Murine atrial HL-1 cells express highly active peptidylglycine alpha-amidating enzyme.

Peptidylglycine-alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) catalyzes the rate limiting step in peptide alpha-amidation, a posttranslational modification that is essential for receptor recognition and signal transduction. Secretory granules of the cardiac atrium contain the highest natural concentration of PHM and clearly demonstrate regulation of PHM expression and activity. The HL-1 atrial myocyte cell line faithfully maintains the differentiated phenotype of native atrial cells and thus provides an in vitro model system for investigating the mechanisms that regulate PHM. We observed that the specific activity of PHM expressed in HL-1 cells is five times higher than that found in rat atrium. The increased activity of HL-1 cell PHM was not reflected by a difference in Km for peptide substrate, change in copper optimum, altered sensitivity to inactivation by suicide inhibitor or variance in response to limited proteolysis by trypsin. Additionally, mixing experiments indicated that the increased activity in HL-1 cells versus rat atrium was not due to a diffusible factor. Based upon these findings we propose that the increased Vmax of HL-1 cell PHM results from a structural or conformational difference that involves either differential posttranslational modification and/or a high affinity chaperone that serves to regulate enzymatic activity by protein-protein interaction. The mechanism involved may participate in physiologic regulation of PHM.

Proteomic analysis of rat atrial secretory granules: a platform for testable hypotheses.

The recent development of powerful proteomic tools has enabled investigators to directly examine the population of proteins present in defined biological systems. We report here the first proteomic analysis of atrial secretory granules. Approximately 100 distinct protein components of the atrial secretory granule proteome were detected using subcellular fractionation and one-dimensional SDS-PAGE in conjunction with peptide mass fingerprinting by MALDI-TOF mass spectrometry. Of this number, 61 proteins were clearly identified by high probability data matches and repeated observation. The majority of the proteome was found to be membrane-associated with the most prominent proteins being peptidylglycine alpha-amidating monooxygenase (PAM) and pro-atrial natriuretic peptide (pro-ANP). This proteomic analysis of the rat atrium secretory granule produced an assembly of proteins with a diverse array of reported functions. The identified proteins fall into seven functional categories: (1) granular transport, docking and fusion; (2) signal transduction; (3) calcium-binding/calcium-dependent; (4) cellular architecture/chaperoning; (5) peptide/protein processing; (6) hormone; (7) proton transport. The novel finding of several protein processing enzymes and signal transduction proteins offer new perspectives on how pro-ANP is stored and processed to ANP during release. Accordingly, defining the proteome of the atrial secretory granule provides a framework for the development of new hypotheses that address key mechanisms governing granule function and ANP secretion.