Mehta and Gunst (1999) and Jones et al (1999) reported the existence of G-actin in smooth muscle, based on the method of DNase I inhibition and phalloidin staining, respectively. Subsequently, Barany, et al (2001) showed the exchange of the actin-bound nucleotide in intact smooth muscle. This was based on the separation of the actin bound nucleotides from the cytoplasmic nucleotides with 50% ethanol (Fig. SM12).
Fig. SM12. Extraction of nucleotides and radioactivity from 32P-labeled arterial smooth muscles (From Barany et al., 2001).The percentage of the total absorbance and counts eluted from the muscles in 8 extractions is shown on the ordinate.
32P-labeled arterial smooth muscles were extracted in the refrigerator several times for elutions of the nucleotides (Absorbance at 260 nm) and the 32P-label (Counts). The absorbance and the counts are very high in the first extract and drop in the successive five extractions virtually to zero. In the 7th and 8th elution the muscles were extracted with 1.5% perchloric acid (PCA). New absorbance and radioactivity appeared, corresponding to the release of the bound-nucleotide and the associated 32P-label from actin.
The composition of the PCA extract is shown on Fig. SM13.
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