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At least 523 records · Page 29Linked to original sources
PubMed1991

Incomitant dissociated vertical deviation and superior oblique overaction.

When superior oblique overaction coexists with dissociated vertical deviation, it may produce a pattern of disparity in the amount of dissociated vertical deviation in different horizontal gaze fields, which the authors have termed incomitance. Seven patients with the constellation of esotropia, bilateral superior oblique overaction, "A"-pattern, and incomitant dissociated vertical deviation are presented. In each case, a distinctive incomitant pattern of dissociated vertical deviation was noted in which dissociated vertical deviation was least in adduction, moderate in primary position, and greatest in abduction. Four patients underwent asymmetric superior rectus recessions and partial tenotomy of the superior oblique tendons at their insertions as the initial treatment for their incomitant dissociated vertical deviation. Recognition of such a pattern of incomitant dissociated vertical deviation is important in choosing appropriate surgical management.

Child↗
PubMed2001

Synaptic development in semi-dissociated cultures of rat retina.

Cultured neurons provide a simpler and more accessible environment to study the synaptic physiology. However, it is not clear if development of synapses in culture is similar to that in the in vivo condition. We studied the developmental sequence and morphological differentiation of chemical synapses in semi-dissociated rat retinal cultures that consisted of dissociated neurons as well as undissociated retinal aggregates. Synapses were quantified by synaptophysin immunoreactive puncta. During second week of in vitro development the average number of chemical synapses on the cell body decreased while that on the neurites increased significantly. Conventional synapses appeared both in aggregate and in dissociated neurons, with the developmental profile similar to that reported for in vivo retina. In contrast, the development of ribbon synapses was adversely affected by the in vitro microenvironment as suggested by following observations. The ribbon synapses were more frequently found in aggregate than in dissociated neurons, and were not associated with dyadic or triadic synaptic arrangement. The photoreceptor ribbons did not contact a postsynaptic process while bipolar ribbons made single (monadic) synapses. Further, photoreceptor ribbons in dissociated neurons were late to form and took more time to mature as compared to those in the aggregate cultures. Most of the rod bipolar cells, identified by their immunoreactivity to protein kinase C (PKC), had three or more neurites. Unlike in the in vivo retina, the dissociated rod bipolar cells did not show any PKC immunoreactive varicosities, suggesting that they failed to develop a well-differentiated synaptic terminal. Interestingly, we did not find any parvalbumin positive AII amacrine cells that are normally postsynaptic to rod bipolar cells. These results show that the conventional synapses of retina, which are similar to chemical synapses in other parts of the brain, develop normally both in aggregate and dissociated neurons. However, the highly specialized ribbon synapses have more stringent developmental requirements, and their normal development may require the presence of postsynaptic neurons in their close vicinity.

Animals↗
PubMed1998

New method to study the effects of peptide sequence on the dissociation energetics of peptide ions.

A new method has been developed to study the dissociation patterns of singly protonated peptides by using a quadrupole ion trap mass spectrometer. The new approach involves using boundary-activated dissociation to characterize the ease of dissociation of peptide ions. Insight can be gained into the effect of specific peptide sequences on the dissociation energetics of protonated peptides. Increased knowledge of the effects of specific sequences on the dissociation patterns of peptide ions should improve the ability to interpret complex spectra from tandem mass spectrometry (MS/MS) experiments. This method has confirmed the previously observed increase in the energy needed for the dissociation of peptide ions containing basic residues. In addition, this technique has revealed the effect of the location of proline residues on the dissociation energetics of peptides with this amino acid.

Algorithms↗
PubMed1997

Results of anterior transposition of the inferior oblique muscle in incomitant dissociated vertical deviation.

PURPOSE: Recession with anterior transposition of the inferior oblique muscle has been shown to effectively decrease dissociated vertical deviation in primary position. However, studies to date have not addressed the long-term postoperative results with respect to residual deviation in lateral gaze, development of A-pattern strabismus, and the effect of the procedure on upgaze. METHODS: Twenty-three eyes in 12 patients were treated with recession with anterior transposition of the inferior oblique muscle for dissociated vertical deviation greater in adduction than in abduction (termed incomitant dissociated vertical deviation) associated with inferior oblique muscle overaction. Before the operation, dissociated vertical deviation was measured in primary position and lateral gaze, oblique muscle dysfunction was graded, and A or V patterns were measured. Similar measurements were made after the operation. All patients have been followed up for a minimum of 4 years after the operation. RESULTS: Recession with anterior transposition of the inferior oblique muscle effectively eliminated the dissociated vertical deviation in primary position and in adduction. The operation was less effective in reducing small amounts of dissociated vertical deviation in abduction. No significant A patterns developed after the operation. Postoperative inferior oblique muscle function ranged from -1 underaction to +2 overaction, and postoperative upgaze in abduction was normal to mildly deficient. CONCLUSIONS: Recession with anterior transposition of the inferior oblique muscle results in long-term improvement of incomitant dissociated vertical deviation, with a low incidence of late development of A patterns and upgaze deficiency.

Eye Movements↗
PubMed1998

Analysis of isomeric mixtures using blackbody infrared radiative dissociation: determining isomeric purity and obtaining individual tandem mass spectra simultaneously.

A new method that makes possible, for the first time, simultaneous acquisition of individual dissociation mass spectra of isomeric ions in mixtures is presented. This method exploits the exquisite sensitivity of blackbody infrared radiative dissociation kinetics to minor differences in ion structure. Instead of separating precursor ions based on mass (isomers have identical mass), fragment ions are related to their original precursor ions on the basis of rate constants for dissociation. Mixtures of the peptide isomers des-R1 and des-R9 bradykinin are dissociated simultaneously at several temperatures. By fitting the kinetic data to double-exponential functions, the dissociation rate constant and abundance of each isomer in the mixture are obtained. To overcome the difficulty of fitting double-exponential functions, a novel global analysis method is used in which several dissociation data sets collected at different temperatures are simultaneously fit. The kinetic data measured at multiple temperatures are modeled with the preexponentials (corresponding to the abundance of each isomer) as "global" parameters which are constant for all data sets and the exponentials (rate constants) as "local" variables which differ for each data set. The use of global parameters significantly improves the accuracy with which abundances and dissociation rate constants of each individual compound can be obtained from the mixture data. Fragment ions produced from a mixture of these two isomers are related back to their respective precursor ions from the kinetic data. Thus, not only can the composition of the isomeric mixture be determined but an individual tandem mass spectrum of each component in the mixture can be obtained.

Bradykinin↗
PubMed1995

Subunit dissociation and unfolding of macrophage NO synthase: relationship between enzyme structure, prosthetic group binding, and catalytic function.

Macrophage NO synthase is a homodimer of 130 kDa subunits. Each subunit contains an oxygenase domain that binds iron protoporphyrin IX (heme) and tetrahydrobiopterin (H4biopterin) and a reductase domain that binds FAD, FMN, and calmodulin (CaM) [Ghosh & Stuehr (1995) Biochemistry 34, 801-807]. We have studied the dissociation and unfolding reactions of dimeric iNOS in urea to learn how enzyme structure relates to catalysis and prosthetic group binding. The iNOS dimer dissociated between 0 and 2.5 M urea, and the subunits partially unfolded at 2.5 M urea and above. Dimer dissociation was accompanied by loss of NO synthesis activity and release of bound H4biopterin from the protein. However, the dissociated subunits maintained their cytochrome c and ferricyanide reductase activities and retained near stoichiometric quantities of bound heme. The subunit unfolding transition was accompanied by loss of reductase activities and partial loss of bound heme but retention of bound flavins and CaM. The heme iron in the dissociated subunits remained coordinated through axial cysteine thiolate ligation. Kinetic analysis of dimer dissociation showed that loss of NO synthesis correlated with a loss of heme Soret absorbance at 398 nm and an appearance of absorbance bands at 377 and 460 nm, which were attributed to DTT coordination to the sixth position of the heme iron to form a mixed bisthiolate complex. Subunits could reassociate into a dimer when incubated with L-arginine and H4biopterin. Dimer formation correlated with proportional recoveries of NO synthesis and heme Soret absorbance at 398 nm. Thus, dimeric iNOS undergoes separate dissociation and unfolding transitions in urea, and each transition is accompanied by a loss of a specific catalytic function.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1991

Dissociation of calcium from the phosphorylated calcium-transporting adenosine triphosphatase of sarcoplasmic reticulum: kinetic equivalence of the calcium ions bound to the phosphorylated enzyme.

The internalization of 45Ca by the calcium-transporting ATPase into sarcoplasmic reticulum vesicles from rabbit muscle was measured during a single turnover of the enzyme by using a quench of 7 mM ADP and EGTA (25 degrees C, 5 mM MgCl2, 100 mM KCl, 40 mM MOPS.Tris, pH 7.0). Intact vesicles containing either 10-20 microM or 20 mM Ca2+ were preincubated with 45Ca for approximately 20 s and then mixed with 0.20-0.25 mM ATP and excess EGTA to give 70% phosphorylation of Etot with the rate constant k = 300 s-1. The two 45Ca ions bound to the phosphoenzyme (EP) become insensitive to the quench with ADP as they are internalized in a first-order reaction with a rate constant of k = approximately 30 s-1. The first and second Ca2+ ions that bind to the free enzyme were selectively labeled by mixing the enzyme and 45Ca with excess 40Ca, or by mixing the enzyme and 40Ca with 45Ca, for 50 ms prior to the addition of ATP and EGTA. The internalization of each ion into loaded or empty vesicles follows first-order kinetics with k = approximately 30 s-1; there is no indication of biphasic kinetics or an induction period for the internalization of either Ca2+ ion. The presence of 20 mM Ca2+ inside the vesicles has no effect on the kinetics or the extent of internalization of either or both of the individual ions. The Ca2+ ions bound to the phosphoenzyme are kinetically equivalent. A first-order reaction for the internalization of the individual Ca2+ ions is consistent with a rate-limiting conformational change of the phosphoenzyme with kc = 30 s-1, followed by rapid dissociation of the Ca2+ ions from separate independent binding sites on E approximately P.Ca2; lumenal calcium does not inhibit the dissociation of calcium from these sites. Alternatively, the Ca2+ ions may dissociate sequentially from E approximately P.Ca2 following a rate-limiting conformational change. However, the order of dissociation of the individual ions can not be distinguished. An ordered-sequential mechanism for dissociation requires that the ions dissociate much faster (k greater than or equal to 10(5) s-1) than the forward and reverse reactions for the conformational change (k-c = approximately 3000 s-1). Finally, the Ca2+ ions may exchange their positions rapidly on the phosphoenzyme (kmix greater than or equal to 10(5) s-1) before dissociating. A Hill slope of nH = 1.0-1.2, with K0.5 = 0.8-0.9 mM, for the inhibition of turnover by binding of Ca2+ to the low-affinity transport sites of the phosphoenzyme was obtained from rate measurements at six different concentrations of Mg2+.

Adenosine Diphosphate↗
PubMed1993

Kinetics of assembly and dissociation of the mitochondrial creatine kinase octamer. A fluorescence study.

The dissociation of octameric mitochondrial creatine kinase (Mi-CK) into dimers induced by the transition-state analogue complex (TSAC) mixture (creatine+Mg(2+)+ADP+NO3-) is accompanied by a large (25.2%) decrease in Trp fluorescence. This effect is caused by a Trp residue situated at the dimer-dimer interface within the octamer, which becomes susceptible to solvent quenching upon octamer dissociation. Octamer formation, induced by adding excess EDTA to TSAC-dissociated Mi-CK, involves a transient tetrameric species, whereas the dissociation reaction proceeds in a one-step, all-or-none fashion. From fluorescence spectroscopic investigations of the octamer formation and dissociation reactions, a first-order dissociation rate constant of 0.19 min-1 and a bimolecular association rate constant of 318 M-1 s-1 at 30 degrees C were obtained. The octamers formed after EDTA addition can be dissociated again by lowering the temperature to 4 degrees C, indicating a substantial hydrophobic contribution to the interactions stabilizing the octamer.

Animals↗
PubMed1991

Binding of ethidium to the nucleosome core particle. 1. Binding and dissociation reactions.

We have examined binding properties of and dissociation induced by the intercalating dye ethidium bromide when it interacts with the nucleosome core particle under low ionic strength conditions. Ethidium binding to the core particle results in a reversible dissociation which requires the critical binding of 14 ethidium molecules. Under low ionic strength conditions, dissociation is about 90% completed in 5 h. The observed ethidium binding isotherm was corrected for the presence of free DNA due to particle dissociation. The corrected curve reveals that the binding of ethidium to the core particle itself is a highly cooperative process characterized by a low intrinsic binding constant of KA = 2.4 X 10(4) M-1 and a cooperativity parameter of omega = approximately 140. The number of base pairs excluded to another dye molecule by each bound dye molecule (n) is 4.5. Through the use of a chemical probe, methidiumpropyl-EDTA (MPE), we have localized the initial binding sites of ethidium in the core particle to consist of an average of 27 +/- 4 bp of DNA that are distributed near both ends of the DNA termini. MPE footprint analysis has also revealed that, prior to dissociation, the fractional population of core particles which bind the dye (f) may be as low as 50%. Comparison of the binding and dissociation data showed that the cooperative maximum of the binding curve occurred at or near the critical value, i.e., at the point where dissociation began. The data were used to generate a detailed model for the association of ethidium with chromatin at the level of the nucleosome.

Animals↗
PubMed1989

Ordered disruption of subunit interfaces during the stepwise reversible dissociation of Escherichia coli phosphofructokinase with KSCN.

The reversible inactivation and dissociation of the allosteric phosphofructokinase from Escherichia coli has been studied in relatively mild conditions, i.e., in the presence of the chaotropic agent KSCN. At moderate KSCN concentration, the loss of enzymatic activity involves two separated phases: first, a rapid dissociation of part of the tetramer into dimers, second, a slower displacement of the dimer-tetramer equilibrium upon further dissociation of the dimer into monomers. These two reactions can no longer be distinguished above 0.3 M KSCN since complete inactivation occurs in a single reaction. Different changes are observed for the fluorescence and the activity of the enzyme in KSCN: the fluorescence is not affected by the dissociation into dimers which is responsible for inactivation. The decrease in fluorescence reflects the change in environment of the unique tryptophan residue, Trp 311, during the dimer to monomer dissociation. This residue belongs to the interface containing the regulatory site, and its native fluorescence indicates that this interface is still present in the dimer. The substrate fructose 6-phosphate protects phosphofructokinase from inactivation by binding to the tetramer and prevents its dissociation into dimers. The presence of phosphoenolpyruvate prevents the slow dissociation of the dimer into monomers, which shows the ability of the dimer to bind the inhibitor. Two successive processes can be observed during reassociation of the protein upon KSCN dilution. First, a fast reaction (k1 = 2 x 10(5) M-1.s-1) is accompanied by a fluorescence increase and results in the formation of the dimeric species.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1981

Light-scattering investigation of the subunit dissociation of Homarus americanus hemocyanin. Effects of salts and ureas.

The subunit dissociation of the hemocyanin of the lobster, Homarus americanus, by the various salts of the Hofmeister series and the hydrophobic reagents of the urea-guanidinium chloride (GdmCl) class was investigated by laser light scattering molecular weight measurements. The dissociations of the hemocyanin dodecamers to hexamers by the various salts and the lower members of the urea series are found to be rapid and reversible, as predicted by the mass action law for monomer-dimer type of reactions. The salts are found to be very effective dissociating reagents with the unusual order of increasing effectiveness, Cl- less than Br- less than I- less than ClO4-, SCN-. The ureas and GdmCl are found to be relatively ineffective dissociating agents. In addition, the ureas show a decreasing order of effectiveness in going from urea to methyl-, ethyl-, and propylurea. This suggests that hydrophobic interactions are not the dominant stabilizing forces between the pairs of hexamers that form the dodecameric structure. Polar and ionic interactions appear to be the major stabilizing forces of the dodecameric structure. The use of equations derived for predicting the effects of dissociating reagents and salts on the structure of subunit proteins [Herskovits, T. T., & Ibanez, V. S. (1976) Biochemistry 15, 5715-5721] together with binding and Setschenow constants based on model amino acid data is found to give good account of the dissociation behavior observed with the salts, urea, and methylurea in the presence of calcium ion at both pH 7.8 and pH 9.5. The apparent number of amino acids at the contact areas of the hexamers, Napp, required to fit the dissociation data were found to be 24 +/- 8 at pH 7.8 and 23 +/- 4 at pH 9.5. However, because of the possible effects of molecular microheterogeneity, the estimates of amino acids at the contact areas must be viewed with caution, depending on further investigations.

Animals↗
PubMed1977

Light-scattering investigations of the subunit dissociation of human hemoglobin A. Effects of various neutral salts.

The effectiveness of various salts of the Hofmeister series as dissociating agents for human hemoglobin A tetramers has been investigated by light-scattering molecular-weight measurements. Dissociation of hemoglobin to half-molecules of alpha beta dimers follows the order of the series dictated predominantly by the sequence of the anions F- less than Cl- less than Br- less than ClO4- less than SCN-, I-, with the cations Na+ and K+ having relatively little effect on the observed dissociation. The use of equations derived for predicting the effects of dissociating reagents on the structure of subunit proteins [Herskovits, T. T., and Ibanez, V. S. (1976), Biochemistry 15, 5715] together with Setschenow constants based on the model amino acid data of Nandi and Robinson were found to give a satisfactory account of the dissociation behavior observed with many of the salts, giving reasonable estimates of the number of amino acids that form the smaller contact area of the alpha beta subunits of hemoglobin shown by the Perutz crystallographic model. The analysis of the dissociation data also extends the utility of the Setschenow constants tested for the characterization of the dissociation behavior of other subunit proteins.

Anions↗
PubMed1975

A light scattering investigation of the propylurea dissociation of human hemoglobin A.

The subunit dissociation of human hemoglobin A by propylurea in several liganded and chemically modified states was investigated by light scattering molecular weight methods. The dissociation data were analyzed by means of the equation developed in our earlier studies: deltaF degrees D EQUALS TO DELTAF degrees D, W - 2N'RTKB[D], where deltaF degrees D and deltaf degreees D,W represent the free energy of dissociation of hemoglobin tetramers into half-molecules consisting of alpha beta dimers in the presence and in the absence of propylurea, KB is the binding constant of the urea to the average peptide unit, [D] is its concentration, and N' is the number of amino acid sites exposed per half-molecule on dissociation. It is found that the dissociation of oxyhemoglobin, cyanmethemoglobin, and N-ethylmaleimide oxyhemoglobin is characterized by essentially the same N' value of 15 to 21 plus or minus 3, that are close to the 19 amino acid residues per surface which comprise the smaller alpha beta contact area, seen in the X-ray crystallographic model of horse hemoglobin of Perutz and coworkers. Due to the very low degree of dissociation of deoxyhemoglobin, only a very approximate estimate of N' of about the same order of magnitude could be obtained for this form of the protein. In contrast, a significantly lower value of N' was obtained with bis(maleimidomethyl) ether modified oxyhemoglobin of 8 plus or minus 3, that is cross-linked at cysteine residue F9 (93)beta and histidine residue FG4 (97) beta in the same beta chains. Our results suggest that alterations caused by the presence of the cross-linking reagent reflect both the loss in amino acid residues that can interact with the urea at the blocked segments of the polypeptide chains in the dissociated state of hemoglobin and the changes in accessibility of some of the amino acid residues perturbed by the introduction of the reagent in the parent tetrameric form.

1-Propanol↗
PubMed2002

Uncoupling Kapbeta2 substrate dissociation and ran binding.

Karyopherinbeta2 (Kapbeta2) imports a variety of mRNA binding proteins into the nucleus. Release of import substrates in the nucleus involves formation of a high-affinity Kapbeta2-RanGTP complex and concomitant dissociation of import substrates. The crystal structure of the Kapbeta2-RanGppNHp complex shows that Ran binds in the Kapbeta2 N-terminal arch and substrate most likely binds its C-terminal arch. The structure suggested a mechanism for Ran-mediated substrate dissociation where a long internal acidic loop in Kapbeta2 transmits structural information between the GTPase and substrate sites, leading to displacement of substrate by the loop when Ran is bound. To study the molecular mechanism of substrate dissociation, we have cleaved the acidic loop of Kapbeta2 proteolytically (cl-Kapbeta2) and also constructed a mutant of Kapbeta2 with a truncated loop (TL-Kapbeta2). Both modified Kapbeta2s are unable to undergo Ran-mediated substrate dissociation. We have also mapped the boundaries of the Kapbeta2 binding site of substrate mRNA binding protein A1 using a widely applicable method employing NMR spectroscopy. This has allowed design of reagents to quantitate the affinities of the Kapbeta2 proteins for Ran and substrate. cl-Kapbeta2, TL-Kapbeta2, and native Kapbeta2 have comparable affinities for both RanGppNHp and import substrates, indicating that perturbation of the loop has not altered the strength of binary Kapbeta2-Ran or Kapbeta2-substrate interactions. The TL-Kapbeta2 mutant also binds RanGppNHp and substrate simultaneously to form a ternary complex, indicating that in addition to the loss of coupling between Ran binding and substrate dissociation, the two ligand sites on Kapbeta2 are spatially distinct. The uncoupling of Ran binding and substrate dissociation in the TL-Kapbeta2 mutant is further evident in significant loss of Ran-mediated nuclear uptake of fluorescent substrate in digitonin-permeabilized HeLa cells. These results support our previously proposed GTPase-mediated Kapbeta2-substrate dissociation mechanism where the acidic loop of Kapbeta2 physically couples distinct Ran and substrate binding sites.

Active Transport, Cell Nucleus↗
PubMed2002

Yeast pyruvate decarboxylase tetramers can dissociate into dimers along two interfaces. Hybrids of low-activity D28A (or D28N) and E477Q variants, with substitution of adjacent active center acidic groups from different subunits, display restored activity.

The tetrameric enzyme yeast pyruvate decarboxylase (YPDC) has been known to dissociate into dimers at elevated pH values. However, the interface along which the dissociation occurs, as well as the fundamental kinetic properties of the resulting dimers, remains unknown. The active sites of YPDC are comprised of amino acid residues from two subunits, a property which we utilize to address the issue as to which dimer interface is cleaved under different conditions of dissociation. Hydroxide-induced dissociation of the active site D28A (or D28N) and E477Q variants, each at least 100 times less reactive than wild-type YPDC, followed by reassociation of D28A (or D28N) and E477Q variants led to a remarkable 35-50-fold increase in activity. This result is possible only if the hydroxide-induced dissociation results in a cleavage along the interface between two subunits so that residues D28 and E477 are now separated. Upon reassociation, one of the two active sites of the hybrid dimer will have both residues substituted, whereas the second one will be of the wild-type phenotype. In contrast to the hydroxide-induced dimers, the urea-induced dissociation recently proposed results in dissociation along dimer-dimer interfaces, without separating the active sites, and therefore, on reassociation, these dimers do not regain activity. The significance of the results is discussed in light of a recently proposed alternating sites mechanism for YPDC. A preparative ion-exchange method is reported for the separation and purification of hybrid enzymes.

Amino Acid Substitution↗
PubMed2002

Dissociation of the single-ring chaperonin GroEL by high hydrostatic pressure.

We investigated the dissociation of single-ring heptameric GroEL (SR1) by high hydrostatic pressure in the range of 1-2.5 kbar. The kinetics of the dissociation of SR1 in the absence and presence of Mg2+, KCl, and nucleotides were monitored using light scattering. The major aim of this investigation was to understand the role of the double-ring structure of GroEL by comparing its dissociation with the dissociation of the single ring. At all the pressures that were studied, SR1 dissociates much faster than the GroEL 14mer. As observed with the GroEL 14mer, SR1 also showed biphasic kinetics and the dissociated monomers do not reassociate readily back to the oligomer. Unlike the GroEL 14mer, the observed rates for SR1 dissociation are independent of the concentrations of Mg2+ and KCl in the studied range. The effects of nucleotides on the observed rates, in the absence or presence of Mg2+ and KCl, are not very significant. The heterogeneity induced in the GroEL molecule with the double-ring structure by ligands such as Mg2+, KCl, and nucleotides is not observed in the case of SR1. This indicates that the inter-ring negative cooperativity in the double-ring GroEL has a major role in this regard. The results presented in this investigation demonstrate that the presence of a second ring in the GroEL 14mer is important for its stability in an environment where the functional ligands of the chaperonin are available.

Adenosine Diphosphate↗
PubMed1997

Agonist binding to the Torpedo acetylcholine receptor. 1. Complexities revealed by dissociation kinetics.

Examination of the kinetics of dissociation of [3H]acetylcholine and [3H]suberyldicholine from the membrane-bound acetylcholine receptor from Torpedo californica has revealed complexities in the high-affinity binding of nicotinic agonists. Each agonist binds to two high-affinity sites per receptor with an equilibrium dissociation constant of approximately 15 nM. When dissociation of [3H]acetylcholine from the receptor complex was triggered by dilution, dissociation occurred as a monophasic process with an apparent rate of 0.023 +/- 0.010 s(-1). However, when micromolar concentrations of unlabeled agonists (acetylcholine, carbamylcholine or suberyldicholine) were included in the dilution buffer this rate increased about 5-fold. This accelerating effect occurred even when the two high-affinity sites were initially saturated with the radioligand. This suggested the presence of an additional site (or subsite) for agonist with affinity in the micromolar range. However, at concentrations of 0-20 microM, no additional sites for [3H]acetylcholine were detected at equilibrium. To explain these results, we propose that each high-affinity site is made up of two subsites, A and B, which are mutually exclusive at equilibrium. With [3H]acetylcholine initially occupying site A, occupancy of site B by unlabeled ligand reduces the affinity for site A and accelerates the dissociation of the radioligand. Studies of dissociation of [3H]suberyldicholine, a large bis-quaternary agonist, provide some clue as to the possible physical nature of these subsites. Whereas its dissociation rate was similar to that of [3H]acetylcholine (0.028 +/- 0.012 s(-1)), this rate was only marginally, if at all, affected by the presence of unlabeled ligands. These results, in addition to those presented in the accompanying manuscript, lead to the proposal that [3H]suberyldicholine is able to cross-link the two subsites or at least sterically occlude the second site.

Acetylcholine↗
PubMed1996

Dissociation of long and very long chain fatty acids from phospholipid bilayers.

Dissociation of fatty acids (FA) from and transbilayer movement (flip-flop) in small unilamellar phosphatidylcholine vesicles (SUV) were monitored by measuring the pH inside the vesicle with an entrapped water-soluble fluorophore, pyranin. With a pH gradient imposed upon SUV preloaded with FA, the rate of flip-flop of saturated very long chain FA (C20:0, C:22:0, and C24:0) was shown to be fast (t1/2 < 1 s); previously, we showed by stopped flow measurements that flip-flop of long chain (14-18 carbons) FA is very fast [t1/2 < 10 ms; Kamp, F., et al. (1995) Biochemistry 34, 11928-11937]. The rates of dissociation of FA from SUV were evaluated by incorporating FA into donor vesicles and measuring transfer to acceptor vesicles. The transfer was followed by changes in internal pH of either donor or acceptor vesicles with stopped flow (C14:0, C16:0, C17:0, C18:0, C18:1, and C18:2) or on-line (C20:0, C22:0, and C24:0) fluorescence. All FA showed a single-exponential transfer process that was slower than the lower limits established for the rate of flip-flop, with t1/2 of dissociation ranging from 20 ms for C14:0 to 1900 s for C24:0. The pseudo-unimolecular rate constant (koff) for dissociation of C14:0 to C26:0 showed a 10-fold decrease for each addition of two CH2 groups to the acyl chain and a delta (delta G) of -740 cal/CH2. The dissociation rate constants for oleic acid (18:1) and linoleic acid (18:2) were 5 and 10 times faster, respectively, than that of C18:0. The rates of dissociation for typical dietary FA are sufficiently rapid that complex mechanisms (e.g. protein-mediated) may not be required for their desorption from biological membranes. The very slow dissociation rates for C24:0 and C26:0 may accentuate their pathological effects in diseases in which they accumulate in tissues.

Arylsulfonates↗