Ultracytochemical localization of X-prolyl-dipeptidyl (amino)peptidase in microglobules and endoplasmic membranes accumulated in pep4-3 mutant of Saccharomyces cerevisiae

The ultracytochemical localization of X-prolyl-dipeptidyl (amino)peptidase (DPP) activity was studied in a late exponential culture of a haploid (alpha) wild-type strain of Saccharomyces cerevisiae and its pep4-3 mutant. Yeast cells were fixed for 20 min in cold 1% glutaraldehyde buffered with 50 mM TES buffer to pH 7.0 and then incubated for 80 min with 1.2 mM L-alanyl-L-proline-4-methoxy-2-naphthylamide (Ala-Pro-MNA) or Lys-Pro-MNA as cytochemical substrates plus 0.06% hexazonium p-rosaniline (HPR) buffered with 160 mM cacodylate to pH 7.0. The osmiophilic azoindoxyl complex was formed by coupling HPR with MNA liberated by DPP activity and was then osmicated during an overnight post-fixation of cells in cold 1% OsO4. In the wild-type strain, conspicuous deposits of DPP reaction product were observed in vacuolar membranes. When compared with the parent strain, the pep4-3 mutant cells were enriched in endoplasmic reticulum (ER), cytoplasmic lipoprotein, and microcompartments: membranous vesicles and microglobules. In the mutant, DPP reaction product was found in about 50% of non-vacuolated cells at the following sites: the nuclear envelope, polar layers of ER sheets and of membranous vesicles (diameter, 40-90 nm), the surface or the lumen of these vesicles, the cytoplasmic membrane (under some bud scars) and the periplasmic space. The largest amount of reaction product was found in microglobules (diameter, 20-50 nm) that were mainly observed in the cytoplasmic matrix but were also present in nuclei (nucleoli) and mitochondria. These microglobules had a single-line boundary and appeared to be composed of lipoprotein. The surface ultrastructure of sectioned microglobules in the cytoplasmic matrix was similar to that of the coated vesicles found in mammalian cells. Only sparse amounts of DPP reaction product were seen in budding yeast. In all pep4-3 cells with electron-lucent vacuoles, the reaction product was confined to the vacuolar membranes (i.e. homologous to the ER), microglobules and the periplasmic space. Polysaccharides with free vic-groups were shown by the cytochemical reaction to be present on the surface of ER membranes, in microglobules, in the periplasmic space and in the cell wall. Our cytochemical results indicate that microglobules participate in the exocytosis of both DPP and glycoproteins, and reveal new features of vacuolar morphogenesis in yeast.     

Cell wall autolysis in log phase cells of Micrococcus lysodeikticus (luteus).

Log phase cells of Micrococcus lysodeikticus (luteus) IFO 3333 autolyzed when incubated at 37 C in 0.01 M sodium-phosphate buffer pH 7.5. The enzyme involved in the autolysis was recovered mainly in an aqueous phase from cytoplasmic membranes and cytoplasmic materials treated with n-butanol, and proved to be an N-acetylmuramyl-L-alanine amidase. The autolysis of log phase cells suspended in autolyzing buffer was depressed by the addition of trypsin to the buffer.     

Conformational changes in surface structures of isolated connexin 26 gap junctions

Gap junction channels mediate communication between adjacent cells. Using atomic force microscopy (AFM), we have imaged conformational changes of the cytoplasmic and extracellular surfaces of native connexin 26 gap junction plaques. The cytoplasmic domains of the gap junction surface, imaged at submolecular resolution, form a hexameric pore protruding from the membrane bilayer. Exhibiting an intrinsic flexibility, these cytoplasmic domains, comprising the C-terminal connexin end, reversibly collapse by increasing the forces applied to the AFM stylus. The extracellular connexon surface was imaged after dissection of the gap junction with the AFM stylus. Upon injection of Ca(2+) into the buffer solution, the extracellular channel entrance reduced its diameter from 1.5 to 0.6 nm, a conformational change that is fully reversible and specific among the divalent cations tested. Ca(2+) had a profound effect on the cytoplasmic surface also, inducing the formation of microdomains. Consequently, the plaque height increased by 0.6 nm to 18 nm. This suggests that calcium ions induce conformational changes affecting the structure of both the hemichannels and the intact channels forming cell-cell contacts.     

Cytoplasmic surface structure in postsynaptic membranes from electric tissue visualized by tannic-acid-mediated negative contrasting

In this study, acetylcholine receptor-rich postsynaptic membranes from electric tissues of the electric rays Narcine brasiliensis and Torpedo californica are negatively contrasted for thin-section electron microscopy through the use of tannic acid. Both outer (extracellular) and inner (cytoplasmic) membrane surfaces are negatively contrasted, and can be studied together in transverse sections. The hydrophobic portion of the membrane appears as a thin (approximately 2 nm), strongly contrasted band. This band is the only image given by membrane regions which are devoid of acetylcholine receptor. In regions of high receptor density, however, both surfaces of the membrane are seen to bear or be associated with material which extends approximately 6.5 nm beyond the center of the bilayer. The material on the outer surface can be identified with the well-known extracellular portion of the receptor molecule. A major portion of the inner surface image is eliminated by extraction of the membranes at pH 11 to remove peripheral membrane proteins, principally the 43,000 Mr (43K) protein. The images thus suggest a cytoplasmic localization of the 43K protein, with its distribution being coextensive with that of the receptor. They also suggest that the 43K protein extends farther from the cytoplasmic surface than does the receptor.     

The structure of Gloeobacter violaceus and its phycobilisomes

The fine structure of the atypical cyanobacterium Gloeobacter violaceus has been studied on frozen-etched replicas and compared to that of a typical unicellular strain: Synechocystis 6701. The complementary fracture faces of G. violaceus cytoplasmic membrane contain particles less numerous and more heterogenous in size than either the cytoplasmic membrane or the thylakoid membranes of Synechocystis. The most frequently observed particles of the exoplasmic fracture (EF) face of the G. violaceus cytoplasmic membrane are 11 nm in diameter and occasionally form short alignments. This particle class is similar in appearance to the numerous, aligned EF particles of Synechocystis thylakoid membranes. In replicas of cross-fractured G. violaceus, a layer 50–70 nm thick, composed of rod-like elements, underlies the inner surface of the cytoplasmic membrane. The rods, 12–14 nm in diameter, are oriented perpendicularly to the cytoplasmic membrane and show a 6 nm repeat along their length.Isolated phycobilisomes of G. violaceus appear, after fixation and negative staining, as bundles of 6 parallel rodshaped elements connected to an ill-defined basal structure. The bundles are 40–45 nm wide and 75–90 nm long. The rods are 10–12 nm in width; their length varies between 50 and 70 nm. These rods are morphologically similar to those observed at the periphery of hemidiscoidal phycobilisomes of other cyanobacteria, with a strong repeat at 6 nm intervals and a weaker one at 3 nm intervals along their length.The calculated molar ratio of phycobiliproteins in isolated G. violaceus phycobilisomes corresponds to 1:3.9:2.9 for allophycocyanin, phycocyanin and phycoerythrin respectively. When excited at 500 nm, isolated phycobilisomes exhibit a major fluorescence emission band centered at 663 nm.Abbreviations PBS phycobilisome(s) - PBP phycobiliprotein(s) - AP allophycocyanin - PC phycocyanin - PE phycoerythrin - K–PO₄ buffer KH₂PO₄ titrated with KOH to a given pH     

Monolayer Film of Phycobilisome-Thylakoid Membrane Complexes from Spirulina platensis

Monolayer films of phycobilisome-thylakoid membrane complexes isolated from Spirulina platensis were prepared at air/aqueous solution interface by using the Langmuir-Blodgett technique. The film preparation was optimized with 0.5 M phosphate buffer (pH 7.0) as sub-phase at 20 °C. The monolayer was transferred into grids and into mica surface for observing the surface image of the complexes by transmission electron microscopy and atomic force microscope, respectively. The shape of complexes was disk-like with the diameter of about 50 nm and the thickness of about 35 nm. The absorption and fluorescence spectra of the complexes in the monolayer were consistent with those in buffer solution, which suggests that the complexes in the monolayer preserve the basic functional groups of photosynthetic apparatus and can be used as a model to investigate the structural connection and functional association of the light-harvesting antenna with the reaction centres.     

Kinetics of adsorption,desorption, and exchange of alpha-chymotrypsin and lysozyme on poly(ethyleneterephthalate) tracked film and track-etched membrane

Adsorption kinetics of (125)I-radiolabeled alpha-chymotrypsin at pH 8.6 was studied in a laminar regime between two walls of poly(ethyleneterephthalate) tracked films and membranes. Adsorption kinetics in the presence of solution (10 microg/mL), desorption by rinsing with buffer, and the following exchange of proteins by flowing unlabeled solution were measured. At pH 8.6, alpha-chymotrypsin is almost neutral and can be mostly removed from the film surface, contrary to positive lysozyme adsorbed at pH 7.4. Results suggest that alpha-chymotrypsin is irreversibly adsorbed in pores, while desorption and exchange occur on membrane flat faces. A method is proposed to determine adsorption kinetics in the pores. Kinetics of desorption and exchange of alpha-chymotrypsin from the film surface can be described by stretched exponential functions in the examined time domain with the same exponent, beta approximately 0.62, which does not depend also on the former adsorption duration. However, the mean residence time at the interface is about 2.5 times greater in the presence of only the buffer than that in the presence of solution. This effect could be explained by a fast exchange at the arrival of unlabeled solution for a part of the adsorbed population.     

CYTOLOGICAL REACTIONS OF NORMAL AND TMV-INFECTED TOBACCO LEAF CELLS TO ACID AND ALKALINE SOLUTIONS

Acid and basic buffer solutions were applied on slides under cover glasses to thin, hand sections of leaf tissue from normal tobacco plants and equivalent plants infected with tobacco mosaic virus (TMV). The effects on living cells were observed and photographed through phase optics. First, reversible changes and, later, irreversible changes were produced in the cells. Movement of the cytoplasm in a cell could be stopped completely and started again by replacing the unfavorable buffer solution with a favorable medium. Of the organelles, plastids became static first, then mitochondria, and lastly spherosomes. Spherosomes often moved actively when all other organelles were still. Translucent virus monolayers, consisting of particles aggregated side by side, provided markers in the parietal cytoplasm of recently infected cells. Mitochondria generally moved across their surface on the side adjacent to the tonoplast, spherosomes across the surface facing the cell wall. Alkaline buffer solution caused little change of texture in nucleus and cytoplasm or change of form in mitochondria. Clear areas appeared in some plastids. The acid buffer emphasized a cytoplasmic network representing flow lines and eliminated many cytoplasmic vesicles; mitochondria became shorter or spherical in outline, grana of chloroplasts more obvious. Virus inclusions, even the fragile monolayers, were not greatly altered until irreversible changes began. In pH 11–treated cells, the final changes included violent bubbling of cytoplasm, and in pH 2.2–treated cells, snapping of flow lines and coagulation of the cytoplasm. In either case, disintegration of cell structure and virus inclusions was rapid.     

Schistosoma mansoni: surface membrane isolation by polycationic beads

The Schistosoma mansoni surface membrane complex was isolated by binding polycationic beads to the worm surface in a sucrose- or sorbitol-acetate buffer, pH 5.0, at 4 C. The ratio of incorporation [3H]cholesterol/[14C]arachidonic acid was measured as well as the specific activities of the alkaline phosphatase (EC 3.1.3.1), Type I phosphodiesterase (EC 3.1.4.1), and Ca2+-adenosine triphosphatase (EC 3.6.1.3). The results indicated that membranes isolated on beads were of comparable or greater purity than membranes isolated by sucrose gradient centrifugation. The isolation procedure was rapid (30 min) and produced membrane fractions whose cytoplasmic surfaces were probably exposed.     

New Method for the Isolation of Membranes from Mycoplasma gallisepticum

Mycoplasma gallisepticum lysed readily in carbonate bicarbonate buffer at pH 9.2 to 10.5. The hemagglutination titer of the lysates was 2- to 16-fold greater than a cell suspension at the same protein concentration in buffered saline. Membranes prepared from cells lysed by this method at pH 10 were relatively free from cytoplasmic contaminants as shown by electron microscopy of thin sections. The membranes retained their hemagglutination activity, gave reactions in immunodiffusion tests identical to those obtained by osmotic lysis and sonic treatment, and showed a similar pattern of protein bands by polyacrylamide disk electrophoresis. When inoculated into rabbits, the membranes gave rise to antibodies active in growth-, metabolic- and hemagglutination-inhibition tests. On the average, membranes obtained by lysis at pH 10 contained 44% of the original cell protein. The method is simple, giving high yields of membranes, and may be adaptable to other mycoplasmas.     

Effects of Sodium and Bicarbonate Ions on γ-Aminobutyric Acid Receptor Binding in Synaptic Membranes of Rat Brain

Crude synaptic membranes treated with Triton X-100 (TX) bound gamma-aminobutyric acid (GABA) to two classes of receptor site in Na+-free 10 mM-Tris-sulfate buffer (pH 7.4), but to only a single class of receptor site in 10 mM Tris-sulfate buffer (pH 7.4), containing 150 mM-NaCl. The high-affinity receptor site in TX membranes was specifically masked in the presence of Na+. However, TX membranes incubated in Krebs-Ringer bicarbonate solution (pH 7.4) bound GABA to two classes of receptor site despite the presence of Na+. It was found that addition of bicarbonate ions to the Na+-containing 10 mM-Tris-sulfate buffer (pH 7.4) could restore that high-affinity class of GABA receptors, rendering both classes detectable. This finding suggests that both Na+ and HCO-3 may have a regulatory function on GABA binding to the receptor.     

Maintenance of Acidic Lateral Intercellular Spaces by Endogenous Fixed Buffers in MDCK Cell Epithelium

The lateral intercellular spaces (LIS) of MDCK cell epithelia grown on permeable supports are about 0.4 pH units acidic to the bathing solutions, presumably because of buffering by the fixed negative charges on the surface of the lateral cell membranes. To test the hypothesis that fixed buffers are responsible for the acidity, a theoretical and experimental approach was developed for the determination of the concentration and pK of the fixed buffer constituted by the glycocalyx. The pH of the solution in the LIS was measured by ratiometric fluorescence microscopy while the buffer concentration or composition of the bathing solutions was altered. In addition, the divalent cation Sr²⁺ was added to the perfusion solutions to displace protons from the fixed buffer sites for the determination of the fixed buffer properties. We conclude that the LIS contain 3.7 mm of pK 6.2 fixed buffer and that this buffer is responsible for the acidic microenvironment in the LIS. Received: 9 April 1998/Revised: 28 July 1998     

Clathrin-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity

Coated pits will assemble onto purified plasma membranes that are attached to a poly-L-lysine coated substratum (Moore, M. S., Mahaffey, D. T., Brodsky, F. M., and Anderson, R. G. W. (1987) Science 236, 558-563; Mahaffey, D. T., Moore, M. S., Brodsky, F. M., and Anderson, R. G. W. (1989) J. Cell Biol. 108, 1615-1624). To better understand the assembly reaction, we have purified both clathrin triskelion and AP-2 subunits from bovine brain and assayed for their ability to bind to the cytoplasmic surface of attached membranes. Two types of membranes were analyzed: those washed with a high pH buffer that selectively removes triskelions and those washed with a high salt buffer that removes both the AP-2 and the triskelion subunits. We found that purified AP-2 subunits bind with high affinity (Kd approximately 3 x 10(-8) M) to salt stripped membranes. Binding is saturable and abolished by treating membranes with less than 20 micrograms/ml of elastase. When membranes were treated with elastase before the salt wash and then salt washed and assayed for AP-2 binding, normal binding was seen, which indicates that the presence of clathrin-coated pits protects the binding site from the protease. Membranes that had rebound AP-2 did not bind purified triskelions, even though high pH buffer-washed membranes that bear endogenous AP-2 bound triskelions with high affinity (Kd approximately 3 x 10(-9) M) and supported lattice assembly. We conclude that coated pit assembly is initiated by the binding of AP-2 to an integral membrane protein but that the AP-2 complex must be activated by an unknown process before the coated pit lattice will assemble.     

Proton flux measurements from tissues in buffered solution

Proton movement across plant cell membranes is part of many important physiological processes. The net proton flux to or from tissues can be determined non-invasively by measuring the proton electrochemical potential gradient in the adjacent solution. In buffered solution, some of the protons crossing the tissue boundary diffuse as proto-nated buffer whose flux is not included in the flux calculated from the proton (hydrogen ion) electrochemical gradient. In this theoretical paper, it is shown how experimenters can calculate the protonated buffer flux from the measured proton flux in solution. The ratio of these two components of total proton flux depends on the pH of the solution and on the concentration and pK of the buffer. For a given concentration of a buffer which has a single pK, the flux ratio rises with pH when the solution pH is lower than the buffer pK. The slope is about 2 on a log₁₀ scale. As the pH increases above the pK, the flux ratio levels off to approach its maximum. With mixed buffers, or one having two or more pK values, the flux ratios are additive: each buffer acts independently based on its concentration and its pK value. Unbuffered solutions always have the buffering effects of water itself and also of carbonates due to carbon dioxide dissolved from the atmosphere. In unbuffered solutions at pH 6, the flux carried by water and carbonate is about 1 % of the measured proton flux. This validates measurements of proton flux from tissues, made by a number of workers, in unbuffered solutions below pH 6.     

Staphylococcal acid phosphatase: extensive purification and characterization of the loosely bound enzyme

Acid phosphatase of Staphylococcus aureus PS55 was eluted from the surface of these cells with 1.0 m KCl at pH 8.5 by gentle agitation at 25 C and was purified 44-fold (51% recovery) by two cycles of dialysis and gel filtration. The eluted enzyme which had a 280/260 (nm) absorbancy ratio of 0.71 required at least 0.5 m salt solution for solubilization; however, most of the purified product which had a 280/260 (nm) absorbancy ratio of 1.72 was soluble in dilute buffer solution [0.01 m tris(hydroxymethyl)aminomethane chloride, pH 8.5]. Purified acid phosphatase appeared homogeneous according to the criteria of gel filtration, starch-block electrophoresis, and analytical ultracentrifugation. In a starch block, migration was toward the cathode at pH 8.0. Maximal activity occurred at pH 5.2 to 5.3 and salt concentration had little effect on phosphatase activity up to 1.0 m KCl or NaCl. Progressive loss of enzymatic acitivity occurred at higher salt concentrations. Molecular weight of purified acid phosphatase was estimated to be 58,000.     

Boar sperm cytoplasmic droplets: Their ultrastructure,their numbers in the epididymis and at ejaculation and their removal during isolation of sperm plasma membranes

Cytoplasmic droplets of the boar are progressively lost from the flagellum of boar spermatozoa during epididymal transit, at ejaculation and during the nitrogen cavitation technique for isolation of plasma membranes. Apparently very fragile, these structures are broken up in the fluids of the reproductive tract and in the buffer used during the nitrogen cavitation procedure. The maximal potential contamination of cytoplasmic droplet internal vesicular membranes in plasma membrane fractions was determined to be 2.2% of the entire membrane surface area collected. The highly sensitive silver-stained, two-dimensional (2-D) polyacrylamide (PAGE) gels of boar sperm plasma membranes did not reveal cytoplasmic droplet, internal membrane, marker polypeptides, further demonstrating the high purity of plasma membrane preparations. In addition, freeze-fracture demonstrates that the internal membranes of the cytoplasmic droplet show few intramembranous particles and these may contribute little protein to plasma membrane preparations. The presence of two forms of vesicular elements in boar sperm Cytoplasmic droplets (typical vesicles and collapsed vesicles) is described.     

Competition between ligands of glycosyltransferases and horseradish peroxidase for binding sites on intracellular and plasma membranes of HeLa cells. Application of a micro-method for the semi-quantitation of surface-bound HRP

A micro-method for the semi-quantitation of surface-bound horseradish peroxidase (HRP) was developed and was applied to study the competition between ligands of glycosyltransferases and HRP for binding sites on the surface of HeLa cells. Dried coverslip cultures of HeLa cells, fixed in methanol, were placed on 0.3 ml of the incubation medium on parafilm and were incubated for 45 min at 37 degrees C. The incubation medium contained HRP, lysozyme and Ca2+ in HEPES buffer, pH 7.2. After washing, the cells were incubated for 60 min at 37 degrees C in HEPES buffer containing 20 mM Ca2+. After this treatment, the plasma membranes showed a strong cytochemical reaction for HRP. Most of the HRP was released into buffer solution during a 5 h incubation at 37 degrees C in the absence of Ca2+, and was measured by spectrophotometry. The addition of 20 mM Ca2+ to the buffer solution prevented the release of most of the HRP from the plasma membranes thus showing that the binding of HRP required Ca2+. Ligands of glycosyltransferases were added to the incubation medium with HRP. The amount of HRP released from the cells decreased in relation to the competing potency and concentration of these ligands. The method was applied to estimate the concentration of some ligands of galactosyltransferase and sialyltransferase that caused a 50% decrease in the release of previously-bound HRP. CMP-neuraminic acid and gangliosides showed a higher competing potency to the surface binding of HRP than UDP-galactose and chitotriose. The spectrophotometric analysis was correlated (on duplicate samples) with cytochemical observations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation ofMycoplasma gallisepticum membranes by a mild alkaline-induced lysis of nonenergized cells

A procedure is described for the preparation of purified membranes from late-exponential or stationary phase cells ofMycoplasma gallisepticum. NonenergizedM. gallisepticum cells were lysed in an isosmotic NaCl-Tris buffer at pH 8.5, conditions that seem to interfere with cell volume regulation. Electron microscopy, chemical, density gradient, and enzymatic analyses of the membrane preparation showed the membranes to be free of cytoplasmic contaminants and partially sealed.     

Electrical properties and molecular architecture of the channel formed by Escherichia coli hemolysin in planar lipid membranes

A 107 kDa hemolysin from Escherichia coli is able to open pores in lipid membranes. By studying its interaction with planar phospholipid bilayers we have derived some structural information on the organization of the pore. We measured the current-voltage characteristic and the ion selectivity of the channel both in neutral membranes, made of egg phosphatidylcholine (PC) and in negatively charged membranes, made of a 1:1 mixture of PC with phosphatidylserine (PS). Experiments were performed varying both the pH and the salt concentration of the bathing KCl solution. In neutral membranes the pore is ohmic and its conductance increases almost linearly with the salt concentration. The channel is cation-selective at high pH but nearly unselective at low pH. We interpret these results in terms of a minimal model based on classical electro-diffusional theories assuming that the pore is wide and bears a negative charge at its entrances. In membranes containing the acidic lipid the current-voltage curve is non-linear in such a way to suggest that the trans (but not the cis) entrance of the pore is affected by the surface potential of the membrane. Applying our model we find that the trans and cis entrances are located, respectively, about 0.5 nm and more than 5 nm apart from the plane of the membrane. We confirmed the asymmetric disposition of the channel by enzymatic digestion of preformed pores. This was effective only when the enzyme was applied on the cis side.