Effects of centrifugal force and centrifugation time on the sedimentation of plant organelles

The effect of centrifugal force and length of centrifugation time on the sedimentation of plant organelles was determined for corn (Zea mays L.) root homogenates. A centrifugal force of 6000g for at least 20 minutes was necessary to pellet 90% of the mitochondrial marker (cytochrome c oxidase). This initial centrifugation step is optimal for separating mitochondria from microsomes, since cross-contamination of endoplasmic reticulum and plasma membrane vesicles with mitochondria is minimized. Centrifugal forces of 8000g or 10,000g for 20 minutes and 13,000g for 15 minutes pellet 90% of the mitochondrial marker; however, these centrifugation conditions also sediment more plasma membrane and endoplasmic reticulum.     

Isolation and partial characterization of rat brain synaptic plasma membranes

Abstract— Synaptic plasma membranes from the cortices of adult rat brain were isolated from synaptosomes prepared by flotation of a washed mitochondrial pellet (P2) in a discontinuous Ficoll-sucrose gradient. Contamination of the synaptosome fraction by microsomes was estimated by enzymic and chemical analysis to be less than 15 per cent. (2) The purified synaptosome fraction was subjected to osmotic shock, subfractionated on a discontinuous sucrose gradient and the distribution of enzymic and chemical markers for synaptic plasma membranes, microsomal membranes and mitochondria was determined. (3) Comparison of synaptosome subfractions prepared in the presence and absence of 1 mM NaH₂ PO₄/0.1 mM EDTA buffer pH 7.5, indicated that the ionic composition of the isolation medium markedly affected the distribution and enzymic composition of the subfractions. (4) Synaptic plasma membranes prepared in the presence of PO₄/EDTA exhibited a 10-fold enrichment in [Na⁺+ K⁺] ATPase and were characterized by less than 15 and 10 per cent contamination by microsomes and mitochondria respectively. (5) The polypeptide composition of the purified synaptic plasma membranes was compared with the microsomes and mitochondria by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. No differences between the protein and glycoprotein composition of the synaptic plasma membranes and microsomes were detected. The mitochondria, in contrast, possessed a unique protein composition.     

A phosphatidyl choline exchange protein isolated from germinated castor bean endosperms

A phospholipid exchange protein (PLEP) functioning between theendoplasmic reticulum and the mitochondrion was purified fromthe cytosolic fraction of germinated castor bean endosperms.In the protein fraction eluted from Sephadex G-100 column, theexchange rate reached 7.3µg phospholipids exchanged/mgprotein/15 min, which was 60-fold that of pota to tuber PLEP.The lipid transfer by this protein was specific for phosphatidylcholine and the transfer rate from microsomes to mitochondriawas as high as that from mitochondria to microsomes. Castorbean PLEP transferred phospholipid from castor bean microsomesto mitochondria from other sources such as potato tubers, cauliflowerinflorescences, pumpkin hypocotyls and rat livers, and to liposomes,but not to Avena etioplasts. In addition, it transferred phospholipidfrom potato microsomes to potato mitochondria. (Received November 17, 1978; )     

Separation of Particulate Phytochrome and Plasma Membranes of Maize Coleoptiles by Density Gradient Centrifugation

Phytochrome, cross linked in situ to its receptor by glutaraldehydefixation, and radioactively-labelled membrane material, obtainedby lactoperoxidase-catalysed ¹²⁵I-treatment of maize coleoptilescould be separated from each other according to sedimentationvelocity and by sucrose density gradient centrifugation. Bindingof iodine to membrane material in maize coleoptiles increasedseveral-fold on the addition of reaction-specific enzymes, i.e.lactoperoxidase and glucose oxidase. Mitochondria were considerednot labelled because mito-chondrial purification reduced ¹²⁵Iincorporation. Membrane material containing incorporated iodineappeared quite heterogenous and sedimented to an equilibriumdensity position close to, but slightly lighter than that ofthe mitochondria; participate phytochrome banded at a heavierposition. Results obtained therefore suggest that the receptorfor phytochrome may not be on the plasma membrane as envisagedin recent hypotheses.     

Nature of the phytochrome effect on the binding of glycolate oxidase to peroxisomes in vitro

The attachment of glycolate oxidase to the peroxisomal fraction derived from etiolated barley leaves (Hordeum vulgare L. cr. Dvir) is affected by light. The effect of red irradiation is reversed by subsequent far-red irradiation, indicating the involvement of phytochrome. This phytochrome effect is assumed to be related to phytochrome binding. Indeed, prevention by filipin (1.2·10^-6 mol g^-1 f wt) or cholesterol of phytochrome binding to membranes abolishes the effect of light on the interaction between glycolate oxidase and the peroxisomal fraction. Glycolate oxidase binding is affected by addition of quasi-ionophores such as gramicidin and filipin at a concentration of 0.6·10^-3 mol g^-1 f wt. This fact indicates that peroxisome-glycolate oxidase interaction may be affected by membrane potential. Since both ion transport and membrane potential are known to be affected by phytochrome, it is proposed that phytochrome acts in the light-induced modulation of glycolate oxidase attachment as a quasi-ionophore.Abbreviations GO glycolate oxidase - Pr and Pfr phytochrome forms absorbing in red and far-red, respectively - R and F red and far-red irradiation - Cumulative 20 Kp 20,000 g pellet obtained by centrifugation of the crude extract - 1 Kp 1,000 g pellet - 20 Kp 20,000 g pellet, obtained by centrifugation of 1 Kp supernatant - 1 Kp, 20 Kp and cumulative 20 Kp pellets obtained after density centrifugation through a sucrose cushion     

Effects of divalent cations and EDTA on spectral properties of phytochrome in particulate fractions from etiolated pea epicotyls

The photoreversible absorbance change of phytochrome in suspensionsof a 20,000xg particulate fraction (20kP) prepared from a 1,000xgsupernatant (1kS) of etiolated pea epicotyl extracts decreasedremarkably in the presence of 5 mM Cu²⁺, Zn²⁺ and Co²⁺, butremained unchanged in 5 mM Ca²⁺, Mg²⁺, Fe²⁺ or Mn²⁺. This spectraldistortion of phytochrome was more evident in soluble preparationsand in suspensions of pellets prepared from red light (R)-irradiatedtissues than it was in suspensions of pellets prepared in thedark from etiolated tissues that received no actinic irradiation. When Cu²⁺ was added to the red-light-absorbing form of phytochrome(Pr) in resuspended pellets prepared from R-irradiated tissues,the distortion of its difference spectrum took place after irradiationwith the first actinic R. In contrast, when Cu²⁺ was added tothe far-red-light-absorbing form of phytochrome (Pfr) in thesame resuspended pellet, no distortion was seen, unless thePfr in the pellet was first photoconverted to Pr and then photoconvertedback to Pfr. Spectral distortion of Pr remained small during dark incubationat 25°C when suspensions of 20kPs were prepared and incubatedwith a buffer containing EDTA, whether the 20kP was preparedfrom nonirradiated tissue or from R-irradiated tissues. But,when EDTA was added to a suspension of 20kP prepared from 1kS,after the 1kS was irradiated with R in the presence of 10 mMCaCl₂, the spectral distortion of Pr in 20kP occurred instantaneously. (Received April 14, 1980; )     

PREPARATION OF PLASMA MEMBRANE FROM ISOLATED NEURONS

A bulk fraction enriched with respect to neuronal cell bodies was used as starting material for the isolation of neuronal plasma membrane The cells were gently homogenized in isotonic sucrose and a crude membrane containing fraction sedimented at 3000 g. Subsequently, the membrane fraction was purified on a discontinuous sucrose density gradient between 35% and 25 5% sucrose (w/w). Enzymatic analyses showed a 4–5-fold enrichment in plasma membrane markers, and a 10–15% contamination of mitochondrial and microsomal material. Electron micrographs of the membrane fraction confirmed the enzymatic data Fragmented membranes were found, mainly in vesicular form No ribosomes, but a few mitochondria and some multilamellar membranes were seen     

Cell Fractionation of Anterior Pituitary Glands from Beef and Pig

Fresh anterior pituitary glands from beef and pig were separated by differential centrifugation into subcellular fractions. Nuclei and debris were obtained at 700 g for 15 minutes, secretory granules at 7000 g for 20 minutes, mitochondria at 34,000 g for 15 minutes, and microsomes at 78,000 g for 3 hours. Electron micrographs were taken of the individual fractions. Each fraction was analyzed for nitrogen, pentosenucleic acid (PNA), and phospholipide. Beef and pig anterior lobes were quite similar in their intracellular composition as seen in the subcellular fractions. Succinic dehydrogenase was localized in mitochondria, while alkaline phosphatase was concentrated in the microsomes. A proteinase with pH optimum at 8.2 was exclusively localized. in microsomal and supernatant fractions. Acid phosphatase, acid ribonuclease, and acid proteinase were distributed among the subcellular fractions in another pattern, indicating the presence of a particle type distinct from mitochondria and microsomes. The distribution of cytoplasmic PNA paralleled that of alkaline phosphatase.     

Subcellular distribution of intraportally injected 125I-labeled insulin in rat liver

Time course studies revealed that at 30 s after intraportal injection of 200 μU of ¹²⁵I-labeled insulin per 100 g rat 47.9 ± 2.8% of the injected radioactivity was recovered from the liver homogenate by precipitation with trichloroacetic acid. Trichloroacetic acid precipitable radioactivity declined to very low levels during the next 30 min whereas trichloroacetic acid soluble radioactivity reached a peak value of 9.56 ± 1.9% at 5 min and declined gradually thereafter. At 30 s mean peak accumulations ±SE of 6.83 ± 0.42, 5.06 ± 0.27, 14.90 ± 1.85, and 3.58 ± 0.58% of injected radioactivity were recovered in trichloroacetic acid precipitates from the 700g (nuclei + debris), 10,000g (mitochondria + lysosome), 105,000g (microsomes), and supernatant (cytosol) subfractions, respectively. Mean peak values of 0.72 ± 0.08, 0.12 ± 0.02, and 1.11 ± 0.16% of injected radioactivity were recovered in the partially purified mitochondrial fraction, purified nuclei, and plasma membranes, respectively, as trichloroacetic acid precipitable material. Most of the trichloroacetic acid precipitable activities in the subfractions were immunoprecipitable. Trichloroacetic acid soluble radioactivity was found mainly in the cytosol and microsomal fractions. Peak specific activity (percentage of injected dose/mg protein × 10^?3) was highest in the microsomes, intermediate in the plasma membranes, and very low in the purified nuclei and partially purified mitochondrial fraction. The specific activity of the microsomes remained at or near peak levels for 5 min after ¹²⁵I-labeled insulin injection and then declined, whereas specific activity of the plasma membranes dropped precipitously to 25% of peak values at 5 min. Sephadex gel filtration of the radioactivity in the deoxycholate soluble fraction of microsomes at 5 min after ¹²⁵I-labeled insulin injection resulted in the elution of a major peak (Peak I) in the region of ¹²⁵I-labeled insulin and a minor peak (Peak II) in the region of the labeled A and B chains. Incubation of the fraction for 30 min at 37 °C with 3 mm reduced glutathione and 15 mm EDTA resulted in a reciprocal fall in Peak I and rise in Peak II. The data suggest that intraportally injected ¹²⁵I-labeled insulin is rapidly internalized and concentrated in the rat liver microsomes. The time courses of appearance and disappearance of trichloroacetic acid precipitable radioactivity in plasma membrane and microsomes further suggest, although do not prove, that insulin binds to plasma membranes before it is internalized. They also provide presumptive evidence suggesting that the sequential degradative pathway is operative in vivo.     

Influence of magnesium in the homogenization medium on the state of a divalent cation-stimulated,diethystilbestrol-sensitive adenylnucleotidyl phosphatase activity from pea stem microsomal preparations

The amount of divalent cation-activated, diethylstilbestrol-sensitive adenylnucleotidyl phosphatase activity recovered in the ‘microsomes’ ($\text{13 000–80 000 x g}\text{sediment}$) from pea stem tissue is strongly influenced by the concentration of Mg²⁺ in the homogenization medium. The absence of Mg²⁺ during homogenization results in a marked decrease of the activity found in the microsomal fraction, compensated by its increase in the soluble fraction. Part of the solubilized activity becomes sedimentable at $\text{80 000 × g}$ upon addition of 5–10 mM Mg²⁺ (or Mn²⁺, Ca²⁺, Zn²⁺) to the supernatant. This sediment shows a very high specific activity, and can be re-solubilized by treatment with either EDTA or 0.3 M monovalent salts, or deoxycholate. When the supernatant containing the solubilized activity is incubated together with low-adenylnucleotidyl phosphatase microsomes and with 10 mM MgCl₂ the activity recovered in the sediment is much larger than the sum of the activity of the microsomes plus that of the sediment obtained by incubating the same supernatant with Mg²⁺. Microsomes prepared with Mg²⁺ in the homogenization medium do not show this effect. The supernatant/microsomes saturation curves as well as a change of the temperature coefficient of the activity following combination of the soluble preparation with the microsomal particles suggest an at least partial reconstitution of the original enzyme-membrane structure.     

Rapid Enrichment of CHAPS-Solubilized UDP-Glucose: (1,3)-beta-Glucan (Callose) Synthase from Beta vulgaris L. by Product Entrapment : Entrapment Mechanisms and Polypeptide Characterization

Rapid enrichment of CHAPS-solubilized UDP-glucose:(1,3)-β-glucan (callose) synthase from storage tissue of red beet (Beta vulgaris L.) is obtained when the preparation is incubated with an enzyme assay mixture, then centrifuged and the enzyme released from the callose pellet with a buffer containing EDTA and CHAPS (20-fold purification relative to microsomes). When centrifuged at high speed (80,000g), the enzyme can also be pelleted in the absence of substrate (UDP-Glc) or synthesis of callose, due to nonspecific aggregation of proteins caused by excess cations and insufficient detergent in the assay buffer. True time-dependent and substrate-dependent product-entrapment of callose synthase is obtained by low-speed centrifugation (7,000-11,000g) of enzyme incubated in reaction mixtures containing low levels of cations (0.5 millimolar Mg²⁺, 1 millimolar Ca²⁺) and sufficient detergent (0.02% digitonin, 0.12% CHAPS), together with cellobiose, buffer, and UDP-Glc. Entrapment conditions, therefore, are a compromise between preventing nonspecific precipitation of proteins and permitting sufficient enzyme activity for callose synthesis. Further enrichment of the enzyme released from the callose pellet was not obtained by rate-zonal glycerol gradient centrifugation, although its sedimentation rate was greatly enhanced by inclusion of divalent cations in the gradient. Preparations were markedly cleaner when product-entrapment was conducted on enzyme solubilized from plasma membranes isolated by aqueous two-phase partitioning rather than by gradient centrifugation. Product-entrapped preparations consistently contained polypeptides or groups of closely-migrating polypeptides at molecular masses of 92, 83, 70, 57, 43, 35, 31/29, and 27 kilodaltons. This polypeptide profile is in accordance with the findings of other callose synthase enrichment studies using a variety of tissue sources, and is consistent with the existence of a multi-subunit enzyme complex.     

Glycoprotein synthesis in the adult rat pancreas: IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5–10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50–100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and $\text{N-}\text{acetylglucosamine}$. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes < smooth microsomes < zymogen granules.Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptide was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [³H]glucosamine or [³H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules.Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB³H₄. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

THE STRUCTURE OF SOME CYTOPLASMIC COMPONENTS OF PLANT CELLS IN RELATION TO THE BIOCHEMICAL PROPERTIES OF ISOLATED PARTICLES

1. Electron micrographs of thin sections of material fixed with buffered osmium tetroxide have been used for comparison of the fine structure of isolated cytoplasmic particles from silver beet petioles and roots of germinating wheat with that of the cytoplasm of the intact cells. 2. Mitochondria of wheat roots have an external double membrane and poorly oriented internal double membranes. As compared with the structures seen in situ, the isolated mitochondria showed evidence of some disorganisation of the fine internal structure, probably due to osmotic effects. The possible influence of such changes on the enzymic properties of the isolated mitochondria is discussed. 3. The isolated plant microsomes are mainly spherical vesicular structures consisting of (a) an outer membrane enclosing (b) either an homogeneous slightly dense material (wheat root microsomes) or some granular dense material (silver beet microsomes) and (c) small dense particles, mostly associated with the vesicle membranes. 4. The cytoplasm of the wheat root cells does not contain any structures similar to the isolated microsomes but has a very dense reticular network, consisting of membranes with associated small dense particles, here called the endoplasmic reticulum. The observations indicate that the isolated microsomes arise mainly by rupture and transformation of the membranes of this structure. The effects of such extensive changes in the lipoprotein membranes on the enzymic activities of the endoplasmic reticulum, as studied in isolated microsomes, is discussed. 5. Meristematic wheat root cells contain structures which consist of smooth membranes with associated vacuoles and are similar to the Golgi zones of animal cells. The membranes of these zones probably contribute to the microsomal fraction under the conditions of preparation used for the enzymic and chemical studies previously reported.     

Receptors for gonadotropins and prostaglandins in lysosomes of bovine corpora lutea.

The total mitochondrial fraction of bovine corpus luteum specifically bound [³H]prostaglandin (PG) E₁, [³H] PGF_2α, and ¹²⁵I-labeled human lutropin (hLH) despite very little 5′-nucleotidase activity, a marker for plasma membranes. Since the total mitochondrial fraction isolated by conventional centrifugation techniques contains both mitochondria and lysosomes, it was subfractionated into mitochondria and lysosomes to ascertain the relative contribution of these fractions to the binding. Subfractionation resulted in an enrichment of cytochrome c oxidase (a marker for mitochondria) in mitochondria and of acid phosphatase (a marker for lysosomes) in lysosomes. The lysosomes exhibited little or no contamination with Golgi vesicles, rough endoplasmic reticulum, or peroxisomes as assessed by their appropriate marker enzymes. Subfractionation also re ulted in [³H] PGE₁, [³H] PGF_2α, and ¹²⁵I-labeled hLH binding enrichment with respect to homogenate in lysosomes but not in mitochondria. The lysosomal binding enrichment and recovery were, however, lower than in plasma membranes. The ratios of marker enzyme to binding, an index of organelle contamination, revealed that plasma membrane and lysosomal receptors were intrinsic to these organelles. Freezing and thawing had markedly increased lysosomal binding but had no effect on plasma membrane binding. Exposure to 0.05% Triton X-100 resulted in a greater loss of plasma membrane compared to lysosomal binding. In summary, the above results suggest that lysosomes, but not mitochondria, in addition to plasma membranes, intrinsically contain receptors for PGs and gonadotropins. Furthermore, lysosomes overall contain a greater number of PGs and gonadotropin receptors compared to plasma membranes and these receptors are associated with the membrane but not the contents of lysosomes.     

Association of Phytochrome with Rough-surfaced Endoplasmic Reticulum Fractions from Soybean Hypocotyls

Distribution of phytochrome (as Pfr) among membranes from soybean hypocotyls (Glycine max L. cv. Wayne) was determined by the combined techniques of cell fractionation, difference spectrometry, and electron microscopic morphometry. More than 90% of the phytochrome was found in the soluble fraction. With homogenates prepared in the presence or absence of Mg²⁺, the portion associated with membrane was only 6.5% and 1%, respectively. In the presence of Mg²⁺, the content of particulate phytochrome correlated with the amount of endoplasmic reticulum with attached ribosomes in the fractions but not with mitochondria or other membranes (including endoplasmic reticulum membranes from which the ribosomes may have been lost during cell fractionation). In the absence of Mg²⁺, phytochrome was associated with a “heavy” plasma membrane fraction. The phytochrome content was sufficiently low to be accounted for by a contamination of less than 10% by rough-surfaced fragments of endoplasmic reticulum. The findings show association of phytochrome with a particulate fraction enriched in rough-surfaced fragments of endoplasmic reticulum but do not rule out cosedimentation of some unknown or unspecific phytochrome aggregate with this fraction.     

Bovine adrenocortical adenylate cyclase: Some properties of the solubilized,fluoride-activated enzyme

The activity of bovine adrenocortical plasma membrane adenylate cyclase can be maintained at 4°C in the presence of NaF. The half-life of the fluoride-stabilized enzyme is approximately 7 days. Maximal activation by fluoride requires approximately 20 min at 0°C and the level of activity attained is dependent on fluoride concentration. The enzyme from freshly harvested membranes can also be stimulated by ACTH_{1 – 24} and Gpp(NH)p and the stimulatory effects of these two activators are additive. Prolonged exposure to either NaF or Gpp(NH)p precludes hormone activation. Optimal concentration for Gpp(NH)p activation is 10^?4–10^?5M. Treatment of the enzyme with a Tris-HCl buffer containing Lubrol-PX (1%), NaF, dithiothreitol, and MgSO₄ followed by sonication affords a preparation that does not sediment at 100 000 g in 1 hr. This material has a low specific activity; however, removal of the detergent on DEAE cellulose restores specific activity to its original level. A significant improvement in specific activity is observed following dialysis or ultrafiltration of the detergent-free 100 000-g supernatant. At this stage the enzyme can be lyophilized and stored at ?70°C without loss of activity. The enzyme in the detergent-free 100 000-g supernatant behaves as a single peak that is included in Sepharose 6B. Comparison of the elution profile of the enzyme with profiles produced by a standard set of proteins suggests a molecular weight of 1 × 10⁶. Hydrophobic chromatography of the detergent-free 100 000-g supernatant on n-hexyl Sepharose 4B results in a fivefold enhancement of specific activity.     

Subcellular distribution of iodothyronine 5′-deiodinase in cerebral cortex from hypothyroid rats

We have examined iodothyronine deiodination in subcellular fractions of cerebral cortex obtained from hypothyroid rats. Enzymatic activities were measured at 37°C in the presence of 20 mM dithiothreitol with ¹²⁵I-labeled T₄ and ¹²⁵I-labeled rT₃ as substrate for 5′-deiodination and ¹³¹I-labeled T₃ as the substrate for the 5-deiodinase. Reaction products were separated by descending paper and/or ion-exchange chromatography. Cerebral cortex subcellular fractions were also characterized by marker enzyme analysis and electron microscopy. Under optimal reaction conditions more than 80% of the 5′-deiodinase was recovered after fractionation. Both 5′-deiodinase and (Na⁺ +K⁺-ATPase showed similar subcellular distributions and were enriched approx. 3-fold in the easily sedimenting membrane fraction and nerve terminal plasma membranes. Crude microsomal membranes (6·10⁶g·min pellet) also showed 2-fold enrichment of these enzymes. Nuclei and isolated mitochondria were devoid of deiodinating activity. T₄ and T₃ 5-deiodinating activity was absent in the easily sedimenting membranes and present but not enriched in particulate fractions containing microsomal membranes. These data suggest that iodothyronine 5′-deiodinase is associated with plasma membrane fractions in the cerebral cortex.     

Marker enzymes,phospholipids and acyl group composition of a somal plasma membrane fraction isolated from rat cerebral cortex: a comparison with microsomes and synaptic plasma membranes

Subjecting brain homogenates to differential speed and sucrose density gradient centrifugation resulted in the isolation of a membrane fraction from the post-mitochondrial supernatant with properties and marker enzyme profiles typical of plasma membranes. This membrane fraction is compared with the microsomes and the synaptic plasma membranes isolated from synaptosomes. Like the synaptic plasma membranes, membranes obtained from the post-mitochondrial supernatant were enriched five-fold in 5′-nucleotidase activity. However, the latter membranes were lower in (Na⁺, K⁺)-ATPase activity and higher in NADPH-cytochrome C reductase activity as compared to the synaptic plasma membranes. The post-mitochondrial plasma membranes were also different from the microsomes in their respective marker enzyme activities. Electron microscopic examination indicated largely membranous vesicles for both plasma membrane fractions with little contamination by myelin, mitochondra and intact synaptosomes. The phospholipid and acyl group profiles of the two plasma membrane fractions were surprisingly similar, but they were different from the characteristic profiles of myelin and mitochondria. It is concluded that plasma membranes isolated from the post-mitochondrial supernatant fraction are derived largely from neuronal and glial soma and are thus designated the somal plasma membrane fraction.     

Further characterization of the in vitro binding of phytochrome to a membrane fraction enriched for mitochondria

This study employs ¹²⁵I-labeled phytochrome (¹²⁵I-P) from oats to quantitate the binding of phytochrome to a membrane fraction from oats that is highly enriched for mitochondria, and it examines several parameters that influence this attachment. The binding of ¹²⁵I-Pfr to the mitochondrial fraction of unirradiated oat seedlings is significantly higher than that of ¹²⁵I-Pr. However, ¹²⁵I-Pfr and ¹²⁵I-Pr bind in equal quantities to mitochondrial preparations isolated from light-exposed seedlings. Maximum ¹²⁵I-Pfr binding to membranes from light-exposed plants occurs within 30 seconds and is optimized in a reaction buffer containing 5 millimolar MgCl₂ at pH 6.8. Scatchard plots of the binding data for Pfr indicate a single high-affinity site with an affinity constant of 1.79 × 10¹¹ per molar. When optimal binding conditions are used, over 20% of the ¹²⁵I-P added is bound and a stoichiometry of about 100 molecules per mitochondrion is attained. When the specificity of binding is tested using competition experiments with a 15-fold excess of unlabeled phytochrome, ¹²⁵I-Pfr shows no specific binding to rat liver mitochondria.