Mass isolation of cell surface membrane fragments from pigeon heart.

Cell surface membrane fragments were isolated and purified by successive rate zonal and isopycnic centrifugation of calcium oxalate-loaded pigeon heart microsomes in sucrose density gradients. The most highly purified cell membrane fraction sediments at a buoyant density of 1.105 g/ml. Some of the membrane pieces are present as open fragments and leaky vesicles, while others form tightly sealed vesicles of both inside-in and inside-out membrane orientation. The pigeon heart cell membrane preparation exhibits high (Na+ + K+ + Mg2+)-ATPase and adenylate cyclase activities. Additional activity of these enzymes is uncovered by sodium dodecyl sulfate and alamethicin, respectively. Electron microscopic inspection of the cell surface membrane preparation revealed (a) a predominance of thick-walled vesicles with smooth surfaces on negative staining and (b) binding of concanavalin A to the bulk of isolated membrane pieces following their incubation with the lectin.     

Orientation of membrane fragments by electric field

Purple membrane fragments from Halobacterium halobium were oriented by a static electric field in a water suspension. It was found that an electric field of approx. 20 V/cm is sufficient to achieve practically complete orientation; the purple membranes have a permanent electric dipole moment of (6 ±1)· 10^?23 C · m, the orientation of the retinal transition moment relative to the direction of the electric dipole moment, θ, is (59 ± 1)⁰, and the purple membrane rotational diffusion constant D_rot = 0.65 s^?1. It was found that because of the electrophoretic movement of the particles a hydrodynamic velocity gradient builds up which also orients the purple membranes.     

The fractionation and partial characterization of membrane fragments derived from the electric organ of Electrophorus electricus

Two types of membrane particles, both binding α-[¹²⁵I]bungarotoxin, were obtained from electric tissue of Electrophorus electricus. They were both separated from acetyleholinesterase-containing particle by centrifugation in sucrose density gradients. The differing properties of the bungarotoxin-binding particles suggest that they may represent synaptic and extrasynaptic membrane structures containing acetylcholine receptors.     

Tetrodotoxin receptors in membrane fragments: purification from Electrophorus electricus electroplax and binding properties

A tetrodotoxin receptor-rich preparation of membrane fragments from the electric organ of Electrophorus electricus is described. The specific binding of neurotoxins and freeze-fracture electron microscopy are used as tools to identify and to characterize membrane fractions. Freeze-fracture electron micrographs of the electric organ demonstrate a high density of membrane particles in the extrasynaptic regions. Density gradient fractions show a broad distribution of [3H]tetrodotoxin, [3H]saxitoxin and 125I-labelled bungarotoxin binding in the range of 1.04--1.15 g/ml sucrose densities, with specific neurotoxin binding up to approx. 5 pmol/mg protein. Carrier-free column electrophoresis of density gradient fractions yields a subfraction with tetrodotoxin and alpha-neurotoxin binding up to 30 pmol/mg protein. The major part of the membrane fragments forms vesicles, which are separated by lectin chromatography into an outside-out and inside-out population. The latter represents at least 50% of the material of a density gradient fraction. For the association of tetrodotoxin, a bimolecular kinetic constant kf greater than or equal to 3.10(5) M-1.s-1 is determined. The dissociation constant is k'b = 2.5.10(-2)s-1. These data are in agreement with a thermodynamic dissociation constant of Kd = 20 nM as determined earlier for E. electricus membrane fragments by equilibrium methods (Grünhagen, H.H., Rack, M., St?mpfli, R., Fasold, H. and Reiter, P. (1981) Arch. Biochem. Biophys. 206, in the press). However, these association kinetics of tetrodotoxin binding in vitro are significantly different from kinetics determined electrophysiologically in Rana (Wagner, H.H. and Ulbricht, W. (1975) Pflügers Arch. 359, 297--315) or Xenopus (Schwarz, J.R., Ulbricht, W. and Wagner, H.H. (1973) J. Physiol. 233, 167--194).     

Membranes of Rhodopseudomonas sphaeroides. V. Identification of bacteriochlorophyll a-depleted cytoplasmic membrane in phototrophically grown cells

The separation of membrane fragments was investigated in extracts of phototropically grown Rhodopseudomonas sphaeroides to determine if the plasma membrane contains discrete regions. A highly purified fraction of bacteriochlorophyll a-deficient membrane fragments was isolated by differential centrifugation, chromatography on Sepharose 2B, reaggregation, and isopycnic sedimentation on sucrose gradients. Significant levels of b- and c-type cytochromes and succinate dehydrogenase were demonstrated in the isolated membrane fragments and their appearance in electron micrographs, their polypeptide profile in dodecyl sulfate-polyacrylamide gel electrophoresis, and overall chemical composition were essentially identical to a similar fraction isolated from aerobically grown cells. Their polypeptide profiles were distinct from those of the intracytoplasmic chromatophore and outer membranes, and on the basis of bacteriochlorophyll content the phototrophic fraction was contaminated with chromatophores by <9%. The membrane fragments contained no diaminopimelic acid or glucosamine. It is concluded that the membrane fragments isolated from phototrophically growing Rp. sphaeroides have arisen from photosynthetic pigment-depleted regions of the plasma membrane structurally and functionally differentiated from the intracytoplasmic chromatophore membrane. These regions represent conserved chemotrophic cytoplasmic membrane whose synthesis continues under photoheterotrophic conditions.     

Plasma membrane fragments in bovine and caprine skim milks

By either differential or linear gradient ultracentrifugation of bovine or caprine skim milks it was possible to obtain fractions which contained 45–75% of the lipid phosphorus and unesterified cholesterol of the skim milk. Electron microscopy of these fractions revealed the presence of numerous membrane-bound vesicles, microvilli and membrane fragments. Assay of the fractions for certain membrane-bound enzymes; i.e. 5′-nucleotide pyrophosphatase, alkaline phosphatase and ATPases, established the presence of all but the latter in the membrane-rich fractions. The distributions of the enzymes in the various fractions were correlated with their lipid phosphorus and cholesterol contents.Compositions of the phospholipids from skim milk membranes, milk fat globule membranes and the plasma membrane of the lactating mammary cell were observed to be similar and unique for having a relatively high level (20–25%) of sphingomyelin. By virtue of secretory processes, all of these membranes appear to be interrelated with each other and with Golgi vesicle membranes. It is concluded that the membrane material in the skim milk originates primarily from plasma membrane of the lactating cell. The possibiltiy that Golgi vesicle membranes form a substantial part of this material is not precluded by the results of this study.Separation of bovine skim milk on a Sepharose 4B gel column demonstrated that virtually all of the 5′-nucleotidase and lipid phosphorus are recovered together in the void volume of the column. Considering the particle size discriminating characteristics of this gel, the skim milk membrane material appears to be constituted of relatively large structures rather than of discrete subunits.     

Spectral changes in membrane fragments and artificial liposomes of Anacystis induced by chilling.

Isolated membrane fragments from Anacystis nidulans grown at 39 °C undergo visible spectral changes on chilling, suggesting a carotenoid component is altered. No such changes are seen when cells are grown at 25 °C. The magnitude of the decreased absorbance is a function of the chilling temperature and the media in which membrane fragments are suspended. The spectral decrease following chilling develops relatively slowly and is a function of the cooling rate and final temperature. The absorbance change is reversed if the fragments are heated to near 50 °C subsequent to chilling. Liposomes prepared from a total lipid extract of Anacystis undergo a spectral change on chilling which closely resembles that occurring in whole cells or isolated membrane fragments. Liposomes prepared from an extract of cells grown at 25 °C show only about 30% as great a spectral change as those from cells grown at 39 °C. The spectral bleaching is freely reversible when the liposomes are reheated, but shows a pronounced hysteresis. It is suggested that specific phase changes occur in Anacystis membranes and artificial liposomes on cooling which alter the environment of carotenoid. These changes may relate to previous observations that cells grown at 39 °C cannot survive a cold shock while those grown at 25 °C do.     

Mass isolation of cell surface membrane fragments from pigeon heart

Cell surface membrane fragments were isolated and purified by successive rate zonal and isopycnic centrifugation of calcium oxalate-loaded pigeon heart microsomes in sucrose density gradients. The most highly purified cell membrane fraction sediments at a buoyant density of 1.105 g/ml. Some of the membrane pieces are present as open fragments and leaky vesicles, while others form tightly sealed vesicles of both inside-in and inside-out membrane orientation. The pigeon heart cell membrane preparation exhibits high ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ and adenylate cyclase activities. Additional activity of these enzymes is uncovered by sodium dodecyl sulfate and alamethicin, respectively. Electron microscopic inspection of the cell surface membrane preparation revealed (a) a predominance of thick-walled vesicles with smooth surfaces on negative staining and (b) binding of concanavalin A to the bulk of isolated membrane pieces following their incubation with the lectin.     

Diurnal changes in maize leaf photosynthesis : I. Carbon exchange rate, assimilate export rate, and enzyme activities

Diurnal changes in photosynthetic parameters and enzyme activities were characterized in greenhouse grown maize plants (Zea mays L. cv Pioneer 3184). Rates of net photosynthesis and assimilate export were highest at midday, coincident with maximum irradiance. During the day, assimilate export accounted for about 80% of net carbon fixation, and the maximum export rate (35 milligrams CH₂O per square decimeter per hour) was substantially higher than the relatively constant rate maintained through the night (5 milligrams CH₂O per square decimeter per hour). Activities of sucrose phosphate synthase and NADP-malate dehydrogenase showed pronounced diurnal fluctuations; maximum enzyme activities were generally coincident with highest light intensity. Reciprocal light/dark transfers of plants throughout the diurnal cycle revealed that both enzymes were deactivated by 30 minutes of darkness during the day, and they could both be substantially activated by 30 minutes of illumination at night. During 24 hours of extended darkness, sucrose phosphate synthase activity declined progressively to an almost undetectable level, but was activated after 1.5 hours of illumination. Thus, the diurnal fluctuation in maize sucrose phosphate synthase can be explained by some form of light modulation of enzyme activity and is not due to an endogenous rhythm in activity. No diurnal fluctuations were observed in the activities of NADP-malic enzyme or fructose 6-phosphate-2-kinase. Phosphoenolpyruvate carboxylase was activated by light to some extent (about 50%) when activity was measured under suboptimal conditions in vitro. The results suggested that the rates of sucrose formation and assimilate export were closely aligned with the rate of carbon fixation and the activation state of sucrose phosphate synthase.     

Biochemical aspects of the visual process XXVIII. Classification of sulfhydryl groups in rhodopsin and other photoreceptor membrane proteins

Reaction of isolated bovine rod outer segment membrane with radioactiveN-ethylmaleimide, both in the presence and absence of 1% dodecyl sulfate followed by dodecyl sulfate-polyacrylamide gel electrophoresis, shows that six sulfhydryl groups (96% of total sulfhydryl in this membrane) are located on the rhodopsin molecule.On the basis of their reactivity towardsp-chloromercuribenzoate andp-chloromercuribenzene sulfonate in suspensions of outer segment membranes, the sulfhydryl groups of rhodopsin can be divided into three pairs. One pair is rapidly modified, both in light and darkness. This modification does not impair the recombination capacity of opsin with 11-cis retinaldehyde under regeneration of rhodopsin. A second pair is modified upon prolonged interaction with thep-chloromercuriderivatives in darkness. Modification of this pair leaves the typical rhodopsin absorbance at 500 nm intact, but a proportional loss of recombination capacity does occur. The third pair is only modified after illumination and is probably located in the vicinity of the chromophoric center.The difference between these results and those obtained by modification with dithiobis-(2-nitrobenzoic acid) orN-ethylmaleimide in suspension, where even upon prolonged exposure to light as well as in darkness only two sulfhydryl groups of rhodopsin are modified, is explained by the detergent-like character of thep-chloromercuri-derivatives.     

Effect of NADP+ on light-induced cytochrome changes in membrane fragments from a blue-green alga

The effect of NADP⁺ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragments prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP⁺ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH₂) to NADP⁺. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH₂ as the electron donor, NADP⁺ had no effect on the light-induced redox changes of cytochromes: with or without NADP⁺, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an α peak at 549 nm. With 664 nm illumination and water as the electron donor, NADP⁺ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b₅₅₉. Cytochrome b₅₅₉ appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP⁺) for the electron flow from water.     

Quantitation of peroxidase-antibody binding to membrane fragments using column chromatography.

A method which can be used to measure the amount of peroxidase antibody which is specifically bound to placental alkaline phosphatase in membrane fragments is described. The technique uses Sepharose 4B chromatography to separate membrane fragments containing bound peroxidase-antibody from unbound peroxidase-antibody. Specificity is demonstrated by nonbinding to rabbit placental membranes, by a strict correlation between membrane-associated phosphatase and bound peroxidase-antibody, and by preventing binding using pure enzyme. The general utility of this method for membrane antigens is discussed.     

Studies on the Hill reaction of membrane fragments of blue-green algae III. Fluorescence characteristics of membrane fragments of Anabaena variabilis and Anabaena cylindrica

Fluorescence spectra of the pigment system at –196°Cin membrane fragments of Anabaena variabilis and A. cylindricawere investigated. The fluorescence spectra of membrane fragments having four emissionbands at 645–655, 685, 695 and 725 nm were basically similarto those reported for intact cells of blue-green algae, thoughthe emission from phycocyanin (645–655 nm) was far strongerwith membrane fragments than with intact algal cells. Incubation of membrane fragments of A. variabilis in a dilutebuffer (10^–2M, pH 7.5) caused an increase in the 645 nmfluorescence and slight decreases in the 685 and 695 nm fluorescences,but had no influence on the 725 nm fluorescence. The decreasein the 685 and 695 nm fluorescences of A. cylindrica was moremarked and had the same kinetics as the inactivation of photosystemII reaction measured by DPIP-photoreduction. When membrane fragments of A. cylindrica were incubated in thebuffer solution at room temperature or in the presence of MgCl₂(10^–3M) at 0°C; phycobilin aggregates, which emittedthe 655 and 685 nm fluorescence, were solubilized. This solubilizationwas not observed with membrane fragments of A. variabilis. (Received August 31, 1972; )     

Separation of outer and inner membranes of mitochondria from the brown adipose tissue of infant rats.

Digitonin treatment and the swelling-shrinkage-sonication procedure as used to separate mitochondria membranes were applied to mitochondria from the brown adipose tissue (BAT) of infant rats. Digitonin at a concentration of 0.15 mg/mg mitochondrial protein produced disruption of the outer membrane of BAT mitochondria and a complete release of adenylate kinase. However, fragments of the outer membrane remained firmly attached to the inner membrane-matrix particles (mitoplasts) and sedimented at 10 000 g, as indicated by the activity of monoamine oxidase in the pellet. Only at 0.5 mg digitonin/mg protein did outer membrane become almost entirely separated. Oxidation of external cytochrome c by mitoplasts was only 50% of the total cytochrome oxidase at 0.5 mg digitonin/mg protein, indicating an incomplete exposure of the inner membrane to the external medium. Ultrastructural studies revealed that a large proportion of mitoplasts retained the orthodox configuration under these conditions. Outer membrane fragments obtained by the swelling-shrinkage-sonication procedure were of buoyant density corresponding to 20–30% (weight/vol) sucrose. After a 10 sec sonication of mitochondria, a relatively pure outer membrane fraction could be obtained with a yield not exceeding 20%. Longer sonication increased the yield, but also increased the degree of contamination by inner membrane fragments. Optimum conditions for the separation of outer and inner membranes from brown adipose tissue mitochondria are described.     

Light scattering from suspensions of membrane fragments derived from sonication of beef heart mitochondria.

The intensity of light scattering from suspensions of membrane fragments prepared by sonication of beef heart mitochondria in the presence of EDTA at alkaline pH (ESMP) was determined at 45, 90, and 135 degrees with light of wavelength 546 nm. The dissymmetry ratio Z = I45 degrees c/I135 degrees c, where I45 degrees c and I135 degrees c are the scattering intensities at 45 and 135 degrees extrapolated to zero particle concentration and corrected for reflectance effects, was used to calculate particle size from the Rayleigh-Gans-Debye theory. An average particle diameter D of 184-190 nm was obtained, within the range of particle diameter 50-300 nm determined previously by electron microscopy. This average diameter determined by light scattering is a useful parameter for characterization of ESMP particle size. We propose the term: light scattering average particle diameter, DLS, for this parameter. The refractive index of ESMP was determined to be 1.443 by measurement of scattering intensity in buffer solutions of varying sucrose concentration. The value of Z was independent of sucrose concentration in this determination, showing that the particles are osmotically inactive toward sucrose. The values of average particle diameter DLS and of refractive index fall within the range of validity of the Rayleigh-Gans-Debye theory, for which light scattering changes are attributable solely to dimension change, rather than to change in particle refractive index. Uptake of water accompanying energy-linked salt uptake in ESMP was calculated from light scattering changes to be 0.18 mul of H2O/mg of protein, compared with 0.49 mul of H2O/mg of protein measured by dextran inaccessibility. Measurement of light scattering changes provides a rapid and sensitive method for determining volume changes of ESMP. The magnitude of the volume change observed during energy-linked water and salt uptake and the initial degree of hydration suggests that ESMP are analogous to polyelectrolyte gels with regard to sorption of strong electrolytes and that the Donnan formulation for ion exchange equilibria may be usefully applied to these processes in ESMP.     

ATP-dependent proton-pumping activities of zucchini fruit microsomes. A study of tonoplast and plasma membrane activities

The mesocarp tissue of zucchini (Cucurbita pepo L. cv. Black Beauty, zucchini) fruit exhibits ATP-dependent H⁺-pumping activities associated with tonoplast (nitrate-sensitive) and plasma membrane (vanadate-sensitive) vesicles. The two activities are easily separated on step gradients with isopycnic densities lower than usually reported (< 20% (w/w) sucrose for tonoplast; 25–35% (w/w) sucrose for plasma membrane). The tonoplast is relatively impermeable to H⁺ (the half-time for equilibration of a pH gradient is 23–36 min) compared to plasma membrane (half-time of 4–6 min). Anion permeability was measured by adding ATP in the absence of an accompanying K⁺ salt, then measuring the increase in the pH gradient caused by the addition of a K⁺ salt. The increase in the pH gradient is presumably due to alleviation of the Δψ component (positive inside) and consequent increase in the Δ pH component (acid inside) of the electrochemical gradient by movement of the anion into the vesicle interior. Cl⁻ and NO₃⁻ are permeable, SO₄²⁻ is not. The anion permeabilities of the tonoplast and plasma membrane were similar. This is inconsistent with the marked difference in the H⁺ permeabilities, but might be explained by the presence of anion channel(s) associated with tonoplast-derived vesicles.     

Characterization of solute transport in plasma membrane vesicles isolated from cotyledons ofRicinus communis L.

Evidence is presented for the proton-coupled transport of sucrose and glutamine in purified plasma membrane vesicles isolated from cotyledons ofRicinus communis. Imposition of a pH gradient (internal alkaline) across the plasma membrane resulted in a rapid uptake of sucrose and glutamine which was inhibited in the presence of carbonyl cyanide-m-chlorophenyl hydrazone. Imposition of a pH gradient plus an internal negative membrane potential stimulated uptake further. Glucose and fructose uptakes were negligible under these conditions. Sucrose uptake into the vesicles demonstrated saturation kinetics with a K_m of 0.87 mol·m^-3, indicating carrier-mediated transport. In support of this, uptake was very sensitive to the protein-modifying reagentp-chloromercuribenzenesulphonic acid. N-Ethylmaleimide, another sulphydryl reagent, was only slightly inhibitory. However, both reagents strongly inhibited sucrose uptake into intact cotyledons; the possible reasons for the difference between the intact and isolated systems are assessed. The value of this system for the study of sucrose and amino acid carriers is discussed.     

Phytochrome mediated regulation of sucrose phosphate synthase activity in maize

The extractable activity of sucrose phosphate synthase was determined in etiolated seedlings of maize (Zea mays L.), soybean (Glycine max [L.] Merr.), and sugar beet (Beta vulgaris L.) following treatments of changing light quality. A 30-minute illumination of 30 microeinsteins per square meter per second white light produced a three-fold increase in sucrose phosphate synthase activity at 2 hours postillumination when compared to seedlings maintained in total darkness. Etiolated maize seedlings treated with 3.6 microeinsteins per square meter per second of red and far-red light showed a 50% increase and a 50% decrease in sucrose phosphate synthase activity, respectively, when compared to etiolated maize seedlings treated with white light. Maize seedlings exposed for 30 minutes to red followed by 30 minutes to far-red showed an initial increase in sucrose phosphate synthase activity followed by a rapid decrease to control level. Neither soybean or sugar beet sucrose phosphate synthase responded to the 30-minute illumination of white light. Phytochrome is involved in sucrose phosphate synthase regulation in maize, whereas it is not responsible for changes in sucrose phosphate synthase activity in soybean or sugar beet.     

Preferential release of new outer membrane fragments by exponentially growing Escherichia coli

We have examined whether the outer membrane fragments released by normally growing Escherichia coli contain relatively old or new outer membrane.Double-label experiments show that after incorporation of radioactive leucine into E. coli protein, there is a preferential release of outer membrane material which contains a high percentage of newly labeled protein. This implies that outer membrane fragments are preferentially released from those regions where newly synthesized proteins are inserted into the outer membrane. We estimate that these insertion regions cover no more than 13% of the total outer membrane, and that newly inserted proteins diffuse in the plane of the outer membrane with a diffusion constant ? 5 · 10^?13 cm²/s.