Purification of the muscarinic acetylcholine receptor from rat forebrain.
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Animals↗
Biomedical subjects
R J Morris
Publications and source records attributed to R J Morris.
Evidence that the centrally and peripherally located cells in the murine epidermal proliferative unit are two distinct cell populations.
The purpose of this investigation was to characterize the [3H]thymidine label-retaining and the "maturing" classes of basal cells from the dorsal epidermis of adult SENCAR mice and to compare their early cellular kinetic responses to topical application of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Autoradiography of epidermal whole mounts and cross sections demonstrated that injection of [3H]thymidine every 6 h for 1 week labeled 95% of the basal nuclei, including those in the central region of the epidermal proliferative units. One month later, the labeling index was reduced to 2%; 90% of the label-retaining cells were within a nuclear diameter of the central suprabasal column of the proliferative units. When mice were treated with 2 micrograms of TPA 1 month after labeling, mitotic label-retaining cells were found within 22 h after treatment. Seventy-five percent of the label-retaining cells remained on the basal layer through the 28-h experimental period. In contrast, the basal labeling index following a 1-h pulse of [3H]thymidine was 5%. Eighty-five percent of the labeled cells were found in the periphery of the proliferative units. By 4 days after pulse labeling, most of the originally labeled cells had divided, although vertical cross sections indicated that 92% remained on the basal layer. When mice were treated with TPA on day 4, labeled cells were rarely found in mitosis. Instead, about 60% of the labeled cells were displaced to the suprabasal layers. These observations suggest that 2 classes of epidermal basal cells have different early responses to TPA treatment: the label-retaining cells proliferate, and most of the "maturing" cells continue to differentiate.
Animals↗
Office diagnostic methods for allergic diseases.
The practice of allergy has been revolutionized in the past 20 years and has undergone a welcome transformation from an arcane art to an immunology-based clinical science. (It is hoped that the best features of the art will be retained, however.) An understanding of allergy diagnosis should provide the requisite information for the primary care physician to choose an allergy consultant wisely, and to understand the scientific basis of his methods and recommendations.
Bronchial Provocation Tests↗
Muscarinic cholinergic receptor of rat brain. Factors influencing migration in electrophoresis and gel filtration in sodium dodecyl sulphate.
The muscarinic cholinergic receptor present in synaptosomal membranes of rat brain was covalently labelled with the alkylating muscarinic antagonist, tritiated propylbenzilylcholine mustard. The labelled receptor was then solubilized in sodium deoxycholate and sodium dodecyl sulphate, and its migration in polyacrylamide gel electrophoresis and gel filtration in the presence of sodium dodecyl sulphate analysed. Provided both proteolysis and inter-chain disulphide bond formation were vigorously prevented, the receptor from rat forebrain (cerebral cortex plus caudate putamen) migrated, in sodium dodecyl sulphate/polyacrylamide gel electrophoresis, as a broad band of apparent Mr 66000-76000. Two dominantly labelled polypeptides, of apparent Mr 68000 and 73000, could be distinguished as the major components of this band. These multiple species seen in electrophoresis may reflect a structural diversity related to the different binding properties, and modes of action, of this receptor. In electrophoresis using discontinuous buffer systems the labelled receptor readily formed intermolecular disulphide bonds and so aggregated. In particular, if solubilized membranes were reduced with 2-mercaptoethanol, and reformation of disulphide bonds during electrophoresis not prevented, then formation of a dimeric species (apparent Mr 119000-128000) occurred. This probably explains previous reports in the literature of larger-Mr species seen in electrophoresis. During gel filtration, the receptor formed intra-chain disulphide bonds which produced conformational heterogeneity, leading to polydisperse migration. In addition, extensive proteolytic degradation of the receptor occurred due to a protease migrating slightly ahead of the receptor. Both effects were eliminated by alkylation of the solubilized membranes with iodoacetamide before gel filtration. Alkylated receptor migrated on Sephacryl S-300 in 0.5% sodium dodecyl sulphate with an equivalent Stokes' radius of 6.1 nm. This is identical to that of reduced ovalbumin, a molecule with an apparent Mr in gel electrophoresis of 43000. On a different gel matrix, TSK HW 55(S), the receptor migrated with a somewhat larger Stokes' radius, eluting just behind reduced bovine serum albumin (Stokes' radius 8.5 nm; apparent Mr in electrophoresis 67000). Thus the receptor appears to adsorb to the Sephacryl matrix, although even on the TSK gel the receptor eluted as a somewhat smaller protein than expected from its behaviour in gel electrophoresis. Solubilized, alkylated receptor, partly purified by gel filtration so that it was not degraded by endogenous proteases, was not cleaved by mild hydroxylamine treatment.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
The endemic faunae of Lake Baikal: their general biochemistry and detailed lipid composition.
The biochemical composition of the important endemic faunal groups in Lake Baikal, Siberia, have been examined. The major biochemical components were determined and detailed analyses of the lipid fractions are presented. The lipids are characterized by high levels of long-chain omega 3 polyunsaturated fatty acids in the glyceride-esters, the presence of large amounts of triglyceride in pelagic and bathy-pelagic species with a complete absence of wax esters, and a very simple sterol composition dominated almost exclusively by cholesterol. The results are discussed in relation to present knowledge concerning both marine and freshwater lipids and the possible origin and evolution of the Baikalian fauna. Present theories concerning the likely role of long-chain polyunsaturates and wax esters in lipids are considered in the light of these present results and a link is suggested between sterol diversity and type of evolution.
Animals↗
Geminate combination of oxygen with iron-cobalt hybrid hemoglobins.
Photodissociation of oxygen from the ferrous subunits of hybrid hemoglobins in which the heme of either the alpha or the beta chain has been replaced by cobalt protoporphyrin IX shows large differences between the subunits. With a 25-ns light pulse, the apparent quantum yield at the end of the flash is greater for the beta-iron hybrid than for the alpha-iron hybrid. With the beta-iron hybrid, the yield is greater when solution conditions favor the T-state. After the flash, a part of the oxygen which has been dissociated recombines with a half-time of the order of tens of nanoseconds. The proportion is greatest in the R-state at low temperature and least in the T-state. With the alpha-iron hybrid, oxygen is much less readily removed, and the rapid recombination is slight or absent. It is seen most clearly at low temperatures in conditions which favor the T-state. The long term (greater than 100 ns) effect is that oxygen is much more readily removed from the beta-iron hybrid in the T-state than under any other condition. Analogous flash experiments performed with human hemoglobin A may be closely simulated by superposition of the results obtained with the two hybrid hemoglobins under the same conditions. Isolated human alpha and beta--SH chains show differences similar to, but less marked than, those of the iron-cobalt hybrids.
Hemoglobins↗
Kinetics of ligand binding and quaternary conformational change in the homodimeric hemoglobin from Scapharca inaequivalvis.
The kinetics of the reaction with oxygen and carbon monoxide of the homodimeric hemoglobin from the bivalve mollusc Scapharca inaequivalvis has been extensively investigated by flash and dye-laser photolysis, temperature jump relaxation, and stopped flow methods. The results indicate that cooperativity in ligand binding, already observed for oxygen at equilibrium, finds its kinetic counterpart in a large decrease of the oxygen dissociation velocity in the second step of the binding reaction. In the case of carbon monoxide, cooperativity is clearly evident in the increase of the combination velocity constant as the reaction proceeds. Therefore, the ligand-binding kinetics of this dimeric hemoglobin shows the characteristic features of the corresponding reactions of tetrameric hemoglobins. Analysis of the data in terms of the allosteric model proposed by Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118) has shown that the values of the allosteric parameters cannot be fixed uniquely for a dimeric hemoglobin. The rapid changes in absorbance observed at the isosbestic points of unliganded and liganded hemoglobin following laser photolysis provided a value of 7 X 10(4) S-1 at 20 degrees C for the rate of the ligand-free quarternary conformational change, postulated on the basis of cooperative ligand binding. Comparison of the rapid absorbance changes observed during ligand rebinding in this hemoglobin with those observed in tuna hemoglobin indicate that, at full photolysis, binding to the T state is followed by further binding and conversion to the liganded R state; at partial photolysis, population of the liganded T state occurs immediately and is followed by a decay to the liganded R state upon further ligand binding. These new results, in conjunction with previous equilibrium data on the same system, show unequivocally that the presence of two different types of chain is not an absolute prerequisite for cooperativity in hemoglobins, contrary to currently accepted ideas.
Animals↗
The apparent quantum yield of T-state human hemoglobin. Contribution of protein and heme to rates of oxygen reactions.
The apparent quantum yield for dissociation of oxygen from T-state human hemoglobin has been determined using pulses of light 350 ns long at 540 nm. Two quantum yields were found. One was the same as for the R-state, and, like it, strongly temperature- and viscosity-dependent. The other, only slightly influenced by temperature and viscosity, was 10 times larger at 20 degrees C. Previous work (Sawicki, C. A., and Gibson, Q. H. (1977) J. Biol. Chem. 252, 7538-7547) has shown two distinct phases in binding of oxygen by T-state human hemoglobin at pH 7, 20 degrees C. When the apparent quantum yield was followed with time, the species with high quantum yield correlated with the rapidly reacting T-state species. The hemoglobin chains have different quantum yields in the T-state. Quantum yield data may serve as a measure of population of the liganded T-state in human hemoglobin, supplementing absorbance and circular dichroism data, and permit calculation of the rates of reaction at the heme in both R- and T-states.
Chemical Phenomena↗
Is Thy-1 expressed only by ganglion cells and their axons in the retina and optic nerve?
The distribution of Thy-1 in the retina and optic nerve has been examined immunohistochemically, and compared to that of the astrocytic marker glial fibrillary acidic protein. The axons and cell bodies of ganglion cells were found to be Thy-1 positive as were processes within the inner plexiform layer. Transection of the optic nerve in the neonatal rat results in the rapid degeneration of the ganglion cells but some Thy-1 staining remains in the inner plexiform layer. We have estimated using an immunoassay of normal and optic nerve transected retinae that about 70% of the Thy-1 in the retina is on ganglion cells and their axons and the remainder is on cells which contribute processes to the inner plexiform layer, presumably amacrine, bipolar or Müller cells. In the optic nerve the Thy-1 was found to be limited to the fascicles of optic nerve fibres and the intrafascicular spaces, containing astrocytes and their processes, were not stained. Axotomy of the adult nerve, which produced axonal degeneration and astrocytic proliferation, led to a loss of over 95% of the Thy-1 from the nerve. We found no evidence that the astrocytes of the retina or optic nerve were Thy-1 positive in normal animals or during degeneration.
Animals↗
Differential expression of Thy-1 on the various components of connective tissue of rat nerve during postnatal development.
The glycoprotein Thy-1 is found on the surface of both neurons, and most fibroblasts, in tissue culture of embryonic or neonatal rat nervous tissue. In adult rat nerves, however, we find the antigen restricted in vivo to neurons and their axons (R. Morris, P. Barber, J. Beech, and G. Raisman, 1983, J. Neurocytol., 12, 1017-1039). We show here that this discrepancy is due to a loss of Thy-1 from neural connective tissue during postnatal development. Moreover, the different elements of connective tissue loose Thy-1 at different times. Epineurial fibroblasts, for instance, express Thy-1 for at least 2 weeks after endoneurial fibroblasts lack detectable antigen. As judged by their loss of Thy-1 antigen, therefore, the different components of neural connective tissue mature at different rates. One important practical implication of this is that Thy-1 cannot be used as a cell surface "marker" for all classes of fibroblasts in vitro. Unusual problems encountered in the immunohistochemical staining of Thy-1 on perineurium, but not on the other elements of the nerve, are also described.
Aging↗
Leghemoglobin. Kinetic, nuclear magnetic resonance, and optical studies of pH dependence of oxygen and carbon monoxide binding.
The rate of dissociation of oxygen from soybean oxyleghemoglobin a increases about 5-fold between pH 4 and pH 7, with apparent pK = 5.46 and n = 1. The rate of dissociation of carbon monoxide from carbon monoxyleghemoglobin a and the rates of combination of oxygen and carbon monoxide with ferrous leghemoglobin a are all invariant in this range of pH. The optical spectrum of oxyleghemoglobin in the visible region and the resonances of the four heme meso protons (protons of the bridge carbon atoms) in the NMR spectrum of oxyleghemoglobin are also dependent on pH with pK near 5.5. We suggest that protonation of the imidazole of the distal histidine residue (His 61) leads to formation of a hydrogen bond to the bound oxygen molecule which affects the electronic configuration or conformation of the heme and decreases the rate of oxygen dissociation.
Carbon Monoxide↗
The distribution of Thy-1 antigen in the P.N.S. of the adult rat.
The distribution of the cell surface glycoprotein Thy-1 in the P.N.S. of adult rats was examined using immunohistochemical and experimental techniques. In the hypoglossal nerve the pattern of Thy-1 labelling suggested the antigen was on the plasma membrane of all axons, not only in their major myelinated course but also on their fine terminal branches and at the motor end plate itself. Similarly in other peripheral nerves examined [phrenic and vagus nerves, dorsal and ventral roots, and both the preganglionic and postganglionic trunks of the superior cervical ganglion (SCG) and the submandibular ganglion] Thy-1 was always associated with axons, but the resolution obtained with immunohistochemical techniques was not in itself sufficient to exclude the possibility that the antigen was on the surface of the ensheathing Schwann cell where it apposed the axons. However, in the hypoglossal nerve the antigen was found to accumulate proximal to a ligation of the nerve, suggesting it was made by the neurons and transported down the nerve by axoplasmic flow. This impression was supported by examining neuronal cell bodies in the SCG, dorsal root ganglia and submandibular ganglion, all of which contain readily detectable cytoplasmic Thy-1. In the SCG this cytoplasmic antigen was shown to include the pool of newly synthesized Thy-1. It was increased by treatment of the ganglion with colchicine, and decreased by cycloheximide. Conversely, treatment of hypoglossal nerve trunk with colchicine did not lead to the appearance of the antigen around the non-neuronal perikarya. It is therefore concluded that in those parts of the adult rat P.N.S. examined, Thy-1 is made by neurons and occurs generally on the plasma membrane of axons.
Animals↗
Density of Thy-1 on axonal membrane of different rat nerves.
The density of the cell surface antigen, Thy-1, has been measured on the axonal plasma membrane of the optic and hypoglossal nerves, and on the sympathetic (preganglionic) chain of the superior cervical ganglion in the rat. For each, the amount of Thy-1 in a standard length of nerve was determined. The amount of axonal plasma membrane in a similar length was then measured by use of a computer graphics system which determined the total length of membrane in electron micrographs of cross sections of nerve. The axons of the two peripheral nerves were found to have a similar density (1100-1500 molecules/microns2) of Thy-1 on their surface, and this was two- to threefold higher than the density on axons of optic nerve (500 molecules/microns2). These figures indicate that the density of Thy-1 on the surface of these axons is somewhat lower than that found on the cells of the lymphoid system. Moreover, contrary to the impression gained from previous determinations of Thy-1 levels, axons in the peripheral nervous system do not necessarily have a lower density of Thy-1 on their surface than do those of the central nervous system.
Animals↗
Estimation of Thy-1 in cryostat sections of nervous tissue.
The conventional assay for measuring cell surface antigens--the quantitative absorption of antibody by tissue homogenates--proved inadequate when used to determine the level of Thy-1 glycoprotein in rat nerves and peripheral ganglia. In this paper we report that the binding of 125I-labelled Fab fragments of a monoclonal anti-Thy-1 antibody to cryostat sections is sufficiently sensitive to give consistent estimates of the Thy-1 level on single samples of even small nerves. Observed levels of Thy-1 were generally higher than had previously been thought, and in particular we found no nerves totally lacking the antigen. The lowest levels (6-10 pmol/mg protein) were in peripheral nerves with a large motor component. Autonomic and sensory nerves had higher levels (15-20 pmol/mg protein). The highest levels were on the optic nerve (34 pmol/mg protein), superior cervical sympathetic ganglion (40 pmol/mg protein), and the cerebellar vermis (46 pmoles/mg protein; the only brain region examined in this study). From a practical point of view, the cryostat assay has the advantage that measurements of Thy-1 can be done on sections from the same series as is used for immunohistochemical localisation.
Animals↗
Fixation of Thy-1 in nervous tissue for immunohistochemistry: a quantitative assessment of the effect of different fixation conditions upon retention of antigenicity and the cross-linking of Thy-1.
It has proved difficult to obtain good immunohistochemical localization of cell surface antigens in nerve for a number of reasons, prominent among which are problems of fixing this class of molecule without destroying their antigenicity. In the course of developing a fixation procedure suitable for one such antigen. Thy-1, we have quantitatively assessed the effect of different fixation parameters upon the retention of Thy-1 antigenicity and upon the extent of cross-linking of the antigen in the tissue. The former was measured using radioimmunoassays adapted for membrane antigens in fixed tissue, the latter by measuring the proportion of antigen rendered insoluble to the detergent, sodium deoxycholate, and by examining the size of the antigen on sodium dodecyl sulfate--polyacrylamide gels. These approaches demonstrated that minor modifications of the standard vascular perfusion fixation of brain, using both glutaraldehyde and paraformaldehyde, were sufficient to fix the Thy-1 molecule, and at the same time substantially spare its antigenicity. In this study we measured Thy-1 using both a conventional rabbit antiserum and a mouse monoclonal antibody to the Thy-1.1 antigenic determinant. The multiple antigenic determinants recognized by the rabbit antibodies were cumulatively more resistant to fixation than the single antigenic determinant recognized by the monoclonal antibody.
Animals↗
Recombination kinetics following nanosecond laser photolysis of carbonmonoxyhaemogloblin.
The kinetics of the ultrafast ligand recombination following 347 nm laser photolysis of aqueous solutions of carbonmonoxyhaemogloblin have been investigated. The process is biphasic and the rate constants for the two processes as functions of temperature have been used to give activation energies of 6 +/- 3.9 kJ . mol-1 for the fast process and 31 +/- 4.8 kJ . mol-1 for the slow process. Frequency factors have also been calculated. The two processes are discussed in relation to both low-temperature studies and model calculations on the rate of entry of carbon monoxide into haem proteins.
Carbon Dioxide↗
Cooperative ligand binding to hemoglobin. Effects of temperature and pH on a hemoglobin with spectrophotometrically distinct chains (Tunnus thynnus).
Binding of CO to the hemoglobin of Tunnus thynnus has been studied by kinetic and static methods in the range pH 6.75-8.0 and at temperatures between 1.5-40 degrees C. The results may be described in terms of the two-state model of cooperativity (Monod. J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118) extended to include chain differences. The rate constants for CO binding to alpha and beta chains in the R state, at 20 degrees C, were 8.4 and 1.9 microM-1 s-1 with associated apparent activation energies of 3.5 and 10.5 kcal mol-1. Dissociation from the R state was monophasic with a rate constant, at 20 degrees C, of 0.016 s-1 and Ea of 26.4 kcal mol-1. The difference spectrum for binding of CO to the alpha chain in the T state is displaced 2 nm to the red as compared to the spectra associated with CO binding to the beta chain in the T state and to both chains in the R state. The proportion of T state hemoglobin present at different fractional saturations can thus be measured and compared with the results predicted from the model. When the temperature is raised, the value of L decreases greatly with an enthalpy of +80 kcal mol-1. The effects of the change in L override the decrease in intrinsic CO affinity of T. thynnus hemoglobin in the R and T states and result in a reverse temperature dependence of CO equilibria compared to mammalian hemoglobins.
Animals↗
Multiple T state conformations in a fish hemoglobin. Carbon monoxide binding to hemoglobin of Thunnus thynnus.
The blood of the Atlantic bluefin tuna (Thunnus thynnus) contains four hemoglobin components separable by chromatography on diethylaminoethylcellulose. These components are stable and functionally identical in their reactions with carbon monoxide. At low pH they remain in the T state even when liganded, and show two kinetic components in binding and in dissociation, with rates of 1) 1.2 microM-1 s-1 and 0.095 s-1 and 2) 0.013 microM-1 s-1 and 0.195 s-1, respectively, at pH 6, 20 degrees C, 0.1 m KPi. These components have difference spectra separated by more than 2 nm and are present in equal amounts. After CO has bound, there is a conformation change to an altered T state, in terms of the model of Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118), with a half-time of 65 s. At equilibrium, one-third of the slow kinetic component is changed into the new conformer, which binds CO at a rate of 0.14 microM-1 s-1.
Animals↗