Abstract
The heavy chain fragments generated by restricted proteolysis of the smooth chicken gizzard myosin subfragment-1 (S-1) with trypsin, Staphylococcus aureus V8 protease, and chymotrypsin were isolated and submitted to partial amino acid sequencing. The comparison between the smooth and striated muscle myosin sequences permitted the unambiguous structural characterization of the two protease-vulnerable segments joining the three putative domain-like regions of the smooth head heavy chain. The smooth carboxyl-terminal connector is a serine-rich region located around positions 632-640 of the rabbit skeletal sequence and would represent the "A" site that is conformationally sensitive to the myosin 10 S-6 transition and to its interaction with actin (Ikebe, M., and Hartshorne, D. J. (1986) Biochemistry 25, 6177-6185). A third site which undergoes a nucleotide-dependent chymotryptic cleavage which inactivates the Mg2+-ATPase (Okamoto, Y., and Sekine, T. (1981) J. Biochem. (Tokyo) 90, 833-842, 843-849) was identified at Trp-31/Ser-32. It is vicinal to Lys-34 that is monomethylated in the skeletal heavy chain but not at all in the smooth sequence. However, the two trimethyl lysine residues present in the skeletal sequence are conserved in the same regions of the smooth S-1 and may play a general functional role in myosin. The smooth central 50-kDa segment could be selectively destroyed by a mild tryptic digestion in the absence of any unfolding agent, with a concomitant inhibition of the ATPase activities. This feature is in line with the proposed domain structure of the S-1 heavy chain and also suggests a relationship between the specific biochemical properties of the smooth S-1 and the particular conformation of its 50-kDa region.
Highlights
Subfragment- 1(S-1)with trypsin, Staphylococcus au- Previous reports describing the proteolytic fragmentation reus VS protease, and chymotrypsin were isolated and of native gizzard smooth muscle myosin, heavy meromyosin, submitted to partial amino acid sequencing
Primary Structures of the Connector I and II Regions of the Smooth S-I Heavy Chain-In the gizzard papain S-1, the connector I segment would represent a narrow part of the heavy chainjoiningthecentral50-kDadomaintothe terminal 24-kDa fragment, the N-terminasl equence of which is homologous to the tryptic skeletal20-kDa peptide [17]
The yield of the S-1 so obtained did not exceed about lo%, but it contained a t least 95% of intact 97-kDa heavy chain (Fig. l),in contrast to the usual papSa-i1npreparation which consistently included a significantamount of anicked75
Summary
Skeletal MyoHsineads structural featuresof the protease-labileregions in the smooth Conditions for K+ EDTA assay were: 1 M KCI, 5 mM EDTA; for. Gizzard papain subfragment-1 and its tryptic derivative (29 kDa50 kDa-25 kDa)-Sl were obtained as reported previously [5], except that thedigestion of myosin with papain was conducted a t a protease to substrate weight ratio of 1:6000 for 15 min a t 20 "C. Proteolytic Digestions-The limited cleavage of gizzard S-1 with the various proteases was performed a t a protein concentrationof 12 mg/ml in 25 mM Tris-HCI, pH 8.0, a t 25 "C for 20-90 min, using enzyme tosubstrate weight ratios of1:20 (Staphylococcus aureus protease), 1:40 (chymotrypsin),and 1:lOOO (trypsin and papain). Amino acid compositions were determined on protein samples hydrolyzed at 110"C under vacuum in 5.7 N HCI for 24 and 48 h using a Beckman analyzer (Model 119B).The analyses of methylated lysine and histidine were performed as previously described [25]. ATPase Measurements-ATPase activities were determined a t 25'C in 50 mM Tris-HCI, pH 7.5, in the presence of2.5 mM ATP
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