Isolation of stable pancreatic zymogen granules

Lisle, Robert C. De; Schulz, Irene; Tyrakowski, Tomasz; Haase, Winfried; Hopfer, Ulrich

doi:10.1152/ajpgi.1984.246.4.g411

Cited by 37 publications

(31 citation statements)

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“…ZGs were purified on self-forming Percoll gradients by a modification of earlier procedures (8,27). ZGs, lysed in alkaline buffer at pH 11.2, yielded ZGMs with <0.1% amylase contamination after purification by sedimentation in discontinuous sucrose gradients and aqueous buffers.…”

Section: Resultsmentioning

confidence: 99%

“…Isolation of ZG membranes (ZGMs) after pH-induced lysis (5) revealed that membrane proteins account for only 1-2% of total granule protein (6). Characterization of ZGMs by SDS/PAGE indicated a relatively simple protein structure (-10 components), whose composition appeared to be distinct from other cytological compartments (7), and a relatively high phospholipid/ protein ratio, 2.49 (8). Analysis of ZGM proteins from dog, rat, rabbit, pig, and beef indicated, in each case, that a single glycoprotein (75-92 kDa, depending upon the species) represented the major protein (>25-30% of protein) (7,(9)(10)(11).…”

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confidence: 99%

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A single gene encodes membrane-bound and free forms of GP-2, the major glycoprotein in pancreatic secretory (zymogen) granule membranes.

Fukuoka

Freedman

Scheele

1991

Proc. Natl. Acad. Sci. U.S.A.

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A single gene encodes membrane-bound and free forms of GP-2, the major glycoprotein in pancreatic secretory (zymogen) granule membranes (phosphatidylinositol-glycan-linked Communicated by George E. Palade, January 3, 1991 (received for review July 27, 1990) ABSTRACT GP-2, a 75-kDa glycoprotein, was isolated from dog pancreatic zymogen granule membranes (ZGMs). In a carbohydrate-shift strategy, N-terminal and internal peptide sequences were obtained on glycosylated and deglycosylated forms of GP-2, respectively, by gas-phase sequencing. Sets of mixed oligonucleotides and the polymerase chain reaction were used to obtain a double-stranded cDNA probe, which was used to isolate overlapping cDNA clones from a dog pancreatic cDNA library. The sequence of these clones revealed an open reading frame that encodes a protein of 509 amino acids, eight N-linked oligosaccharide attachment sites, and an N-terminal signal sequence absent from the mature form of GP-2 associated with ZGMs. The C terminus shows a 20-residue hydrophobic transmembrane domain preceded by a decapeptide containing potential phosphatidylinositol-glycan attachment sites. GP-2 completely released from ZGMs by exogenous phospholipase C showed similar immunochemical properties and electrophoretic mobilities compared to the form associated with ZGMs. A similar form of GP-2 was released from zymogen granules permeabilized with saponin and incubated in the absence of added phospholipase C. Kinetic analysis of GP-2 release at 0°C and 3rC suggested the presence of a granule enzyme responsible for endogenous release of GP-2 to granule contents and into the apical medium. The data indicate that GP-2 is a phosphatidylinositol-glycan-linked membrane protein released from the membrane of mature zymogen granules by an enzymatic mechanism. The cDNA structure presented here thus encodes both membrane-bound and free forms of GP-2.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

A single gene encodes membrane-bound and free forms of GP-2, the major glycoprotein in pancreatic secretory (zymogen) granule membranes.

Fukuoka

Freedman

Scheele

1991

Proc. Natl. Acad. Sci. U.S.A.

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“…Zymogen Granule Enrichment-Pancreatic acinar cell zymogen granules were prepared from adult mice as previously described (13,14). Two to three pancreata were placed in ice-cold 250 mM sucrose, 5 mM MOPS (pH 7.0), 0.1 mM MgSO 4 containing one MiniComplete tablet (Roche Molecular Biochemicals, Indianapolis, IN) per 10 ml of buffer.…”

Section: Generation Of Itmap1mentioning

confidence: 99%

Protection from Pancreatitis by the Zymogen Granule Membrane Protein Integral Membrane-associated Protein-1

Imamura¹,

Asada²,

Vogt

et al. 2002

Journal of Biological Chemistry

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Pancreatitis is a common disease with substantial morbidity and mortality. To better understand the mechanisms conferring sensitivity or resistance to pancreatitis, we have initiated the analysis of novel acinar cell proteins. Integral membrane-associated protein-1 (Itmap1) is a CUB (complement subcomponents C1r/C1s, sea urchin Uegf protein, bone morphogenetic protein-1) and zona pellucida (ZP) domain-containing protein we find prominently expressed in pancreatic acinar cells. Within the acinar cell, Itmap1 localizes to zymogen granule membranes. Although roles in epithelial polarity, granule assembly, and mucosal protection have been postulated for CUB/ZP proteins, in vivo functions for these molecules have not been proven. To determine the function of Itmap1, we generated Itmap1-deficient mice. Itmap1؊/؊ mice demonstrate increased severity of secretagogue-and diet-induced pancreatitis in comparison to Itmap1 ؉/؉ mice. In contrast to previous animal models exhibiting altered severity of pancreatitis, Itmap1 deficiency results in impaired activation of trypsin, an enzyme believed critical for initiating a cascade of digestive zymogen activation during pancreatitis. Itmap1 deficiency does not alter zymogen granule size, appearance, or the composition of zymogen granule contents. Our results demonstrate that Itmap1 plays an essential role in trypsinogen activation and that both impaired and augmented trypsinogen activation can be associated with increased severity of pancreatitis.

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“…The procedure used for the preparation of rat zymogen granules was that described by DeLisle et al [22]. The pancrei were homogenized in an ice-cold homogenization buffer: 0.25 M sucrose, 5 mM Mops and 0.1 mM MgS04, pH 7.0.…”

Section: Preparation Of Rat Zymogen Granulesmentioning

confidence: 99%

Effect of a high‐protein diet on the gene expression of a trypsin‐sensitive, cholecystokinin‐releasing peptide (monitor peptide) in the pancreas

Tsuzuki

Fushiki

Kondo

et al. 1991

European Journal of Biochemistry

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The adaptation to a high protein diet of the concentration and mRNA level of a trypsin-sensitive, cholecystokinin-releasing peptide (monitor peptide), which was proposed to be the mediator of the cholecystokinin release in response to protein intake, was investigated in the rat pancreas. Adult rats were placed on one of two isocaloric diets. One group was fed a 22% casein diet (control diet) and the other a 64% casein diet (high-protein diet) for 14 days. In order to quantify the monitor peptide separately from pancreatic secretory trypsin inhibitor (PSTI-11), which is highly similar in its amino acid and mRNA nucleotide sequences to the monitor peptide but has less cholecystokinin-releasing activity, we used specific assay methods: HPLC was used for determining the monitor peptide concentration in zymogen granules and a synthetic oligonucleotide probe for determining the mRNA of the monitor peptide in the pancreas. The concentrations in the zymogen granules and the mRNA levels in the pancreas of the two peptides increased in parallel during the adaptation to the high protein diet, indicating that these two peptides were under the same control during the adaptation. The concentration and mRNA level of the monitor peptide, which were measured after 0,3, and 14 days, increased throughout the experiment period, as did the concentration of trypsin. This suggested that the monitor peptide and trypsin may respond to similar signals during the adaptation to a high protein diet and that this apparent coordination may facilitate the adaptation of the pancreas to the diet.Prolonged high protein intake leads to increases in the concentrations [l] and mRNA levels [2] of proteases and decreases in those of amylase 11, 31 in the pancreas. Likewise, a lipid-rich diet causes a high content of Iipase [4] and an increase in its mRNA level [5] in the pancreas. This increase in the rate of pancreatic enzymes synthesis is a clever adaptation by the animal to a new diet.There is growing evidence that cholecystokinin (CCK) may be involved in the adaptative increase in pancreatic proteases in response to prolonged intake of a high protein diet [6 -81. The CCK-releasing level is regulated on the basis of a balance between secreted protease activity and the luminal food protein content [9]. Insufficient protease activity for the digestion of food protein in the lumen may cause a signal to be sent to the pancreas to promote the secretion and synthesis of proteases [lo, 111. The release of CCK is mediated by the CCK-releasing peptide (monitor peptide), which is synthesized in pancreas and secreted into the small intestine [9-171. This peptide is an essential factor for CCK secretion, because when it was absorbed with a specific antibody no more pancreatic enzyme secretion was observed in response to food protein intake [9]. Recently, Goke et al. [18] reported that the CCK response to protease inhibitor feeding was increased after the induction of Correspondence to

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Isolation of stable pancreatic zymogen granules

Cited by 37 publications

References 0 publications

A single gene encodes membrane-bound and free forms of GP-2, the major glycoprotein in pancreatic secretory (zymogen) granule membranes.

A single gene encodes membrane-bound and free forms of GP-2, the major glycoprotein in pancreatic secretory (zymogen) granule membranes.

Protection from Pancreatitis by the Zymogen Granule Membrane Protein Integral Membrane-associated Protein-1

Effect of a high‐protein diet on the gene expression of a trypsin‐sensitive, cholecystokinin‐releasing peptide (monitor peptide) in the pancreas

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