Peter B. Garland

Rotational mobility of the membrane-bound acetylcholine receptor of torpedo electric organ measured by phosphorescence depolarisation

The molecular organisation of the ACh receptor protein in its in situ state has been extensively studied in receptor-rich membranes prepared from fish electric organ, using several different techniques. A hexagonal lattice array of this receptor was deduced from X-ray diffraction data [ 1,2] and from some freeze-fracture [3,4] or negative staining [ 1,3,4] studies, but not some others [.5-71. Thus, in the freeze-etched, rotaryshadowed replicas obtained from fresh, rapidly-frozen Torpedo electrocytes [7], a close-packed lattice was not seen, but rather particles mainly in loosely-packed irregular double rows or random aggregates that might leave the possibility of some rotational mobility of the individual receptors. The method of saturation transfer EPR has also been applied to these membranes, to study the rotational mobility of the ACh receptor (reacted with a spin-labelled maleimide) [8]. The ACh receptor was reported thus to show no rotational motion [8] over a time-range of up to 1 ms. It was also deduced from other EPR studies [9,10] that some fatty acids and steroids can be immobilised near, or on [lo], the ACh receptor protein; in contrast, EPR has also indicated that the lipid phase around the receptors is fluid [lO,ll].

Biophysical properties of the surface lipid of parasitic nematodes

The biophysical properties of the surface lipid layer (the epicuticle) of living parasitic nematodes (Trichinella spiralis and Toxocara canis) were examined using fluorescent lipid analogues. A variety of such probes were screened, and only 5-N-(octadecanoyl)-aminofluorescein was found to insert into the outer lipid layer. Fluorescence quenching experiments showed that this probe was confined to the surface, and the rate of its lateral diffusion was then measured by Fluorescence Recovery After Photobleaching. This showed that the probe was not free to diffuse within the plane of the epicuticle. This structure is, therefore, extraordinary in its selectivity to lipid probes, and in the restricted lateral mobility of inserted lipid components.

Depolarization of fluorescence depletion: A microscopic method for measuring rotational diffusion of membrane proteins on the surface of a single cell

2.1. PrincipZes of method The use of triplet probes for the measurement of macromolecular rotations in the ps-ms time range has been reviewed by Cherry [ 11. The most general method of triplet detection is indirect, by spectrophotometric observation of ground state depletion. Most triplet probes are fluorescent, and in principle depletion of their ground state could as well be measured by a fluorescence depletion method, in which it is not the absorption of a measuring light beam that is measured, but instead the prompt fluorescence that such absorption excites. The signal of interest is then the depletion of fluorescence caused by ground state depletion. This fluorescence depletion is polarized when induced with a suitable bleaching flash, and becomes depolarized by rotational diffusion of the probe or the molecule to which it is attached. Hence we call the method ‘depolarization of fluorescence depletion’. It combines the long lifetime of triplet

Intracellular ph and free calcium changes in single cells using quene 1 and quin 2 probes and fluorescence microscopy.

Photometric fluorescence microscopy has been used to measure intracellular pH (pHi) and free calcium concentrations ([Ca]i) in individual mouse thymocytes and 2H3 rat basophil leukaemic cells containing indicators for pH (quene 1) or calcium (quin 2). The pHi and [Ca]i measurements in individual 2H3 cells and mouse thymocytes and their responses to various stimuli were consistent with the corresponding data obtained from suspensions of these cells measured in a spectrofluorimeter. Photometric fluorescence microscopy of these indicators in individual cells provides a sensitive and fast method of following pHi and [Ca]i responses in individual cells.

Phosphorescence depolarization and the measurement of rotational motion of proteins in membranes.

Rotational relaxation times of the order of many microseconds are typical of the presumably uniaxial rotation of membrane proteins [ 11. So far measurements of rotational relaxation times have been limited to those proteins which are present at high occupancy either in the orginal membrane, as with the band-3 proteins of erythrocyte ghosts [2], or in highly purified fragments of membrane such as the acetylcholine receptor protein of the electric organ of Torpedo mamorata [3], or in reconstituted membranes [4]. This restriction arises from the relative insensitivity of the two measurement methods usually used, either saturation transfer EPR spectroscopy [5] or the decay of linear dichroism following flash photolysis of an attached triplet-forming probe such as eosin [6]. We describe here a method for determining rotational relaxation times

Electron transport in aerobically grown Paracoccus denitrificans: Kinetic characterization of the membrane-bound cytochromes and the stoichiometry of respiration-driven proton translocation

1 ms) by measurement of the depolarization of laser flashinduced phosphorescence of erythrosin (tetraiodoflurorescein) attached to the protein of interest. This new method provides experimental realisation of many earlier suggestions [7,8]: it exceeds the sensitivity of the photodichroism method [6] by a factor of -lo’, and the saturation transfer method [S] by -104. We illustrate the use of our method by describing the slow isotropic rotation of proteins in viscous media, and the anisotropic rotation of Ca”dependent ATPase in sarcoplasmic reticulum membrane.

Changes in the organization of the surface membrane upon transformation of cercariae to schistosomula of the helminth parasite Schistosoma mansoni.

John and Whatley [l] have reviewed the information so far available about the functional organization of the membrane-bound components responsible for electron transport dependent ATP synthesis in aerobically grown Paracoccus denitnficans and drew attention to the similarities that exist between these components and those of the inner mitochondrial membrane. The object of the present work was to extend these studies and to characterize further the following properties of the plasma membrane of l? denitrificans: (a) the cytochrome components identifiable by low-temperature difference spectroscopy; (b) the fast oxidation-reduction kinetics of the membrane-bound cytochromes; and (c) the stoichiometry of respiration-driven proton translocation during the oxidation of different added substrates.

CHANGES IN THE PROPERTIES OF THE SURFACE MEMBRANE OF SCHISTOSOMA MANSONI DURING GROWTH AS MEASURED BY FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING

Merocyanin 540 (Mc540) is a fluorescent compound which is thought to bind to membranes in which there are substantial amounts of lipid in the lipid-crystalline phase. It is shown here to be of value in detecting the transformation by both mechanical and skin-penetration methods of the cercaria to the schistosomulum. The cercaria does not appear to bind Mc540, but the schistosomulum, binds Mc540 initially, in its anterior region, and at later times over the entire surface. The suggestion that transformation involves changes in the surface membrane lipid phase from gel to liquid-crystalline phase is supported by fluorescence recovery after photobleaching results with 5-N-(octadecanoyl)-amino fluorescein, a lipophilic dye which appears to be immobile in the cercaria, but fully mobile in the 40 min schistosomulum.

Identification of cholera toxin-binding sites in the nucleus of intestinal epithelial cells

Schistosomiasis is a widespread tropical disease of human beings. Infection occurs when a free-living, water-borne cercaria penetrates the skin, becomes transformed into a schistosomulum which then migrates through the blood stream of the host to develop into an adult parasite in the portal blood sys- tem [ 11. The surface membrane of both the schisto- somulum and adult worm is unusual; it is a double bilayer, and forms a continuous surface covering the peripheral syncytium [2]. The properties of this membrane change during growth and development in the host so that it becomes progressively less suscepti- ble to damage by a variety of agents [3-81. An under- standing of the nature of these changes is sought in the hope that immunological control or chemotherapy of the disease may be made more effective. The tech- nique of fluorescence recovery after photobleaching (FRAP) can measure the lateral diffusion of protein and lipid in the cell surface [9-131. We describe here its use in comparing the outer surfaces of the schisto- somulum and adult

Biochemistry for medical students: a flexible student-oriented approach.

Post‐embedding immunogold electron microscopy shows several binding sites for cholera toxin in mouse intestinal epithelial cells, particularly in the heterochromatin of the nucleus as well as in the plasma membrane. Anti‐ganglioside GM1 antibodies also bound to the nucleus, but did not interfere with the binding of toxin. 125I‐labelled toxin bound specifically to a nuclear preparation from rabbit intestinal cells.