Preliminary Identification of ATP Compartment Associated Proteins On Erythrocyte Membrane.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3019-3019
Author(s):  
Haiyan Chu ◽  
Joseph F. Hoffman ◽  
Philip S. Low
Keyword(s):  
Erythrocyte Membrane ◽  
Conflicts Of Interest ◽  
Uv Light ◽  
Junctional Complex ◽  
Preliminary Identification ◽  
Atp Pool ◽  
Associated Proteins ◽  
Membrane Bound ◽  
Protein Components ◽  
Blocking Antibodies

Abstract Abstract 3019 Poster Board II-995 A compartment/pool of ATP that is generated by glycolytic enzymes has been reported to exist on the erythrocyte membrane where it channels ATP directly to the Na+/K+-ATPase (Proverbio F and Hoffman JF. J. Gen. Physiol. 1977, 69:605-632. Mercer RW and Dunham PB. J. Gen. Physiol. 1981, 78:547-567). In order to identify the protein components enclosing this compartment of ATP, a photoactivatable probe, 8-azido-[αa-32P]ATP, was loaded into porous erythrocytes under conditions that fill the compartment with ATP, and the radiolabeled ATP was induced to label proximal proteins by illumination with UV light. Analysis of radiolabeled bands reveals that spectrin, adducin, protein 4.1, and actin constitute major components of the compartment. To further verify the involvement of these proteins in the ATP compartment, antibodies against the aforementioned proteins were pre-incubated with porous erythrocytes before attempting to load the compartment with ATP. Analysis of the efficiency of ATP loading in the presence of these blocking antibodies reveals that antibody binding prevents normal filling of the ATP pool. These findings confirm Hoffman's earlier hypothesis that a membrane-bound compartment of ATP exists. The data also suggest that the location of the compartment might reside at the junctional complex, and that the complex of enzymes that fill the ATP pool is different from the complex organized around band 3. Disclosures No relevant conflicts of interest to declare.

scholarly journals Adducin forms a bridge between the erythrocyte membrane and its cytoskeleton and regulates membrane cohesion

Blood ◽  
2009 ◽  
Vol 114 (9) ◽  
pp. 1904-1912 ◽  
Author(s):  
William A. Anong ◽  
Taina Franco ◽  
Haiyan Chu ◽  
Tahlia L. Weis ◽  
Emily E. Devlin ◽  
...  
Keyword(s):  
Erythrocyte Membrane ◽  
Membrane Skeleton ◽  
Band 3 ◽  
Membrane Stability ◽  
Junctional Complex ◽  
C Protein ◽  
Protein 4.1 ◽  
Protein Components ◽  
Actin Protein ◽  
Glycophorin C

Abstract The erythrocyte membrane skeleton is the best understood cytoskeleton. Because its protein components have homologs in virtually all other cells, the membrane serves as a fundamental model of biologic membranes. Modern textbooks portray the membrane as a 2-dimensional spectrin-based membrane skeleton attached to a lipid bilayer through 2 linkages: band 3–ankyrin–β-spectrin and glycophorin C–protein 4.1–β-spectrin.1–7 Although evidence supports an essential role for the first bridge in regulating membrane cohesion, rupture of the glycophorin C–protein 4.1 interaction has little effect on membrane stability.8 We demonstrate the existence of a novel band 3–adducin–spectrin bridge that connects the spectrin/actin/protein 4.1 junctional complex to the bilayer. As rupture of this bridge leads to spontaneous membrane fragmentation, we conclude that the band 3–adducin–spectrin bridge is important to membrane stability. The required relocation of part of the band 3 population to the spectrin/actin junctional complex and its formation of a new bridge with adducin necessitates a significant revision of accepted models of the erythrocyte membrane.

scholarly journals Rapid purification of glycosyl-phosphatidylinositol-anchored alkaline phosphatase from human neutrophils after up-regulation to the cell surface.

1993 ◽  
Vol 41 (9) ◽  
pp. 1367-1372 ◽  
Author(s):  
T J Cain ◽  
Y Liu ◽  
T Kobayashi ◽  
J M Robinson
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Surface ◽  
Human Neutrophils ◽  
Extracellular Medium ◽  
Isolation And Purification ◽  
Glycosyl Phosphatidylinositol ◽  
Associated Proteins ◽  
Membrane Bound ◽  
Pre Treatment ◽  
Rapid Purification

Alkaline phosphatase (APase) belongs to a growing family of membrane-associated proteins tethered to the lipid bilayer via a glycosyl-phosphatidylinositol (GPI) anchor. Human neutrophils contain an intracellular pool of APase associated with a novel membrane-bound compartment. Stimulation of neutrophils with the chemotactic peptide formyl-Met-Leu-Phe (fMLP) leads to rapid up-regulation of essentially all of the APase to sites in continuity with the extracellular medium. Pre-treatment of neutrophils with cytochalasin B (cyto B) followed by fMLP likewise leads to expression of the enzyme on the cell surface and a dramatic alteration in cell morphology, but subsequent internalization of the plasmalemma is minimized. Pre-treatment with cyto B and fMLP has been used for isolation and purification of neutrophil APase. Specifically, neutrophils were treated with phosphatidylinositol-specific phospholipase C to release GPI-anchored proteins from the cell surface. APase was purified from supernatants of these preparations by electrophoresis in a non-denaturing gel system and subsequent electroelution. With this approach we rapidly purified neutrophil APase to homogeneity; this protein was then used for immunization. Immunoblotting, ELISA, and immunocytochemical localization were used to characterize the resulting antibodies.

scholarly journals Identification and Analysis of Bacterial Protein Secretion Inhibitors Utilizing a SecA-LacZ Reporter Fusion System

2000 ◽  
Vol 44 (6) ◽  
pp. 1418-1427 ◽  
Author(s):  
L. E. Alksne ◽  
P. Burgio ◽  
W. Hu ◽  
B. Feld ◽  
M. P. Singh ◽  
...  
Keyword(s):  
Protein Secretion ◽  
Antimicrobial Agents ◽  
Membrane Integrity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Reporter Construct ◽  
Central Protein ◽  
Bona Fide ◽  
Membrane Bound ◽  
Protein Components

ABSTRACT Protein secretion is an essential process for bacterial growth, yet there are few if any antimicrobial agents which inhibit secretion. An in vivo, high-throughput screen to detect secretion inhibitors was developed based on the translational autoregulation of one of the central protein components, SecA. The assay makes use of a SecA-LacZ fusion reporter construct in Escherichia coli which is induced when secretion is perturbed. Several compounds, including two natural product extracts, which had the ability to induce the reporter fusion were identified and the MICs of these compounds forStaphylococcus aureus strain MN8 were found to be ≤128 μg/ml. Enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation techniques were used to analyze the affects of these compounds on protein secretion. Six representative compounds presented here appear to be bona fide secretion inhibitors but were found to have deleterious effects on membranes. It was concluded that, while the method described here for identifying inhibitors of secretion is valid, screens such as this, which are directed against the membrane-bound portion of a pathway, may preferentially identify compounds which affect membrane integrity.

Vinculin but not alpha-actinin is a target of PKC phosphorylation during junctional assembly induced by calcium

1998 ◽  
Vol 111 (23) ◽  
pp. 3563-3571 ◽  
Author(s):  
M. Perez-Moreno ◽  
A. Avila ◽  
S. Islas ◽  
S. Sanchez ◽  
L. Gonzalez-Mariscal
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Actin Binding ◽  
Junctional Complex ◽  
Cell Periphery ◽  
Protein Kinase C Inhibitors ◽  
Kinase C ◽  
Phosphorylation Pattern ◽  
Associated Proteins ◽  
Level 2

The establishment of the junctional complex in epithelial cells requires the presence of extracellular calcium, and is controlled by a network of reactions involving G-proteins, phospholipase C and protein kinase C. Since potential candidates for phosphorylation are the tight junction associated proteins ZO1, ZO2 and ZO3, in a previous work we specifically explored these molecules but found no alteration in their phosphorylation pattern. To continue the search for the target of protein kinase C, in the present work we have studied the subcellular distribution and phosphorylation of vinculin and alpha-actinin, two actin binding proteins of the adherent junctions. We found that during the junctional sealing induced by Ca2+, both proteins move towards the cell periphery and, while there is a significant increase in the phosphorylation of vinculin, alpha-actinin remains unchanged. The increased phosphorylation of vinculin is due to changes in phosphoserine and phosphothreonine content and seems to be regulated by protein kinase C, since: (1) DiC8 (a kinase C stimulator) added to monolayers cultured without calcium significantly increases the vinculin phosphorylation level; (2) H7 and calphostin C (both protein kinase C inhibitors) completely abolish this increase during a calcium switch; (3) inhibition of phosphorylation during a calcium switch blocks the subcellular redistribution of vinculin and alpha-actinin. These results therefore suggest that vinculin phosphorylation by protein kinase C is a crucial step in the correct assembly of the epithelial junctional complex.

scholarly journals Band 3 ectopic expression in colorectal cancer induces an increase in erythrocyte membrane-bound IgG and may cause immune-related anemia

2020 ◽  
Vol 111 (5) ◽  
pp. 657-666
Author(s):  
Akihito Kitao ◽  
Shinichiro Kawamoto ◽  
Keiji Kurata ◽  
Ikuyo Hayakawa ◽  
Takashi Yamasaki ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Erythrocyte Membrane ◽  
Ectopic Expression ◽  
Band 3 ◽  
Membrane Bound

scholarly journals Network Analysis of a Membrane-Enriched Brain Proteome across Stages of Alzheimer’s Disease

Proteomes ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 30 ◽  
Author(s):  
Lenora Higginbotham ◽  
Eric Dammer ◽  
Duc Duong ◽  
Erica Modeste ◽  
Thomas Montine ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Network Analysis ◽  
Proteomic Analysis ◽  
Expression Patterns ◽  
Label Free ◽  
Post Mortem Brain ◽  
Brain Proteome ◽  
Associated Proteins ◽  
Membrane Bound

Previous systems-based proteomic approaches have characterized alterations in protein co-expression networks of unfractionated asymptomatic (AsymAD) and symptomatic Alzheimer’s disease (AD) brains. However, it remains unclear how sample fractionation and sub-proteomic analysis influences the organization of these protein networks and their relationship to clinicopathological traits of disease. In this proof-of-concept study, we performed a systems-based sub-proteomic analysis of membrane-enriched post-mortem brain samples from pathology-free control, AsymAD, and AD brains (n = 6 per group). Label-free mass spectrometry based on peptide ion intensity was used to quantify the 18 membrane-enriched fractions. Differential expression and weighted protein co-expression network analysis (WPCNA) were then used to identify and characterize modules of co-expressed proteins most significantly altered between the groups. We identified a total of 27 modules of co-expressed membrane-associated proteins. In contrast to the unfractionated proteome, these networks did not map strongly to cell-type specific markers. Instead, these modules were principally organized by their associations with a wide variety of membrane-bound compartments and organelles. Of these, the mitochondrion was associated with the greatest number of modules, followed by modules linked to the cell surface compartment. In addition, we resolved networks with strong associations to the endoplasmic reticulum, Golgi apparatus, and other membrane-bound organelles. A total of 14 of the 27 modules demonstrated significant correlations with clinical and pathological AD phenotypes. These results revealed that the proteins within individual compartments feature a heterogeneous array of AD-associated expression patterns, particularly during the preclinical stages of disease. In conclusion, this systems-based analysis of the membrane-associated AsymAD brain proteome yielded a unique network organization highly linked to cellular compartmentalization. Further study of this membrane-associated proteome may reveal novel insight into the complex pathways governing the earliest stages of disease.

Two-dimensional electrophoretic analysis of erythrocyte membranes.

1982 ◽  
Vol 28 (4) ◽  
pp. 925-931 ◽  
Author(s):  
B B Rosenblum ◽  
S M Hanash ◽  
N Yew ◽  
J V Neel
Keyword(s):  
Erythrocyte Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Electrophoretic Analysis ◽  
High Molecular Mass ◽  
Erythrocyte Membranes ◽  
Two Dimensional ◽  
Two Dimensional Electrophoresis ◽  
Protein Components ◽  
Erythrocyte Membrane Proteins ◽  
Dimensional Electrophoresis

Abstract In an effort to maximize the amount of genetic information that can be extracted from a blood sample, we investigated the use of two-dimensional polyacrylamide-gel electrophoresis (PAGE) to resolve the protein constituents of the erythrocyte membrane. Lyophilized membranes were dissolved in various concentrations of urea, NP-40 detergent, and mercaptoethanol and subjected to two-dimensional PAGE by a modification of the O'Farrell procedure, with use of the ISO-DALT apparatus. More than 600 spots were visible in silver-stained gels under conditions that excluded specific cytoskeleton protein components, including spectrin and actin. The reproducibility of the pattern depended highly on the precise composition of the solubilization mixture. Poor resolution was observed in the presence of actin and other proteins of high molecular mass (spectrin bands 1 and 2) when we used high urea concentrations that solubilized the entire erythrocyte membrane. The large number of polypeptides observed could not be attributed to proteolysis, because addition of proteolytic inhibitors to the membrane wash solutions did not alter the pattern on the gel. The pattern also did not appear to include erythrocyte cytosol proteins because, except for globin, none of five purified erythrocyte lysate proteins was visible in the erythrocyte membrane gels. We conclude that two-dimensional electrophoresis provides a powerful tool for the study of non-cytoskeletal erythrocyte membrane proteins.

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