Abstract
The molecular mechanisms underlying the heterogeneity in contractile properties observed among smooth muscle tissues are unknown. We examined whether part of this diversity might be intrinsic to myosin by comparing structural and enzymatic properties of myosins from two physiologically diverse tissues. Using the reverse transcriptase polymerase chain reaction, we compared avian intestinal smooth muscle and vascular smooth muscle myosin heavy chain (MHC) mRNA. We found that intestinal, but not vascular, MHC mRNA contains an insert of 21 nucleotides, encoding 7 amino acids, in a region near the ATP binding site in the myosin head. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified myosin revealed that the relative mobilities of the previously described intestinal MHC isoforms SM1 (204 kDa) and SM2 (200 kDa) were slower than the corresponding vascular SM1 and SM2 isoforms. Furthermore, antibodies raised against a synthetic peptide corresponding to the deduced amino acid sequence of the intestinal insert strongly recognized intestinal SM1 and SM2 but only weakly recognized the vascular isoforms. The presence of the insert in intestinal myosin correlated with a higher velocity of movement of actin filaments in vitro and a higher actin-activated Mg(2+)-ATPase activity, compared with vascular myosin. Other than the MHC insert, one other structural difference distinguished intestinal and vascular myosins: two isoforms of the 17-kDa myosin light chain were found in vascular myosin, whereas a single isoform was found in intestinal myosin. Exchange of the intestinal myosin light chains onto the vascular MHC did not alter its activity in the in vitro motility assay, suggesting that the 7-amino acid MHC insert is responsible for the different enzymatic activities of vascular and intestinal myosins.
Highlights
The molecular mechanisms underlying the hetero- molecular structure of a dimer of two heavy chains noncovalently associated with two pairs of smooth muscle tissues are unknown
Other than the might be intrinsic to heavy chains (MHC) insert, one other structural dif- and how their expression is linked to thpehysiological diverference distinguished intestinaland vascularmyosins: two isoforms of the 17-kDa myosin light chain were found in vascular myosin, whereas a single isoform was found in intestinaml yosin
Two isoforms of the MHC, SM1 and SM2, generated by alternatively splicing at the carboxyl terminus, had been reported in thesetissues [10, 11].We confirmed the presence of mRNAs encoding both SM1 and SM2, by using the same mRNA used in Fig. l A, to obtain the two expected products using the appropriateprimersand reverse transcriptasePCR(datanotshown)
Summary
Isoforms ences between the intestinal and vascular MHC isoforms and determine whether thestructural differencesimpartfunctional differences which might contribute to the unique enzymatic properties of these smooth muscle tissues. Purification of Proteins-Myosin was prepared from turkey gizzards and aortasby low salt ATP extraction followed by ammonium sulfate fractionation and gel filtration as described [21, 22]. Myosin light chain kinase was purified from turkey gizzards as described previously [27]. A solution containing 1 mg/ml aorta myosin, 1mg/ml gizzard LC17, 10 mM MgCl,, 1mM ADP, and 4.7 M NHIC1, pH 7.75, was incubated for 30 min a t 30 "C followed by dialysis in a buffer containing 0.5 M KC1,20 mM MOPS, pH 7.0, 2 mM MgC12,0.2 mM ATP, 0.1% 2-mercaptoethanol, and 20% glycerol. Antibodies specific for the gizzard MHC insert were made against a peptide synthesized based on the deduced amino acid sequence of the insertnear the ATP binding site, QGPSFSY. Conjugation of the peptides to keyhole limpet hemocyanin, immunization of rabbits, and affinity purification of the antisera were performed as described previously [17]
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