Characterization of ferric reducing activity in roots of Fe-deficient Phaseolus vulgaris

Light-induced absorbance changes [LIAC; measured as Δ( A ₄₂₈– A ₄₁₀)] reflecting the reduction of a b -type cytochrome and mediated by an endogenous blue light absorbing receptor have been proposed to be related to blue light physiology of fungi and higher plants. It has also been suggested that the same cytochrome specifically can be reduced by red light in the presence of methylene blue. We have investigated the distribution of LIAC between different membrane fractions from corn ( Zea mays L.) coleoptiles and cauliflower ( Brassica oleracea L.) inflorescences. The membrane fractions were obtained by differential centrifugation followed by partition in an aqueous polymer two-phase system. By this procedure fractions rich in plasma membrane were obtained from both mitochondrial and microsomal fractions obtained by centrifugation. LIAC was by far most enriched in fractions also enriched in plasma membranes (identified by silicotungstic acid staining), but LIAC could be obtained also in other fractions. Our conclusion is that LIAC undoubtedly is caused by a b -cytochrome bound to the plasma membrane, but that LIAC also may be due to other b -cytochromes, one of which is probably located in the endoplasmic reticulum. Thus, the two assay procedures used for LIAC (blue and red light induced) could not disciminate between different b -cytochromes giving rise to LIAC.     

Plasma membranes from oats prepared by partition in an aqueous polymer two-phase system : on the use of light-induced cytochrome B reduction as a marker for the plasma membrane

Presumptive plasma membrane fractions have been prepared from oat (Avena sativa L. cv. Brighton) roots and shoots, respectively, by partition of microsomal fractions in a dextran-polyethylene glycol two-phase system. The plasma membranes had a high affinity for the polyethylene glycol-rich upper phase, whereas membranes from mitochondria and other organelles partitioned in the dextran-rich lower phase or at the interface. Thus, relatively pure plasma membranes were obtained by only two partition steps, and within 3 hours from homogenization of the material.

The plasma membranes from both organs were enriched in K⁺-stimulated Mg²⁺-dependent ATPase and glucan synthetase II, two tentative markers for the plant plasma membrane. Silicotungstic acid, an indicative stain for the plasma membrane, stained the vesicles recovered from the upper phase, but failed to stain the membranes partitioning in the lower phase or at the interface.

The plasma membranes were also enriched in a light-reducible b-cytochrome. This b-cytochrome can be measured by its light-induced absorbance change and may serve as a marker for the plant plasma membrane.

     

Preparation of mammalian plasma membranes by aqueous two-phase partition

Plasma membranes are readily purified from crude mixtures by the technique of aqueous two-phase partition. This procedure has been used widely to prepare plasma membrane fractions, highly purified, from both green and dark-grown plant materials. Only recently, however, has the method been applied to animal cells and tissues to supplant previous protocols where preparative sucrose and other gradient procedures were employed. The method based on aqueous two-phase partition, is rapid, reproducible and facile. It is especially useful for tissue culture cells since gradient methods often are complicated by alterations in plasma membrane density with different culture conditions and the presence of extensive cytoskeleton-membrane interactions. Homogenates prepared either in dilute 1 mM bicarbonate or isotonic sucrose are first centrifuged to concentrate the plasma membrane vesicles. The concentrated membranes are then combined with a mixture of dextran and polyethylene glycol that will of itself spontaneously separate into a polyethylene glycol-rich upper phase and a dextran-rich lower phase. The mixture is usually centrifuged to accelerate phase separation. The plasma membranes enter the upper, polyethylene glycol-rich phase, whereas contaminating membranes remain with the dextran of the lower phase. The yield of plasma membranes is 20% or more of those present in homogenates and the fraction purity is 90% or greater.     

The Isolation of Plasma Membrane from Vicia faba Leaves and Pollen Tubes of Luffs cylindrica by the Two-Polymer Phase System

A method for the isolation of plasma membrane enriched fraction from plant sources is described. An aqueous two-phase system is suitable for the isolation of plasma membrane. Purified plasma membrane fractions have been prepared from Vicia faba leaves and pollen tubes of Luffa cylindrica. The determination of marker enzymes shows that the plasma membrane has a high affinity for the polyethylene glycol-rich upper phase, whereas those membranes from mitochondria, chloroplasts and other organelles prefer the dextran-rich lower phase and the interface. The plasma membrane was identified with the aid of silicotungstic acid-chromic acid staining. In the upper phase there are mainly stained elliptical vesicles, whereas in the lower phase there are many differnet kind of unstained vesicles. This result is coincident with the marker enzyme examination. The two-phase method for the isolation of plasma membrane may be widely applied not only to nongreen tissues but also to green tissues of plants.     

Application of phase partition method for the fractionation of hydrophobic membrane proteins]

Phase partition method in a two-phase polyethylene glycol-dextrane system has been applied to fractionation in Triton X-100 of hydrophobic membrane proteins from Micrococcus lysodeikticus. This method allowed to separate the cytochrome b556 from other cytochromes. Spectral and gel electrophoretic patterns of isolated cytochrome are given.     

Isolation of plasma membrane fractions from green wheat leaves by free-flow electrophoresis or two-phase partition alone or in combination

Plasma membrane vesicles were isolated from shoots of light-grown wheat seedlings by preparative free-flow electrophoresis, aqueous polymer two-phase partition or both. Plasma membrane vesicles were identified from staining of thin sections prepared for electron microscopy with phosphotungstic acid at low pH. The orientation of the plasma membrane vesicles was determined from latency and trypsin sensitivity of K⁺ Mg²⁺ATPase and of glucan synthase II, and concanavalin A-peroxidase binding and membrane asymmetry visualized by electron microscopy. The K⁺Mg²⁺ATPase and of glucan synthase II activities of plasma membrane fractions isolated by two-phase partition were latent and trypsin resistant. The vesicles bound concanavalin A-peroxidase strongly and exhibited a cytoplasmic side-in morphology. These fractions of cytoplasmic side-in vesicles were less than 10% contaminated by cytoplasmic side-out vesicles. By free-flow electrophoresis, two populations of vesicles which stained with phosphotungstic acid at low pH, designated D and E, were obtained. The vesicle population with the lower electrophoretic mobility, fraction E, contained plasma membrane vesicles with properties similar to those of the plasma membrane vesicles obtained after two-phase partition. The phosphotungstic-reactive vesicles with greater electrophoretic mobility, fraction D, were concanavalin A unreactive with the cytoplasmic membrane leaflet outwards. Less than 50% of the K⁺Mg²⁺-ATPase activity of this fraction was latent and trypsin sensitive. The vesicles of fraction D appeared to be preferentially cytoplasmic side-out. The electrophoretic mobilities of cytoplasmic side-out (non-latent glucan synthase II activity) and cytoplasmic side-in (latent glncan synthase II activity) plasma membrane vesicles isolated from a frozen and thawed wheat plasma membrane fraction, corresponded with the mobilities of fraction D and E, respectively, again showing that the plasma membrane vesicles with the lesser electrophoretic mobility were cytoplasmic side-in. The cytoplasmic side-in and cytoplasmic side-out vesicles therefore showed opposite eletrophoretic mobilities compared with a previous free-flow electrophoretic separation of soybean plasma membranes. The majorities of the plasma membrane vesicles of both fractions D and E entered the upper phase upon two-phase partition with the phase composition used for purification of wheat plasma membranes. Thus, neither electrophoretic mobility nor phase partitioning characteristics can be used as the only criteria for assignment of vesicle orientation.     

Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: an anomalous effect of ficoll

An investigation was conducted into the isolation of plasma membrane vesicles from primary roots of corn (Zea mays L., WF9 × M14) by sucrose density gradient centrifugation. Identification of plasma membranes in cell fractions was by specific staining with the periodic-chromic-phosphotungstic acid procedure. Plasma membrane vesicles were rich in K⁺-stimulated ATPase activity at pH 6.5, and equilibrated in linear gradients of sucrose at a peak density of about 1.165 g/cc. It was necessary to remove mitochondria (equilibrium density of 1.18 g/cc) from the homogenate before density gradient centrifugation to minimize mitochondrial contamination of the plasma membrane fraction. Endoplasmic reticulum (NADH-cytochrome c reductase) and Golgi apparatus (latent IDPase) had equilibrium densities in sucrose of about 1.10 g/cc and 1.12 to 1.15 g/cc, respectively. A correlation (r = 0.975) was observed between K⁺-stimulated ATPase activity at pH 6.5 and the content of plasma membranes in various cell fractions. ATPase activity at pH 9 and cytochrome c oxidase activity were also correlated.     

The effect of detergent treatment on methylene blue sensitized cytochrome b photoreduction in fractions from corn coleoptiles

It was previously demonstrated that photoexcited methylene blue can act as electron donor in red light induced reduction of a particulate b-type cytochrome in fraction from etiolated corn coleoptiles (Zea mays L. WF 9 × Bear 38). It was postulated that the same cytochrome as the one active in blue light photoprocesses was involved. This study describes the effect of detergents upon such red light induced reductions in corn coleoptile preparations fractionated after differential centrifugation into 9 KP, 21 KP and 50 KP (500–9000 g pellet, 9000–21,000 g pellet and 21,000–50,000 g pellet, spun for 20, 20, and 45 min, respectively). Both Triton X-100 (more effective) and deoxycholate (less effective and somewhat destructive) could be used as solubilizers if concentrations above the critical micellar concentration were chosen. Tween 40 was ineffective and dodecyl sulphate affected the cytochrome so that it lost its accessibility to electrons from photoexcited methylene blue. The recovery, measured as the ratio between light induced absorbance change (LIAC) in the Soret region after and before solubilization, was highest in 9 KP (70%) and lower in 21 and 50 KP fractions (50% and 43% respectively). The band in the Soret region in light minus dark spectra had its peak at longer wavelengths compared to the dithionite reduced minus no addition absorption difference band, whether the sample was solubilized or not. Similar results were obtained when the material was separated on a discontinuous sucrose gradient (15/28/33/45% w/w sucrose). In such a separation, the distribution of LIAC between fractions (collected at the interfaces) was about the same after solubilization as before (solubilization brought about a slight shift towards heavier fractions). The ratios of LIAC to the dithionite reduced minus no addition absorbance difference decreased upon detergent treatment. The LIAC still had its peak at longer wavelengths compared to the peak obtained upon dithionite reduction. The usefulness of detergents in the purification of the particulate b-type cytochrome is discussed.     

Sulfur dioxide inhibits the sucrose carrier of the plant plasma membrane.

Plasma membrane vesicles were prepared by phase partition from a microsomal fraction of broad bean (Vicia faba L.) leaf. In order to study the effects of sodium sulfite on active uptake of sucrose, the vesicles were artificially energized by a transmembrane pH gradient (delta pH) and/or a transmembrane electrical gradient (delta psi). At 1 mM, sulfite strongly inhibited sucrose uptake but did not affect the two components of the proton motive force, delta pH (measured by dimethyloxazolidine dione) and delta psi (measured by tetraphenylphosphonium). Moreover, sulfite did not inhibit the proton-pumping ATPase of the plasma membrane vesicles. These data demonstrate that sulfite may inhibit transport of photoassimilates in plant by a direct inhibition of the sucrose carrier of the plasma membrane.     

Isolation and Identification of Plasma Membrane from Light-Grown Winter Rye Seedlings (Secale cereale L. cv Puma)

An effective method for the isolation of plasma membrane from light-grown winter rye seedlings (Secale cereale L. cv Puma) was established using a liquid two-polymer phase separation. The conditions for the specific partition of plasma membrane into the polyethylene glycol-enriched upper phase were examined, including variations in the polymer concentration, buffer system, pH, and NaCl addition in the phase partition system. The most effective phase partition system for the isolation of plasma membrane from winter rye consisted of 5.6/5.6% (w/w) polyethylene glycol 4000/dextran T500 in 0.25 molar sucrose-10 millimolar potassium phosphate-30 millimolar NaCl (pH 7.8), repeated once. When the isolated plasma membrane was centrifuged on a linear sucrose density gradient, a single band was found at the 34% (w/w) sucrose layer (1.141 grams per cubic centimeter) which co-fractionated with the pH 6.5-ATPase.

Identification of plasma membrane was performed by the combination of phosphotungstic acid-chromic acid stain and specific binding of N-1-naphthylphthalamic acid. Based on morphometrical observations after phosphotungstic acid-chromic acid stain, the isolated plasma membrane consisted mostly of vesicles of high purity. The isolated plasma membrane also showed extremely high specificity for N-1-naphthylphthalamic acidbinding, 10-fold higher than other membranes. It was also confirmed that there is a distinct difference in properties between plasma membrane and other membranes. The endomembranes such as from chloroplasts, mitochondria, and endoplasmic reticulum were observed to be highly sensitive to Zn²⁺ ion and lower pH, which resulted in an abrupt aggregation of membranes. On the contrary, plasma membrane was very stable to these treatments and no aggregation was observed. These unique properties of isolated plasma membrane are generally observed in a wide variety of plant species and can be utilized for the assessment of the purity of preparations of isolated plasma membranes and for their identification.

     

Plasma membrane vesicles of opposite sidedness from soybean hypocotyls by preparative free-flow electrophoresis

Absolute orientations (sidedness) of plasma membrane vesicles obtained in highly purified fractions by preparative free-flow electrophoresis and by aqueous two-phase partition were determined based on ATPase latency and morphological criteria. Free-flow electrophoresis yielded two plasma membrane fractions. One, the least electronegative and designated fraction `E,' was pure plasma membrane. The other, more electronegative and designated fraction `C,' was heavily contaminated by various other cellular membranes. Plasma membrane vesicles from both fraction C and fraction E partitioned into the upper phase with aqueous two-phase partitioning. Purified plasma membrane obtained from microsomes by two-phase partition (upper phase) when subjected to free-flow electrophoresis also yielded two fractions, one fraction co-migrated with fraction C and another fraction co-migrated with fraction E. Both fractions exhibited an ATPase activity sensitive to vanadate and insensitive to nitrate and azide. ATPase activity was used as a structure-linked latency marker for the inner membrane surface. Concanavalin A binding (linked to peroxidase) was used as an imposed electron microscope marker for the outer membrane surface. Fraction E vesicles showed low ATPase latency (two-fold or less) and weak reactivity with concanavalin A peroxidase. In contrast, fraction C vesicles were characterized by much greater latencies upon detergent treatment (sevenfold) and a strong reaction with concanavalin A peroxidase. Two-phase partition as the initial procedure for plasma membrane isolation, yielded mixtures of vesicles of both inside out and right-side out orientation. Free-flow electrophoresis resolved the plasma membrane isolates into vesicles from fraction C which were right-side out (cytoplasmic side in), and vesicles from fraction E which were wrong-side out (cytoplasmic side out). Therefore, the two methods used in series, provided highly purified membrane preparations of apparently homogenous vesicles of opposite known absolute orientations.     

The role of plasma membrane redox activity in light effects in plants

Stimulations by light of electron transport at the plasma membrane make it possible that redox activity is involved in light-induced signal transduction chains. This is especially true in cases where component(s) of the chain are also located at the plasma membrane. Photosynthetic reactions stimulate transplasma membrane redox activity of mesophyll cells. Activity is measured as a reduction of the nonpermeating redox probe, ferricyanide. The stimulation is due to production of a cytosolic electron donor from a substance(s) transported from the chloroplast. It is unknown whether the stimulation of redox activity is a requirement for other photosynthetically stimulated processes at the plasma membrane, but a reduced intermediate may regulate proton excretion by guard cells. Blue light induces an absorbance change (LIAC) at the plasma membrane whose difference spectrum resembles certainb-type cytochromes. This transport of electrons may be due to absorption of light by a flavoprotein. The LIAC has been implicated as an early step in certain blue light-mediated morphogenic events. Unrelated to photosynthesis, blue light also stimulates electron transport at the plasma membrane to ferricyanide. The relationship between LIAC and transmembrane electron flow has not yet been determined, but blue light-regulated proton excretion and/or growth may depend on this electron flow. No conclusions can be drawn regarding any role for phytochrome because of a paucity of information concerning the effects of red light on redox activity at the plasma membrane.     

Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells

Partitioning in dextran–poly(ethylene)glycol (PEG) aqueous–aqueous phase systems represents a mature technology with many applications to separations of cells and to the preparation of membranes from mammalian cells. Most applications to membrane isolation and purification have focused on plasma membranes, plasma membrane domains and separation of right side-out and inside-out plasma membrane vesicles. The method exploits a combination of membrane properties, including charge and hydrophobicity. Purification is based upon differential distributions of the constituents in a sample between the two principal compartments of the two phases (upper and lower) and at the interface. The order of affinity of animal cell membranes for the upper phase is: endoplasmic reticulum<mitochondria<Golgi apparatus<lysosomes and endosomes<plasma membranes. Salt concentrations and temperature affect partitioning behavior and must be precisely standardized. In some cases, it is more fortuitous to combine aqueous two-phase partition with other procedures to obtain a more highly purified preparation. A procedure is described for preparation of Golgi apparatus from transformed mammalian cells that combines aqueous two-phase partition and centrifugation. Also described is a periodic NADH oxidase, a new enzyme marker for right side-out plasma membrane vesicles not requiring detergent disruptions for measurement of activity.     

Identification of Tetrahymena Ciliary Surface Proteins Labeled with Sulfosuccinimidyl 6-(Biotinamido) Hexanoate and Concanavalin A and Fractionated with Triton X-114

ABSTRACT. Tetrahymena thermophila cells were labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate, a sensitive nonradioactive probe for cell surface proteins, and Western blots of axonemes and ciliary membrane vesicles were compared to cilia fractionated with Triton X-114 (TX-114) in order to study the orientation of ciliary membrane proteins. Greater than 40 ciliary surface polypeptides, from >350 kDa to <20 kDa, were resolved. The major surface 50–60 kDa proteins are hydrophobic and partition into the TX-114 detergent phase. Two high molecular weight proteins, one of which is biotinylated, comigrate with the heavy chains of ciliary dynein, sediment at 14S in a sucrose gradient, and partition into the TX-114 aqueous phase. Fractions containing these high molecular weight proteins as well as fractions enriched in 88-kDa and 66-kDa polypeptides contain Mg²⁺-ATPase activities. Detergent-solubilized tubulins partition into the TX-114 aqueous phase, are not biotinylated, and must not be exposed to the ciliary surface. The detergent-insoluble axoneme and membrane fraction contains a 36-kDa polypeptide and a portion of the 50-kDa polypeptides that otherwise partition into the detergent phase. These polypeptides could not be solubilized by ATP or by NaCl extraction and appear to be associated with pieces of ciliary membrane tightly linked to the axoneme. The ciliary membrane polypeptides were also tested for Concanavalin A binding and at least sixteen Con A-binding polypeptides were resolved. Of the major Con A-binding polypeptides, three are hydrophobic and partition into the TX-114 detergent phase, three partition into the TX-114 aqueous phase, and four partition exclusively in the detergent-insoluble fraction, which contains axonemes and detergent-resistant membrane vesicles.     

Comparison of two methods for the preparation of a membrane fraction of cauliflower inflorescences containing a blue light reducible b -type cytochrome

Presumptive plasma membrane fractions containing a light-sensitive flavocyto–chrome b -protein complex from cauliflower inflorescences were isolated by two different procedures. In the first procedure a density gradient centrifugation was used, while in the second the separation was carried out using an aqueous polymer two phase system based on the specific surface properties of the membranes. This latter method is much faster and its yield is as good as a purification on a linear sucrose density gradient in terms of light inducible cyt b reduction. When the LIAC-containing membranes obtained through a sucrose gradient are further purified on an Urografin gradient, the presence of contaminating cyt b is shown. The distribution of this b type cytochrome, showing no redox change upon illumination, is similar to the distribution as a NADH dependent and antimycin A resistant cytochrome c reduc–tase, a marker enzyme for endoplasmic reticulum. Measurable mitochondrial contamination is indicated by cytochrome oxidase activity. Both contaminants were less pronounced in the fractions obtained after Urografin gradient centrifugation of the membranes prepared by the two phase system method. The isolation procedure based upon the use of the two phase aqueous polymer system allows more rapid preparation of LIAC-containing presumptive plasma membranes than that obtained with the sucrose density gradient centrifugation. It also yields a purer preparation.     

Separation of dense, polysaccharide-containing vesicles from secreting, cultured oat cells. Characterization of a putative secretory vesicle fracton

Suspension cultured oat (Avena sativa L. cv. Garry) cells, which secrete polysaccharides into the medium, were used as starting material for analyses of Golgi-derived vesicle membranes and plasma membranes isolated during cell fractionation. Vesicles collected by a procedure employing ultrafiltration followed by ultracentrifugation into a sucrose step gradient exhibited an equilibrium density of 1.27 g cm^?3 when run on continuous sucrose gradients, a feature which is most likely attributable to the high concentration of enclosed polysaccharides. Brief sonication lowered the density of these vesicles to about 1.15 g cm^?3, as judged from the coincidence of the protein peak and the marker enzymes for Golgi [Triton-stimulated UDPase, cold-storage IDPase (EC 3.6.1.6)] and plasma membrane [vanadate-inhibited K⁺, Mg²⁺-ATPase (EC 3.6.1.3)]. Sonication of these vesicles also greatly diminished the amount of detectable polysaccharide observed in a colorimetric assay for sugars. Fractionation of a plasma membrane-enriched preparation from these cells on continuous sucrose gradients showed the major protein peak and the peak activity for the plasma membrane marker at 1.17 g cm^?3, however, there was also significant overlap with a mitochondrial [cytochrome c oxidase (EC 1.9.3.1)] peak at 1.18 g cm^?3, Smaller peaks of the Golgi markers were seen at 1.14 g cm^?3. Analyses of marker enzymes for ER and mitochondria (EC 1.6.99.3) showed little contamination of the membranes of presumptive secretory vesicles from these sources, and the lack of significant vanadate-insensitive ATPase activity in the density range from 1.13–1.18 g cm^?3 in either fractionation scheme suggests that these membranes do not include material from the tonoplast. The coincidence of markers for Golgi and plasma membrane with from the tonoplast. The coincidence of markers for Golgi and plasma membrane with the membranes of sonicated, dense vesicles, at a density slightly lower than that of plasma membranes prepared from the same cells, supports the possibility that membranes en route to the plasma membrane are incompletely differentiated.     

Distribution of ATPases in wheat root membranes separated by phase partition

The distribution of divalent cation stimulated ATPase activity in relation to the distribution of other enzyme activities was studied for membrane fractions from wheat roots ( Tritium aestivum L . cv. Svenno). A homogenate from dark grown plants was fractionated by differential centrifugation at 1000 g , 10,000 g , 30,000 g and 60,000 g (1, 10, 30 and 60 KP fractions), followed by partition in an aqueous polymer two-phase system, using polyethylene glycol 4000/dextran T500 concentrations of 5.7/5.7, 5.9/5.9, 6.1/6.1, 6.3/6.3 and 6.5/6.5% (w/w). The 30 KP fraction was also separated by counter-current distribution id a 6.3/6.3% two-phase system. Protein and activities of Ca²⁺, Mg²⁺, and Mn²⁺ stimulated ATPases. cytochrome oxidase, light induced absorbance change (LIAC) related to cyt b reductions, inosine diphosphatase and NADH dependent antimycin A insensitive cytochrome c reductase were measured.
The partition of ATPase activities stimulated by Ca²⁺, Mg²⁺ or Mn²⁺ was similar at all polymer concentrations tested, indicating: a low cation specificity of the dominating ATPases. The distribution of ATPases. agreed with different marker enzymes in different centrifuge fractions. Divalent cation stimulated ATPases were evidently related to several of the organelles. In the different fractions the distribution of ATPase activity should then follow that of the marker enzyme of the dominant organelle. From studies with different polymer concentrations the 6.3/6.3-system was selected for further separation of the membranes in the 30 KP fraction by counter-current distribution. By this method one fraction was obtained, which probably consisted of plasmalemma and was free from mitochondrial material. Indications for plasmalemma in this fraction were a) similar partition as protoplasts and b) high LIAC activity.     

The dynamics of phase partition. A study of parameters affecting rat liver organelle partitioning in aqueous two-polymer phase systems.

Separation of subcellular organelles by two-phase partition is thought to reflect differential partition of the organelles between the two phases or between one of the phases and the interface. Studies by Fisher and colleagues [Fisher & Walter (1984) Biochim. Biophys. Acta 801, 106-110] suggest that cell separation by phase partition is a dynamic process in which the partition changes with time. This is mainly due to association of the cells with sedimenting droplets of one phase in the bulk of the other. Rat liver organelle partition was studied to determine whether the same dynamic behaviour is observed. Partition was clearly time-dependent during 24 h at unit gravity, and was also affected by altering the volume ratio of the two phases and the duration of phase mixing. These results indicate that, as with cells, the partition of organelles between phases is a dynamic process, and is consistent with the demonstration that organelles adhere to the phase droplet surfaces. Optimization of the volume ratio between phases may lead to significant processing economies. Organelle sedimentation in the upper phase was significantly faster than in the isoosmotic sucrose. Theoretical modelling of apparent organelle sizes indicates that aggregation occurs in the poly(ethylene glycol)-rich upper phase. This phenomenon is likely to limit the use of this technique in organelle separations unless means can be found to decrease aggregation.     

Preparation of a low-density species of endocytic vesicle containing immunoglobulin A.

Immunoglobulin A is transported across hepatocytes in specialized vesicles. A population of endocytic vesicles of approx. 140 nm diameter, containing immunoglobulin A, has now been separated from all other major cytoplasmic organelles, including plasma membrane and lysosomes, by sequential centrifugation on Ficoll/sucrose and Metrizamide gradients.     

B-type cytochromes in plasma membranes isolated from rat liver, in comparison with those of endomembranes

Fractions of plasma membranes, Golgi apparatus, endoplasmic reticulum (ER), and nuclear envelope were isolated from rat liver and were characterized by electron microsocpe and biochemical methods. The purity of the fractions was controlled by morphometry and by marker enzyme activities. Amounts of cytochromes b5, P-450, and P-420 were measured, as well as the NADPH- and NADPH-cytochrome c reductase activities. The pigments of the microsomal electron transport system were found in all membrane fractions in relatively high amounts, thus excluding an origin by microsomal contamination. Purified preparations of plasma membrane and Golgi apparatus contained approximately 30% of the cytochrome b5 and cytochrome P-450 + P-420 found in ER membranes. Plasma membranes were also characterized by a high ratio of P-420/450. Degradation of cytochromes P-450 and P-420 was relatively rapid in all fractions, except in the ER. Cytochrome b5 extracted from plasma membranes was spectrophotometrically and enzymatically indistinguishable from ER cytochrome b5. However, immunnlogical characterization with rabbit antibodies against the trypsin-resistant core of microsomal cytochrome b5 showed the presence of at least two types of cytochrome b5 in ER membranes, in contrast to the plasma membranes in which only one of these components was detected. This immunological differentiation also demonstrates that the plasma membrane-bound cytochrome b5 is endogenous to this membrane and does not reflect contamination by ER elements. We conclude that cytochromes b5, P-450, and P-420 are not confined only to ER and nuclear membranes but also occur in signficant amounts in Golgi apparatus and plasma membranes. The findings are discussed in relation to observations of similar redox components in Golgi apparatus, secretory vesicles, and plasma membranes of other cells.