Expression of Na-K-ATPase alpha- and beta-subunit mRNA and protein isoforms in the rat nephron
| Autor: | Jeff M. Sands, J. A. Tumlin, Carol A. Hoban, R. M. Medford |
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| Rok vydání: | 1994 |
| Předmět: |
Gene isoform
Male Physiology Macromolecular Substances Blotting Western Molecular Sequence Data Gene Expression In Vitro Techniques Kidney Polymerase Chain Reaction Muscle Smooth Vascular Rats Sprague-Dawley Western blot medicine Animals Northern blot RNA Messenger In Situ Hybridization Southern blot DNA Primers Gel electrophoresis Messenger RNA biology medicine.diagnostic_test Base Sequence Brain DNA Nephrons Molecular biology Rats Isoenzymes Blotting Southern Kidney Tubules Polyclonal antibodies Organ Specificity biology.protein Sodium-Potassium-Exchanging ATPase ATP synthase alpha/beta subunits |
| Zdroj: | The American journal of physiology. 266(2 Pt 2) |
| ISSN: | 0002-9513 |
| Popis: | Na-K-ATPase is a heterodimeric complex composed of an alpha-catalytic and a glycosylated beta-subunit. Previous studies using in situ hybridization and Northern blot analysis to determine alpha- and beta-subunit mRNA isoform expression in the rat kidney have given conflicting results. This heterogeneity may be due to detection of alpha 2- or alpha 3-isoforms arising from nonrenal epithelial sources such as peripheral nerves or vascular smooth muscle. To address this possibility, we investigated alpha-subunit mRNA isoform expression in different nephron segments using tubule microdissection and reverse transcription-polymerase chain reaction amplification. Southern blot analysis of polymerase chain reaction products using isoform-specific primers and probes detected the expression of alpha 1- and alpha 3-mRNA isoforms in whole kidney, cortical collecting ducts (CCD), and proximal tubule S2 subsegments (S2). No evidence for alpha 2 was found in kidney or microdissected CCD or S2. To determine whether alpha 1- or alpha 3-mRNA in the CCD or S2 is translated into protein, Western blot analysis of total protein from microdissected S2, CCD, medullary thick ascending limb (MTAL), or outer medullary collecting duct outer stripe (OMCDos) was performed. Total protein was separated according to size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then probed using polyclonal antibodies specific for the alpha 1-, alpha 2-, alpha 3-, beta 1-, and beta 2-protein isoforms. Rat brain was used as a positive control and demonstrated that the antibodies could detect a single 97- and 35-kDa band for the alpha- and beta-isoforms, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) |
| Databáze: | OpenAIRE |
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