Myosin assay

Figure 13, A schematic diagram of the motility assay. Myosin molecules (HMM or S-1 are also used) stick to glass coverslips coated with nitrocellulose. Actin, in solution, is then added to the glass coverslip and it binds to the myosin molecules. When ATP is added, actin can move over the surface, propelled by the myosin molecules.

The work that follows pertains primarily to actin networks. Many proteins within a cell are known to associate with actin. Among these are molecules which can initiate or terminate polymerization, intercalate with and cut chains, crosslink or bundle filaments, or induce network contraction (i.e., myosin) (A,11,12). The central concern of this paper is an exploration of the way that such molecular species interact to form complex networks. Ultimately we wish to elucidate the biophysical linkages between molecular properties and cellular function (like locomotion and shape differentiation) in which cytoskeletal structures are essential attributes. Here, however, we examine the iri vitro formation of cytoplasmic gels, with an emphasis on delineating quantitative assays for network constituents. Specific attention is given to gel volume assays, determinations of gelation times, and elasticity measurements. 

A modified version of the ECVAM-approved embryonic stem cell test (EST) (52) was recently developed by Hunter and coworkers in NHEERL (31). This assay is capable of quantitatively assessing cytotoxicity and cardiomyocyte differentiation and was used to test the ToxCast Phase I chemical library (32). Briefly, male murine J1 mES cells were seeded onto gelatin-coated 96-well plates at a known density in differentiation media on day 0. On day 1, cells were treated with chemicals ranging from 0.0125 to 12.5 pM, and on day 9 In-Cell Western (Li-Cor Biosciences) assays were assayed for a- and P-cardiac Myosin Heavy Chain (MYH6/MYH7)... 

Myosin light chain kinase is a Ser/Thr-type protein kinase involved in regulation of smooth muscle. The enzyme is also found in smooth muscle and platelets. This assay uses a synthetic substrate that is not radioactively labeled. 

The titration of myosin has been studied by Mihalyi (1950). The coimt of groups in the various regions of the curve agrees with analysis to better than 10%, except for the carboxyl region, where titration indicates 165 groups per 100,000 gm, as compared with the analytical figure of 132. It is likely that the analytical assay for amide groups is the source of the error. 

The following method has been described for the assay of cyclic AMP [144, 145]. Cyclic AMP is converted to 5 -AMP with the aid of phosphodiesterase and then to ATP with myokinase (EC 2.V.4.3 ATP AMP phosphotransferase adenylate kinase) and pyruvate kinase (EC 2.7.1.40 ATP pyruvate phosphotransferase). ATP is measured by determining the orthophosphate which accumulates during incubation of ATP with a cycling system containing myosin, pyruvate kinase, and phosphoenol pyruvate. Alternately, the ATP is determined by its luminescent reaction with firefly luciferin and luciferase [145-147]. With a sensitivity to about 1 pmol/tube of cyclic AMP, this assay is almost as sensitive as the phosphorylase method, but with a linearity over three orders of magnitude, it is linear over a much wider range than the phosphorylase method. 

Troponin—A protein found predominately in cardiac, but not skeletal, muscle which regulates calcium-mediated interaction of actin and myosin. Troponin I and T are released into the blood from the myocytes at the time of myocardial cell necrosis secondary to infarction. These biochemical markers become elevated and are used in the diagnosis of myocardial infarction. Troponin I and T are more sensitive and specific for infarction that creatinine kinase which is found in both skeletal and myocardial cells. The exact value of troponin I or T which is diagnostic of infarction differs based upon assay. 

H9C2 cells were subjected to hypoxic stress in the presence of IL-plas-mid complex and were kept under hypoxia for 6 h. After that, cells were washed and incubated for another 48 h under normoxic conditions. Enzyme-linked immunosorbent assays (ELISAs) assays with rat cardiac myosin were performed to assess the expression of 2G42D7 ScFv. 

Standard ELISA assays with porcine (10 pg/mL) and rat (50 pg/mL) cardiac myosin are performed to assess the expression of 2G42D7 ScFV. 

It has been claimed that the immunological assays are more reliable than x-ray and fragment count methods (Kitto et al., 1994 Quinn et al., 1992). It has been shown that with radiography, one can detect only 50% of second instar larvae and nearly 100% of the other stages of S. granarius, whereas iELISA is sensitive to detect even first instar larvae having 1 jj g of myosin/ insect (Schatzki et al., 1993). In wheat, iELISA can detect hidden infestation of 6 0.8 insects/50 g of grain, and that is of interest to the millers in the United States. 

A EXPERIMENTAL FIGURE 19-17 Sliding-filament assay is used to detect myosin-powered movement. 

Movement of actin filaments by myosin can be direcdy monitored In the sllding-fllament assay (see Figure 19-17). 

The ability of myosin to walk along an actin filament may be observed with the aid of an appropriately equipped microscope. Describe how such assays are typically performed. Why is ATP required in these assays How may such assays be used to determine the direction of myosin movement or the force produced by myosin ... 

Klnesln-dependent movement of vesicles can be tracked by In vitro motility assays similar to those used to study myosin-dependent movements. In one type of assay, a vesicle... 

Mutations of LC20 coupled with motility assays (see Trybus, Chapter 3 this volume) claim major victories in elucidation of the elementary steps in the contractile interaction between myosin and actin. The scope of this approach is rapidly expanding and new discoveries are expected in the future. Application of these methods to LC17 would be of importance. The first problem to be solved is the reversible removal of LC17 from vertebrate smooth muscle myosin. The question arises whether it is possible to dissect LC17 selectively while LC20 remains bound to the HC. 

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