Abstract
Recent investigations have shown that the heart atrium is an endocrine tissue. In the present studies, high levels of peptidylglycine alpha-amidating monooxygenase (PAM), which catalyzes the formation of bioactive alpha-amidated peptides from their glycine-extended precursors, have been found in particulate fractions from bovine and rat heart atrium; only low levels of PAM activity were present in soluble fractions. Corresponding fractions from the ventricles contained 20-fold less activity. Immunocytochemical studies demonstrated that PAM was localized primarily to atrial cardiocytes, with a distribution resembling that of atriopeptin. Following differential centrifugation of rat atrial homogenates, most of the PAM activity was associated with crude granule fractions, with lesser amounts of activity associated with crude microsomal fractions. Upon further subcellular fractionation, PAM activity in the rat atrium was found primarily with immunoactive atriopeptin in fractions enriched in secretory granules. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, antisera to purified bovine pituitary PAM identified a 113,000-dalton protein in bovine atrial microsomes and secretory granules; the protein predicted from the sequence of the cDNA encoding bovine pituitary PAM is of similar size (Eipper, B. A., Park, L. P., Dickerson, I. M., Keutmann, H. T., Thiele, E. A., Rodriguez, H., Schofield, P. R., and Mains, R. E. (1987) Mol. Endocrinol. 1, 777-790). Northern blot analysis using cDNA probes encoding bovine pituitary PAM demonstrated higher levels of PAM mRNA in heart atrium than in anterior pituitary. Rat heart contains PAM mRNA species of 3.6 and 3.8 kilobases, the smaller mRNA species corresponding in size to the PAM mRNA expressed in rat anterior pituitary.
Highlights
Recent investigationshave shown thattheheart 38,000) were purified from the bovine neurointermediate piatrium is an endocrine tissue
PAM was purified from the soluble high levels of peptidylglycine a-amidating monooxy- fraction of the bovine neurointermediate pituitary, analysis genase (PAM), which catalyzes the formation of bioac- of the cDNA encoding PAM revealed the presence of a hydrotive a-amidated peptides from their glycine-extended phobic putative membrane spanning domain near the carprecursors, have been found in particulate fractions boxyl terminus of the initial transcript[1].Subsequent analyfrom bovine and rat heart atrium; only low levels of sis of rat anterior andneurointermediate pituitary revealed a PAM activity were present in soluble fractions
Immunocytochemical studies demonstrated that PAM was localized primarily to atrial cardiocytes,with a distribution resembling that of tissue-specific distribution of PAM activity among soluble and membrane-associated forms ( 5 ) .Analysis of the cDNA encoding PAM indicated the presence of an aminoterminal signal peptide and 10pairs of basic amino acids that atriopeptin
Summary
Identification of PAM in Rat andBovine Heart-With the demonstration of a tissue-specific distribution of pituitary PAM activity between soluble and membrane-associated forms [5] and the availability of cDNA probes for PAM ( l ) , a reexamination of the levels of PAM in various tissues inthe adult male rat was undertaken. High levels of PAM activity were observed in theparticulate fraction from rat atrium, but not in corresponding fractions from rat ventricle. A similar enrichment of PAM activity in particulate fractions was observed in bovine atrium (TableI). Both rightand left atria contained high levelsof membrane-associated PAM activity, with a slightly higher specific activity in the right atrium. The specific activity of PAM in the particulate fraction from bovine atrium was 30-fold greater than in the soluble fraction. Consistent with the levels of PAM activity measured, rat atrium contained approximately 6-fold higher levels of PAM mRNA than the rat anterior pituitary (Fig. 1B);PAM mRNA found in the rat anterior pituitary corresponded in size to the smaller form observed in the atrium.
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