Quantification of myotrophin from spontaneously hypertensive and normal rat hearts.
A novel peptide, myotrophin, has been isolated, purified, cloned, and sequenced from the hearts of spontaneously hypertensive rats (SHR) and from dilated cardiomyopathic human heart tissue. Myotrophin accelerates myocyte growth by stimulating protein synthesis (not by altering myocardial cell division). Our successive studies were conducted to evaluate the pathophysiological significance of myotrophin; a solid-phase radioimmunoassay technique was developed for quantifying the protein in hypertrophied and normal hearts. Specific antipeptide antibody was raised in rabbits against a peptide that represents a selected amino acid sequence of a 17-amino acid myotrophin segment by using the multiple antigenic peptide technique. The specificity of the antibody was evaluated by determining the affinity constant after constructing the Scatchard plot obtained from the ratio of bound to free myotrophin against bound myotrophin. The value obtained was 2.61 x 10(7) L/mol. The specificity was further demonstrated by Western blot analysis, in which a single protein band was obtained in the region of 12 kD. Pretreatment of the antibody with myotrophin completely blocked the binding sites, because no protein band was detected on the immunoblot. The antibody prevented the myocardial protein synthesis induced by myotrophin as revealed by the blockage of the stimulation of [3H]leucine incorporation into myocyte protein. Quantification of myotrophin from different heart tissues was achieved by Western blot and dot blot analyses. Amounts of myotrophin present in different dots were determined by using a video image analyzer. The level of myotrophin in the embryonic tissue was found to be similar in male normal and SHR hearts.(ABSTRACT TRUNCATED AT 250 WORDS)
Sil
P
P
Department of Molecular Cardiology, Cleveland Clinic Foundation, OH 44195, USA.
Mukherjee
D
D
Sen
S
S
D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D000906:Antibodies / Q000032:analysis; D000918:Antibody Specificity; D015153:Blotting, Western; D006332:Cardiomegaly / Q000209:etiology; D002478:Cells, Cultured; D005260:Female; D006133:Growth Substances / Q000032:analysis* / Q000276:immunology; D036341:Intercellular Signaling Peptides and Proteins; D008297:Male; D009124:Muscle Proteins / Q000096:biosynthesis; D009206:Myocardium / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D011817:Rabbits; D011863:Radioimmunoassay; D051381:Rats; D011918:Rats, Inbred SHR; D017208:Rats, Wistar
D000906:Antibodies; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D009124:Muscle Proteins; C065700:myotrophin