scholarly journals Comparison of New ELISA Method With Established SDS-PAGE Method for Determination of Muscle Myosin Heavy Chain Isoforms

2011 ◽  
pp. 899-904 ◽  
Author(s):  
J. ŘÍČNÝ ◽  
T. SOUKUP
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Myosin Heavy Chain Isoforms ◽  
Elisa Method ◽  
Comparative Measurement ◽  
Sds Page ◽  
Myhc Isoforms ◽  
Methodological Approaches ◽  
Muscle Myosin

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 analysis 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 nor faster compared to SDS-PAGE.

scholarly journals Electrophoretic Mobility of Cardiac Myosin Heavy Chain Isoforms Revisited: Application of MALDI TOF/TOF Analysis

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Petra Arnostova ◽  
Petr L. Jedelsky ◽  
Tomáš Soukup ◽  
Jitka Zurmanova
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Myosin Heavy Chain Isoforms ◽  
Cardiac Myosin ◽  
Thyroid Status ◽  
Mobility Rate ◽  
Sds Page ◽  
Myhc Isoforms ◽  
Lower Mobility ◽  
Altered Thyroid

The expression of two cardiac myosin heavy chain (MyHC) isoforms in response to the thyroid status was studied in left ventricles (LVs) of Lewis rats. Major MyHC isoform in euthyroid and hyperthyroid LVs had a higher mobility on SDS-PAGE, whereas hypothyroid LVs predominantly contained a MyHC isoform with a lower mobility corresponding to that of the control soleus muscle. By comparing the MyHC profiles obtained under altered thyroid states together with the control soleus, we concluded that MyHCαwas represented by the lower band with higher mobility and MyHCβby the upper band. The identity of these two bands in SDS-PAGE gels was confirmed by western blot and mass spectrometry. Thus, in contrast to the literature data, we found that the MyHCαpossessed a higher mobility rate than the MyHCβisoform. Our data highlighted the importance of the careful identification of the MyHCαand MyHCβisoforms analyzed by the SDS-PAGE.

Hypertensive renal damage: Modulation expression of smooth muscle myosin heavy chain isoforms

Nephrology ◽  
1997 ◽  
Vol 3 (3) ◽  
pp. 251-259
Author(s):  
Naoe SUZUKI ◽  
Kenjiro KIMURA ◽  
Ryozo NAGAI ◽  
Shigeyoshi OHBA ◽  
Naobumi MISE ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Renal Damage ◽  
Myosin Heavy Chain Isoforms ◽  
Smooth Muscle Myosin ◽  
Muscle Myosin

scholarly journals Association of myosin with the connecting cilium of rod photoreceptors

1992 ◽  
Vol 103 (1) ◽  
pp. 183-190 ◽  
Author(s):  
D.S. Williams ◽  
M.A. Hallett ◽  
K. Arikawa
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Actin Filaments ◽  
Critical Role ◽  
Rod Outer Segments ◽  
Outer Segments ◽  
Vertebrate Photoreceptor ◽  
Sds Page ◽  
Membrane Morphogenesis ◽  
Muscle Myosin

The cilium of a vertebrate photoreceptor cell connects the phototransductive outer segment of the cell to the inner segment. Previous studies have shown that, within the connecting cilium, there is a small cluster of actin filaments, which play a critical role in the formation of new disk membranes. Here, we have detected a polypeptide in rat rod outer segments that is recognized by myosin heavy chain antibodies and was found to possess other characteristics of conventional non-muscle myosin heavy chain: it comigrates in SDS-PAGE with non-muscle myosin heavy chain; it associates with the cytoskeleton of rod outer segments in an ATP-sensitive manner; and it binds to purified actin filaments in the absence of ATP. Myosin ATPase activity was also detected in isolated rod outer segments. Electron immunomicroscopy revealed that myosin is present in the small actin-containing domain within the connecting cilium at the site of disk membrane morphogenesis. These results pose the possibility that an actin-myosin contractile mechanism functions in the formation of new photoreceptor disk membranes.

scholarly journals Electrophoretic separation and quantitation of cardiac myosin heavy chain isoforms in eight mammalian species

1998 ◽  
Vol 274 (3) ◽  
pp. H1048-H1053 ◽  
Author(s):  
Peter J. Reiser ◽  
William O. Kline
Keyword(s):  
Skeletal Muscle ◽  
Sample Preparation ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Mammalian Species ◽  
Myosin Heavy Chain Isoforms ◽  
Glycerol Concentration ◽  
Cardiac Myosin ◽  
Homogenization Step

A protocol for sample preparation and gel electrophoresis is described that reliably results in the separation of the α- and β-isoforms of cardiac myosin heavy chain (MHC-α and MHC-β) in eight mammalian species. The protocol is based on a simple, nongradient denaturing gel. The magnitude of separation of MHC-α and MHC-β achieved with this protocol is sufficient for quantitative determination of the relative amounts of these two isoforms in mouse, rat, guinea pig, rabbit, canine, pig, baboon, and human myocardial samples. The sensitivity of the protocol is sufficient for the detection of MHC isoforms in samples at least as small as 1 μg. The glycerol concentration in the separating gel is an important factor for successfully separating MHC-α and MHC-β in myocardial samples from different species. The effect of sample load on MHC-α and MHC-β band resolution is illustrated. The results also indicate that inclusion of a homogenization step during sample preparation increases the amount of MHC detected on the gel for cardiac samples to a much greater extent than for skeletal muscle samples. Although the protocol described in this study is excellent for analyzing cardiac samples, it should be noted that the same protocol is not optimal for separating MHC isoforms expressed in skeletal muscle, as is illustrated.

Expression and function of COOH-terminal myosin heavy chain isoforms in mouse smooth muscle

2007 ◽  
Vol 293 (1) ◽  
pp. C238-C245 ◽  
Author(s):  
Anne F. Martin ◽  
Sunita Bhatti ◽  
Gail J. Pyne-Geithman ◽  
Mariam Farjah ◽  
Vlasios Manaves ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Immunoblot Analysis ◽  
Myosin Heavy Chain Isoforms ◽  
Smooth Muscle Myosin ◽  
Tail Region ◽  
Protein Ratio ◽  
Muscle Myosin

Isoforms of the smooth muscle myosin motor, SM1 and SM2, differ in length at the carboxy terminal tail region. Their proportion changes with development, hormonal status and disease, but their function is unknown. We developed mice carrying the myosin heavy chain (MyHC) transgenes SM1, cMyc-tagged SM1, SM2, and V5-tagged SM2, and all transgenes corresponded to the SMa NH2-terminal isoform. Transgene expression was targeted to smooth muscle by the smooth muscle α-actin promoter. Immunoblot analysis showed substantial expression of the cMyc-tagged SM1 and V5-tagged SM2 MyHC protein in aorta and bladder and transgene mRNA was expressed in mice carrying unlabeled SM1 or SM2 transgenes. Despite significant protein expression of tagged MyHCs we found only small changes in the SM1:SM2 protein ratio. Significant changes in functional phenotype were observed in mice carrying unlabeled SM1 or SM2 transgenes. Force in aorta and bladder was increased (72 ± 14%, 92 ± 11%) in SM1 and decreased to 57 ± 1% and 80 ± 3% in SM2 transgenic mice. SM1 transgenic bladders had faster (1.8 ± 0.3 s) and SM2 slower (7.1 ± 0.5 s) rates of force redevelopment following a rapid step shortening. We hypothesize that small changes in the SM1:SM2 ratio could be amplified if they are associated with changes in thick filament assembly and underlie the altered contractility. These data provide evidence indicating an in vivo function for the COOH-terminal isoforms of smooth muscle myosin and suggest that the SM1:SM2 ratio is tightly regulated in smooth muscle tissues.

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