Effect of vanadate, molybdate, and azide on membrane-associated ATPase and soluble phosphatase activities of corn roots

The effects of vanadate, molybdate, and azide on ATP phosphohydrolase (ATPase) and acid phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn (Zea mays L. WF9 × MO17) roots were investigated. Azide (0.1-10 millimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial fraction, while molybdate (0.01-1.0 millimolar) was a relatively selective inhibitor of acid phosphatase activity in the supernatant fraction. The pH 6.4-ATPase activity of the plasma membrane fraction was inhibited by vanadate (10-500 micromolar), but vanadate, at similar concentrations, also inhibited acid phosphatase activity. This result was confirmed for oat (Avena sativa L.) root and coleoptile tissues. While vanadate does not appear to be a selective inhibitor, it can be used in combination with molybdate and azide to distinguish the plasma membrane ATPase from mitochondrial ATPase or supernatant acid phosphatase.

Vanadate appeared to be a noncompetitive inhibitor of the plasma membrane ATPase, and its effectiveness was increased by K⁺. K⁺-stimulated ATPase activity was inhibited by 50% at about 21 micromolar vanadate. The rate of K⁺ transport in excised corn root segments was inhibited by 66% by 500 micromolar vanadate.

     

Failure of lactoperoxidase to iodinate specifically the plasma membrane of cucurbita tissue segments

An attempt has been made to use lactoperoxidase-catalyzed iodination of excised Cucurbita hypocotyl hooks to monitor the distribution of plasma membrane fragments relative to that of phytochrome in particulate fractions from this tissue. Upon fractionation, the iodinated tissue yields a 20,000g pellet which contains 58% of the trichloroacetic acid-precipitable ¹²⁵I at a specific radioactivity 12 times that of the proteins in the supernatant. On sucrose gradients, the labeled fraction has a mean isopycnic density of 1.15 g · cm⁻³. The distribution profile is distinct from that of the total particulate protein and does not coincide with either mitochondrial or endoplasmic reticulum markers. These observations satisfy operational criteria commonly accepted in other systems as indices of selective labeling of the cell surface. The sucrose gradient profiles of the phytochrome and ¹²⁵I in the 20,000g pellets are noncoincident. In the absence of more direct evidence, this is readily interpreted to indicate a lack of association of the pigment with the plasma membrane.     

SUBSTRUCTURE OF AMPHIBIAN MOTOR END PLATE : Evidence for a Granular Component Projecting from the Outer Surface of the Receptive Membrane

End-plate membrane has been examined at amphibian myoneural junctions by means of transmission electron microscopy of thin tissue sections. The postjunctional membrane exhibits morphologically specialized dense, convex patches which are located superficially facing the axon terminal but do not extend into the depths of the junctional folds. In the specialized regions the plasma membrane is ~ 120 Å thick and trilaminar. The outer dense lamina is thickened by the presence in it of granular elements ~60–120 Å in diameter which are spaced semiregularly at ~100–150-Å intervals and which border the junctional cleft directly. In these regions the concentration of the granules is of the order of ~ 10⁴/µm², which is in the same range as the estimated concentration of receptor sites at other vertebrate cholinergic junctions. Filamentous projections can sometimes be seen extending from the granules to the overlying basement membrane, and in oblique views a reticular pattern may appear both in these patches and in the basement membrane. The cytoplasmic surface of the specialized membrane is covered with an amorphous and filamentous dense material whose distribution coincides with that of the granules visible in the outer layer and which may be connected to them across the membrane. In unosmicated specimens stained with permanganate and uranyl acetate the specialized regions exhibit the same morphological features but stand out sharply in contrast to adjacent areas of unspecialized membrane which appear only faintly. Such preparations are particularly useful in assessing the extent of the specialized membrane. It is proposed that the granules visible at the outer surface of the end-plate membrane represent acetylcholine receptors and that in amphibians, as in annelids, the receptors at myoneural junctions are concentrated into patches which occupy less than the total postjunctional membrane surface area.     

Headpiece-stalk particles lining membranes of mitochondria isolated from normal and oligomycin-resistant mutants ofSaccharomyces cerevisiae

Mitochondria isolated from the yeastSaccharomyces cerevisiae were negatively stained with ammonium molybdate. Extensive headpiece-stalk projections were observed lining the disrupted mitochondrial membranes. These observations represent the first clear demonstration of headpiecestalk particles on yeast mitochondrial membranes. Phosphotungstic acid was somewhat less satisfactory than ammonium molybdate in the visualization of the headpiece-stalk particles. Mitochondria isolated from glucose-repressed cells and oligomycin-resistant mutant cells were also examined by negative staining and found to show numerous headpiecestalk elements. Gross differences in the morphology of mitochondria from normal, glucose-repressed and oligomycin-resistant cells, as examined by negative staining, were not apparent in the present studies. The nature and expression of the oligomycin mutation is discussed in terms of possible changes in membrane protein and phospholipid.     

Na,K-ATPase activity and epithelial interfaces in gills of the freshwater shrimp Macrobrachium amazonicum (Decapoda,Palaemonidae)

Diadromous freshwater shrimps are exposed to brackish water both as an obligatory part of their larval life cycle and during adult reproductive migration; their well-developed osmoregulatory ability is crucial to survival in such habitats. This study examines gill microsomal Na,K-ATPase (K-phosphatase activity) kinetics and protein profiles in the freshwater shrimp Macrobrachium amazonicum when in fresh water and after 10-days of acclimation to brackish water (21‰ salinity), as well as potential routes of Na⁺ uptake across the gill epithelium in fresh water. On acclimation, K-phosphatase activity decreases 2.5-fold, Na,K-ATPase α-subunit expression declines, total protein expression pattern is markedly altered, and enzyme activity becomes redistributed into different density membrane fractions, possibly reflecting altered vesicle trafficking between the plasma membrane and intracellular compartments. Ultrastructural analysis reveals an intimately coupled pillar cell-septal cell architecture and shows that the cell membrane interfaces between the external medium and the hemolymph are greatly augmented by apical pillar cell evaginations and septal cell invaginations, respectively. These findings are discussed regarding the putative movement of Na⁺ across the pillar cell interfaces and into the hemolymph via the septal cells, powered by the Na,K-ATPase located in their invaginations.     

REGULAR STRUCTURES IN MEMBRANES : I. Membranes in the Endocytic Complex of Ileal Epithelial Cells

An "apical endocytic complex" in the ileal lining cells of suckling rats is described. The complex consists of a continuous network of membrane-limited tubules which originate as invaginations of the apical plasma membrane at the base of the microvilli, some associated vesicles, and a giant vacuole. The lumenal surface of this tubular network of membranes and associated vesicles is covered with a regular repeating particulate structure. The repeating unit is an ~7.5-nm diameter particle which has a distinct subunit structure composed of possibly nine smaller particles each ~3 nm in diameter. The ~7.5-nm diameter particles are joined together with a center-to-center separation of ~15 nm to form long rows. These linear aggregates, when arranged laterally, give rise to several square and oblique two-dimensional lattice arrangements of the particles which cover the surface of the membrane. Whether a square or oblique lattice is generated depends on the center-to-center separation of the rows and on the relative displacement of the particles in adjacent rows. Four membrane faces are revealed by fracturing frozen membranes of the apical tubules and vesicles: two complementary inner membrane faces exposed by the fracturing process and the lumenal and cytoplasmic membrane surfaces revealed by etching. The outer membrane face reveals a distinct array of membrane particles. This array also sometimes can be seen on the outer (B) fracture face and is sometimes faintly visible on the inner (A) fracture face. Combined data from sectioned, negatively stained, and freeze-etched preparations indicate that this regular particulate structure is a specialization that is primarily localized in the outer half of the membrane mainly in the outer leaflet.     

FLUIDITY OF THE SURFACE OF CULTURED MUSCLE FIBERS : Rapid Lateral Diffusion of Marked Surface Antigens

Fluorescent antibody fragments of anti-muscle plasma membrane antibody bound as small fluorescent spots when applied by micropipetting to cultured myotubes. The spots were observed to enlarge with time. The rate of enlargement of fluorescent spots was greater when fragments were applied than when divalent antibody was used. It was also greater at 23°–25°C than at 0°–4°C. With glutaraldehyde-fixed cells no increase in the size of the spots was seen. The observations are consistent with the spread of fluorescent spots due to diffusion of surface protein antigens within the plane of a fluid membrane. From measurements of spot size against time, a diffusion constant of 1–3 x 10^-9 cm² s^-1 can be calculated for muscle plasma membrane proteins of mol wt approximately 200,000. This value is consistent with other observations on the diffusion of surface antigens and of labeled lipid molecules in synthetic and natural membranes.     

Electron microscopic visualization of the arrangement of the two protein components of (Na + K)-ATPase

The information obtained by electron microscopic examination of highly purified membrane preparations of (Na⁺ + K⁺)-ATPase after freeze-fracturing or negative staining suggests the following conclusions. The catalytic 100 000 dalton protein component penetrates with its greater ‘globular’ mass the plasma membrane and protudes with its smaller mass from the protoplasmic surface by a stalked knob carrying the catalytic centre. The 40 000 dalton glycoprotein component is anchored in the membrane interior by a non-polar ‘fibrous’ side chain, whereas its major polar mass projects from the outer membrane surface forming a surface coat of ill-definable substructure.     

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.     

Subfractionation of rat liver plasma membrane: Uneven distribution of plasma membrane-bound enzymes on the liver cell surface

Plasma membranes were islotaed from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5′-nucleotidase and (Na⁺+K⁺)-ATPase were used. The yield of plasma membrane was 0.6–0.9 mg protein per g wet weight of liver. The recovery of 5′-nucleotidase and (Na⁺+K⁺)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the acitvity of glucose-6 phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5′-nucleotidase, alkaline phosphatase, (Na⁺+K⁺)-ATPase and Mg²⁺-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na⁺+K⁺)-ATPase and Mg²⁺-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphatase was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.     

Surface properties of right side-out plasma membrane vesicles isolated from barley roots and leaves

Highly purified plasma membrane vesicles were obtained from roots and leaves of 7-day-old light-grown barley (Hordeum vulgare L. cv Kristina) seedlings by partitioning of crude microsomal fractions in a dextran-polyethylene glycol two-phase system. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed the polypeptide composition of plasma membranes from the two organs to be qualitatively similar, but with different relative amounts of some of the polypeptides. Between 80 and 100% of the K⁺,Mg²⁺-ATPase activity was latent indicating that the vesicles were sealed and right side-out. The isoelectric points of the outer surface of root and leaf plasma membranes as determined by cross-partitioning were similar and quite acidic—about pH 3.6. In contrast, the net negative surface charge density at pH 7.0 as measured by 9-aminoacridine fluorescence differed significantly, being −29 mC·m⁻² for the leaf plasma membrane and only −19 mC·m⁻² for the root plasma membrane. As isolated, both types of plasma membrane vesicles had Ca²⁺ and Mg²⁺ bound to the outer surface as shown by the combined use of chelators and 9-aminoacridine fluorescence; however, the leaf plasma membrane had a relatively higher proportion of Ca²⁺ bound (0.57) than did the root plasma membrane (0.45). This difference probably reflects differences in the in vivo conditions as no chelator was present during the isolation procedure. Also Ni²⁺ could bind to the root vesicles as indicated by the effect of Ni²⁺ on 9-aminoacridine fluorescence, and by the binding of ⁶³Ni²⁺ (44 nanomoles bound per milligram protein) at 100 micromolar NiCl₂.     

Improved yield of plasma membrane from mammalian cells through modifications of the two-phase polymer isolation procedure

Modifications to the two-phase polymer gradient procedure for isolating plasma membrane from mammalian cells have resulted in greatly increased yields of purified plasma membrane. First, the cells were not treated with a membrane stabilizer (ZnCl₂) prior to homogenization. This reduced the severity of homogenization required for disruption and allowed a greater proportion of the surface membrane to form large, flattened sheets that are more easily purified than the smaller fragments formed during more severe homogenization. Second, three crude fractions obtained from the homogenate (600g, 2000g, and 12,000g pellets), rather than a single, low-speed pellet (600g) containing only large sheets of membrane, were subjected to gradient centrifugation to obtain plasma membrane. This modification allowed purification of small as well as large fragments of plasmalemma and greatly increased the yield of purified membrane. Mg⁺²-dependent, Na⁺-K⁺-stimulated ATPase, a marker enzyme for plasma membrane, was enriched in the purified fraction by ≈17-fold relative to homogenate on a specific activity basis, and the yield of isolated plasma membrane averaged 70%, and was occasionally as high as 90%.     

MEMBRANE SPECIALIZATION AT AN INSECT MYONEURAL JUNCTION

Myoneural junctions were examined in the asynchronous basalar flight muscle of the beetle Pachnoda ephippiata. The outer surface of the postjunctional membrane exhibits an array of prominent projections spaced at ~200 Å intervals which arise directly from the outer dense lamina of the plasma membrane and extend part way across the junctional cleft. The projections follow irregularities in the contour of the postjunctional membrane precisely and they end abruptly near the edge of the junctional region. No separation can be resolved between the projections and the underlying trilaminar plasma membrane after a variety of preparative methods, and the projections therefore appear to be a component part of the membrane. This specialization, which is distinctly different from that at desmosomes and hemidesmosomes, occurs nowhere else on the surface of the muscle and is interpreted as a mosaic of specialized membrane subunits which probably include the receptor sites for the transmitter.     

Localization of pyrophosphatase in membranes of cauliflower inflorescence cells

Using a polyclonal antiserum specific for the tonoplastic H⁺-pyrophosphatase (tPPase), significant amounts of antigenic polypeptides of the correct molecular mass were detected in Western blots of plasma membrane isolated from cauliflower (Brassica oleracea L.) inflorescence by phase-partitioning and subsequent sucrose density centrifugation. Potassium iodide-stripped plasma membranes continued to give a strong positive signal, indicating that the PPase antigen detected was not a result of contamination through soluble PPase released during homogenisation. The same preparation contained negligible vacuolar (v)H⁺-ATPase activity and the A subunit of the vATPase could not be detected by immunoblotting. Plasma membrane fractions exhibited a proton-pumping activity with ATP as substrate, but such an activity was not measurable with pyrophosphate, although the hydrolysis of this substrate was recorded. By contrast, pyrophosphate supported proton pumping in tonoplast-containing fractions. Immunogold electron microscopy confirmed the presence of PPase at the plasma membrane as well as at the tonoplast, trans Golgi network, and multivesicular bodies. The density of immunogold label was higher at the plasma membrane than at the tonoplast, except for membrane fragments occurring in the lumen of the vacuoles which stained very conspicuously. Received: 29 June 1998 / Accepted: 9 November 1998     

Tubular invaginations with caveolae and coated pits in the sinus endothelial cells of the rat spleen

The fine structure of plasmalemmal tubular invaginations with caveolae and coated pits in the sinus endothelial cells of the rat spleen has been demonstrated by scanning and transmission electron microscopy. In addition, the three-dimensional structure of the tubular invagination has been revealed by computer-aided reconstruction. The tubular invaginations of the plasma membrane plunged into the cytoplasm everywhere from the apical, lateral, and basal surfaces of the plasma membrane. The invaginations were tubular and branched away, and their plasma membranes were reinvaginated to form numerous caveolae and occasional coated pits. Numerous caveolae were found in clusters that looked similar to a bunch of grapes and the coated pits were present at the base of the clusters. The caveolae and coated pits derived from the tubular invaginations were almost ultrastructurally identical to those derived from the surface plasma membrane. From examination of the fractured surfaces of the endothelial cells treated with the aldehyde prefix osmium-dimethyl sulfoxide-osmium method and of ultrathin sections of those infiltrated by lanthanum nitrate, the tubular invaginations were found to not penetrate any endothelial cells. A computer-aided reconstruction revealed that the caveolae derived from the tubular invaginations were in close apposition to the surface-connected canaliculi. The reaction product of Concanavalin A conjugated to horseradish peroxidase was present on the outer leaflet of the membranes of the coated pits and coated vesicles and also in the contents of the endosomes, but it was absent from any caveolae. Based on our observations, the functional significance of the tubular invaginations in sinus endothelial cells is discussed. Accepted: 13 September 1999     

Characean charasome-complex and plasmalemma vesicle development

Summary We report on an unusual phenomenon which occurs in some characean algae as a normal plasma membrane activity and also in association with charasome formation. The phenomenon of formation of coated invaginations of the plasma membrane was observed in twoChara and 6Nitella species. These invaginations are coated on their cytoplasmic surface, are 50–60 nm in diameter and rarely exceed 60 nm in length. They are abundant in the young cells ofChara andNitella and also occur in mature cells, but at a lower frequency.N. translucent is an exception in that coated invaginations were few in the young cells and absent in mature cells. Coated vesicles (50–60 nm diameter) were closely associated with these invaginations. Our observations suggest the vesicles may be derived from the invaginations by endocytosis.A close relationship was noted between the development of charasomes (plasmalemma modifications) and coated invaginations. Numerous coated invaginations are seen along the membranes of young charasomes; these invaginations appear to be associated with growth of the charasomes. Coated vesicles were not associated with the coated invaginations of the charasome membrane. The tubular network of cytoplasm and wall space seen in the mature charasome may be formed by fusion of coated invaginations of the developing charasomes, leaving cytoplasmic strands between the fused portions. Coated invaginations were not present along charasomes of the mature cells.     

Morphology, Ultrastructure, and Bacteriophage Infection of the Helical Mycoplasma-Like Organism (Spiroplasma citri gen. nov., sp. nov.) Cultured from “Stubborn” Disease of Citrus

The mycoplasma-like organism Spiroplasma citri gen. nov., sp. nov., isolated from citrus infected with “Stubborn” disease and carried in serial cultures in several media, was examined by dark-field microscopy and electron microscopy of negatively-stained and shadowed preparations and of sections. It grows as motile, helical filaments in liquid, but as nonmotile, nonhelical filaments and round bodies in agar cultures. Helicity and motility are lost in old broth cultures and upon addition of a variety of negative stains, fixatives, and other solutions. No organelles accounting for motility are present, but a layer of surface projections is present on the surface of the single, bounding membrane. The mycoplasma produces a tailed, type B bacteriophage which appear to attach to the outer layer. Helical filaments are preserved in ammonium molybdate, but not in sodium phosphotungstate, and by fixation in Formalin or glutaraldehyde made up in medium, but not by osmium nor by glutaraldehyde in cacodylate buffer. This mycoplasma appears similar to the noncultured helical microorganism in corn stunt-diseased tissues and is probably a representative of a new group of mycoplasmas which are in possession of surface projections, rotary motility, and bacteriophage infection.     

Characterization of a k-stimulated adenosine triphosphatase associated with the plasma membrane of red beet

A membrane fraction enriched with a magnesium-dependent, monovalent cation-stimulated ATPase was isolated from red beet (Beta vulgaris L.) storage roots by a combination of differential centrifugation, extraction with KI, and sucrose density gradient centrifugation. This fraction was distinct from endoplasmic reticulum, Golgi, mitochondrial, and possibly tonoplast membranes as determined from an analysis of marker enzymes. The ATPase activity associated with this fraction was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg²⁺ and K⁺. The activity was substrate specific for ATP and had a temperature optimum near 40°C. Kinetics with Mg:ATP followed a simple Michaelis-Menten relationship. However the kinetics of K⁺-stimulation were complex and suggestive of negative cooperativity. When monovalent cations were present at 2.5 millimolarity, ATPase was stimulated in the sequence K⁺ > Rb⁺ > Na⁺ > Li⁺ but when the concentration was raised to 50 millimolarity, the sequence changed to K⁺ ≥ Na⁺ ≥ Rb⁺ > Li. The activity was not synergistically stimulated by combinations of Na⁺ and K⁺. The enzyme was insensitive to NaN₃, oligomycin, ouabain, and sodium molybdate but sensitive to N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, and sodium vanadate. Based on the similarity between the properties of this ATPase activity and those from other well characterized plant tissues, it has been concluded that this membrane fraction is enriched with plasma membrane vesicles.     

EXCITABLE MEMBRANE ULTRASTRUCTURE : I. Freeze Fracture of Crayfish Axons

Cross-sectioned and cross-fractured crayfish axons display regions in which axon and Schwann cell surface membranes are regularly curved and project into the axoplasm. At these regions (projections) the two membranes run precisely parallel, separated by a gap of 130–140 Å. Longitudinal fractures through the axons expose the inner fractured surface of either the internal (face A) or the external (face B) leaflet of axon and adjacent Schwann cell surface membranes. On both membranes the projections appear as elongated structures oriented with the long axis parallel to the long axis of the nerve fiber. On face A of the axon surface membrane they are seen as elongated indentations 0.5–1.2-µm long, 0.12–0.15-µm wide. The indentations contain parallel chains of globules. The chains repeat every 120–125 Å and are oriented obliquely in such a way that if one looks at the axon surface from the extracellular space, the axis of the chains is skewed counterclockwise to the long axis of the indentations by an acute angle (most often 55–60°). The globules repeat along the chain every 80–85 Å. Globules of adjacent chains are in register in such a way that the axis on which globules of neighboring chains are aligned forms an angle of 75–85° with the axis of the chains. The complex structure can be defined as a globular array with a rhomboidal unit cell of 80–85 x 120–125 Å. On face B of the axon surface membrane the complementary image of these structures is seen. The projections of the Schwann cell surface membrane also contain groupings of globules; however, these differ from those in the axonal projections in size, pattern of aggregation, and fracture properties. Several possible interpretations of the meaning of these membrane specializations could be proposed. They could be: (a) structures involved in the mechanism of excitation, (b) regions of presumed metabolic couplings, and (c) areas of cell-to-cell adhesion.