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.
A tumor cell line from a patient with hyponatremia was able ectopically to produce, process, and secrete ANP in the same immunoreactive form as the biologically active molecule. Preliminary studies show that tumor cell line NCI-H1284 contains an enzyme that can cleave precursors at the same amino acid sequences needed to produce ANP-(S99-Y126) from pro-ANP.
Tumor associated antigen (TAA) on oral squamous cell carcinoma (SCC) was characterized using the monoclonal antibody (MAb) 3F8E3. Flow cytometric analysis revealed a varying degree of reactivity of MAb 3F8E3 to TAA on oral tumor cells. Pretreatment of SCC cells with pronase and trypsin annulled the reactivity of MAb 3F8E3. Sodium metaperiodate (NaIO4) and neuraminidase marginally enhanced the binding of 3F8E3 on oral SCC cells. The studies indicate that the TAA recognized by MAb 3F8E3 on oral tumors is a protein moiety. On Western blotting MAb 3F8E3 showed reactivity to proteins with a molecular weight of 60-66 kDa on oral tumor lysates. MAb 3F8E3 reacted strongly to recombinant human hsp60 and 70 in ELISA. The results suggest that MAb 3F8E3 may react to an epitope expressed on a family of heat shock proteins.
Pseudo-peptide bond inhibitors (psi-bond inhibitors) and peptide-aldehyde inhibitors of atrial granule serine proteinase, the candidate processing enzyme of pro-atrial natrieuretic factor, are prepared in high yield and purity by novel synthetic routes. The psi-bond compounds retain essential residues for enzyme binding, but place the enzyme inhibition site in the midst of the peptide sequence. Thus, Bz-APR-psi-LR and Bz-APR-psi-SLRR can be considered "readthrough inhibitors" of atrial granule serine proteinase. The most potent psi-peptide, Bz-APR-psi-SLRR (IC50=250 microM), is about fivefold less potent than the best peptide-aldehyde inhibitor (EACA-APR-CHO), and both the psi-bond and peptide-aldehyde compounds are competitive, reversible inhibitors of the enzyme. The psi-bond peptides containing two C-terminal Arg residues are three- to tenfold more potent than the analogous compounds containing only one C-terminal Arg residue, confirming the importance of both Arg residues in the enzyme processing recognition site. As expected, because of their moderate potencies, the psi-peptides are not useful affinity ligands for purification of atrial granule serine proteinase, but both peptide aldehydes are effective affinity ligands.
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