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A highly sensitive technique to measure myosin regulatory light chain phosphorylation: the first quantification in renal arterioles
- Source :
- The American Journal of Physiology. June, 2008, Vol. 294 Issue 6, pF1487, 6 p.
- Publication Year :
- 2008
-
Abstract
- Phosphorylation of the 20-kDa myosin regulatory light chains ([LC.sub.20]) plays a key role in the regulation of smooth muscle contraction. The level of [LC.sub.20] phosphorylation is governed by the relative activities of myosin light chain kinase and phosphatase pathways. The regulation of these two pathways differs in different smooth muscle types and in the actions of different vasoactive stimuli. Little is known concerning the regulation of [LC.sub.20] phosphorylation in the renal microcirculation. The available pharmacological probes are often nonspecific, and current techniques to directly measure [LC.sub.20] phosphorylation are not sensitive enough for quantification in small arterioles. We describe here a novel approach to address this important issue. Using SDS-PAGE with polyacrylamide-bound [Mn.sup.2+]-phosphate-binding tag and enhanced Western blot analysis, we were able to detect [LC.sub.20] phosphorylation using as little as 5 pg (250 amol) of isolated [LC.sub.20]. Phosphorylated and unphosphorylated [LC.sub.20] were detected in single isolated afferent arterioles, and [LC.sub.20] phosphorylation levels could be accurately quantified in pooled samples of three arterioles ( afferent arteriole; Phos-tag SDS-PAGE
- Subjects :
- Myosin -- Physiological aspects
Myosin -- Health aspects
Myosin -- Research
Phosphorylation -- Physiological aspects
Phosphorylation -- Health aspects
Phosphorylation -- Research
Renal artery -- Physiological aspects
Renal artery -- Health aspects
Renal artery -- Research
Biological sciences
Subjects
Details
- Language :
- English
- ISSN :
- 00029513
- Volume :
- 294
- Issue :
- 6
- Database :
- Gale General OneFile
- Journal :
- The American Journal of Physiology
- Publication Type :
- Academic Journal
- Accession number :
- edsgcl.180471213