyperthermia, fishes demonstrate a range of species-specific maneuvers, all of which appear to provide some mitigation of the deleterious effects of obligatory thermoconformation and to ...
Research Interests:
Research Interests:
Research Interests:
The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, alpha, of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb).... more
The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, alpha, of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb). One fragment, corresponding to residues 17-484 (PhbN'), contained the regulatory domain of the protein, but in missing the first 16 residues was devoid of the sole phosphorylation site of Phb, Ser14; the second fragment corresponded to residues 485-843 (PhbC) and contained the catalytic domain of Phb. Truncation fragments of the alpha subunit were screened for interactions against these two substrate fragments. PhbC was not found to interact with any alpha constructs; however, PhbN' interacted with a region of alpha (residues 864-1014) that is near the phosphorylatable region of that subunit. PhbN' was also screened for interactions against a variety of fragments of the catalytic gamma subunit of PhK; however, no interactions were detected...
Research Interests:
The kinetics of the interaction of rabbit skeletal muscle phosphorylase kinase with glycogen was studied by the turbidimetric method at pH 6.8 and 8.2. Binding of phosphorylase kinase by glycogen occurs only in the presence of Ca2+ and... more
The kinetics of the interaction of rabbit skeletal muscle phosphorylase kinase with glycogen was studied by the turbidimetric method at pH 6.8 and 8.2. Binding of phosphorylase kinase by glycogen occurs only in the presence of Ca2+ and Mg2+. The initial rate of complex formation is proportional to the enzyme and polysaccharide concentration; this suggests the formation of a complex with 1:1 stoichiometry in the initial step of phosphorylase kinase binding by glycogen. The kinetic data suggest that phosphorylase kinase substrate--glycogen phosphorylase b--favors the binding of phosphorylase kinase with glycogen. This conclusion is supported by direct experiments on the influence of phosphorylase b on the interaction of phosphorylase kinase with glycogen using analytical sedimentation analysis. The kinetic curves of the formation of the complex of phosphorylase kinase with glycogen obtained in the presence of ATP are characterized by a lag period. Preincubation of phosphorylase kinase...
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests: Biochemistry, Kinetics, Enzyme Inhibitors, Fluorescent Dyes and Reagents, Animals, and 10 moreNicotinic Acetylcholine Receptors, Phencyclidine, Quaternary Ammonium Compounds, Structure activity Relationship, Biochemistry and cell biology, Calcium Channel Blockers, binding sites, fluorescent dyes, torpedo ink, and Medical biochemistry and metabolomics
Research Interests:
Research Interests: Chemical Engineering, Mathematics, Thermodynamics, Kinetics, Biological Sciences, and 14 morePhysical sciences, Iontophoresis, Anti-Bacterial Agents, Lipid bilayers, Reaction Rate, Ion Transport, Lipid Bilayer, Electric Conductivity, Chlorpromazine, Hydrostatic Pressure, Biochemistry and cell biology, Valinomycin, Macrolides, and Cell membrane permeability
Research Interests: Chemical Engineering, Mathematics, Thermodynamics, Kinetics, Biological Sciences, and 14 morePhysical sciences, Iontophoresis, Anti-Bacterial Agents, Lipid bilayers, Reaction Rate, Ion Transport, Lipid Bilayer, Electric Conductivity, Chlorpromazine, Hydrostatic Pressure, Biochemistry and cell biology, Valinomycin, Macrolides, and Cell membrane permeability
The kinetic behaviour of rabbit skeletal muscle phosphorylase kinase at variable concentrations of the enzyme and the substrate (glycogen phosphorylase b) has been studied. The kinetic curves reveal a lag period whose duration decreases... more
The kinetic behaviour of rabbit skeletal muscle phosphorylase kinase at variable concentrations of the enzyme and the substrate (glycogen phosphorylase b) has been studied. The kinetic curves reveal a lag period whose duration decreases with a rise in the phosphorylase kinase concentration (when the reaction is initiated by an addition of the ATP + MgCl2 mixture to the enzyme preincubated with phosphorylase b, CaCl2, glycogen and glucose-1-phosphate or inorganic phosphate). A decrease of the phosphorylase b concentration eliminates the lag period. Under these conditions the specific activity of phosphorylase kinase decreases with a rise in the enzyme concentration. The kinetic behaviour of phosphorylase kinase is interpreted in terms of a model of a linearly associating system, such as M reversible M2 reversible M3 reversible ...Mi, where M is the dexadecameric molecule of phosphorylase kinase. Acceleration of the phosphorylase kinase-catalyzed reaction in the course of the enzymatic process seems to be due to the breakdown of inactive enzyme associates (Mi) caused by phosphorylase b. The short gamma-subunit of phosphorylase kinase devoid of the calmodulin-binding domain does not display any hysteretic properties.
Research Interests:
The changes in the quaternary structure of chicken skeletal muscle phosphorylase kinase during limited proteolysis by trypsin and chymotrypsin were studied. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of... more
The changes in the quaternary structure of chicken skeletal muscle phosphorylase kinase during limited proteolysis by trypsin and chymotrypsin were studied. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the products of phosphorylase kinase limited proteolysis revealed a similarity in the structure of the alpha'- and beta-subunits and some differences in the structure of the gamma-subunits of the chicken and rabbit enzymes. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol of 32P/mol of alpha' beta gamma' sigma monomer) and autophosphorylation (up to 8 mol of 32P/mol alpha' beta gamma' delta monomer) increased the activity of chicken phosphorylase kinase 1.5-fold and 2.0-fold, respectively. The incorporation of phosphate into the alpha' and beta-subunits in the course of the protein kinase-catalyzed reaction was demonstrated.
Research Interests:
The activation of different forms of muscle phosphorylase kinase by actin has been studied. F-actin which is polymerized by 2 mM MgCl2 is a more effective activator of phosphorylase kinase than F-actin polymerized by 50 mM KCl. There is... more
The activation of different forms of muscle phosphorylase kinase by actin has been studied. F-actin which is polymerized by 2 mM MgCl2 is a more effective activator of phosphorylase kinase than F-actin polymerized by 50 mM KCl. There is evidence suggesting that the activation of phosphorylase kinase b by actin is not due to the presence of trace amounts of calmodulin in actin preparations: (1) Troponin I and trifluoperazine inhibit the activation of phosphorylase kinase by calmodulin but do not inhibit the activation by actin. (2) The activation induced by saturating concentrations of calmodulin and actin is additive. (3) The activation of phosphorylase kinase by calmodulin and actin has different pH profiles. An addition of F-actin does not affect the apparent Km value for ATP but increases the sensitivity to phosphorylase b and the value of V. F-actin has no stimulating effect on the phosphorylated form (a) of phosphorylase kinase or on the form a previously activated by proteolysis.
Research Interests:
Red and white avian skeletal muscles (chicken and pigeon) contain the same alpha'-isoenzyme of phosphorylase kinase. According to data from gradient polyacrylamide slab electrophoresis in the presence of SDS, the molecular masses... more
Red and white avian skeletal muscles (chicken and pigeon) contain the same alpha'-isoenzyme of phosphorylase kinase. According to data from gradient polyacrylamide slab electrophoresis in the presence of SDS, the molecular masses of beta- and gamma-subunits of phosphorylase kinase from rabbit, chicken and pigeon muscles are not identical. Electron microscopy data suggest that the quaternary structure of chicken and pigeon phosphorylase kinase is of the same type. The alpha'-isozyme of chicken and pigeon phosphorylase kinase is strongly activated by calmodulin and troponin C. Avian phosphorylase kinase is activated 2--3-fold by phosphorylation with cAMP-dependent protein kinase and by autophosphorylation. This activation is associated with the phosphorylation of both alpha'- and beta-subunits. The affinity of pigeon phosphorylase kinase a for Ca2+ is 20 times as high as that of phosphorylase kinase b.
Research Interests:
Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native... more
Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme as determined by gel filtration on Sepharose 4B is close to that of rabbit skeletal muscle phosphorylase kinase (i. e., approximately 1300 000). The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 140 000 beta, 129 000; gamma', 44 000; delta, 17 000 (cf. the Mr values of the alpha- and gamma-subunits of the rabbit muscle isoenzyme are 146 000 and 42 000). The four subunits, alpha', beta, gamma' and delta, were found to exist in equimolar amounts as shown by a densitometric analysis of acrylamide gels; hence, the subunit formula of the chicken skeletal muscle isoenzyme is (alpha' beta gamma' delta)4. Rabbit antisera against a mixture of alpha'- and beta-subunits of chicken phosphorylase kinase yield a single precipitin line with this enzyme, do not show cross reactions of identity with the rabbit muscle enzyme but strongly inhibit the activity of the chicken enzyme and partially inhibit the activity of the rabbit muscle isoenzyme.
Research Interests:
The main kinetic parameters for purified phosphorylase kinase from chicken skeletal muscle were determined at pH 8.2: Vm = 18 micromol/min/mg; apparent Km values for ATP and phosphorylase b from rabbit muscle were 0.20 and 0.02 mM,... more
The main kinetic parameters for purified phosphorylase kinase from chicken skeletal muscle were determined at pH 8.2: Vm = 18 micromol/min/mg; apparent Km values for ATP and phosphorylase b from rabbit muscle were 0.20 and 0.02 mM, respectively. The activity ratio at pH 6.8/8.2 was 0.1-0.4 for different preparations of phosphorylase kinase. Similar to the rabbit enzyme, chicken phosphorylase kinase had an absolute requirement for Ca2+ as demonstrated by complete inhibition in the presence of EGTA. Half-maximal activation occurred at [Ca2+] = 0.4 microM at pH 7.0. In the presence of Ca2+, the chicken enzyme from white and red muscles was activated 2-4-fold by saturating concentrations of calmodulin and troponin C. The C0.5 value for calmodulin and troponin C at pH 6.8 was 2 and 100 nM, respectively. Similar to rabbit phosphorylase kinase, the chicken enzyme was stimulated about 3-6-fold by glycogen at pH 6.8 and 8.2 with half-maximal stimulation occurring at about 0.15% glycogen. Protamine caused 60% inhibition of chicken phosphorylase kinase at 0.8 mg/ml. ADP (3 mM) at 0.05 mM ATP caused 85% inhibition with Ki = 0.2 mM. Unlike rabbit phosphorylase kinase, no phosphorylation of the chicken enzyme occurred in the presence of the catalytic subunit of cAMP-dependent protein kinase. Incubation with trypsin caused 2-fold activation of the chicken enzyme.
Research Interests:
Using DEAE-Toyopearl column chromatography, a preparation of pigeon skeletal muscle phosphorylase kinase was obtained in a state approaching homogeneity. The molecular mass of the native enzyme (1320 kDa) and the subunit formula (alpha... more
Using DEAE-Toyopearl column chromatography, a preparation of pigeon skeletal muscle phosphorylase kinase was obtained in a state approaching homogeneity. The molecular mass of the native enzyme (1320 kDa) and the subunit formula (alpha beta gamma delta)4 are similar to those of rabbit and chicken counterparts. Both red and white pigeon skeletal muscle isozymes contain the alpha'-subunit instead of alpha. Gradient SDS-PAGE electrophoresis revealed small but well-reproducible differences in the molecular masses of rabbit, chicken and pigeon muscle beta- and gamma-subunits. The activity ratio at pH 6.8/8.2 is 0.06-0.15 for different preparations of phosphorylase kinase b. The activity of pigeon muscle phosphorylase kinase b is Ca2+-dependent. The [Ca2+]0.5 value at pH 7.0 is 20 microM, which exceeds that for the chicken muscle enzyme by two orders of magnitude. In the presence of Ca2+, pigeon phosphorylase kinase b is activated 4-fold by saturating concentrations of calmodulin and troponin C. Pigeon muscle phosphorylase b is activated 3-5-fold during autophosphorylation or phosphorylation by the catalytic subunit of cAMP-dependent protein kinase.
Research Interests:
The activation of muscle phosphorylase kinase b by actin has been studied. F-actin which is polymerized by 2 mM MgCl2 is a more effective activator of phosphorylase kinase than F-actin polymerized by 50 mM KCl. There is evidence... more
The activation of muscle phosphorylase kinase b by actin has been studied. F-actin which is polymerized by 2 mM MgCl2 is a more effective activator of phosphorylase kinase than F-actin polymerized by 50 mM KCl. There is evidence suggesting that the activation of phosphorylase kinase by actin is not due to trace contamination of actin preparations with calmodulin: (1) Troponin I and trifluoperazine inhibit the activation of phosphorylase kinase by calmodulin but do not inhibit the activation of phosphorylase kinase by F-actin. (2) The activation induced by saturating concentrations of calmodulin and actin is additive both at pH 8.2 and at pH 6.8. (3) The activation of phosphorylase kinase by calmodulin and actin has different pH profiles. An addition of F-actin does not affect the apparent Km value for ATP but increases the sensitivity to phosphorylase b and the value of Vmax.
Research Interests:
The kinetic behaviour of rabbit skeletal muscle phosphorylase kinase at variable concentrations of the enzyme and the substrate (glycogen phosphorylase b) has been studied. The kinetic curves reveal a lag period whose duration decreases... more
The kinetic behaviour of rabbit skeletal muscle phosphorylase kinase at variable concentrations of the enzyme and the substrate (glycogen phosphorylase b) has been studied. The kinetic curves reveal a lag period whose duration decreases with a rise in the phosphorylase kinase concentration (when the reaction is initiated by an addition of the ATP + MgCl2 mixture to the enzyme preincubated with phosphorylase b, CaCl2, glycogen and glucose-1-phosphate or inorganic phosphate). A decrease of the phosphorylase b concentration eliminates the lag period. Under these conditions the specific activity of phosphorylase kinase decreases with a rise in the enzyme concentration. The kinetic behaviour of phosphorylase kinase is interpreted in terms of a model of a linearly associating system, such as M reversible M2 reversible M3 reversible ...Mi, where M is the dexadecameric molecule of phosphorylase kinase. Acceleration of the phosphorylase kinase-catalyzed reaction in the course of the enzymati...
Research Interests:
Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on... more
Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme...
Research Interests: Kinetics, Calcium, Animals, Muscles, Trypsin, and 14 morePhosphorylation, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Skeletal Muscle, Chickens, Calmodulin, Molecular weight, Troponin, Rabbits, Hydrolysis, Hydrogen-Ion Concentration, Glycogen, Biochemistry and cell biology, Adenosine Diphosphate, and Sodium Dodecyl Sulfate
Without Abstract
Research Interests:
Research Interests: Calcium, Molecular Recognition, Humans, Magnesium, Animals, and 4 moreProline, CHEMICAL SCIENCES, Glycogen, and Betaine
Research Interests:
Research Interests:
Research Interests:
Research Interests: Chemical Engineering, Mathematics, Thermodynamics, Kinetics, Biological Sciences, and 14 morePhysical sciences, Iontophoresis, Anti-Bacterial Agents, Lipid bilayers, Reaction Rate, Ion Transport, Lipid Bilayer, Electric Conductivity, Chlorpromazine, Hydrostatic Pressure, Biochemistry and cell biology, Valinomycin, Macrolides, and Cell membrane permeability
Research Interests:
The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, α, of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb). One... more
The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, α, of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb). One fragment, corresponding to residues 17-484 (PhbN"), contained the regulatory domain of the protein, but in missing the first 16 residues was devoid of the sole phosphorylation site of Phb, Ser14; the second fragment corresponded to residues 485-843 (PhbC) and contained the catalytic domain of Phb. Truncation fragments of the α subunit were screened for interactions against these two substrate fragments. PhbC was not found to interact with any α constructs; however, PhbN" interacted with a region of α (residues 864-1014) that is near the phosphorylatable region of that subunit. PhbN" was also screened for interactions against a variety of fragments of the catalytic γ subunit of PhK; however, no interactions were detected, even with fulllength γ. Our results support the idea that amino acid residues proximal to the convertible serine of Phb are important for its specific interaction with the catalytic subunit of PhK, but that regions distinct from the convertible serine residue of Phb and from the catalytic domain of PhK may also be involved in the interaction of these two proteins.
Research Interests:
Research Interests: Molecular Biology, Kinetics, DNA replication, Biophysical Chemistry, Anisotropy, and 14 moreBiological Sciences, Helicases, Physical sciences, Plasmids, Enzyme, Steady state, CHEMICAL SCIENCES, Analytical Ultracentrifugation, DNA binding, Fluorescence anisotropy, Protein Conformation, Protein Binding, Biochemistry and cell biology, and Nucleic Acid
Research Interests:
Research Interests:
Research Interests:
The interaction of flavin adenine dinucleotide (FAD) with rabbit skeletal muscle phosphorylase kinase has been studied. Direct evidence of binding of phosphorylase kinase with FAD has been obtained using analytical ultracentrifugation. It... more
The interaction of flavin adenine dinucleotide (FAD) with rabbit skeletal muscle phosphorylase kinase has been studied. Direct evidence of binding of phosphorylase kinase with FAD has been obtained using analytical ultracentrifugation. It has been shown that FAD prevents the formation of the enzyme-glycogen complex, but exerts practically no effect on the phosphorylase kinase activity. The dependence of the relative rate of phosphorylase kinase-glycogen complex formation on the concentration of FAD has cooperative character (the Hill coefficient is 1.3). Under crowding conditions in the presence of 1 M trimethylamine-N-oxide (TMAO), FAD has an inhibitory effect on self-association of phosphorylase kinase. The data suggest that the complex of glycogen metabolism enzymes in protein-glycogen particles may function as a flavin depot in skeletal muscle.
Research Interests:
Agonist-binding kinetics to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were measured using sequential-mixing stopped-flow fluorescence methods to determine the contribution of each individual site to... more
Agonist-binding kinetics to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were measured using sequential-mixing stopped-flow fluorescence methods to determine the contribution of each individual site to agonist-induced opening and desensitization. Timed dansyl-C6-choline (DC6C) binding followed by its dissociation upon mixing with high, competing agonist concentrations revealed four kinetic components: an initial, fast fluorescence decay, followed by a transient increase, and then two characteristic decays that reflect dissociation from the desensitized agonist sites. The transient increase resulted from DC6C binding to the open-channel based on its prevention by proadifen, a noncompetitive antagonist. Further characterization of DC6C channel binding by the inhibition of [3H]phencyclidine binding and by equilibrium measurements of DC6C fluorescence yielded KD values of 2-4 microM for the desensitized AChR and approximately 600 microM for the closed state. At this site, DC6C displayed a strongly blue-shifted emission spectrum, higher intrinsic fluorescence, and weaker energy transfer from tryptophans than when bound to either agonist site. The initial, fast fluorescence decay was assigned to DC6C dissociation from the alphadelta site of the AChR in its closed conformation, on the basis of inhibition with the site-selective antagonists d-tubocurarine and alpha-conotoxin MI. Fast decay amplitude data indicated an apparent affinity of 0.9 microM for the closed-state alphadelta site; the closed-state alphagamma-site affinity is inferred to be near 100 microM. These values and the known affinities for the desensitized conformation show that the alphagamma site drives AChR desensitization to a approximately 40-fold greater extent than the alphadelta site, undergoes energetically larger conformational changes, and is the primary determinant of agonist potency.
Research Interests:
Fluorescent energy transfer measurements of dansyl-C6-choline binding to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were used to determine binding characteristics of the alpha gamma and alpha delta binding sites.... more
Fluorescent energy transfer measurements of dansyl-C6-choline binding to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were used to determine binding characteristics of the alpha gamma and alpha delta binding sites. Equilibrium binding measurements show that the alpha gamma site has a lower fluorescence than the alpha delta site; the emission difference is due to differences in the intrinsic fluorescence of the bound fluorophores rather than differences in energy transfer at the two sites. Stopped-flow fluorescence kinetics showed that dissociation of dansyl-C6-choline from the AChR in the desensitized conformation occurs 5-10-fold faster from the alpha gamma site than from the alpha delta site. The dissociation rates are robust for distinct protein preparations, in the presence of noncompetitive antagonists, and over a broad range of ionic strengths. Equilibrium fluorescent binding measurements show that dansyl-C6-choline binds with higher affinity to the alpha delta site (K = 3 nM) than to the alpha gamma site (K = 9 nM) when the AChR is desensitized. Similar affinity differences were observed for acetylcholine itself. The distinct dissociation rates permit the extent of desensitization to be measured at each site during the time course of binding. This sequential mixing method of measuring the desensitized state population at each agonist site can be applied to study the mechanism of AChR activation and subsequent desensitization in detail.
Research Interests:
Research Interests:
The effect of trimethylamine N-oxide (TMAO) on self-association of phosphorylase kinase (PhK) has been studied by analytical ultracentrifugation and turbidimetry in 40 mM N-(2-hydroxyethyl)piper-azine-N′-ethanesulfonic acid buffer, pH 6.8... more
The effect of trimethylamine N-oxide (TMAO) on self-association of phosphorylase kinase (PhK) has been studied by analytical ultracentrifugation and turbidimetry in 40 mM N-(2-hydroxyethyl)piper-azine-N′-ethanesulfonic acid buffer, pH 6.8 and 8.2. PhK is a hexade-camer (αβγδ)4 with a molecular mass of 1,300 kDa. The oligomeric state of the native enzyme is dependent on the protein concentration and the concentrations of Ca2+ and Mg2+, which are essential for the enzymatic activity. In the absence of Ca2+ and Mg2+ the enzyme exists in the monomeric and dimeric forms (with s 20,w = 23 and 36.5 S); however, the addition of 0.1 mM Ca2+ and 10 mM Mg2+ results in the appearance of associates of higher order. TMAO (0.6–1.0 M) was found to favor greatly self-association of PhK. In the presence of TMAO, apart from the association products, consisting of a rather low number (n) of PhK molecules (n = 2, 3, 4,...), two distinct rapidly moving boundaries with S 20,w = 189 and 385 S are registered on the sedimentation profiles. These boundaries correspond to 24-mers and 70-mers (the molecular masses of associates were estimated using the Mark-Houwink—Kuhn—Sakurada equation, assuming a spherical form. The kinetics of TMAO-induced association of PhK was monitored by following the increase in the turbidity of the enzyme solution at varying concentrations of the protein and TMAO. The initial rate of the association is proportional to the enzyme concentration squared, suggesting that the initial step of PhK association is the stage of dimerization.
Research Interests:
The effect of trimethylamine N-oxide (TMAO) on self-association of phosphorylase kinase (PhK) has been studied by analytical ultracentrifugation and turbidimetry in 40 mM N-(2-hydroxyethyl)piper-azine-N′-ethanesulfonic acid buffer, pH 6.8... more
The effect of trimethylamine N-oxide (TMAO) on self-association of phosphorylase kinase (PhK) has been studied by analytical ultracentrifugation and turbidimetry in 40 mM N-(2-hydroxyethyl)piper-azine-N′-ethanesulfonic acid buffer, pH 6.8 and 8.2. PhK is a hexade-camer (αβγδ)4 with a molecular mass of 1,300 kDa. The oligomeric state of the native enzyme is dependent on the protein concentration and the concentrations of Ca2+ and Mg2+, which are essential for the enzymatic activity. In the absence of Ca2+ and Mg2+ the enzyme exists in the monomeric and dimeric forms (with s 20,w = 23 and 36.5 S); however, the addition of 0.1 mM Ca2+ and 10 mM Mg2+ results in the appearance of associates of higher order. TMAO (0.6–1.0 M) was found to favor greatly self-association of PhK. In the presence of TMAO, apart from the association products, consisting of a rather low number (n) of PhK molecules (n = 2, 3, 4,...), two distinct rapidly moving boundaries with S 20,w = 189 and 385 S are registered on the sedimentation profiles. These boundaries correspond to 24-mers and 70-mers (the molecular masses of associates were estimated using the Mark-Houwink—Kuhn—Sakurada equation, assuming a spherical form. The kinetics of TMAO-induced association of PhK was monitored by following the increase in the turbidity of the enzyme solution at varying concentrations of the protein and TMAO. The initial rate of the association is proportional to the enzyme concentration squared, suggesting that the initial step of PhK association is the stage of dimerization.