Phosphatase activities and osmium reduction in cell organelles ofMicrasterias americana

Summary Organelles in resting and growing cells ofMicrasterias americana were examined using electron microscopy after cytochemical procedures for four kinds of phosphatases, acid phosphatase (ACPase), alkaline phosphatase (ALPase), thiamine pyrophosphatase (TPPase), and inosine diphosphatase (IDPase), and osmium tetroxide reduction. Special attention was paid to activities in the Golgi apparatus.In resting cells, positive reactions for ACPase and TPPase were observed in all cisternae of the dictyosome, especially in the peripheral parts. A positive IDPase reaction was seen in one central cisterna and was frequent in the distal-most cisterna. Reduction of osmium tetroxide was seen in the proximal cisternae.In early growing cells, the dictyosomes gave positive reactions for ACPase in the proximal cisternae and the distal cisterna, while in late growing cells only in proximal cisternae. Both in early and late growing cells, the dictyosomes were positive for TPPase and IDPase in the distal cisternae and vesicles derived from the distal cisternae, and for the reduction of osmium tetroxide in the proximal cisternae. ALPase activity was detected in the growing cell wall but not in the dictyosome.     

Identification of secretory vesicles in homogenates of pea stem segments

In homogenates of stem sections from etiolated pea (Pisum sativum L.) seedlings, secretory vesicles can be separated from Golgi-apparatus cisternae by rate-zonal centrifugation in renografin gradients. Optically, two bands of turbidity are observed, the uppermost containing the secretory vesicles and the lower one the Golgi-apparatus cisternae. The absence of glutaraldehyde in the homogenizing medium has allowed the effective characterization of marker-enzyme activities. Golgi-apparatus cisternae have been recognized by the presence of inosine-diphosphatase and glucan-synthase I activities as well as by electron microscopy. In contrast, although secretory vesicles also bear inosine diphosphatase they do not appear to possess glucan-synthase activity. Three plasma-membrane markers, NPA-binding, glucan synthase II, and KCl,Mg²⁺-adenosine triphosphatase (pH 6.5), were not detected in secretory vesicles. Pulse-chase experiments with [³H]glucose support our designation of secretory vesicles and Golgi-cisternal fractions.Abbreviations ER endoplasmic reticulum - GSI, GSII glucan, synthase I, II, respectively - IDPase inosine diphosphatase - PM plasma membrane - SV(s) secretory vesicle(s)     

Golgi apparatus activity and membrane flow during scale biogenesis in the green flagellateScherffelia dubia (Prasinophyceae). I: Flagellar regeneration

Summary Cells ofScherffelia dubia regenerate flagella with a complete scale covering after experimental flagellar amputation. Flagellar regeneration was used to study Golgi apparatus (GA) activity during flagellar scale production. By comparing the number of scales present on mature flagella with the flagellar regeneration kinetics, it is calculated that each cell produces ca. 260 scales per minute during flagellar regeneration. Flagellar scales are assembled exclusively in the GA and abstricted from the rims of thetrans-most GA cisternae into vesicles. Exocytosis of scales occurs at the base of the anterior flagellar groove. The central portion of thetrans-most cisterna, containing no scales, detaches from the stack of cisternae and develops a coat to become a coated polygonal vesicle. Scale biogenesis involves continuous turnover of GA cisternae, and scale production rates indicate maturation of four cisternae per minute from each of the cells two dictyosomes. A possible model of membrane flow routes during flagellar regeneration, which involves a membrane recycling loop via the coated polygonal vesicles, is presented.     

Pentachlorophenol affects mitochondria and induces formation of golgi apparatus-endoplasmic reticulum hybrids in tobacco pollen tubes

Summary The toxic effect of pentachlorophenol (PCP) on the growth and ultrastructure of tobacco pollen tubes was tested using a semivivo technique of tube culture. In this technique the pollen tubes were allowed to grow in the pistilin situ for 24 hr before they protruded from the cut end of the style and came into contact with the medium containing PCP. The inhibitory effect of different PCP concentrations was determined by measuring the length of tube bundles. The intracellular action of PCP was analysed by electron microscopy. This biocide caused four obvious alterations in the pollen tube ultrastructure: (1) swelling of the mitochondrial saccules; (2) enlargement of the dictyosomes by the increase of the cisternal diameter and the number of cisternae per stack; (3) formation of cup-shaped Golgi apparatus-endoplasmic reticulum hybrid structures (GER hybrids) showing continuities of ER and Golgi cisternae; (4) formation of stacked and/or concentric arrangements of rough ER cisternae. It is suggested that swelling of saccules was directly due to the uncoupling of oxidative phosphorylation whereas the changes of the endomembrane system were caused by energy depletion due to the inhibition of ATP synthesis. These changes are consistant with dynamic concepts of dictyosome and ER function when membrane formation exceeds membrane use in the production of secretory and transition vesicles. Thus, the enlargement of the dictyosomes and the formation of GER hybrids are thought to result from inhibition of budding of vesicles from the Golgi apparatus or from both the ER and the Golgi apparatus, respectively.     

Characterization,enzymatic and lectin properties of isolated membranes from Phaseolus aureus

Cellular membranes were prepared from the non-extending part of dark grown hypocotyls of Phaseolus aureus. The relative effectiveness of continuous and discontinuous sucrose gradient centrifugation for the separation of membranes was investigated. Characteristic densities of membranes were determined by the localization of enzyme activities on continuous sucrose gradients: NADH-cytochrome c-reductase for endoplasmic reticulum, β-1-3-glucan synthetase for plasma-membrane and IDPase for dictyosomes. The diffuculties involved in the application of ATPase and IDPase as specific membrane markers are discussed. Negative staining of isolated fractions indicated that intact dictyosomes could be prepared from this tissue without the use of chemical fixatives in the homogenization medium.Extraction of isolated membranes showed that carbohydrate-binding proteins (lectins) were present both in an easily removable and in a more strongly bound form. In vivo incorporation of d-[U-¹⁴C]glucose and subsequent isolation and solubilization of the different membranes showed that sugar-containing polymers could be released without hydrolytic techniques and were present in the equivalent extracts that exhibited lectin activity. The possibility of lectin-polysaccharide complexes in endoplasmic reticulum and dictyosomes and their involvement in the synthesis and transport of secretory substances by the membranes is discussed.     

Characterization, enzymatic and lectin properties of isolated membranes from Phaseolus aureus.

Cellular membranes were prepared from the non-extending part of dark grown hypocotyls of Phaseolus aureus. The relative effectiveness of continuous and discontinuous sucrose gradient centrifugation for the separation of membranes was investigated. Characteristic densities of membranes were determined by the localization of enzyme activities on continuous sucrose gradients: NADH-cytochrome c-reductase for endoplasmic reticulum, beta-1-3-glucan synthetase for plasma-membrane and IDPase for dictyosomes. The difficulties involved in the application of ATPase and IDPase as specific membrane markers are discussed. Negative staining of isolated fractions indicated that intact dictyosomes could be prepared from this tissue without the use of chemical fixatives in the homogenization medium. Extraction of isolated membranes showed that carbohydrate-binding proteins (lectins) were present both in an easily removable and in a more strongly bound form. In vivo incorporation of D-[U-14C]glucose and subsequent isolation and solubilization of the different membranes showed that sugar-containing polymers could be released without hydrolytic techniques and were present in the equivalent extracts that exhibited lectin activity. The possibility of lectin-polysaccharide complexes in endoplasmic reticulum and dictyosomes and their involvement in the synthesis and transport of secretory substances by the membranes is discussed.     

Structure,differentiation, and multiplication of Golgi apparatus in fungal hyphae

Summary Golgi apparatus in subapical regions of hyphae consist of paranuclear dictyosomes with 4–5 cisternae each. Transverse and tangential sections provide ultrastructural evidence for a three-dimensional architectural model of the Golgi apparatus and a stepwise mechanism for dictyosome multiplication. The dictyosomes are polarized, with progressive morphological and developmental differentiation of cisternae from the cis to the trans pole. Small membrane blebs and transition vesicles provide developmental continuity between the nuclear envelope and the adjacent dictyosome cisterna at the cis face. Cisternae are formed as fenestrated plates with extended tubular peripheries. The morphology of each cisterna depends on its position in the stack, consistent with a developmental gradient of progressive maturation and turnover of cisternae. Mature cisternae at the trans face are dissociated to produce spheroid and tubular vesicles. Evidence in support of a schematic sequence for increasing the numbers of dictyosomes comes from images of distinctive and unusual forms of Golgi apparatus in hyphal regions where nuclei and dictyosomes multiply, as follows: (a) The area of the nuclear envelope exhibiting forming-face activity next to a dictyosome expands, which in turn increases the size of cisternae subsequently assembled at the cis face of the dictyosome. (b) As subsequent large cisternae are formed and mature as they pass through the dictyosome, an entire dictyosome about twice normal size is built up. The number of cisternae per stack remains the same because of continuing turnover and loss of cisternae at the trans face, (c) This enlarged dictyosome becomes separated into two by a small region of the nuclear envelope next to the cis face that acquires polyribosomes and no longer generates transition vesicles, (d) As a consequence, assembly of new dictyosomes is physically separated into two adjacent regions, (e) As.the enlarged cisternae are lost to vesiculation at the trans pole, they are replaced by two separate stacks of cisternae with typical normal diameters, (f) The net result is two adjacent dictyosomes where one existed previously. Dictyosome multiplication is thus accomplished as part of the normal developmental turnover of cisternae, without interrupting the functioning of the Golgi apparatus as it continues to produce new secretory vesicles from mature cisternae at the trans face. Coordination of Golgi apparatus multiplication with nuclear division ensures that each daughter nucleus receives a complement of paranuclear dictyosomes.     

Acid phosphatase activity during spore differentiation of the red algaeGigartina teedii andChondria tenuissima

The acid phosphatase activity during carposporogenesis inGigartina and tetrasporogenesis inChondria was studied using the Gomori technique. During the first steps of gonimoblast maturation ofGigartina, portions of cytoplasm are ensheathed by ER cisternae with acid phosphatase activity, giving rise to autolysosomal concentric membrane bodies. In a similar way large mucilage sacs are severed. They extrude their contents in a kind of exocytosis. Multivesicular bodies, concentrically arranged cisternae and extracytoplasmic compartments, each with acid phosphatase activity, remain in young carpospores for some time, probably as remnants of the autophagocytotic and exocytotic events. The Golgi apparatus is poorly developed in gonimoblast cells and young carpospores. It becomes a prominent cell component in maturing carpospores and then participates in cell wall formation. Only some of the dictyosomal cisternae contain acid phosphatase; these are irregularly distributed in the dictyosome. — In pre- and postmeiotic tetraspore mother cells ofChondria massive lead deposits are found in the dictyosomes and in adjacent Golgi vesicles. Finer lead precipitates occur in ER cisternae, especially in those which are sequestering starch-grain-containing portions of the cytoplasm to give rise to autolysosomes. During cell cleavage, the dictyosomes aggregate. They become devoid of acid phosphatase activity with the exception of vesicles at the trans face. Later, Golgi stacks associate and have common, Gomori positively reacting, narrow cisternae at the cis face. The Golgi apparatus derived cored vesicles do not contain lead precipitates whereas the Golgi cisternae in the final stage of tetrasporogenesis show acid phosphatase activity. Variations in acid phosphatase distribution are explained in the light of current models of membrane flow.Dedicated to Univ.-Prof. DrO. Härtel on the occasion of his 80th birthday.     

Effects of induced pinocytotic activity and extreme temperatures on the morphology of golgi bodies in Amoeba proteus

Summary The morphology of the Golgi apparatus of Amoeba proteus can be influenced by substances inducing pinocytotic activity as well as by extreme temperatures. During the ingestion of a solution of 0.5% egg white the number of Golgi bodies decreases from 100% measured in control cells to 82% measured in cells showing induced pinocytosis. Simultaneously the ratio of the surface area of the cisternae at the proximal face to that of the vesicles at the distal face of single dictyosomes remains constant (1.74–1.72).The decrease and increase of the temperature of the culture medium to 4° C and 30° C respectively, causes the disappearance of most of the dictyosomes. After keeping the cells for 3–10 h at these temperatures the number of Golgi bodies was only 5–10% of the controls. A continued treatment with cold or warm culture medium leads to a partial reorganization of dictyosomes. After 15 h the number of Golgi bodies counted per cell returned to 57% at 4° C and 38% at 30° C. The ratio of the surface area of the Golgi cisternae to the surface area of the Golgi vesicles also alters under the influence of extreme temperatures. The values measured after treating the cells for 3 h, 4 h 10 h and 15 h at 4° C and 30° C amounted to 0.75, 0.85, 1.14 1.53 and 0.93, 0.38, 0.88, 1.60, respectively, compared to 1.72 of control amoebae.The different values of the ratio of the surface area of cisternae to that of vesicles indicate that there are strong morphological changes of single dictyosomes.     

Numerical and structural changes in dictyosomes during zygospore germination ofClosterium ehrenbergii

Summary Numerical and structural changes in dictyosomes during the germination of zygospores inClosterium ehrenbergii were examined by electron microscopy. In the dormant mature zygospores, two parallel cisternac were seen which were derived from the disorganization of dictyosomes during the maturation of zygospores. After the induction of germination, the two parallel cisternae developed into dictyosomes with ten or eleven cisternae. The dictyosomes doubled in number by division every day for four days and reached, at the time of germination, a density of distribution similar to that found in the youngest zygospore. On the 4th day after the induction of germination, dictyosomes produced two kinds of vesicles which appear to be involved in the formation of new cell wall layers. The germination of the zygospore was effected by the escape of the cell covered with the new cell wall layers through the broken old cell wall layers.     

Reversible inhibition of secretion in root cap cells of cress after treatment with cytochalasin B: Support for the membrane flow concept

After treatment of cress roots with cytochalasin B (cytB) (25 μg/ml. 5.2 × 10^?5 M) for 4 h, marked structural changes are observed in the peripheral secretory calyptra cells. Deposits of slime outside the plasma membrane are smaller than in cells of untreated roots, whereas secretory vesicles accumulate within the treated cells. Dictyosomes are no longer present and the number of cisternae of rough endoplasmic reticulum surrounding the nucleus is increased at least three-fold. After an 8 h leaching of the drug, the structure of the secretory cells changes again. Accumulation of secretory vesicles no longer takes place, slime is deposited outside the plasma membrane and the number of ER cisternae surrounding the nucleus decreases. On the other hand, dictyosomes are now present. However, they are different from those in the hypertrophied stage of cells exhibiting high secretory activity, but are similar to those of an early developmental stage found at the beginning of the secretion process. This indicates that the dictyosomes are rebuilt during the leaching procedure. The results show that ER membranes accumulate near the nuclear envelope. They also indicate that bulk membrane material is transferred from the RER to the plasma membrane via dictyosome membranes and secretory vesicles, i.e. that membrane flow occurs in secretory cells of higher plants.     

Distribution of golgi apparatus-associated polyribosomes across the polarity axis of dictyosomes of wild carrot (Daucus carota L.)

Summary Endoplasmic reticulum-polyribosome-Golgi apparatus associations were a general feature of cells of suspension cultures of wild carrot (Daucus carota L.). Free polyribosomes occurred within the Golgi apparatus zone for all dictyosomes and with equal frequency at all levels within the stack including the most mature or trans face. When evaluated and quantified from electron micrographs, approximately 60% of the dictyosome profiles were characterized by a system of transition elements consisting of part smooth-part rough endoplasmic reticulum. These were encountered most frequently in the immediate vicinity of the immature, forming or cis face, usually toward the periphery of the stacked cisternae. Analysis of serial sections showed that those dictyosome profiles not exhibiting this characteristic did so primarily because of an unfavorable plane of sectioning. All dictyosomes examined in 5 or more serial sections revealed some type of close association with endoplasmic reticulum. Some of the associations were so close that direct connections between Golgi apparatus and endoplasmic reticulum tubules could not be excluded. Also present, especially at the forming or cis face, were small 600 nm transition vesicles with nap-like surface coats on nearly 90% of the dictyosomes examined. More than 50% exhibited spiny (clathrin-)coated vesicles at the mature or trans face.     

The fine structure of aleurone cells in the soybean seed coat

Summary The morphology and fine structure of aleurone cells of soybean [Glycine max (L.) Merr.] seed coats were analyzed with transmission electron microscopy for the period of rapid seed fill up to physiological maturity. Thin sections and freeze-fracture replicas were prepared for each stage. The aleurone is a tissue lining the embryo sac and consists of a single layer of cells attached to the aerenchyma of the seed coat proper. During seed fill, aleurone cells contained numerous Golgi-derived vesicles in the basal region of the cytoplasm that were either free or attached to the plasma membrane along the lateral and basal regions of the cell wall. Correspondingly, the Golgi apparatus were well developed with individual dictyosomes having 5 to 8, highly fenestrated stacked cisternae. The degree of fenestration along the periphery of each cisterna increased from the cis to trans region. Rough endoplasmic reticulum (RER) was also abundant, often consisting of up to 30, stacked swollen cisternae which occupied large regions of cytoplasm. Plasmodesmata which connected adjacent aleurone cells was not observed along the dorsal walls of aleurone cells that faced aerenchyma. At physiological maturity, dictyosome cisternae were less fenestrated and had fewer associated secretory vesicles. Stacked lamellae of RER were absent, being replaced by short tubular cisternae and small vesicles. At physiological maturity, the aleurone cells had thick walls, and contained numerous lipid bodies in apposition to the plasma membrane. The cytoplasm appeared densely stained in thin-sections and contained protein bodies and amyloplasts with large starch grains. We conclude that during the period of rapid seed fill aleurone cells produce, package, transport and secrete vesicular contents toward the embryo, that is followed at physiological maturity by the storage of lipid, protein and starch in the same cells. The embryo is the most likely destination for secretory products during the period of rapid seed fill. The fate of the stored food reserves in aleurone cells at physiological maturity may be analogous to that of aleurone tissue of grasses, being utilized during imbibition for processes important to germination.     

Selective staining of dictyosome-like structures (DLS) from spermatocytes of the guinea pig using phosphotungstic acid at low pH

Dictyosome-like structures (DLS) occur abundantly in primary spermatocytes of the guinea pig. DLS superficially resemble dictyosomes of Golgi apparatus in that they consist of stacked cisternae and react similarly to some cytochemical markers. DLS saccules are also present in residual bodies and in the cytoplasmic droplet of the sperm, but the stacked configuration (or dictyosome form) is seldom present at these stages of development. A mixture of 1% phosphotungstic acid in 10% chromic acid selectively stains the DLS and DLS saccules of guinea pig germ cells. The thick cisternae of spermatid Golgi apparatus and the sperm plasma membrane also stain, but endoplasmic reticulum and the parts of the Golgi apparatus other than the thick cisternae do not stain. The specificity of the stain is retained in crude homogenates as well as in purified cell fractions and may be helpful in identification of DLS in cell fractionation studies. Additionally, the information obtained provides clues to the origin and fate of DLS in the developing mammalian germ cells.     

Structural differentiation of membranes involved in the secretion of polysaccharide slime by root cap cells of cress (Lepidium sativum L.)

The peripheral secretion tissue of the root cap of Lepidium sativum L. was investigated by electronmicroscopy and freeze-fracturing in order to study structural changes of membranes involved in the secretion process of polysaccharide slime. Exocytosis of slime-transporting vesicles occurs chiefly in the distal region of the anticlinal cell walls. The protoplasmic fracture face (PF) of the plasmalemma of this region is characterized by a high number of homogenously distributed intramembranous particles (IMPs) interrupted by areas nearly free of IMPs. Near such areas slime-transporting vesicles are found to be underlying the plasma membrane. It can be concluded that areas poor in particles are prospective sites for membrane fusion. During the formation of slime-transporting vesicles, the number of IMPs undergoes a striking change in the PF of dictyosome membranes and their derivatives. It is high in dictyosome cisternae and remarkably lower in the budding region at the periphery of the cisternae. Slime-transporting vesicles are as poor in IMPs as the areas of the plasmalemma. Microvesicles rich in IMPs are observed in the surroundings of dictyosomes. The results indicate that in the plasmalemma and in membranes of the Golgi apparatus special classes of proteins — recognizable as IMPs — are displaced laterally into adjacent membrane regions. Since the exoplasmic fracture face (EF) of these membranes is principally poor in particles, it can be concluded that membrane fusion occurs in areas characterized by a high quantity of lipid molecules. It is obvious that the Golgi apparatus regulates the molecular composition of the plasma membrane by selection of specific membrane components. The drastic membrane transformation during the formation of slime-transporting vesicles in the Golgi apparatus causes the enrichment of dictyosome membranes by IMPs, whereas the plasma membrane probably is enriched by lipids. The structural differentiations in both the plasma membrane and in Golgi membranes are discussed in relation to membrane transformation, membrane flow, membrane fusion, and recycling of membrane constituents.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face - IMP intramembranous particle     

Unusual transfer cells in the epithelium of the nectaries ofAsclepias curassavica L.

Summary The secretory cells of the nectaries ofAsclepias curassavica form a glandular epithelium in the inner parts of the stigmatic chambers. They resemble transfer cells in having many infoldings of the plasmalemma. The wall protuberances, however, are poorly developed and often lacking. The plasmalemma is highly convoluted and forms, in places, external compound membranes where the extracytoplasmic space is collapsed completely. Active glands contain dilated cisternae of the ER and large vesicles which are mainly associated with the cis face of the dictyosomes. In addition, small vesicles are observed in high number. It is discussed whether the secretion is granulocrine or eccrine and whether the enlargement of the plasmalemma is the cause or the consequence of the high secretory activity. After the secretory phase the outer peripheral part of the cytoplasm disintegrates. The remaining part of the protoplast is covered by a new plasmalemma.     

Fine structure of the wet,detached cell stigma of the orchid Dendrobium speciosum Sm.

Summary The stigmatic surface of the orchid Dendrobium speciosum is a cup containing detached cells suspended in a mainly carbohydrate mucilage. The fine structure of the detached cells and their organelles is indicative of secretory cells. The cells contain numerous mitochondria with well-developed cristae, dictyosomes containing extensive cisternae, an extensive network of rough and smooth endoplasmic reticulum and free polysomes throughout. There are many amyloplasts in the vicinity of the nucleus. Vesicles are seen arising from the dictyosomes and endoplasmic reticulum. The plasmalemma is undulating, and vesicles can be seen in its vicinity, giving the typical appearance of a granulocrine secretory system. Cetylpyridinium chloride (CPC) fixation to immobilise acidic carbohydrates detected a highly electron-opaque layer surrounding each cell and globules dispersed through the cell wall. The walls of the detached cells show irregular surface projections which are the remains of pitfields. Biochemical analysis showed that carbohydrates and arabinogalactan proteins are major components of the mucilage.     

Further studies of the glandular tissue of the Sauromatum guttatum (Araceae) appendix

Electron microscopic studies showed that the trans-Golgi network (trans indicates the polarity of cisternae within the Golgi apparatus; it is opposite to the cis-face that is adjacent to the rough endoplasmic reticulum) was involved in the processing of the osmiophilic material present in the appendix of the inflorescence of Sauromatum guttatum. This material accumulated in the rough endoplasmic reticulum and in special pockets of the plasma membrane prior to heat production. Associations between the endoplasmic reticulum and trans-Golgi network were observed. The Golgi apparatus was composed of 5–6 dictyosomes on one side and one or two somewhat detached cisternae on the other side. Various nonosmiophilic Golgi-derived vesicles were observed: small ones covered with spike-like material, large ones with a smooth surface, and irregularly shaped ones. These electron-translucent vesicles seemed to accumulate in specific localities at the plasma membrane surface in the vicinity of the osmiophilic material; they were not found when the aroma was released. During heat production, the Golgi structures shrank and the activity of the trans-Golgi network seemed to be reduced. At the same time, coated pits were seen at the plasma membrane surface. In some cells, hypertrophic Golgi apparatuses were seen with only 2–3 dictyosomes that contained granulated material in their lumens. Finally, the osmiophilic material was also found in the plasmodesmata.     

CYTOLOGY OF DIFFERENTIATING TRACHEARY ELEMENTS I. ORGANELLES AND MEMBRANE SYSTEMS

The distribution of organelles, membrane systems, and ribosomes is not at any time obviously related to the pattern of secondary wall in helically thickened tracheary elements in leaves of Beta vulgaris L. (sugar beet) and Cucurbita maxima Duchesne, fixed with potassium permanganate and osmium tetroxide. During the differentiation of the secondary wall, cisternae of the endoplasmic reticulum and dictyosomes are particularly conspicuous, and the dictyosomes are associated with numerous vesicles. Similar vesicles appear to be in various stages of fusion with the secondary wall thickenings. The tracheary elements contain plastids which may include starch granules. Ribosomes occur free in the cytoplasm and in association with endoplasmic membranes.     

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.