Myristoyl-coA:protein N-myristoyltransferase from bovine cardiac muscle: molecular cloning, kinetic analysis, and in vitro proteolytic cleavage by m-calpain.
Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the attachment of myristate onto the amino terminal glycine residue of select polypeptides. Cardiac tissue expresses high levels of cAMP-dependent protein kinase whose catalytic subunit is myristoylated; however, cardiac muscle extracts were found to contain low NMT activities. Northern blot analysis of bovine heart poly(A)+ RNA probed with bovine spleen NMT cDNA revealed a 1.7-kb mRNA. Western blot analysis of cardiac muscle extracts with human NMT antibody indicated a prominent immunoreactive band with a molecular mass of 50 kDa. The expression of mRNA and protein levels in cardiac muscle is not correlated with NMT activities, suggesting the presence of regulators of the enzyme activity. We have isolated the cDNA encoding bovine cardiac muscle NMT (cNMT) by reverse transcription polymerase chain reaction. The single long open reading frame of 1248 bp of bovine cNMT specifies a protein of 416 amino acids with a predicted mass of 46,686 Da. The cDNA clone expressed in Escherichia coli resulted in the production of functionally active 50-kDa NMT. Ultrastructural and immunolocalization of NMT utilizing the immunogold labeling technique demonstrated cytoplasmic distribution with occasional mitochondrial and myofilaments localization of the NMT antibody. Cardiac muscle NMT has a higher affinity for myristoyl-CoA than toward palmitoyl-CoA. Substrate specificity indicated that cNMT has a higher affinity toward pp60src and M2 gene segment of reovirus type 3-derived peptide substrates than toward cAMP-dependent protein kinase-derived peptide. Primary translational product of cNMT sequence contained several regions rich in proline, glutamic acid, serine, and threonine, which are known as "PEST" regions. PEST-FIND analysis of the amino acid sequences indicated eight PEST regions were present in the cNMT. These PEST regions are suggested to be recognized by specific proteases, particularly Ca(2+)-dependent neutral proteases, calpains, which are responsible for the degradation of PEST-containing proteins. We have demonstrated the abolishment of NMT activity and NMT protein degradation in vitro by m-calpain. The proteolysis of cNMT by m-calpain and the abolishment of NMT activity was prevented by the calpain inhibitor, calpastatin. These observations indicate that calpains may regulate NMT activity.
Raju
R V
RV
Department of Pathology, College of Medicine, Royal University Hospital, University of Saskatchewan, Saskatoon, Canada.
Kakkar
R
R
Datla
R S
RS
Radhi
J
J
Sharma
R K
RK
D000217:Acyltransferases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D002154:Calpain / Q000494:pharmacology; D002417:Cattle; D003001:Cloning, Molecular; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006868:Hydrolysis / Q000187:drug effects; D007150:Immunohistochemistry; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry / Q000201:enzymology* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000737:chemistry / Q000201:enzymology
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D012333:RNA, Messenger; C033668:calpastatin; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D002154:Calpain