XAIP1: a Xenopus homologue of yeast actin interacting protein 1 (AIP1), which induces disassembly of actin filaments cooperatively with ADF/cofilin family proteins.
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We carried out affinity column chromatography using Xenopus ADF/cofilin (XAC), identified several polypeptides in oocytes specifically bound to this column with actin, and isolated a full-length cDNA clone for a 65 kDa protein in this fraction. The predicted amino acid sequence revealed that the 65 kDa protein has seven obvious WD repeats and exhibits striking homology with yeast actin interacting protein 1 (AIP1). Thus, we designated this protein Xenopus AIP1 (XAIP1). We purified XAIP1 from Xenopus oocytes, and its interaction with actin was characterized by a pelleting assay, photometrical analysis and electron microscopy. Although XAIP1 itself cosedimented with F-actin and increased unsedimented actin to some extent, it induced a rapid, drastic disassembly of actin filaments associated with XAC. Electron microscopic observation revealed that XAIP1 severs actin filaments in the presence of XAC. To elucidate the in vivo effects of XAIP1, the purified protein was injected into blastomeres at the two-cell stage. Although the localization of XAIP1 was similar to that of XAC, at the cortical cytoskeleton and diffusely in the cytoplasm, injection of a large amount of XAIP1 arrested development and abolished the strong cortical staining of both actin and XAC. From these results, we concluded that XAIP1 regulates the dynamics of the cortical actin cytoskeleton cooperatively with XAC in eggs. (+info)
Identification, purification, and characterization of the rat liver golgi membrane ATP transporter.
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Phosphorylation of secretory and integral membrane proteins and of proteoglycans also occurs in the lumen of the Golgi apparatus. ATP, the phosphate donor in these reactions, must first cross the Golgi membrane before it can serve as substrate. The existence of a specific ATP transporter in the Golgi membrane has been previously demonstrated in vitro using intact Golgi membrane vesicles from rat liver and mammary gland. We have now identified and purified the rat liver Golgi membrane ATP transporter. The transporter was purified to apparent homogeneity by a combination of conventional ion exchange, dye color, and affinity chromatography. An approximately 70,000-fold purification (2% yield) was achieved starting from crude rat liver Golgi membranes. A protein with an apparent molecular mass of 60 kDa was identified as the putative transporter by a combination of column chromatography, photoaffinity labeling with an analog of ATP, and native functional size determination on a glycerol gradient. The purified transporter appears to exist as a homodimer within the Golgi membrane, and when reconstituted into phosphatidylcholine liposomes, was active in ATP but not nucleotide sugar or adenosine 3'-phosphate 5'-phosphosulfate transport. The transport activity was saturable with an apparent Km very similar to that of intact Golgi vesicles. (+info)
Identification of megalin as the sole rat kidney sialoglycoprotein containing poly alpha2,8 deaminoneuraminic acid.
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Recently, poly alpha2,8 deaminoneuraminic acid (poly alpha2,8 KDN) was demonstrated in various embryonic and adult mammalian tissues. This study reports the purification and characterization of the single poly alpha2,8 KDN-bearing glycoprotein from rat kidney. Amino acid sequences of proteolytic fragments shared homology with megalin, a member of the LDL receptor family. Immunochemical analysis supported this finding, since immunoprecipitated poly alpha2,8 KDN-bearing glycoprotein was immunoreactive with anti-megalin antibodies in Western blotting and conversely immunoprecipitated megalin was immunoreactive with the monoclonal anti-poly alpha2,8 KDN antibody. Furthermore, receptor-associated protein affinity-purified megalin reacted with the anti-poly alpha2,8 KDN antibody. By immunoelectron microscopy, labeling for both poly alpha2,8 KDN and megalin coincided in the brush border, endocytic invaginations and vesicles, and apical dense tubules of proximal convoluted tubules. Immunoreactivity for poly alpha2,8 KDN on purified megalin was abolished by beta-elimination reaction but not by N-glycosidase F treatment. These data identified megalin as the sole glycoprotein of rat kidney, which contains poly alpha2,8 KDN present on O-glycosidically linked oligosaccharides. Furthermore, this study shows that megalin carries N-glycosidically linked hybrid and complex-type oligosaccharides terminating with sialic acid. Both poly alpha2,8 KDN and sialic acids on megalin may contribute to the binding of Ca2+ and cationic ligands. (+info)
IgA interaction with carboxy-terminal 43-kD fragment of fibronectin in IgA nephropathy.
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IgA deposition in the glomerular mesangial matrix is a prerequisite for the diagnosis of IgA nephropathy, and circulating IgA-containing complex has been implicated in this process. Since fibronectin is known to be involved in the assembly of extracellular matrix, this study was conducted to investigate whether fibronectin and its fragments are present in sera of patients and are capable of binding IgA1. Sera from patients with IgA nephropathy were purified by heparin-affinity chromatography, and column eluate were analyzed for the presence of fibronectin using Western blot and a set of anti-fibronectin monoclonal antibodies. Native fibronectin was digested with cathepsin D to obtain fragments similar to those of serum fibronectin. The capacity of fibronectin to bind IgA was examined with a mixture of purified IgA1 and cathepsin D-digested fibronectin fragments. A 43-kD carboxy-terminal fragment of fibronectin was detected in samples derived from sera of patients with IgA nephropathy but not in healthy control subjects. A similar-sized fragment was generated by cathepsin D digestion of the native molecule and was shown to bind to IgA1 in vitro. Since the carboxy-terminal domain is known to be critical in assembling exogenous fibronectin into the extracellular matrix, the affinity to IgA1 to a fragment found in patients may have pathogenic potential to mediate extracellular IgA deposition in IgA nephropathy. (+info)
Interaction of a lectin from Psathyrella velutina mushroom with N-acetylneuraminic acid.
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A lectin from the fruiting body of Psathyrella velutina has been used as a specific probe for non-reducing terminal N-acetylglucosamine residues. We reveal in this report that P. velutina lectin recognizes a non-reducing terminal N-acetylneuraminic acid residue in glycoproteins and oligosaccharides. Binding of biotinyl P. velutina lectin to N-acetylneuraminic acid residues was prevented by desialylation of glycoconjugates and was distinguished from the binding to N-acetylglucosamine. Sialooligosaccharides were retarded or bound and eluted with N-acetylglucosamine on a P. velutina lectin column, being differentiated from each other and also from the oligosaccharides with non-reducing terminal N-acetylglucosamine which bound more strongly to the column. (+info)
Expression of two glutathione S-transferase genes in the yeast Issatchenkia orientalis is induced by o-dinitrobenzene during cell growth arrest.
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Glutathione S-transferases (GSTs) Y-1 and Y-2 from the yeast Issatchenkia orientalis were purified by passage through a glutathione-agarose column, and the cDNA for GST Y-1 was cloned and sequenced. The deduced amino acid sequence consisted of 188 residues with a total calculated molecular mass of 21,001 Da and showed 36.7% identity to that of GST Y-2, another GST isoenzyme expressed in this strain. Escherichia coli DH5alpha transformed with pUC119 harboring the GST Y-1 gene under the control of the lac promoter exhibited 29-fold-higher GST activity than the same strain with pUC119. Northern blot analysis revealed that both genes were highly expressed in cells cultured in the presence of 200 microM o-dinitrobenzene (DNB), one of the substrates of GST, while only the GST Y-1 gene was expressed, and only slightly, under normal (DNB-free) culture conditions. The DNB in the medium arrested cell growth until it was reduced by conjugation with reduced glutathione. Kinetic analysis of GST gene expression during detoxification of DNB revealed that the levels of expression of both genes were elevated within 3 h after the addition of DNB and that they further increased until 12 h postaddition. The levels of expression of both genes were decreased markedly when the DNB concentration in the culture medium was lowered. These results suggest that I. orientalis cells sense xenobiotics and arrest cell growth as a mechanism for preventing the induction of mutations by these compounds, while the levels of expression of the GST genes are up-regulated for detoxification. (+info)
Affinity isolation of imidazoline binding proteins from rat brain using 5-amino-efaroxan as a ligand.
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We have employed an amino derivative of the imidazoline ligand, efaroxan, to isolate imidazoline binding proteins from solubilised extracts of rat brain, by affinity chromatography. A number of proteins were specifically retained on the affinity column and one of these was immunoreactive with an antiserum raised against the ion conducting pore component of the ATP-sensitive potassium channel. Patch clamp experiments confirmed that, like its parent compound, amino-efaroxan blocks ATP-sensitive potassium channels in human pancreatic beta-cells and can stimulate the insulin secretion from these cells. The results reveal that a member of the ion conducting pore component family is strongly associated with imidazoline binding proteins in brain and in the endocrine pancreas. (+info)
Isolation and identification of the third subunit of mammalian DNA polymerase delta by PCNA-affinity chromatography of mouse FM3A cell extracts.
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Using proliferating cell nuclear antigen affinity chroma-tography and glycerol gradient centrifugation of partially purified fractions from mouse FM3A cells we have been able to isolate novel complexes of DNA polymerase delta and DNA ligase 1 containing clearly defined subunit compositions. In addition to the well known catalytic subunit of 125 kDa and accessory subunit of 48 kDa, the DNA polymerase delta complex contained three supplementary components, one of which reacted with antibodies directed against the p40 and p37 subunits of RF-C. Of the two remaining components, one termed p66 turned out to be coded by a gene whose putative C-terminal domain displayed significant homology with that of the Cdc27 subunit of Schizosaccharomyces pombe polymerase delta. On the basis of these and other observations, we propose p66 to be the missing third subunit of mammalian DNA polymerase delta. The DNA ligase 1 complex was made up of three novel components in addition to the 125 kDa catalytic subunit, two of which, p48 and p66, were common to DNA polymerase delta. We discuss the implications of our findings within the current framework of our understanding of DNA replication. (+info)