Characterization and regulation of peptidylglycine α-amidating monooxygenase (PAM) expression in H9c2 cardiac myoblasts

Abstract

Peptidylglycine α-amidating monooxygenase (PAM), which catalazyes the two-step formation of bioactive α-amidated peptides from their glycine-extended precursors, has been found in H9c2 myoblasts. The expression of PAM has been evaluated in H9c2 cells. Northern blot analysis and amplification of fragments derived from rat PAM by the reverse transcription/polymerase chain reaction method has demonstrated the presence of rPAM-1, -2, -3, -3a and -3b mRNA transcripts. These forms of PAM mRNA may be generated by alternative splicing. PAM mRNA levels are increased to 160±12% of control values by treatment with dexamethasone but are unchanged during triiodothyronine incubation of the cells. PAM activity is very low, which is not comparable to the high levels found in adult atrium tissue. Western blot analysis has demonstrated 86-, 76-, and 46-kDa PAM proteins in the particulate fraction. The soluble fraction contains major PAM proteins of 110, 86, and 46 kDa. In situ hybridization studies with 35 S-labeled full length RNA antisense transcripts of rat PAM-1 cDNA have localized autoradiographic grains around the nucleus. Our data clearly demonstrate PAM expression in H9c2 rat heart cells, suggesting the ability of these cardiac cells to make bioactive α-amidated hormones and/or neuropeptides.

Aqueous Kinetics Of Α-Hydroxyhippuric Acid Derivatives Bearing Electron-Withdrawing Groups As A Function Of Ph And Buffer Catalysts

“…A final example of the importance of carbinolamide chemistry is in the generation of peptide hormones where a carbinolamide is generated by the bifunctional enzyme peptidylglycine--hydroxylating monooxygenase (PHM, EC 1.14.17.3). 11,16,[33][34][35][36][37][38][39][40][41][42][43][44][45] The fact that an enzyme exists that is capable of catalyzing the breakdown of carbinolamides was interesting given that the accepted mechanisms of carbinolamide reaction would not be easily catalyzed. [46][47][48][49][50] This observation has led to the study detailed herein.…”