Reconstitution of ciliary membranes containing tubulin.

Membranes from the gill cilia of the mollusc Aequipecten irradians may be solubilized readily with Nonidet P-40. When the detergent is removed from the solution by adsorption to polystyrene beads, the proteins of the extract remain soluble. However, when the solution is frozen and thawed, nearly all of the proteins reassociate to form membrane vesicles, recruiting lipids from the medium. The membranes equilibrate as a narrow band (d = 1.167 g/cm3) upon sucrose density gradient centrifugation. The lipid composition of reconstituted membranes (1:2 cholesterol:phospholipids) closely resembles that of the original extract, as does the protein content (45%). Ciliary calmodulin is the major extract protein that does not associate with the reconstituted membrane, even in the presence of 1 mM calcium ions, suggesting that it is a soluble matrix component. The major protein of reconstituted vesicles is membrane tubulin, shown previously to differ hydrophobically from axonemal tubulin. The tubulin is tightly associated with the membrane since extraction with 1 mM iodide or thiocyanate leaves a vesicle fraction whose protein composition and bouyant density are unchanged. Subjecting the detergent-free membrane extract to a freeze-thaw cycle in the presence of elasmobranch brain tubulin or forming membranes by warming the extract in the presence of polymerization-competent tubulin yields a membrane fraction with little incorporated brain tubulin. This suggests that ciliary membrane tubulin specifically associates with lipids, whereas brain tubulin preferentially forms microtubules.

Effects of tumbling and lateral diffusion on phosphatidylcholine model membrane 31P-NMR lineshapes.

Two factors determining the isotropic motional averaging of NMR spectra obtained from lipids in model and biological membranes systems are particle tumbling and lateral diffusion. The influence of these motions (of which the magnitudes are detemined by the medium viscosity and temperature) on the 31P-NMR spectra arising from unilamellar dioleoyl phosphatidylcholine vesicles of a defined size are examined. It is shown that the lineshapes obtained are in good agreement with those predicted by the theory of motional narrowing. These results are discussed with regard to order parameter determinations and polymorphic phase identifications as obtained by NMR techniques.

Sub-thylakoid fractions containing ribosomes.

A sub-membrane fraction which contains a large portion of any thylakoid-bound ribosomes can be obtained when thylakoids are treated with the detergents Nonidet P-40, or Triton X-100. These 'pseudopolysome' fractions contain 50% of thylakoid-bound ribosomes, but less than 0.5% of thylakoid chlorophyll. Triton and Nonidet psuedopolysomes contain about 10%, and 3% of thylakoid protein, respectively. Pseudopolysomes, prepared from thylakoids with low levels of ribosomes, contain about the same proportion of thylakoid protein but proportionately less ribosomes. Pseudopolysomes contain thylakoid polypeptides in addition to chlorophyll, but lack a major membrane polypeptide of Mr 50 000. Pseudopolysome chlorophyll, and RNA band at the same buoyant density. However, they band at different densities after pseudopolysomes are treated with trypsin (a procedure which strips thylakoids of ribosomes). Pseudopolysome fractions from thylakoids with low levels of ribosomes have a lower density than the corresponding fractions from thylakoids with high levels of ribosomes. Ribosomes are released from thylakoids, and pseudopolysomes by the same treatments. Subunits are released with KCl and puromycin. Polysomes are released with trypsin. It was concluded the pseudopolysomes consist of ribosomes and a membrane fragment containing the sites to which ribosomes are bound.

Localization of photosynthetic reaction centers by antibody binding to chromatophore membranes from Rhodopseudomonas spheroides strain R26.

Rabbit antiserum against highly purified reaction center preparations was shown to react specifically with a single component of chromatophore membranes from Rhodopseudomonas spheroides strain R-26. The conjugate of purified gamma globulin and ferritin prepared with toluene diisocyanate was used to determine the localization of reaction centers in the chromatophore membranes. Virtually no antibody was bound by intact membranes. After removing the 9nm ATPase from these membranes by dilute EDTA treatment, a considerable amount of antibody was bound to the exposed outer membrane surface. The reaction center binding sites were estimated to be uniformly distributed with approx. 1 reaction center per 200 nm-2 of membrane surface. These results indicate that the reaction centers are located near the outer membrane surface but below the ATPase particles. Since the distribution of reaction centers and particles on rough faces seen by freeze-fracture particle may be a complex of a reaction center and other electron transfer components localized within the hydrophobic region of the membrane.

Concanavalin A-reactive protein of rabbit thymocyte plasma membranes: analysis by crossed immune electrophoresis and sodium dodecylsulfate/polyacrylamide gel electrophoresis.

1. Thymocyte plasma membrane extracts, prepared with the non-ionic detergent Triton X-100, show 10 major protein components upon sodium dodecysulfate/polyacrylamide gel electrophoresis and at least 11 immunologic components upon crossed immune electrophoresis. 2. Concanavalin A reactive membrane proteins have been identified using crossed immune electrophoresis with receptor-ligand interaction. 3. These proteins are absorbed from Triton X-100-solubilized membranes onto immobilized concanavalin A. They are eluted in stepwise fashion, using increasing concentrations of alpha-methyl-d-glucoside, between 0.0004 M and 0.1 M. The predominant proteins eluted in each step are components with high electrophoretic mobility in crossed immune electrophoresis and are identical with a glycosylated component in sodium dodecysulfate/polyacrylamide gel electrophoresis with molecular weight of 55 000. 4. This component forms multimers in the presence of Triton X-100 which are not totally dissociated in sodium dodecylsulfate. 5. Neuramidase treatment followed by crossed immune electrophoresis of total plasma membrane isolates, as well as the purified glycoprotein fraction, indicates that the concanavalin A-reactive proteins are sialoglycoproteins. 6. Sodium dodecylsulfate component 5.1 comprises at least two different populations of glycoproteins (6 and 9) in crossed immune electrophoresis, one of which exclusively exhibits heterogenous carbohydrate antigenic sites (component 9). 7. Present data, taken together with previously published experiments, indicate that concanavalin A binding to intact thymocytes induces an increased turnover and release of the receptor protein(s).

Acyl composition and biosynthesis of acylated steryl glucosides in Calendula officinalis.

Fatty acids C12-C22 are components of acylated steryl glucosides in Calendula officinalis. Various particulate fractions from 14-day-old seedlings catalyze the esterification of the steryl glucosides with utilization of endogenous acyl donors. The activity seems to be associated mainly with the membranous structures being fragments of Golgi complex, as it has previously been suggested for UDPG: sterol glucosyltransferase. Succesive treatment of the particulate enzyme fraction with Triton X-100 and acetone affords a soluble acyltransferase preparation partly depleted of endogenous lipids. As a source of acyl groups for the synthesis of steryl acylglucosides this preparation utilizes various phospholipids obtained from the same plant in the following sequence: phosphatidylinositol greater than phosphatidylethanolamine greater than phosphatidylcholine. It does not utilize triacylglycerols and monogalactosyldiacylglycerols.

Limited proteolysis of the vasoactive intestinal peptide receptor: comparison of its folded structure in the membrane-bound and detergent-solubilized states.

Limited proteolysis was used to probe and compare the conformation of the rat lung vasoactive intestinal peptide (VIP) receptor in membrane-bound and detergent-solubilized states. It had been shown previously that the activity of the detergent-solubilized VIP receptor is sensitive to the nature of the detergent used for extraction (Patthi, S., Simerson S. and Velicelebi, G. (1988) J. Biol. Chem., 263, 19363-19369). Receptors that were extracted from the membrane using digitonin retained the ability to bind 125I-VIP, while those solubilized in Triton X-100 displayed little or no detectable activity. In order to correlate the differences observed in the activity of the receptor with its folded state, membrane-bound and detergent-solubilized receptors were covalently labeled with 125I-VIP and subjected to limited proteolysis using trypsin, chymotrypsin or carboxypeptidase Y. Digitonin-solubilized receptors most closely resembled the membrane-bound protein in terms of protease sensitivity and proteolytic cleavage products. By contrast, receptors solubilized in Triton X-100 displayed increased sensitivity to proteases and produced distinctly different proteolytic patterns. Thus, the differences observed in the activities of receptors solubilized in digitonin and those solubilized in Triton X-100 could be correlated with detectable differences in the conformation of the protein in each respective detergent solution. These results suggest that digitonin provides an environment that is more compatible with the native folded state of the receptor, similar to its conformation in the membrane.

Quantitative method for studying orientation of transition dipoles in membrane vesicles of spherical symmetry.

Pigmented vesicular membranes embedded in polyacrylamide gel exhibit linear dichroism when the gel sample is squeezed [Abdourakhmanov, I.A., Ganago, A.O., Erokhin, Yu.E., Solov'ev, A.A. and Chugunov, V.A. (1979) Biochim. Biophys. Acta 546, 183-186]. The orientation technique of gel-squeezing was modified to enhance polarization effects in membrane vesicles of spherical symmetry. Model calculations were carried out to provide a tool for the quantitative evaluation of the dichroism of squeezed gel samples. The orientation angles of the dipoles can be calculated with reasonable precision by measuring two quantities: (i) the macroscopic deformation parameter of the gel sample, and (ii) a parameter (e.g. the polarization ratio of the fluorescence emission) characterizing the orientation of the transition dipoles in the membranes embedded in the squeezed gel. The validity of the model was confirmed through a series of polarization measurements relating to the fluorescence of chlorophyll a in membranes of osmotically shocked chloroplasts, 'blebs'.

[The use of the cationic detergent dodecyltrimethylammonium bromide for the isolation of influenza virus glycoproteins with subsequent integration of the protein into liposome membrane]. / Ispol'zovanie kationnogo detergenta dodetsiltrimetilammonii bromida dlia vydeleniia glikoproteidov virusa grippa s posleduiushchim vstraivaniem belka v membranu liposom.

Glycoproteins were isolated from influenza virus with the use of cationic detergent dodecyltrimethylammoniumbromide and attached to preformed liposomes. Liposomal vesicles, thus, acquired their ability for hemagglutination and lysis of chicken erythrocytes. The possibility of using these liposomes for transfer of alien agents into eucaryotic cells is discussed.

[Free iron in intracellular membrane structures]. / Svobodnoe zhelezo vo vnutrikletochnykh membrannykh strukturakh.

Possibility of long-term existence of closed model membrane structures loaded with low molecular compounds of bivalent ferrum was shown. Liposomes prepared from phospholipids of egg yolk were used as a model. There were found similar effects of heating and detergents on the formation of nitrosyl complexes of non-heme iron (complexes 2.03) in the model system--liposomes loaded with ferrum on the one hand and mitochondria, hepatocytes and liver on the other. Heating and detergents intensify the formation of complexes 2.03, which is conditioned by iron liberation from the liposomes and closed intracellular membrane structures as a result of their destruction. A conclusion is drawn that free iron in animal liver cells is localized in the close membrane structures.