Profilaggrin is an intermediate filament-associated protein of cornified epithelia. It consists of multiple copies of similar filaggrin domains joined by peptide linker regions; during terminal differentiation of the epidermis, the linker regions are processed away in a regulated manner. In order to characterize the sites of proteolysis in rat profilaggrin, tryptic peptides of filaggrin and profilaggrin were fractionated by reverse-phase HPLC, and the HPLC fractions were analyzed by nebulization-assisted electrospray ionization mass spectrometry. Peptide sequences were confirmed or corrected by tandem mass spectrometry; in several cases, this was achieved by collisional activation of multiply charged precursor ions of peptides exceeding 3 kDa in mass. The tryptic peptides accounted for all of the sequence predicted by a partial cDNA sequence, with the exception of six arginines or dipeptides. Although the cDNA sequence predicted eight sites of heterogeneity among the filaggrin domains, only one of these was observed. An additional unpredicted site of heterogeneity was also seen. Comparison of the peptides from filaggrin with those of profilaggrin revealed several peptides unique to filaggrin, specifically at the new amino- and carboxyl-termini, that result from proteolytic processing of the linker region of profilaggrin. Both the amino- and carboxyl-termini were "ragged", suggesting that processing may involve exopeptidase action after an initial endopeptidase cleavage. The average mass of this mixture of filaggrins was determined by electrospray mass spectrometry to be 42 452 Da, in reasonable agreement with that predicted from the mass spectrometric analysis of the terminal sequences. The linker peptide of rat profilaggrin was found in two forms, which differed only in the phosphorylation state of serine 22.

The amino acid sequence of spinach ferredoxin: NADP+ oxidoreductase was determined by using overlapping sets of peptides derived by cleavage at arginyl or methionyl residues. The protein from different preparations varied in its length at the amino terminus. In the longest form the amino terminus is blocked with a pyroglutamyl residue, as determined by NMR. A single disulfide bond was placed between cysteine residues 132 and 137. The 314-residue sequence corresponds to a molecular weight of 35 317. The carboxyl-terminal half of the sequence has been fit to the electron density map of the NADP binding domain, revealing that this portion of the chain forms a typical nucleotide binding fold.

Partial protein sequences from the 59-kDa bovine heart and the 63-kDa bovine brain calmodulin-dependent phosphodiesterases (CaM-PDEs) were determined and compared to the sequence of the 61-kDa isozyme reported by Charbonneau et al. [Charbonneau, H., Kumar, S., Novack, J. P., Blumenthal, D. K., Griffin, P. R., Shabanowitz, J., Hunt, D. F., Beavo, J. A. & Walsh, K. A. (1991) Biochemistry (preceding paper in this issue)]. Only a single segment (34 residues) at the N-terminus of the 59-kDa isozyme lacks identity with the 61-kDa isozyme; all other assigned sequence is identical in the two isozymes. Peptides from the 59-kDa isozyme that correspond to residues 23-41 of the 61-kDa protein bind calmodulin with high affinity. The C-terminal halves of these calmodulin-binding peptides are identical to the corresponding 59-kDa sequence; the N-terminal halves differ. The localization of sequence differences within this single segment suggests that the 61- and 59-kDa isozymes are generated from a single gene by tissue-specific alternative RNA splicing. In contrast, partial sequence from the 63-kDa bovine brain CaM-PDE isozyme displays only 67% identity with the 61-kDa isozyme. The differences are dispersed throughout the sequence, suggesting that the 63- and 61-kDa isozymes are encoded by separate but homologous genes.

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A pentadecapeptide derived from the N-terminal portion of bovine carboxypeptidase Ay was isolated by the action of cyanogen bromide on the protein. Its sequence was established as Asn.Tyr.Ala.Thr.Tyr.His.Thr.Leu.Asp.Glu.Ile.Tyr.Asp.Phe.Met. An analogous peptide, derived from carboxypeptidase Aa, differs from carboxypeptidase A7 by seven residues at the N terminus, rather than the five residues previously indicated by . The correct N-terminal sequence is proved to be: