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PubMed1990

Cation selectivity of and cation binding to the cGMP-dependent channel in bovine rod outer segment membranes.

The properties of the cGMP-dependent channel present in membrane vesicles prepared from intact isolated bovine rod outer segments (ROS) were investigated with the optical probe neutral red. The binding of neutral red is sensitive to transport of cations across vesicular membranes by the effect of the translocated cations on the surface potential at the intravesicular membrane/water interface (Schnetkamp, P. P. M. J. Membr. Biol. 88: 249-262). Only 20-25% of ROS membrane vesicles exhibited cGMP-dependent cation fluxes. The cGMP-dependent channel in bovine ROS carried currents of alkali and earth alkali cations, but not of organic cations such as choline and tetramethylammonium; little discrimination among alkali cations (K greater than Na = Li greater than Cs) or among earth alkali cations (Ca greater than Mn greater than Sr greater than Ba = Mg) was observed. The cation dependence of cGMP-induced cation fluxes could be reasonably well described by a Michaelis-Menten equation with a dissociation constant for alkali cations of about 100 mM, and a dissociation constant for Ca2+ of 2 mM. cGMP-induced Na+ fluxes were blocked by Mg2+, but not by Ca2+, when the cations were applied to the cytoplasmic side of the channel. cGMP-dependent cation fluxes showed a sigmoidal dependence on the cGMP concentration with a Hill coefficient of 2.1 and a dissociation constant for cGMP of 92 microM. cGMP-induced cation fluxes showed two pharmacologically distinct components; one component was blocked by both tetracaine and L-cis diltiazem, whereas the other component was only blocked by tetracaine.

Animals↗
PubMed1981

Induction of acrosomal reaction in sperm with ionophore A23187 and calcium.

The acrosome reaction was induced in ram, rabbit, and human sperm by incubating with the ionophore A23187 plus calcium ions, although the percentage of reacted sperm was different for each species: 90% in ram, 38% in rabbit, and 69% in man. Fusion of the outer acrosomal and plasma membranes of ram and rabbit sperm formed vesicles, but in human sperm only the outer acrosomal membrane was involved in vesiculation. Acrosin was lost from ram, rabbit, and human sperm after treatment with ionophore plus calcium. Ionophore alone induced the reaction in only 35% of ram sperm, 20% of human sperm, and 12% of rabbit sperm. On exposure to A23187 alone, the acrosome of unreacted rabbit sperm bulged and the acrosome and nuclear membrane of human sperm were often lifted away from the nucleus. The percentage of mammalian sperm undergoing the acrosome reaction in the presence of calcium alone, was very low.

Acrosin↗
PubMed1978

Development of the hamster retina: a morphologic study.

Hamster retinas were examined by light and electron microscopy during the first 15 postnatal days and at the adult stage. At birth, these retinas were more primitive than those of other rodents, but eventually they became very similar. Initial postnatal growth and development were more rapid than in other rodents of comparable ages. Mitotic figures were present throughout the first 9 days. The primitive inner plexiform layer formed during days 2 to 4; on day 7, several developmental changes became apparent: outer segment disks of the photoreceptor cells formed from flattened, smooth vesicles, horizontal cells became readily identifiable, the outer plexiform layer appeared, and ribbon synapses formed in the receptor cell axon terminals. By day 11, there were numerous conventional synapses in the inner plexiform layer, and ribbon synapses were beginning to appear in the terminal portions of the bipolar axons. The retinal tissue was progressively invaded by developing vasculature. The first photoreceptor outer segment disks formed from smooth membranous vesicles flattening radially around the ends of the connecting cilium. After the outer segment assumed a more mature appearance, with a stacked disk arrangement, further disk formation occurred by plasma membrane infoldings at the outer segment base. The adult hamster retinas were similar to those of other rodents.

Animals↗
PubMed1991

Increased matrix vesicle protein in rachitic rat epiphyseal growth plates.

Extracellular, membrane-bound vesicles are widely regarded to be the initial site of calcification in a variety of tissues under normal and pathological conditions. Alkaline phosphatase is believed to play a vital role in this process by hydrolysing ester phosphates or mineral inhibitors, e.g. inorganic phosphates. In the present study, matrix vesicles from normal and rachitic rat growth plates were compared with regard to specific activity of alkaline phosphatase, total vesicle protein and ultrastructural distribution of alkaline phosphatase activity. Matrix vesicles were released from normal or rachitic growth plates by collagenase digestion and isolated by differential centrifugation. Enzyme cytochemical localization involving a cerium capture method was performed on vesicles collected by vacuum filtration on Millipore filters. SDS gels and Western blots on fractions of both normal and rachitic matrix vesicles showed major proteins to be almost identical and confirmed the presence of alkaline phosphatase in both. Total matrix vesicle protein ((mg total matrix vesicle protein/rat) x 10(2)) per rat was significantly greater for the rachitic animals (9.0 +/- 2.0 vs. 4.0 +/- 1.0), P less than 0.0001. Alkaline phosphatase specific activity (units alkaline phosphatase/mg vesicle protein) in the rachitic and normal matrix vesicles was 25.29 +/- 9.36 and 18.78 +/- 3.37, respectively (0.05 less than P less than 0.1). Electron dense cerium phosphate deposits were localized to the outer membrane surface of matrix vesicles derived from both types of rats. This data, the first to quantify the relationship between rickets, matrix vesicle protein and alkaline phosphatase specific activity, suggests that matrix vesicles from rachitic and normal rats have biochemical and morphological similarity.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed2006

Mpp4 recruits Psd95 and Veli3 towards the photoreceptor synapse.

Membrane-associated guanylate kinase (MAGUK) proteins function as scaffold proteins contributing to cell polarity and organizing signal transducers at the neuronal synapse membrane. The MAGUK protein Mpp4 is located in the retinal outer plexiform layer (OPL) at the presynaptic plasma membrane and presynaptic vesicles of photoreceptors. Additionally, it is located at the outer limiting membrane (OLM) where it might be involved in OLM integrity. In Mpp4 knockout mice, loss of Mpp4 function only sporadically causes photoreceptor displacement, without changing the Crumbs (Crb) protein complex at the OLM, adherens junctions or synapse structure. Scanning laser ophthalmology revealed no retinal degeneration. The minor morphological effects suggest that Mpp4 is a candidate gene for mild retinopathies only. At the OPL, Mpp4 is essential for correct localization of Psd95 and Veli3 at the presynaptic photoreceptor membrane. Psd95 labeling is absent of presynaptic membranes in both rods and cones but still present in cone basal contacts and dendritic contacts. Total retinal Psd95 protein levels are significantly reduced which suggests Mpp4 to be involved in Psd95 turnover, whereas Veli3 proteins levels are not changed. These protein changes in the photoreceptor synapse did not result in an altered electroretinograph. These findings suggest that Mpp4 coordinates Psd95/Veli3 assembly and maintenance at synaptic membranes. Mpp4 is a critical recruitment factor to organize scaffolds at the photoreceptor synapse and is likely to be associated with synaptic plasticity and protein complex transport.

Adaptor Proteins, Signal Transducing↗
PubMed1975

Light dependence of osmium reactivity in mouse photoreceptor cells.

Mouse photoreceptor cells exhibit local accumulations of osmium deposits after prolonged osmic staining at slightly elevated temperatures. Deposits were evident along the membranes of outer segment lamellae, Golgi cisternae and vesicles, nuclear envelopes, and synaptic vesicles. Other membranes within the photoreceptor cells were unreactive. No osmium reactivity was seen in other cells of the retina except for osmiophilic outer segment material which had been phagocytized by the pigment epithelium. In the outer segments, inner segments, and synaptic regions of the photoreceptor cells, the amount of osmium reactivity was increased by light stimulation and decreased following extended dark adaptation. The possible significance of the localized osmium reactivity is discussed.

Animals↗
PubMed1995

Immunocytochemical localization of opsin in rod photoreceptors during periods of rapid disc assembly.

Transport of opsin from photoreceptor inner to outer segments has been assumed to occur via the connecting cilium, the only permanent structural connection between these two regions. However, in prior work, little or no immunoreactive opsin has been detected in the cilium, despite the high rate of transport of this protein. This suggests that immune epitopes are masked during passage through the cilium or that opsin is transported via an extra-ciliary route. In this study, we stained the photoreceptors of Xenopus laevis with well-characterized monoclonal antibodies directed at the N-terminal, C-terminal, and 5-6 loop regions of bovine opsin. This was done on isolated retinas incubated in vitro under conditions that support rapid disc assembly, to insure that opsin transport to forming discs was occurring at the time of fixation. Five MAbs that gave robust staining of Xenopus rod inner segment/rod outer segment preparations with the light microscope were utilized for electron microscopic studies on LR White embedded or cryo-ultrathin sections. Four of these stained outer segment discs and inner segment vesicles and plasma membrane. However, no significant staining of the connecting cilium was found. Furthermore, freeze-fractured mouse photoreceptors prepared by the 'fracture-label' technique showed extensive labelling of membrane compartments but lacked staining of the connecting cilium. Isolated retinas incubated under conditions that support robust rod disc synthesis contained many finger-like and vesicular projections of the apical inner segment plasma membrane and inner segment vesicles extending into them. Rod outer segment nascent discs usually made close contact with the inner segment. Both the vesicular profiles associated with the inner segment plasma membrane and the basal discs extending to the inner segment were heavily stained with all four anti-opsin antibodies. This suggests an alternate route for bulk transport of opsin to newly forming discs that involves direct transfer from apical inner segment plasma membrane to nascent discs.

Animals↗
PubMed1994

Precursor-specific requirements for SecA, SecB, and delta muH+ during protein export of Escherichia coli.

We compare translocation into inside-out plasma membrane vesicles (INV) of the in vitro synthesized outer membrane proteins LamB and OmpA and the periplasmic protein Skp of Escherichia coli and demonstrate a precursor-specific dependence on the export factors SecA, SecB, and the proton-motive force (delta mu H+). A partial reduction in soluble SecA caused a 50% decrease in translocation of preLamB. In contrast, removal of INV-bound SecA by urea extraction was required to see a decrease in translocation of preOmpA and preSkp, with 8% of preSkp still being translocated into urea-treated INV. Translocation of the three precursors into INV showed a corresponding differential sensitivity toward dissipation of delta mu H+ following removal of the F1-ATPase from the INV. While depletion of both F1 and SecA or simply lowering of the reaction temperature resulted in an inhibition of complete transmembrane translocation, it interfered less severely with signal sequence cleavage, indicating the formation of translocation intermediates under these conditions. The relative amounts of intermediate obtained were also different for the three preproteins correlating a low requirement for SecA and delta mu H+ with a facilitated initiation of translocation. Whereas preSkp was translocated independently of SecB, preLamB was not even targeted to the INV in its absence. Functional targeting of preOmpA required the presence of SecB during incubation of the precursor with INV and not during its synthesis. SecB, exogenously added during the period of synthesis, did not prevent the formation of translocation-incompetent preLamB. The latter results are consistent with an important targeting function of SecB, which so far has mostly been described as a molecular chaperone. The findings are discussed with respect to current models of bacterial protein export usually derived from the analysis of a single precursor.

Adenosine Triphosphatases↗
PubMed1989

Decrease in the fluidity of brush-border membrane vesicles induced by gentamicin. A spin-labeling study.

In our previous paper (Horio et al., Biochim Biophys Acta 858: 153-160, 1986), we reported that the addition of gentamicin in vitro to rabbit renal brush-border membrane vesicles decreases the apparent Vmax of Na+-dependent D-glucose transport without affecting the apparent Km. In the present study, we investigated the effects of gentamicin on the physical state of spin-labeled rabbit renal brush-border membranes, using electron spin resonance spectrometry. Brush-border membrane vesicles were prepared from outer cortex (mainly contains early proximal tubule) and outer medulla (containing primarily late proximal tubule), and the gentamicin toxicities in both preparations were compared. Significant decreases were observed in the membrane fluidity of 5 mM gentamicin-treated brush-border membranes. The fluidity of outer cortical brush-border membranes was affected at both 25 degrees and 35 degrees, whereas that of outer medullary membranes was affected only at 35 degrees. Two different stearic acid spin labels revealed that gentamicin affected the fluidity only in the superficial region of the membranes. We also demonstrated that the gentamicin-induced decreases in Na+-dependent D-glucose transport and in the membrane fluidity were recovered by washing gentamicin-treated brush-border membranes. We suggest that gentamicin binds to the superficial region of brush-border membranes and inhibits Na+-dependent D-glucose transport across brush-border membranes through the decrease in the membrane fluidity.

Animals↗
PubMed1991

Role of the Yersinia outer membrane protein YadA in adhesion to rabbit intestinal tissue and rabbit intestinal brush border membrane vesicles.

The Yersinia virulence plasmid confers on strains of Yersinia pseudotuberculosis and Y. enterocolitica an adhesive potential superior to the one encoded by the chromosome alone. We have evaluated the role of the plasmid-encoded outer membrane protein YadA (formerly called Yopl) in adhesion. Insertional inactivation of the yadA gene (formerly called yopA), which encodes YadA, led to a reduction in the capacity of plasmid-carrying strains of Y. pseudotuberculosis 0:III and Y. enterocolitica 0:9 to adhere to intestinal tissue, brush border membranes and polystyrene surfaces. The adhesive characteristics of the mutants were comparable to those of their plasmid-cured counterparts. When the yadA gene from Y. pseudotuberculosis serotype 0:III or Y. enterocolitica serotype 0:3 or 0:8 was cloned into an Escherichia coli strain, increased ability to adhere to intestinal tissue, brush border membrane vesicles and polystyrene was transferred concomitantly. The introduction of the yadA gene from Y. pestis, which is unable to express YadA due to a one base pair deletion, did not change the adhesive characteristics of E. coli. Expression of YadA in the outer membrane may, therefore, make an important contribution to intestinal adherence of the two enteropathogenic members of the Yersinia species, Y. pseudotuberculosis and Y. enterocolitica.

Animals↗
PubMed2000

Targeting and insertion of C-terminally anchored proteins to the mitochondrial outer membrane is specific and saturable but does not strictly require ATP or molecular chaperones.

A distinct class of proteins contain a C-terminal membrane anchor and a cytoplasmic functional domain. A subset of these proteins is targeted to the mitochondrial outer membrane. Here, to probe for the involvement of a saturable targeting mechanism for this class of proteins, and to elucidate the roles of chaperone proteins and ATP, we have utilized an in vitro targeting system consisting of in vitro-synthesized proteins and isolated mitochondria. To establish the specificity of targeting we have used a closely related protein pair. VAMP-1A and VAMP-1B are splice variants of the vesicle-associated membrane protein/synaptobrevin-1 (VAMP-1) gene. In intact cells VAMP-1B is targeted to mitochondria whereas VAMP-1A is targeted to membranes of the secretory pathway, yet these isoforms differ by only five amino acids at the extreme C-terminus. Here we demonstrate that, in vitro, VAMP-1B is imported into both intact mitochondria and mitochondrial outer-membrane vesicles with a 15-fold greater efficiency than VAMP-1A. We generated and purified bacterially expressed fusion proteins consisting of the C-terminal two-thirds of VAMP-1A or -1B proteins fused to glutathione S-transferase (GST). Using these fusion proteins we demonstrate that protein targeting and insertion is saturable and specific for the VAMP-1B membrane anchor. To elucidate the role of cytosolic chaperones on VAMP-1B targeting, we also used the purified, Escherichia coli-derived fusion proteins. (33)P-Labelled GST-VAMP-1B(61-116), but not GST-VAMP-1A(61-118), was efficiently targeted to mitochondria in a chaperone-free system. Thus the information required for targeting is contained within the targeted protein itself and not the chaperone or a chaperone-protein complex, although chaperones may be required to maintain a transport-competent conformation. Moreover, ATP was required for transport only in the presence of cytosolic chaperone proteins. Therefore the ATP requirement of transport appears to reflect the participation of chaperones and not any other ATP-dependent step. These data demonstrate that targeting of C-terminally anchored proteins to mitochondria is sequence specific and mediated by a saturable mechanism. Neither ATP nor chaperone proteins are strictly required for either specific targeting or membrane insertion.

Adenosine Triphosphate↗
PubMed1987

Characterization of translocation contact sites involved in the import of mitochondrial proteins.

Import of proteins into the mitochondrial matrix requires translocation across two membranes. Translocational intermediates of mitochondrial proteins, which span the outer and inner membrane simultaneously and thus suggest that translocation occurs in one step, have recently been described (Schleyer, M., and W. Neupert, 1985, Cell, 43:339-350). In this study we present evidence that distinct membrane areas are involved in the translocation process. Mitochondria that had lost most of their outer membrane by digitonin treatment (mitoplasts) still had the ability to import proteins. Import depended on proteinaceous structures of the residual outer membrane and on a factor that is located between the outer and inner membranes and that could be extracted with detergent plus salt. Translocational intermediates, which had been preformed before fractionation, remained with the mitoplasts under conditions where most of the outer membrane was subsequently removed. Submitochondrial vesicles were isolated in which translocational intermediates were enriched. Immunocytochemical studies also suggested that the translocational intermediates are located in areas where outer and inner membranes are in close proximity. We conclude that the membrane-potential-dependent import of precursor proteins involves translocation contact sites where the two membranes are closely apposed and are linked in a stable manner.

Biological Transport↗
PubMed1998

Identification of hemolytic activity in Prevotella intermedia.

Hemolysin production was measured in strains of Prevotella intermedia. Zones of beta-hemolysis were detected on agar plates supplemented with either sheep, rabbit or human erythrocytes. A standard tube assay was performed on cell suspensions of the organism to measure hemolytic activity, which was found to be dose dependent, eliminated by heat treatment, and saturable with increasing concentrations of blood. Growth-phase experiments suggested that hemolysin production was increased during logarithmic growth and was reduced during stationary phase. Cell fractionation, performed on several strains of P. intermedia, localized the activity in the outer membrane and in cell vesicles. The biological implication of this study is that P. intermedia, by virtue of its hemolytic activity, is capable of liberating the hemoglobin from erythrocytes, thereby acquiring an essential nutrient, iron, for its metabolism.

Animals↗
PubMed1996

Interaction of transducin with light-activated rhodopsin protects It from proteolytic digestion by trypsin.

The tryptic cleavage pattern of transducin (Gt) in solution was compared with that in the presence of phospholipid vesicles, rod outer segment (ROS) membranes kept in the dark, or ROS membranes containing light-activated rhodopsin, metarhodopsin II (Rh*). When Gt was in the high affinity complex with Rh*, the alphat subunit was almost completely protected from proteolysis. The protection of alphat at Arg310 was complete, while Arg204 was substantially protected. The cleavage of alphat at Lys18 was protected in the presence of phospholipid vesicles, ROS membranes kept in the dark, or ROS membranes containing Rh*. The cleavage of betat was slower in the presence of ROS membranes or phospholipid vesicles. When the Rh*. Gt complex was incubated with guanyl-5'-yl thiophosphate, a guanine nucleotide analog known to release the high affinity interaction between Gt and Rh*, the protection at Arg310 and Arg204 was diminished. From our results, we propose that Rh* either physically blocks access of trypsin to Arg204 and Arg310 or maintains the heterotrimer in such a conformation that these cleavage sites are not available. Since Arg204 is involved in the switch interface with betagammat (Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319), it may be that betagammat is implicated in protecting this cleavage site in the receptor-bound, stabilized heterotrimer. Arg310 is not near the betagammat subunit, thus we believe that the high affinity binding of Gt to Rh* physically or sterically blocks access of trypsin to this site. Thus, Arg310, only a few angstroms away from the carboxyl terminus of alphat, which is known to directly bind to Rh*, is likely to also be a part of the Rh* binding site. This is in agreement with other studies and has implications for the mechanism by which receptors catalyze GDP release from G proteins. The protection of Lys18 in the presence of phospholipid vesicles suggests that the amino-terminal region is in contact with the membrane, consistent with the crystal structure of the heterotrimer (Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319).

Amino Acid Sequence↗
PubMed1979

The effects of triton X-100 on the transfer of mannose, glucose and n-acetylglusomine phosphate to dolichol monophosphate by preparations of rough and smooth endoplasmic reticulum and of mitochondria of rat liver.

Triton X-100 and exogenous dolichol monophosphate have been used to investigate the nature of enzymes responsible for the transfer of mannose, glucose and N-acetylglucosamine phosphate from nucleotide donors to dolichol monophosphate in vesicles derived from rough and smooth endoplasmic reticulum and mitochondria. Mitochondria were shown to contain the highest specific activities of these enzymes. The responses of the glycosyltransferases to increasing concentrations of Triton X-100 and the effect on these responses of exogenous dolichol monophosphate suggest that the enzymes for mannose and glucose transfer are less hydrophobic, and therefore less intrinsic, in the membrane than the enzyme for N-acetylglucosamine phosphate transfer. In smooth vesicles the results are consistent with mannosyl- and glucosyl-transferases being located at both inner and outer faces of the membrane. In rough vesicles and in mitochondria mannosyl- and glucosyl-transferases were confirmed at the outer face. There is, however, only one site of N-acetylglucosamine phosphate transfer, this being more hydrophobically located in the membrane than the other sites of glycosyl transfer. Mitochondrial enzyme activity closely resembled that of rough endoplasmic reticulum in response to Triton X-100 and exogenous dolichol monophosphate, and is probably associated with the outer membrane.

Acetylglucosamine↗
PubMed2006

Fluorescence microscopic investigation on morphological changes of giant multilamellar vesicles induced by amphiphilic additives.

Adding an artificial bolaamphiphile to a dispersion of giant multilamellar vesicles (GMVs) made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) induced a cup-shaped deformation in GMVs accompanied by partial extrusion of the inner vesicle; thereafter, the deformed vesicles returned to their original shape. On the other hand, when the artificial bolaamphiphile together with a surfactant was added to the vesicular dispersion, these deformation and reformation dynamics were transmitted from the outer membranes in GMVs to the inner membranes until an intact inner vesicle was extruded out of the outer membrane. The microscopic aspects of these processes were investigated using amphiphiles tagged with individual fluorophores.

Journal Article↗
PubMed1984

The static head method for determining the charge stoichiometry of coupled transport systems. Applications to the sodium-coupled D-glucose transporters of the renal proximal tubule.

The static head method for determining the charge stoichiometry (the number of moles of charge translocated per mole of substrate) of a coupled transport system is presented. The method involves establishing experimental conditions under which a membrane potential exactly balances the thermodynamic driving force of a known substrate gradient. The charge stoichiometry can then be calculated from thermodynamic principles. In contrast to the usual steady-state method for determining charge stoichiometry in cell suspensions and vesicle preparations, the static head method is applicable to systems which are not capable of maintaining a constant membrane potential over time. The charge stoichiometries of two renal sodium coupled D-glucose transporters previously identified in brush-border membrane vesicle preparations from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) are determined. The charge stoichiometries of these transporters are in good agreement with their sodium/glucose coupling ratios arguing against the possibility that glucose transport is coupled to ions other than sodium in these membranes.

Animals↗