Partial amino acid sequence of human pancreatic stone protein, a novel pancreatic secretory protein

Abstract

Pancreatic stone protein (PSP) is the major organic component of human pancreatic stones. With the use of monoclonal antibody immunoadsorbents, five immunoreactive forms (PSP-S) with close Mr values (14,000-19,000) were isolated from normal pancreatic juice. By CM-Trisacryl M chromatography the lowest-Mr form (PSP-S1) was separated from the others and some of its molecular characteristics were investigated. The Mr of the PSP-S1 polypeptide chain calculated from the amino acid composition was about 16,100. The N-terminal sequences (40 residues) of PSP and PSP-S1 are identical, which suggests that the peptide backbone is the same for both of these polypeptides. The PSP-S1 sequence was determined up to residue 65 and was found to be different from all other known protein sequences.

The disulfide bridges of the immunoreactive forms of human pancreatic stone protein isolated from pancreatic juice

“…The absence of free thiol in PSP-S1 and PSP-$2-5 [4] was confirmed by titration with 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of EDTA and SDS. Moreover, a mild reduction of the proteins with sodium arsenite was performed.…”

“…Most of the methods have been described: preparation of PSP SI and PSP $2-5 [4], dansylation [7], succinylation [6], amino acid analysis [7,8], automated Edman degradation [6], 5,5 '-dithiobis(2-nitrobenzoic acid) reaction in the presence of 10 mM EDTA and 1070 SDS [9], location of the half-cystinecontaining peptides eluted from column [10] and pepsin digestion [5]. The chymotryptic digestion was performed on 15 mg succinylated PSP $1 or PSP $2-5 in 1 ml of 60 mM NH4HCO3 buffer (pH 7.8) for 5 h at 37°C (enzyme/substrate, 1:50, w/w).…”

“…Various forms of human pancreatic stone protein deriving from the same native protein, synthesized in pancreatic acinar cells [1], have been revealed: PSP, the form prepared from calculi [2]; PSP S1 one of the immunoreactive forms (Mr 15000) [3,4]; and PSP $2-5, other immunoreactive forms (Mr 16000-19000) detected in pancreatic juice but not separated from each other [3,4]. Both PSP and PSP S1 have the same Mr as well as N-terminal sequence and C-terminal residues [4,5].…”

“…Both PSP and PSP S1 have the same Mr as well as N-terminal sequence and C-terminal residues [4,5]. PSP S1 derives from PSP $2-5 through the tryptic cleavage of an Arg-Ile bond [5].…”

“…The 6 half-cystines of the molecule were located at positions 3, 14, 31, 104, 121 and 129 in the alignment of the 133 amino acid residues of PSP-S1. Since no free thiol was detected in PSP S1 [4], the presence of three Correspondence address: P. Rouimi, Centre de Biochimie et de Biologie Mol~culaire, CNRS, 31 Chemin J. Aiguier, BP 71, 13402 Marseille Cedex 9, France disulfide bridges was assumed. In a preceding paper [5] the first S-S bridge connecting Cys 3 to Cys 14 was demonstrated in the course of an investigation performed in view of revealing the nature of the bond cleaved in the conversion of PSP $2-5 to PSP S1.…”