We developed a new method for the quantitative determination of myosin heavy chain (MyHC) isoforms taking advantage of immunochemical differences and based on the ELISA principle. In the present paper we compare anal ysis of MyHC isoforms using the SDS-PAGE and the ELISA methods in the same samples of adult female inbred Lewis strain euthyroid, hyperthyroid and hypothyroid rats. In all thyroid states, the same composition and corresponding changes of MyHC isoforms were determined using both methodological approaches in the slow soleus and the fast extensor digitorum longus muscles. Our results showed that ELISA can be used for a “semi-quantitative” or “comparative” measurement of MyHC isoforms in multiple muscle samples, but that it is neither more exact no r faster compared to SDS-PAGE., Jan Říčný., and Obsahuje bibliografii a bibliografické odkazy
Muscarinic acetylcholine receptors in the heart have been shown to display agonist-independent spontaneous (constitutive) activity which causes changes in the opening of cardiac ion channels and in the activity of G proteins. We investigated whether an inhibition of the constitutive activity of muscarinic receptors induced by the binding of antagonist brings about a change in the synthesis of cyclic AMP in rat cardiac membranes, and whether the action of the antagonist is stereospecific. Atropine and S-(-)-hyoscyamine were indeed found to enhance the forskolin-stimulated synthesis of cyclic AMP in rat cardiac (both atrial and ventricular) membranes by up to 24%. The effect was stereospecific and the potency of R-(+)-hyoscyamine was 30 fold lower than that of the S-(-) enantiomer, confirming that the action of hyoscyamine is receptor-mediated. The effect did not depend on the presence of endogenous acetylcholine in the system used. The results strongly suggest that the adenylyl cyclase in the heart is exposed to continuous mild inhibition by constitutively active muscarinic receptors in the membranes of cardiomyocytes., J. Říčný, F. Gualtieri, S. Tuček., and Obsahuje bibliografii
We have separated 2b myosin heavy chain (MyHC) isoform from the rat extensor digitorum longus muscle by SDS-PAGE and analyzed it by two subsequent mass spectrometry techniques. After tryptic digestion, the obtained peptides were identified by Matrix-Assisted Laser Desorption/Ionisation reflectron Time of Flight mass spectrometry (MALDI-TOF MS) and sequenced by Liquid chromatography tandem mass spectrometry (ESI LC/MS/MS). The analyzed peptides proportionally covered 30 % of the 2b MyHC isoform sequence. The results suggest that the primary structure is identical with the highest probability to a NCBI database record ref|NP_062198.1|, representing the last updated record of rat 2b isoform. Nonetheless, four peptides carrying amino acid substitution(s) in comparison with the NCBI database record were identified., J. Žurmanová, D. Maláčová, F. Půta, P. Novák, J. Říčný, T. Soukup., and Obsahuje bibliografii a bibliografické odkazy