Abstract
Two cardiac myosin heavy chain cDNA clones, pMHC alpha 252 and pMHC beta 174, were constructed using rabbit ventricular mRNA isolated from adult thyrotoxic and normal hearts, respectively. The complete DNA sequences of the 2.2- and 1.4-kilobase inserts of pMHC beta 174 and pMHC alpha 252, respectively, were obtained. The 736 amino acids specified by pMHC beta 174 begin 439 (1.3 kilobases) residues from the heavy chain NH2 terminus and include a 400-amino acid segment of subfragment 1 and the entire subfragment 2 region. Clone pMHC alpha 252 encodes 465 amino acids encompassing all of subfragment 2 and a portion of light meromyosin. Comparison of these two clones revealed extensive sequence overlap which included 1107 nucleotides specifying a 369-amino acid segment corresponding to subfragment 2. Within this region 78 (7%) base and 32 (8.7%) amino acid mismatches were noted. These differences were clustered within discrete regions, with the subfragment 1/subfragment 2 junctional region being particularly divergent. Structural differences between pMHC alpha 252 and pMHC beta 174 indicate that these two clones represent two similar but distinct myosin heavy chain genes whose expression is responsible for ventricular myosin heavy chain isoforms alpha and beta, respectively. The derived amino acid sequences of both clones exhibit extensive homology (greater than 81%) with sequences obtained by direct analysis of adult rabbit skeletal muscle myosin heavy chain protein. The sequences corresponding to the subfragment 2 region are consistent with an alpha-helical conformation with a characteristic 7-residue periodicity in the linear distribution of nonpolar amino acids. Conversely, subfragment 1 sequences specified by pMHC beta 174 suggest a folded highly irregular structure.
Highlights
Public Health Service Grants HL09172 and HL20592 from the National Institutes of Health and by a grant from the Louis Block Foundation, the University of Chicago
Analysis of fine structure [1], enzymatic activity [1, 3, 4], and amino acid [5, 6] and DNA sequences [7,8,9] have established that the COOH-terminal half or rod portion of the HC is a-helical in conformation. This region contains an abundance of a-helix-forming amino acids with a regular 3,4 residue periodicity in the linear distribution of hydrophobic amino acids
In this paper the complete DNA and derived amino acid sequences of clones pMHC,252 and pMHCiJ174 are compared. These sequences are used to determine structural differences between the two mRNAs and to correlate these differences with the primary structural and functional variability observed between VI and V3 ventricular isomyosins, In addition, implications of the derived amino acid sequence on the conformational structure of the myosin HC are considered
Summary
National Research Service Award 5 T32 GM07281 from the National Institutes of Health. Analysis of fine structure [1], enzymatic activity [1, 3, 4], and amino acid [5, 6] and DNA sequences [7,8,9] have established that the COOH-terminal half or rod portion of the HC (containing light meromyosin and subfragment 2 HC segments) is a-helical in conformation. In this paper the complete DNA and derived amino acid sequences of clones pMHC,,252 and pMHCiJ174 are compared These sequences are used to determine structural differences between the two mRNAs and to correlate these differences with the primary structural and functional variability observed between VI and V3 ventricular isomyosins, In addition, implications of the derived amino acid sequence on the conformational structure of the myosin HC are considered
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