Cloning of the cDNA and nucleotide sequence of a skeletal muscle protease from myopathic hamsters

John C. Holt, Victor Bernard Hatcher, James B. Caulfield, Paul Norton, Patrick K. Umeda, J. Andres Melendez, Leon Martino, Stanley P. Mudzinsky, Frank Blumenstock, Henry S. Slayter, Sarkis S. Margossian

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

A neutral protease with an estimated M(r) of about 26 kD and responsible for cleavage of myosin LC2 was isolated from hamster skeletal muscle. Complementary DNAs were generated by RT-PCR using total hamster muscle RNA and degenerate oligonucleotide primers based on the sequences of two internal peptides. The nucleotide sequences of the resultant cDNAs were subsequently determined and the complete amino acid sequence of the protease deduced. Although the hamster protein shared 63-85% identity in nucleotide and amino acid sequences with rat and mouse mast cell proteases, it had a higher degree of specificity for myosin LC2 than mast cell proteases which also digested myosin LC1 and myosin heavy chains. As a result, the hamster protease was designated mekratin because of its unique enzymatic specificities to distinguish it from other mast cell proteases. A polyclonal antibody was raised specific to the hamster muscle and human cardiac muscle mekratins without apparent cross-reaction with rat mast cell proteases. We have earlier demonstrated the presence in excess of a neutral protease that specifically cleaves LC2 in human hearts obtained at end stage idiopathic dilated cardiomyopathy (IDC). Western analyses revealed that heart tissue from patients with IDC contained 5-10 fold more mekratin than control samples. Furthermore, the level of the protease in human IDC tissues was similar to that seen in myopathic hamster skeletal muscle. No bands were recognized by the antibody when IDC myofibrils were probed due to the removal of soluble proteins during sample preparation. Thus, these results strongly suggest that the anti-mekratin antibody will provide positive identification of IDC in many cases and diagnosis by exclusion may be replaced.

Original languageEnglish (US)
Pages (from-to)125-135
Number of pages11
JournalMolecular and Cellular Biochemistry
Volume181
Issue number1-2
DOIs
StatePublished - 1998

Fingerprint

Cloning
Cricetinae
Muscle
Organism Cloning
Skeletal Muscle
Peptide Hydrolases
Nucleotides
Complementary DNA
Dilated Cardiomyopathy
Mast Cells
Myosins
Antibodies
Rats
Amino Acid Sequence
Tissue
Amino Acids
Muscles
DNA Primers
Myosin Heavy Chains
Myofibrils

Keywords

  • Cardiomyopathy
  • cDNA cloning
  • Mast cells
  • Mekratin
  • Myosin light chain 2
  • Serine proteases

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Holt, J. C., Hatcher, V. B., Caulfield, J. B., Norton, P., Umeda, P. K., Andres Melendez, J., ... Margossian, S. S. (1998). Cloning of the cDNA and nucleotide sequence of a skeletal muscle protease from myopathic hamsters. Molecular and Cellular Biochemistry, 181(1-2), 125-135. https://doi.org/10.1023/A:1006842332340

Cloning of the cDNA and nucleotide sequence of a skeletal muscle protease from myopathic hamsters. / Holt, John C.; Hatcher, Victor Bernard; Caulfield, James B.; Norton, Paul; Umeda, Patrick K.; Andres Melendez, J.; Martino, Leon; Mudzinsky, Stanley P.; Blumenstock, Frank; Slayter, Henry S.; Margossian, Sarkis S.

In: Molecular and Cellular Biochemistry, Vol. 181, No. 1-2, 1998, p. 125-135.

Research output: Contribution to journalArticle

Holt, JC, Hatcher, VB, Caulfield, JB, Norton, P, Umeda, PK, Andres Melendez, J, Martino, L, Mudzinsky, SP, Blumenstock, F, Slayter, HS & Margossian, SS 1998, 'Cloning of the cDNA and nucleotide sequence of a skeletal muscle protease from myopathic hamsters', Molecular and Cellular Biochemistry, vol. 181, no. 1-2, pp. 125-135. https://doi.org/10.1023/A:1006842332340
Holt, John C. ; Hatcher, Victor Bernard ; Caulfield, James B. ; Norton, Paul ; Umeda, Patrick K. ; Andres Melendez, J. ; Martino, Leon ; Mudzinsky, Stanley P. ; Blumenstock, Frank ; Slayter, Henry S. ; Margossian, Sarkis S. / Cloning of the cDNA and nucleotide sequence of a skeletal muscle protease from myopathic hamsters. In: Molecular and Cellular Biochemistry. 1998 ; Vol. 181, No. 1-2. pp. 125-135.
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AU - Andres Melendez, J.

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