The macrostructure of mucus glycoproteins in solution.
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Amino Acid Sequence↗
Biomedical subjects
S E Harding
Publications and source records attributed to S E Harding.
On the interaction in solution of a candidate mucoadhesive polymer, diethylaminoethyl-dextran, with pig gastric mucus glycoprotein.
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Animals↗
Contractile responses of isolated adult rat and rabbit cardiac myocytes to isoproterenol and calcium.
Myocytes were isolated by Langendorff perfusion of rat or rabbit hearts with low calcium solution followed by collagenase and hyaluronidase, or by incubation of chunks of rat ventricular tissue in similar media. Cells were then placed in a bath on a microscope stage, superfused and electrically stimulated. Contraction amplitude and rate of change of length during contraction were measured using a video camera and edge detection monitor. Cells were selected for study using a number of criteria developed to identify and define a cell population able to give consistent inotropic responses over a long period. The maximum contraction amplitude with isoproterenol in rabbit cells was 0.244 micron (sarcomere length change) or 13.1% (percentage change in cell length), and the EC50 was 12.8 nM. The maximum contraction amplitude with isoproterenol did not differ significantly between rat and rabbit, between cells prepared by perfusion and those made from chunks, or when determined from non-cumulative rather than cumulative curves. The EC50 for isoproterenol in rat cells made by the perfusion method (cumulative curves) was 3.81 nM, significantly lower than in rabbit. The maximum amplitude obtained with increasing concentrations of calcium was not significantly different from that with isoproterenol under any condition. The EC50 for calcium averaged 2.78 mM in rat cells made by the perfusion method (cumulative curves) and was significantly greater than that in rabbit (1.4 mM). Maximum rates of contraction for rat cells averaged 4.59 micron/s in 8 mM calcium. Rat cells contracted faster than they relaxed, whereas rabbit cells in 8 mM calcium relaxed faster than they contracted. Rat cells, maximally activated by either calcium or isoproterenol, contracted significantly faster than rabbit. There was no difference in rates of contraction (or relaxation) between rat cells prepared by perfusion and those made from chunks of tissue.
Animals↗
Pertussis toxin reduces the antiadrenergic effect of 2-chloroadenosine on papillary muscle and the direct negative inotropic effect of 2-chloroadenosine on atrium.
2-Chloroadenosine reduced the contractile tension of guinea-pig atria directly, and inhibited the increase in tension produced by beta-adrenergic stimulation of guinea-pig papillary muscle. Both effects were reduced by 8-phenyltheophylline, a competitive antagonist at extracellular P1-purinoceptors. Treatment of guinea-pigs with pertussis toxin reduced the sensitivity of both atria and ventricles to 2-chloroadenosine. Atria were significantly affected after treatment with 125 micrograms/kg toxin, but not 100 micrograms/kg. 60 micrograms/kg toxin had no effect on the sensitivity of the ventricles, but 100 and 125 micrograms/kg significantly decreased the antiadrenergic effect of 2-chloroadenosine. We conclude that both the direct and antiadrenergic effects are mediated by an inhibitory guanine nucleotide binding protein.
2-Chloroadenosine↗
Analysis of the molecular size of tomato (Lycopersicon esculentum Mill) fruit polyuronides by gel filtration and low-speed sedimentation equilibrium.
The cell-wall structures of tomato (Lycopersicon esculentum Mill) and other fruit are intimately linked with the nature of their polyuronides. Cell-wall polyuronides from unripe and ripe tomato fruit were isolated and purified and their molecular size and molecular-size distributions were compared. It was demonstrated that there is a considerable decrease in the weight-average Mr upon ripening (from 160,000 +/- 10,000 to 96,000 +/- 4000) and a corresponding increase in polydispersity, particularly at the low-Mr end of the distribution. The estimates of polyuronide molecular size and molecular-size distribution were obtained without the need for polyuronide standards of known Mr by using gel-filtration chromatography combined with the absolute method of low-speed sedimentation equilibrium.
Centrifugation, Density Gradient↗
An antiadrenergic effect of adenosine on guinea-pig but not rabbit ventricles.
The antiadrenergic effect of adenosine was investigated using isolated guinea-pig heart and guinea-pig and rabbit papillary muscle. Adenosine, 15 microM, completely abolished the increased tension stimulated by 0.1-1.0 nM isoprenaline in Langendorff-perfused guinea-pig hearts. With guinea-pig papillary muscles, adenosine decreased by 40% the increased force stimulated by 1-10 nM isoprenaline. When 5 microM 2-chloroadenosine was used in conjunction with 1 unit ml-1 adenosine deaminase, a complete inhibition of the isoprenaline-stimulated tension was seen in guinea-pig papillary muscles. The antiadrenergic effect of 2-chloroadenosine was blocked by 8-phenyltheophylline. In rabbit, there was little effect of 2-chloroadenosine (plus deaminase) on isoprenaline-stimulated tension. (-)-N6 (R-phenylisopropyl)-adenosine (PIA) had no effect on basal or isoprenaline-stimulated adenylate cyclase activity of guinea-pig or rabbit sarcolemmal membranes. We conclude that the antiadrenergic effect of adenosine is mediated by A type receptors and is seen in guinea-pig but not rabbit. Production of adenosine by superfused papillary muscle may obscure the effect of added adenosine. We find no evidence that the antiadrenergic effect is mediated by inhibition of adenylate cyclase.
2-Chloroadenosine↗
Contamination of a cardiac sarcolemmal preparation with endothelial plasma membrane.
Preparation of sarcolemma from whole rabbit heart using the method of Jones et al. (Jones,L.R., Besch, H.R., Fleming, J.W., McConnaughey, M.M. and Watanabe, A.M. (1979) J. Biol. Chem. 254, 530-539) results in a 46-fold purification of the endothelial plasmalemma-specific marker angiotensin converting enzyme. This implies contamination of the sarcolemma with vascular endothelial plasmalemma. During preparation of sarcolemma from sheep heart, using the same method, angiotensin converting enzyme copurified with the general plasma membrane marker (Na+ + K+)-ATPase. The ratio of myocyte to endothelial plasma membrane in the final preparation is therefore similar to that in the whole heart homogenate. Ultrastructural analysis has shown that the myocyte/endothelial surface area is 70:30 in whole cardiac muscle. Comparison of angiotensin converting enzyme activity of an endothelial plasma membrane fraction with that of whole heart sarcolemma suggests an upper limit of 42% for endothelial contamination. Contamination by endothelial plasmalemma was dramatically reduced by preparing sarcolemma from myocytes produced by proteolytic disruption of whole hearts. Following disruption, myocytes were separated from non-muscle cells by sedimentation through 0.5 M sucrose. Sarcolemma prepared from sheep cardiac myocytes had approximately 15-fold less angiotensin converting enzyme activity than whole sheep heart sarcolemma but comparable ouabain-inhibitable (Na+ + K+)-ATPase activity.
Animals↗
GDP activates rabbit heart adenylate cyclase, but does not support stimulation by isoproterenol: a re-appraisal of the control mechanism.
The effect of GDP on rabbit heart adenylate cyclase has been determined under conditions where only 0.08% to 0.26% of an added 100 microM was converted to GTP in the course of the assay. At concentrations of 100 microM, GDP stimulated basal cyclase activity to the same extent as GTP and guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). Isoproterenol increased activity in the presence of GTP or guanylyl-imidodiphosphate (Gpp(NH)p), but not in the presence of GDP or GDP beta S. It is suggested that the hydrolysis of GTP to GDP is the "turn-off" mechanism for beta-receptor stimulation of cardiac adenylate cyclase, but not for stimulation by GTP alone. The effects of GDP and GDP beta S are readily removed by washing, implying that their binding to Ns (the guanine nucleotide binding protein) is weak. GDP beta S initially competes with Gpp(NH)p, reducing Gpp(NH)p-stimulated activity. As stimulation of cyclase activity by Gpp(NH)p develops, in the course of 30 min, Gpp(NH)p becomes no longer displaceable by GDP beta S. Isoproterenol does not release 3H-Gpp(NH)p or reduce Gpp(NH)p-stimulated activity, once the nucleotide has become tightly bound. Nor does isoproterenol change the relative affinities of GDP beta S and Gpp(NH)p when these analogs are given together. There is, therefore, no evidence that isoproterenol acts by releasing tightly bound GDP from Ns, or that it 'unlocks' the guanine nucleotide binding site in the myocardial sarcolemma. In this, the cardiac adenylate cyclase system differs from the avian erythrocyte system. The action of isoproterenol is best explained by an increased dissociation of alpha(GTP) and beta,gamma-subunits of the Ns protein.
Adenylyl Cyclases↗
The molecular actions of beta-agonists in the cardiac sarcolemma.
Although alpha-receptors are known to exist in the myocardium, the predominant effect of catecholamines on the force of contraction of the heart is mediated through beta-receptors. This review deals only with the latter, and is particularly concerned with the sarcolemmal molecular mechanisms by which beta-stimulation increases the rate of cyclic adenosine monophosphate (cAMP) production by adenylate cyclase.
Adenylyl Cyclases↗
Applications of light scattering in microbiology.
Applications of the three principal light scattering techniques of turbidimetry, differential light scattering, and quasi-elastic light scattering to systems of microorganisms are reviewed. The relation between the three techniques is demonstrated and it is shown how these techniques can yield basic structural, optical, and even hydrodynamic properties for a wide range of microorganisms, with particular emphasis on changes in such properties. Such applications include antibiotic susceptibility testing, the effects of inhibitors on trypanosome motility, spore structure, virus self-assembly, and bacterial motility on the surface of fermentation reactors.
Animals↗
The concentration-dependence of macromolecular parameters.
Theories concerning the concentration-dependence of sedimentation and diffusion coefficients for macro-molecules in dilute solution are compared and discussed, together with their experimental basis. An attempt has been made to clarify an important uncertainty still present in the literature as to whether sedimentation coefficients should be corrected for solvent or solution density. It is pointed out that the two processes yield the same extrapolation limit but different concentration-dependencies, which have, however, been related. A general expression is derived thermodynamically for the concentration-dependence of diffusion that includes the coefficient of the concentration term involved in sedimentation (on the basis of sedimentation coefficients corrected from solution density). For rigid spherical particles the expression is shown to be exactly equivalent to one given by Batchelor [(1976) J. Fluid Mech. 74, 1-29], which was derived on the basis of sedimentation coefficients corrected from solvent density. Finally, we discuss the concentration-dependence of apparent weight-average relative molecular masses ('molecular weights') (from, e.g., sedimentation equilibrium) and note an important omission in some earlier representations.
Centrifugation↗
Physicochemical studies on turnip-yellow-mosaic virus. Homogeneity, relative molecular masses, hydrodynamic radii and concentration-dependence of parameters in non-dissociating solvents.
Turnip-yellow-mosaic virus, with its stable, highly spherical and monodisperse character, was chosen as a suitable model substance with which to test hydrodynamic theories of transport. Sedimentation coefficients, diffusion coefficients (obtained through photon correlation spectroscopy) and viscosities were measured accurately as a function of concentration in well-defined and nearly neutral buffer systems. Ancillary information was also obtained from very-low-speed sedimentation-equilibrium experiments. The coefficients expressing the variation in sedimentation and diffusion coefficients with weight concentration were obtained, and by combination with other data it was possible to avoid assumptions concerning solvation and transform such regression coefficients into the form appropriate to volume fractions. Some measure of support for Batchelor's [(1972) J. Fluid Mech. 52, 245-268] calculations was thus obtained, but over most of the pH range the coefficients were significantly smaller than those calculated from his theory. It seems likely that electrostatic interactions are responsible for the discrepancies. Hydrodynamic radii (from diffusion coefficients) were in very fair agreement with those calculated from the thermodynamic excluded-volume term, but were higher than indicated by electron microscopy and X-ray diffraction, a discrepancy ascribable to solvation.
Chemical Phenomena↗
The representation of equilibrium solute distributions for nonideal polydisperse systems in the analytical ultracentrifuge. Application to mucus glycoproteins.
It is relatively easy to represent by computer simulation the observed Rayleigh equilibrium fringe data for systems that are both associative and nonideal in the thermodynamic sense, and to extract the determinant parameters (see, for example, Roark, D., and D. A. Yphantis, 1969, Ann. NY Acad. Sci., 164:245-278; and Johnson M. L., J. J. Correia, D. A. Yphantis, and H. R. Halvorson, 1981, Biophys. J., 36:575-588). It is, however, considerably more difficult to represent systems that are both polydisperse (namely, those that consist of noninteracting species of different molecular weight) and nonideal, although the ideal case has been well described (see, for example, Tindall, S. H., and K. C. Aune, 1982, Anal. Biochem. 120:71-84). Here we show that the representation of nonideal polydisperse systems is now possible, after certain assumptions, by using a two-part interdependent minimization routine that uses readily available numerical packages. The method is applied to a well-characterized mucus glycoprotein (Mr approximately 2 X 10(6)) from the bronchial secretion of a cystic fibrosis patient. An excellent fit to the observed fringe data is obtained for a polydisperse three-component system, with a value for the second virial coefficient, B, of 0.57 ml mol g-2.
Biophysical Phenomena↗
Inhibition of rabbit cardiac adenylate cyclase by theophylline.
Theophylline inhibits basal adenylate cyclase activity as well as cyclase stimulated by sodium chloride, sodium fluoride, GTP or 5'-guanylimidodiphosphate. This inhibition, is dose-dependent and shows non-competitive inhibition, with respect to MgATP. The presence of adenosine deaminase does not alter the effect of theophylline. The inhibition produced by theophylline is not additive with that due to 2'-deoxyadenosine 3'-monophosphate (a P-site agonist). It is suggested that theophylline may act at the P-site to reduce adenylate cyclase activity.
Adenosine Deaminase↗
Quasi-elastic light scattering studies on dormant and germinating Bacillus subtilis spores.
Spores of Bacillus subtilis in suspension, both dormant and germinating, have been examined by light-scattering methods, both integrated intensity and correlation versions. When intensity of scatter at constant volume was plotted against angle, curves possessing a maximum at about 20 degrees were regularly obtained but without any noticeable features at higher angles. This indicated polydispersity and/or asymmetry among the population. Curves of the intensity correlation function for both dormant and germinating spores at different angles, theta, did not superimpose at the lower angles when plotted appropriately, but did so for theta greater than 35 degrees. This was considered to arise from asymmetry of the spores. By using the high-angle data the apparent diffusion coefficient was determined for both dormant spores and for germinating spores from 1 min after germinant addition. No appreciable difference was observed, from which volume changes greater than 6% during germination could be excluded. The occurrence of germination was confirmed by both absorbance and phase-contrast-microscopy observations.
Alanine↗
An analysis of the heterogeneity of mucins. No evidence for a self-association.
There are two possible contributions to the heterogeneity of mucus glycoproteins as observed in the analytical ultracentrifuge: firstly, from associative phenomena, and, secondly, from a polydisperse distribution of non-interacting species of different molecular masses. It is shown from the non-superposability of plots of point-weight-average relative molecular masses against concentration for differing initial cell-loading concentrations that polydispersity must be significant. It is further shown, by attempting to block any associative phenomena by competitive inhibition of potential sites for hydrophobic inter-particle interaction, that the observed heterogeneity is primarily a result of polydispersity and not a self-association.
Chemical Phenomena↗
The mode of action of chloride on rabbit heart adenylate cyclase.
The mechanism by which chloride stimulates adenylate cyclase was investigated. Depletion of GDP increased basal adenylate cyclase activity and reduced the stimulation by isoprenaline. Restoration of bound GDP partially reversed these effects. Chloride stimulated cyclase activity by the same proportion in control, GDP-depleted and GDP-restored preparations, as did Gpp(NH)p. Fluoride increased adenylate cyclase activity to the same final level in both GDP-depleted and GDP-restored membranes; addition of Gpp(NH)p as well as fluoride had no further effect. Solubilisation of adenylate cyclase reduced the stimulatory effect of Gpp(NH)p only slightly, but greatly attenuated the activation by chloride. We conclude that chloride does not stimulate cyclase activity by an action on GDP exchange. Activation by chloride may be due to a disrupting or chaotropic effect on membrane/protein interactions.
Adenylyl Cyclases↗
The effect of anions and cations on cardiac adenylate cyclase: interactions with isoprenaline and GTP.
Anions stimulated rabbit heart adenylate cyclase in the order chloride greater than phosphate greater than acetate, while the cations sodium and potassium had little effect. Activation by KCI was multiplicative with that of fluoride. Isoprenaline (10(-4) M) and GTP(10(-4) M) stimulated the cyclase independently, and their combined effects were multiplicative. GTP stimulated less than chloride but more than acetate. Activation by chloride or acetate was more than additive with that by GTP. The effect of isoprenaline was reduced in the presence of chloride or acetate, and this reduction was not modified by sodium or potassium. It was concluded that ionic influence may be biologically important in modifying cyclase activity and the extent of beta-receptor stimulation.
Acetates↗