scholarly journals Characterization of the pyrophosphate-dependent proton transport in microsomal membranes from maize roots

1988 ◽  
Vol 74 (4) ◽  
pp. 643-650 ◽  
Author(s):  
Alain Chanson ◽  
Paul-Emile Pilet
Keyword(s):  
Proton Transport ◽  
Maize Roots ◽  
Microsomal Membranes

Effects of pH on proton transport by vacuolar pumps from maize roots

1992 ◽  
Vol 86 (1) ◽  
pp. 63-70 ◽  
Author(s):  
David Brauer ◽  
DeNea Conner ◽  
Shu-I Tu
Keyword(s):  
Proton Transport ◽  
Maize Roots ◽  
Effects Of Ph

scholarly journals Identification and characterization of a second 4,4′-dibenzamido-2,2′-stilbenedisulphonate (DBDS)-binding site on band 3 and its relationship with the anion/proton co-transport function

2005 ◽  
Vol 388 (1) ◽  
pp. 343-353 ◽  
Author(s):  
James M. SALHANY ◽  
Karen S. CORDES ◽  
Renee L. SLOAN
Keyword(s):  
Binding Site ◽  
Proton Transport ◽  
Chloride Concentration ◽  
Band 3 ◽  
Low Ph ◽  
Binding Mechanism ◽  
Access Channel ◽  
Binding Kinetic ◽  
Transport Site

Band 3 mediates both electroneutral AE (anion exchange) and APCT (anion/proton co-transport). Protons activate APCT and inhibit AE with the same pK (∼5.0). SDs (stilbenedisulphonates) bind to a primary, high-affinity site on band 3 and inhibit both AE and APCT functions. In this study, we present fluorescence and kinetic evidence showing that lowering the pH activates a second site on band 3, which binds DBDS (4,4′-dibenzamido-2,2′-stilbenedisulphonate) independently of chloride concentration, and that DBDS binding to the second site inhibits the APCT function of band 3. Activation of the second site correlated with loss of chloride binding to the transport site, thus explaining the lack of competition. The kinetics of DBDS binding at the second site could be simulated by a slow-transition, two-state exclusive binding mechanism (R0↔T0+D↔TD↔RD, where D represents DBDS, R0 and T0 represent alternate conformational states at the second DBDS-binding site, and TD and RD are the same two states with ligand DBDS bound), with a calculated overall Kd of 3.9 μM and a T0+D↔TD dissociation constant of 55 nM. DBDS binding to the primary SD site inhibited approx. 94% of the proton transport at low pH (KI=68.5±11.8 nM). DBDS binding to the second site inhibited approx. 68% of the proton transport (KI=7.27±1.27 μM) in a band 3 construct with all primary SD sites blocked through selective cross-linking by bis(sulphosuccinimidyl)suberate. DBDS inhibition of proton transport at the second site could be simulated quantitatively within the context of the slow-transition, two-state exclusive binding mechanism. We conclude that band 3 contains two DBDS-binding sites that can be occupied simultaneously at low pH. The binding kinetic and transport inhibition characteristics of DBDS interaction with the second site suggest that it may be located within a gated access channel leading to the transport site.

scholarly journals Purification and characterization of a major glycoprotein in rat hepatoma plasma membranes. One of the membrane proteins released by phosphatidylinositol-specific phospholipase C

1987 ◽  
Vol 241 (1) ◽  
pp. 63-70 ◽  
Author(s):  
Y Ikehara ◽  
Y Hayashi ◽  
S Ogata ◽  
A Miki ◽  
T Kominami
Keyword(s):  
Alkaline Phosphatase ◽  
Phospholipase C ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Soluble Form ◽  
Microsomal Membranes ◽  
Hydrophobic Nature ◽  
Rat Hepatoma

A major glycoprotein of rat hepatoma plasma membranes was selectively released as a soluble form by incubating the membrane with phosphatidylinositol-specific phospholipase C. The soluble form corresponding to the glycoprotein was also prepared by butan-1-ol extraction of microsomal membranes at pH 5.5, whereas extraction at pH 8.5 yielded an electrophoretically different form with a hydrophobic nature. The soluble glycoprotein extracted at pH 5.5 was purified by sequential chromatography on concanavalin A-Sepharose, Sephacryl S-300 and anti-(alkaline phosphatase) IgG-Sepharose, the last step being used to remove a contaminating alkaline phosphatase. The glycoprotein thus purified was a single protein with Mr 130,000 in SDS/polyacrylamide-gel electrophoresis, although it behaved as a dimer in gel filtration on Sephacryl S-300. The glycoprotein was analysed for amino acid and carbohydrate composition. The composition of the carbohydrate moiety, which amounted to 64% by weight, suggested that the glycoprotein contained much larger numbers of N-linked oligosaccharide chains than those with O-linkage. It was confirmed that the purified glycoprotein was immunologically identical not only with that released by the phospholipase C but also with the hydrophobic form extracted with butan-1-ol at pH 8.5. The results indicate that the glycoprotein of rat hepatoma plasma membranes, which has an unusually high content of carbohydrate, is another membrane protein released by phosphatidylinositol-specific phospholipase C, as documented for alkaline phosphatase, acetylcholinesterase and Thy-1 antigen.

Characterization of cell wall oxalate oxidase from maize roots

Plant Science ◽  
2000 ◽  
Vol 157 (2) ◽  
pp. 257-263 ◽  
Author(s):  
Mirjana Vuletić ◽  
Vesna Hadži-Tašković Šukalović
Keyword(s):  
Cell Wall ◽  
Oxalate Oxidase ◽  
Maize Roots

Characterization of the proteins of the intestinal Na(+)-K(+)-2Cl- cotransporter

1994 ◽  
Vol 267 (2) ◽  
pp. C375-C384 ◽  
Author(s):  
W. Suvitayavat ◽  
P. B. Dunham ◽  
M. Haas ◽  
M. C. Rao
Keyword(s):  
Protein Kinase ◽  
Subcellular Fractionation ◽  
Inhibitory Effect ◽  
Polyclonal Antiserum ◽  
Calyculin A ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Microsomal Membranes ◽  
Dependent Protein

Absorptive intestinal epithelia, such as that of the winter flounder, absorb salt via a bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport mechanism on the brush-border membrane (BBM). The present study demonstrates the first molecular characterization of the intestinal Na(+)-K(+)-2Cl- cotransporter and its unique regulation. The photoaffinity bumetanide analogue, 4-[3H]benzoyl-5-sulfamoyl-3- (3-thenyloxy)benzoic acid, specifically labeled three groups of proteins in flounder intestinal microsomal membranes (MM): a approximately 180-kDa peptide, prominently labeled, and diffuse bands at approximately 110-70 and 50 kDa, less intensely labeled. Subcellular fractionation revealed a single prominently labeled protein of approximately 170 kDa in BBM but not in basolateral membranes (BLM) and little or no labeling of proteins of approximately 110-70 or 50 kDa. Polyclonal antiserum raised against the Ehrlich ascites cell cotransporter identified a 180-kDa peptide in MM and a 175-kDa peptide (pI approximately 5.4) in BBM but none in BLM or in the cytosol of flounder intestine. As predicted from the regulation of cotransport in this tissue, phosphorylation of this protein is increased by guanosine 3',5'-cyclic monophosphate (cGMP)-dependent but not by adenosine 3',5'-cyclic monophosphate-dependent protein kinase. In addition, phosphorylation of the protein is not increased by protein kinase C or Ca2+/calmodulin-dependent protein kinase but is increased by the phosphatase inhibitor calyculin A. Finally, calyculin A preserves the inhibitory effect of cGMP on ion transport, even in the absence of the nucleotide, suggesting that phosphorylation-dephosphorylation mechanisms are crucial in cotransporter regulation. Thus the flounder intestinal cotransporter is a approximately 175-kDa BBM protein that can be regulated by phosphorylation.

scholarly journals Purification of mouse brain (Na+ + K+)-ATPase catalytic unit, characterization of antiserum, and immunocytochemical localization in cerebellum, choroid plexus, and kidney.

1984 ◽  
Vol 32 (12) ◽  
pp. 1309-1318 ◽  
Author(s):  
G J Siegel ◽  
C Holm ◽  
J H Schreiber ◽  
T Desmond ◽  
S A Ernst
Keyword(s):  
Choroid Plexus ◽  
Mouse Brain ◽  
Molecular Layer ◽  
Salt Gland ◽  
Catalytic Unit ◽  
Antibody Method ◽  
Microsomal Membranes ◽  
Intense Activity ◽  
Ependymal Lining

The denatured catalytic polypeptide of mouse brain (Na+ + K+)-adenosine triphosphatase(ATPase) was separated from microsomal membranes on polyacrylamide gels and used as an immunogen. The antiserum, characterized by immunoblots, recognizes the polypeptide corresponding to the catalytic unit in various fractions of mouse brain and cross-reacts with the catalytic unit from lamb kidney, duck salt gland, and electroplax. The same polypeptide in brain and salt gland is recognized by antiserum raised against purified lamb kidney enzyme. Light microscopy was performed with the peroxidase-conjugated second antibody method. In mouse cerebellum, immunochemical staining outlines Purkinje cell and granule cell perikarya. Intense activity is associated with regions of high synaptic content including the pericellular basket meshes and preaxonal regions of Purkinje cells and the glomeruli in the granular layer. In the molecular layer, the neuropil is diffusely reactive with distinct vertically oriented processes evident. White matter exhibits light stain deposition. Choroid plexus presents abundant reaction product only at ependymal apical surfaces, while the ependymal lining of the fourth ventricle displays little or no immunoreactivity. Specificity of the antiserum was demonstrated further in mouse kidney where staining conforms to the well-characterized localization of the enzyme along basolateral surfaces of cortical and medullary tubules. The biochemical and immunocytochemical data show the efficacy of generating antisera to brain (Na+ + K+)-ATPase using catalytic polypeptide as an immunogen.

scholarly journals Characterization of the interaction between p100, a novel G-protein-related protein, and rat liver endosomes

1991 ◽  
Vol 280 (1) ◽  
pp. 171-178 ◽  
Author(s):  
L M Traub ◽  
E Shai ◽  
R Sagi-Eisenberg
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Trypsin Treatment ◽  
Putative Receptor ◽  
Microsomal Membranes ◽  
Direct Binding ◽  
Triton X ◽  
Binding Sequence

p100 is a recently identified 100 kDa protein which shares a putative receptor-binding sequence with the signal transducing G-proteins Gt and Gi. In liver, p100 immunoreactivity is distributed between the cytosolic and the microsomal fractions [Traub, Evans & Sagi-Eisenberg (1990) Biochem. J. 272, 453-458; Udrisar & Rodbell (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6321-6325]. More specifically, we have localized the membrane-associated form of p100 to an endosomal subfraction of rat liver microsomes. In this study we have investigated the nature of the interaction between p100 and microsomal membranes. p100 was located on the cytoplasmic surface of the microsomal vesicles, and could be released by treatment with 0.5 M-NaCl or 0.5 M-Tris/HCl, pH 7.0. However, p100 was not released by non-ionic detergents, such as Triton X-100. Binding of p100 to the membrane was reversible, as both membrane-released and cytosolic p100 could re-bind stripped (Tris-washed) microsomes. Soluble p100 could not, however, bind to untreated microsomes. Binding to stripped microsomes approached saturation and was inhibited by up to 60% by either heat treatment or mild trypsin treatment of the vesicles. This implies that the interaction between p100 and the microsomal vesicles involves the direct binding of p100 to vesicular proteins. This binding was regulated by both adenine and guanine nucleotides. As p100 contains a region similar to the C-terminal decapeptide of alpha i, (the alpha-subunit of Gi) and has a localization that is restricted to an endosomal subfraction, we propose that cytosolic p100 may bind to cytoplasmically exposed domains of internalized receptors. Thus, like the adaptins, p100 may be involved in the process of sorting and receptor trafficking through the endosomal compartment of the cells.

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