N-terminal sequence analysis of atrial granule serine proteinase purified by affinity chromatography.
Atrial granule serine proteinase is considered the leading candidate endoproteolytic processing enzyme of pro-atrial natriuretic factor. Its cleavage specificity is directed toward a monobasic amino acid processing site, and as such, the atrial enzyme is distinguished from the family of prohormone convertases which act at dibasic amino acid processing sites. To delineate the molecular mechanisms which distinguish monobasic from dibasic amino acid-directed processing enzymes, pure atrial enzyme is needed for sequence determination leading to molecular cloning, and for preparation of antisera. An affinity chromatography purification scheme seemed a logical modification of our established procedures to yield suitable amounts of enzyme for further studies [Damodaran and Harris (1995, J. Protein Chem., this issue] formed ineffective affinity ligands, even though these compounds contain essential residues on either side of what would be the scissile bond in a peptide substrate. On the other hand, tripeptide aldehydes (based on the substrate recognition sequence of the atrial enzyme) linked to Sepharose formed effective affinity matrices, permitting purification of the enzyme in a single step from a subcellular fraction enriched for atrial granules and lysosomes. Hence, the enzyme was purified 2000-fold in 90% overall yield, and subjected to N-terminal sequence analysis through 26 residues. The sequence determined, XXPEAAGLPG[R,L]GNPVP[F,G]R[Q,I]XY[G,E]XR(N,A]V, indicates that the atrial enzyme is unique, showing little sequence homology to other proteins in the database.
Damodaran
A
A
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Medical College of Virginia, Richmond 23298-0614, USA.
Harris
R B
RB
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D002846:Chromatography, Affinity / Q000379:methods; D003594:Cytoplasmic Granules / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification
D011092:Polyethylene Glycols; D012697:Serine Endopeptidases; C098098:atrial granule serine proteinase