Cloning of the cDNA and nucleotide sequence of a skeletal muscle protease from myopathic hamsters.
A neutral protease with an estimated Mr of about 26 kD and responsible for cleavage ofmyosin 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.
Holt
J C
JC
Rhone-Poulenc Rorer Central Research, King of Prussia, PA, USA.
Hatcher
V B
VB
Caulfield
J B
JB
Norton
P
P
Umeda
P K
PK
Melendez
J A
JA
Martino
L
L
Mudzinsky
S P
SP
Blumenstock
F
F
Slayter
H S
HS
Margossian
S S
SS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D024722:Cardiac Myosins; D002311:Cardiomyopathy, Dilated / Q000201:enzymology*; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics*; D004285:Dogs; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology*; D009135:Muscular Diseases / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D009210:Myofibrils / Q000201:enzymology; D018994:Myosin Light Chains / Q000378:metabolism; D051381:Rats; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013045:Species Specificity
D018076:DNA, Complementary; D018994:Myosin Light Chains; C107184:myosin light chain 2; D012697:Serine Endopeptidases; C112160:mekratin; D024722:Cardiac Myosins