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  • Key words: Phosphate transport —X. laevis oocytes — TCEP — Disulfide bonds — Degradation  (1)
  • Phosphate  (1)
  • 1
    Electronic Resource
    Electronic Resource
    Studies on the topology of the renal type II NaPi-cotransporter (1999)
    Springer
    Pflügers Archiv 437 (1999), S. 972-978 
    Details
    ISSN: 1432-2013
    Keywords: Key words FLAG ; Phosphate ; Sodium phosphate cotransport ; Topology ; Transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The rat type II sodium/phosphate cotransporter (NaPi-2) is a 85- to 90-kDa glycosylated protein located at the proximal tubular brush border membrane. Hydropathy predictions suggest eight transmembrane domains (sTM) with a large glycosylated loop between sTM 3 and sTM 4. We have studied the membrane topology of NaPi-2 expressed in oocytes. A 33-amino-acid fragment containing the FLAG epitope was inserted into seven loops connecting the sTMs and into the NH2- and COOH-ends of the protein. FLAG-antibody binding suggested that the loops connecting sTM 1 and sTM 2 as well as sTM 3 and sTM 4 are located extracellularly. Based on the lack of FLAG-antibody binding we suggest intracellular locations for the NH2- and COOH-termini and the region connecting sTM 4 and sTM 5. Immunoprecipitation studies of in vitro translated protein also suggest that the NH2-terminus is sited extracellularly. In immunohistochemical studies with NaPi-2-transfected MDCK cells, an interaction with NH2- and COOH- terminal antipeptide antibodies could only be obtained after membrane permeabilization. The presented data are an experimental documentation of the intracellular location of the NH2- and COOH-termini, and of the extracellular location of extracellular loops 1 and 2.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050869
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    Paper (German National Licenses)
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  • 2
    Electronic Resource
    Electronic Resource
    Cleavage of Disulfide Bonds Leads to Inactivation and Degradation of the Type IIa, But not Type IIb Sodium Phosphate Cotransporter Expressed in Xenopus laevis Oocytes (2000)
    Springer
    The journal of membrane biology 176 (2000), S. 143-149 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Phosphate transport —X. laevis oocytes — TCEP — Disulfide bonds — Degradation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Tris(2-carboxyethyl)phosphine (TCEP) reduces (cleaves) disulfide bonds of the renal proximal tubule type IIa Na/Pi- cotransporter (rat NaPi IIa) and thereby inhibits its function. We tested the effect of TCEP on the murine type IIa Na/P i -cotransporter and the corresponding IIb intestinal isoform both expressed in Xenopus laevis oocytes. After incubation with TCEP the function of NaPi IIa was inhibited and protein amount was decreased. Injection of the lysosomal inhibitor leupeptin prevented degradation of the protein. Exposure of oocytes to TCEP at 0°C led to a reduction in transport function without concomitant loss in Na/Pi IIa protein. In contrast to NaPi type IIa, the type IIb isoform was neither inhibited, nor degraded after incubation with TCEP. These results suggest that cleavage of disulfide bonds led to changes within the confirmation of the type IIa transporter that result in (i) inhibition of the transport activity and (ii) internalization and subsequent lysosomal degradation of transporter protein. Sequence comparisons suggest the involvement/presence of different disulfide bonds in type IIa and type IIb Na/P i -cotransporters.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s00232001083
    Library Location Call Number Volume/Issue/Year Availability
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    Paper (German National Licenses)
    Fulltext
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