cDNA Cloning and Tissue‐specific Regulation of Expression of Rat Calcium‐binding Protein 65/67

Fan, Hao; Josić, Djuro; Lim, Yow‐Pin; Reutter, Werner

doi:10.1111/j.1432-1033.1995.0741h.x

Cited by 18 publications

(15 citation statements)

References 76 publications

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“…To validate the experiment, vesicles bound on magnetic beads with [21,40]. In rat liver, annexin A6 is a membrane-associated protein [12,41]. Some annexins can interact with integral membrane proteins, and they also seem to be part of signaling pathways in the cell [42].…”

Section: Discussionmentioning

confidence: 99%

Use of magnetic beads with immobilized monoclonal antibodies for isolation of highly pure plasma membranes

et al. 2006

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In plasma membrane proteome research, contamination of the isolated plasma membrane fraction with proteins from other organelles is still a problem. Even if highly specific isolation methods are used, such as density gradient centrifugation combined with selective extraction, contaminating proteins cannot be completely removed. To solve this problem, a protocol for the isolation of highly pure plasma membrane fractions from rat liver and two different hepatocellular carcinoma cell lines was developed. Magnetic beads with immobilized mAb's against highly expressed membrane proteins were used for specific binding of membrane vesicles and their separation from other organelles. Isolated plasma membranes were further selectively solubilized with different reagents and analyzed by use of different methods, such as Western blotting, 1- and 2-DE, and MS. Purification and further selective solubilization was validated by use of mAb's against the marker integral plasma membrane protein carcinoembryonic antigen cell adhesion molecule 1, and identification of isolated proteins by MS. The method presented here minimizes contamination with other organelles and enables further identification of membrane proteins.

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Section: Discussionmentioning

confidence: 99%

Use of magnetic beads with immobilized monoclonal antibodies for isolation of highly pure plasma membranes

et al. 2006

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“…The coding region was polymerase chain reaction-amplified and generated a 2.0-kilobase pair annexin VI cDNA fragment (26) that was cloned into a mammalian expression vector (pcDNA3.1ϩ; Invitrogen) to generate pcDNAanx6. DNA sequence analysis using a Dye Terminator Cycle Sequencing reaction kit (PerkinElmer Life Sciences) together with T7, SP6 (Promega), and internal annexin VI primers confirmed the full-length and wild type rat annexin VI cDNA sequence in pcDNAanx6.…”

Section: Methodsmentioning

confidence: 99%

Annexin VI Stimulates Endocytosis and Is Involved in the Trafficking of Low Density Lipoprotein to the Prelysosomal Compartment

Grewal¹,

Heeren²,

Mewawala³

et al. 2000

Journal of Biological Chemistry

126

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Annexins are calcium-binding proteins with a wide distribution in most polarized and nonpolarized cells that participate in a variety of membrane-membrane interactions. At the cell surface, annexin VI is thought to remodel the spectrin cytoskeleton to facilitate budding of coated pits. However, annexin VI is also found in late endocytic compartments in a number of cell types, indicating an additional important role at later stages of the endocytic pathway. Therefore overexpression of annexin VI in Chinese hamster ovary cells was used to investigate its possible role in endocytosis and intracellular trafficking of low density lipoprotein (LDL) and transferrin. While overexpression of annexin VI alone did not alter endocytosis and degradation of LDL, coexpression of annexin VI and LDL receptor resulted in an increase in LDL uptake with a concomitant increase of its degradation. Whereas annexin VI showed a wide intracellular distribution in resting Chinese hamster ovary cells, it was mainly found in the endocytic compartment and remained associated with LDL-containing vesicles even at later stages of the endocytic pathway. Thus, data presented in this study suggest that after stimulating endocytosis at the cell surface, annexin VI remains bound to endocytic vesicles to regulate entry of ligands into the prelysosomal compartment.Annexins are a family of highly conserved proteins, which are characterized by their Ca 2ϩ -dependent binding to phospholipids (1). Each annexin consists of a conserved core domain with four or eight repeats (70 amino acids) and a nonconserved, short, NH 2 -terminal domain. More than 10 different family members, several of which exist as multiple isoforms, have been described in higher vertebrates (2). Since annexins are expressed in many tissues and are located in the same cellular compartments, the understanding of the distinct physiological role of each annexin still remains elusive (1, 3). In recent years, the involvement of annexins in membrane traffic has emerged as one of their predominant functions (1, 4). Several annexins including annexin I, II, IV, VI, VII, and XIIIb have been directly implicated in different steps of the intracellular trafficking pathways (5-14) and, despite some controversy, essentially due to the variety of cells and antibodies used, they are all associated with the endocytic compartment.The enrichment of annexin VI in rat liver endosomes (12, 13), its polarized localization in the apical endosomes in rat hepatocytes (14) and WIF-B cells (15), and the colocalization with lgp120, a prelysosomal marker in normal rat kidney cells (15), indicate a potential role for annexin VI in the endocytic pathways of polarized and nonpolarized cells.In support of this hypothesis, annexin VI has been found to bind ␤-spectrin at the cell surface, which in turn recruits and activates a calpain-like protease. This cascade of events seems to open the actin-cortical cytoskeleton to facilitate the initial steps of endocytosis (16,17). The complexity of these interactions has recently...

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“…In our previous work, one of these proteins was identified as annexin A6 [45]. Identification of other proteins from this fraction is ongoing.…”

Section: Discussionmentioning

confidence: 99%

“…Figure 3 also shows a comparison between the crude membrane fraction and highly purified liver plasma membranes with regard to the content of CEACAM 1. If the Triton X-100 insoluble pellet is further treated with suitable reagents such as EGTA, urea/4% CHAPS mixture, or SDS, socalled detergent-insoluble proteins can be extracted [44,45]. In previous experiments, we showed that membraneassociated annexin A6 can be solubilized by simple calcium-complexing with EDTA or EGTA (not shown here, cf.…”

Section: Fractionation Of Purified Liver Plasma Membranesmentioning

confidence: 99%

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Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

et al. 2005

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Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteinsA model system for selective solubilization and fast separation of proteins from the rat liver membrane fraction and purified rat liver plasma membranes for their further proteomic analysis is presented. For selective solubilization, high-pH solutions and a concentrated urea solution, combined with different detergents, are used. After extraction, proteins are separated by anion-exchange chromatography or a combination of anion-and cation-exchange chromatography with convective interaction monolithic supports. This separation method enables fast and effective prefractionation of membrane proteins based on their hydrophobicity and charge prior to one-dimensional (1-D) and 2-D electrophoresis and mass spectrometry. By use of this sample preparation method, the less-abundant proteins can be detected and identified.

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cDNA Cloning and Tissue‐specific Regulation of Expression of Rat Calcium‐binding Protein 65/67

Cited by 18 publications

References 76 publications

Use of magnetic beads with immobilized monoclonal antibodies for isolation of highly pure plasma membranes

Use of magnetic beads with immobilized monoclonal antibodies for isolation of highly pure plasma membranes

Annexin VI Stimulates Endocytosis and Is Involved in the Trafficking of Low Density Lipoprotein to the Prelysosomal Compartment

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

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