An unusual bovine pancreatic protein exhibiting pH-dependent globule-fibril transformation and unique amino acid sequence.

Abstract

An unusual hitherto unreported protein, extracted in acid from fresh bovine pancreas, has been purified and characterized biochemically. It precipitates in the neutral pH range in the form of uniform double-helical threads, each strand of which is smooth and of uniform diameter, about 7-8 nm. The threads dissolve to a nonviscous solution below pH 3.6 and above pH 9.4, and they reconstitute reversibly in the pH range in between. The monomer in acid has an apparent molecular weight of 17,800 and consists of two disulfide-linked nonidentical polypeptide chains of different lengths. It is rich in aromatic amino acids, particularly tryptophan. There is no significant content of carbohydrate, fatty acid, or bound phosphate. The amino acid sequences of the first NH2-terminal 48 residues of the A chain and 35 residues of the B chain appear to be unique, differing from all other reported animal proteins, including those of the pancreas. Thus far, a function has not been found.In the course of early studies on the structure of elastic fibers by electron microscopy utilizing trypsin digestion, the appearance of 12-nm-wide, uniformly helical threads in the "digestate" led to the erroneous conclusion that these coiled elements were structural components of elastin (1). Subsequent reexamination (2) led the author to conclude that the helical threads were either a conversion product of trypsinogen or a contaminant of the various commercial crystallized trypsin preparations, not observed earlier in the trypsin control for technical reasons. High-speed centrifugation of dissolved trypsin or trypsinogen alone at neutral pH produced a pellet consisting largely of straight, tightly wound, double-helical, 12-nm threads of variable length with a pitch of 47-58 nm and a right-handed screw axis. A variable proportion of these threads were uncoiled single filtments or parallel pairs. They dissolved it acid pH and rapidly reconstituted to helical threads on neutralization in a reversible manner (3). They were not a component of elastin as originally proposed (1) or of bacterial flagella (4).Although there have been numerous studies of the composition of the pancreatic exocrine secretion from various mammals, including bovines (5-9), the protein described here has not been previously recognized.We report here the partial molecular characterization of this unusual bovine pancreatic thread protein (PTP) with an as-yet-unrecognized function. MATERIALS AND METHODSWhole pancreases from freshly slaughtered calves were collected in cold 0.25 M sulfuric acid. Within 24 hr the tissue was homogenized in a blender in the cold and the acid extract was separated from the residue by filtration and sedimentation. It was then processed by sequential ammonium sulfate precipitation and re-solution in borate buffers as outlined by Kunitz and Northrup (10). At that stage in the described procedure where the third 70% saturated ammonium sulfate precipitate is obtained, the solution at pH 8 was allowed to stand at 40C until a cloudy precipitate form...

A Family of 16-kDa Pancreatic Secretory Stress Proteins Form Highly Organized Fibrillar Structures upon Tryptic Activation

“…7, B (PTP) and C (PATP III), shows filaments that were negatively stained with phosphotungstic acid. This procedure, which causes extensive fragmentation of filaments, compares favorably with negatively stained micrographs that appear in the literature (22,23).…”

“…In both species thread proteins were isolated and shown to be fibrillar by negative staining in the electron microscope (22,23). Purified PSP/lithostathine has been shown to form similar fibrils after tryptic cleavage in vitro (43), and prolonged incubation of PSP, purified by column chromatography, suggested that activation may occur by autoproteolysis.…”

“…Recent studies have demonstrated that PSP/reg and PAP both act as acute phase reactants in pancreatic juice under a variety of conditions including acute pancreatitis (20), chronic pancreatitis in male WBN/Kob rats, and during the post-weaning period (21). Trypsin cleavage of PSP/reg and PAP has resulted in the appearance of precipitated proteins believed to represent insoluble thread structures in humans (22) and cows (23). However, it remains difficult to understand how precipitation properties could serve useful functions in pancreatic physiology, and it is not clearly known whether these precipitated protein forms demonstrate specific or nonspecific structures.…”

The Role of Reg Proteins, a Family of Secreted C-Type Lectins, in Islet Regeneration and as Autoantigens in Type 1 Diabetes

“…Protein data bank accession numbers are given after each member. Abbreviations: Reg=regenerating islet-derived; INGAP=islet neogenesis associated protein; PTP=pancreatic thread protein; PAP=pancreatitis associated protein; HIP=hepatocellular carcinoma/intestine/pancreas; PSP=pancreatic stone protein www.intechopen.com pancreatic protein that precipitates at neutral pH in the form of double helical threads and was subsequently identified as a member of the Reg family (Gross, Brauer et al 1985) and 'HIP' (for hepatocellular carcinoma, intestine, pancreas) -a name assigned to a human family member after Lasserre at al. found that it was overexpressed in a proportion of the hepatocellular carcinoma samples they had investigated (Lasserre, Christa et al 1992).…”

Pathogenesis of Chronic Pancreatitis

“…A degraded form. insoluble at pH 7. has been discovered in the ox pancreas and in the human pancreas by Gross and co-workers (34,35 [36], Elisa with a monoclonal antibody as first antibody and a polyclonal antibody as che second antibody [37]), its concentration is constantly decreased in patients presenting with CCP compared co normal controls. This suggests that a decrease of PSP secretion could be responsible for the formation of calcite calculi.…”