Pancreozymin and caerulein stimulate in vitro protein phosphorylation in the rat pancreas

Lambert, Monique; Camus, Jean Claude; Christophe, Jean

doi:10.1016/0006-291x(73)91027-9

Cited by 29 publications

(8 citation statements)

References 11 publications

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“…If we assume that this kinase remained unaltered during membrane isolation, this observation might suggest that the in vivo phosphorylation of zymogen granule membrane is not under hormonal control. This conclusion would contradict our previous hypothesis, based on intact cell préparation [1], according to which phosphorylation of spécifie proteins could change granule membrane properties and facilitate the fusion with the plasma membrane during exocytosis. It is however possible to reconcile the data obtained on pancréas fragments and pancréas extracts if we admit that rises in the intracellular levels of cyclic GMP or cyclic AMP entail a dissociation of cytoplasmic protein kinase(s) and that the liberated catalytic subunit(s) are secondarily transferred on zymogen granule membranes, resulting in 'in situ' phosphorylation of 8 to 10 constituent proteins.…”

Section: Resultscontrasting

confidence: 93%

“…Our earlier results have already documented some aspects of protein phosphorylation in rat pancréas fragments [1]. The stimulation of amylase sécrétion by pancreozymin and caerulein was accompanied in vitro by an increase in ^^P orthophosphate incorpo ration into proteins, and especially se in the membrane proteins of zymogen granules.…”

Section: Introductionmentioning

confidence: 56%

“…Zymogen granules were isolated from rat pancréas as described previously [1]. Secretory proteins were released in 0.2 M bicarbonate at pH 8.2.…”

Section: Methodsmentioning

confidence: 99%

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Phosphorylation of protein components of isolated zymogen granule membranes from the rat pancreas

1974

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Section: Resultscontrasting

confidence: 93%

Section: Introductionmentioning

confidence: 56%

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Phosphorylation of protein components of isolated zymogen granule membranes from the rat pancreas

1974

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“…The following differential centrifugation was based on data from de Duve et al [7] and Meldolesi et al [8] and has already been described [9,10]. In brief, the total homogenate was separated into six crude fractions: a fraction sedimenting with 180 x g for 10 min and containing nuclei and cell debris ; a "zymogen granule" fraction sedimenting with 1000 x g for 10 min; a "mitochondrial" fraction sedimenting with 4000xg for 15 min; a "heavy microsome" fraction sedimenting with 15000 x g for 15 min; a "light microsome" fraction sedimenting with 150000 x g for 30 min; and the 150000 x g postmicrosomal supernatant.…”

Section: Subcellulav Fractionation Of Rat Pancreasmentioning

confidence: 99%

Subcellular Distribution and Response to Gastrointestinal Hormones of Adenylate Cyclase in the Rat Pancreas Partial Purification of a Stable Plasma Membrane Preparation

Svoboda¹,

Robberecht²,

Camus³

et al. 1976

European Journal of Biochemistry

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1. The subcellular distribution of adenylate cyclase activity in rat pancreatic homogenates was examined after differential centrifugation. Divalent cations exerted significant effects on this distribution. In addition, the ratio of adenylate cyclase activities in the presence of the C-terminal octapeptide of cholecystokinin-pancreozymin and secretin was lower in the crude 'mitochondrial' fraction than in 'microsomal' fractions. This difference was due to the lability of cholecystokinin-pancreozymin receptors compared to secretin receptors. The K,, app of activation was affected more than the V by this lability. Such a degradation of cholecystokinin-pancreozymin receptors was markedly delayed by isolation and storage in the presence of a phospholipid mixture.2. A simple, reasonably rapid (6 h), and easily reproducible method was developed to prepare a stable semi-purified plasma membrane fraction, characterized by a 10-fold increase in the specific activity of adenylate cyclase with respect to the whole homogenate. At variance with data obtained on crude subcellular fractions, the V of adenylate cyclase activity observed in this preparation, under maximal concentration of the C-terminal octapeptide of cholecystokinin-pancreozymin, was higher than that obtained with secretin or the vasoactive intestinal polypeptide.The role exerted by cyclic AMP as a second messenger of hormone stimulation on the exocrine pancreas is still controvertial. Indeed, cyclic AMP levels increase in rat pancreatic fragments only when cholecystokinin-pancreozymin concentrations are higher than 0.1 pM, whereas the rate of secretion of hydrolases is already maximal with a 0.01 pM cholecystokinin-pancreozymin concentration [l ,2]. In addition, the activation of rat pancreatic adenylate cyclase in acellular systems, by cholecystokininpancreozymin or its analogs, is also a matter of controversy. Previous reports considered the enzyme as being less sensitive towards cholecystokinin-pancreozymin than to secretin [3,4]. However, a recent publication indicates that this relative insensitivity might be due to the lability of the hormone receptor [5]. Our own preliminary attempts to purify plasma membranes from the rat pancreas amounts of preparations with varying hormone responsiveness. After having reexamined the subcellular distribution and stability of adenylate cyclase under various conditions, we were able to design a convenient method giving a good yield of such a preparation with high sensitivity towards the C-terminal octapeptide of cholecystokinin-pancreozymin, secretin and the vasoactive intestinal polypeptide. MATERIALS AND METHODS Subcellulav Fractionation of Rat PancreasBatches of five male Wistar albino rats, 2-3months old, with free access to standard diet and water, were sacrificed by cervical dislocation. Their pancreases were carefully dissected. All subsequent steps were performed at 2-4 "C. The tissue was homogenized in 20 volumes of buffer A at 900 rev./min for 2 min by 5 up-and-down strokes in a glass-teflon homogenizer with a...

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“…It is possible that membrane-associated, cyclic AMP-dependent protein kinases could alter the structure and function of cell membranes by catalyzing the phosphorylation of specific polypeptides in plasma membranes (7)(8)(9)(10)(11)(12)(13).…”

mentioning

confidence: 99%

Studies on the Orientation of Cyclic AMP-Dependent Protein Kinase in Human Erythrocyte Membranes

Rubin

Rosenfeld

Rosen

1973

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

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The topographic location of cyclic AMPdependent protein kinase has been studied in preparations of permeable and sealed membranes derived from human erythrocytes. Both the catalytic and cyclic AMP-binding components of protein kinase are localized on the inner, cytoplasmic, surface of the plasma membrane.3':5'-Cyclic AMP has been implicated in the regulation of a variety of membrane-associated functions including: contact inhibition (1, 2), hormone secretion (3), cellular permeability (4), and synaptic transmission (5, 6). It is possible that membrane-associated, cyclic AMP-dependent protein kinases could alter the structure and function of cell membranes by catalyzing the phosphorylation of specific polypeptides in plasma membranes (7-13).Plasma membranes derived from human erythrocytes contain greater than 70% of the total protein kinase activity found in these cells (14), and catalyze the cyclic AMPcontrolled phosphorylation of two endogenous polypeptide chains (9-11). These observations suggest that erythrocyte membranes provide an appropriate system for investigations on protein kinase-mediated alterations in the properties of membranes.Since the protein components of the erythrocyte membrane are asymmetrically positioned with respect to the cytoplasmic and exterior membrane surfaces (15), the biological role of cyclic AMP in membranes may be uniquely determined by the topographic distribution of the enzymes involved in the metabolism and action of the cyclic nucleotide. In the present experiments the spatial orientations of the catalytic and cyclic AMP-binding components of protein kinase were studied in permeable ghosts, sealed rightside-out ghosts, and sealed inside-out vesicles prepared according to the methods of Steck and Kant (16,17) and Bodemann and Passow (18). The disposition of protein kinase in the membrane was determined by comparing its accessibility in the various membrane preparations to the accessibility of enzymes known to be associated with either the cytoplasmic or the exterior surfaces of the membrane (15-17). (EC 1.6.4.3) by the procedure of Wallach and Kamat (22). Assays of enzymic and cyclic AMP-binding activities in salt-sealed ghosts were performed in saline buffered with 10 mM Tris -HCl, pH 7.4, to prevent lysis. Substitution of buffered saline for the standard buffers had no effect on the enzymic and binding activities in any of the membrane preparations. Inside-out vesicles and Mg2+-sealed ghosts were not lysed by incubation in the standard assay buffers. MATERIALS AND METHODS RESULTSThe data on the accessibility of cyclic AMP-dependent protein kinase and three reference enzymes are presented in Table 1. Acetylcholinesterase, a marker for the exterior surface of the erythrocyte membrane (23), is completely accessible in permeable ghosts and sealed, rightside-out membranes, but is relatively inaccessible in sealed, inside-out membranes. Conversely, glyceraldehyde-3-phosphate dehydrogenase, an indicator of the inner surface of the membrane (24), is completely available i...

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Pancreozymin and caerulein stimulate in vitro protein phosphorylation in the rat pancreas

Cited by 29 publications

References 11 publications

Phosphorylation of protein components of isolated zymogen granule membranes from the rat pancreas

Phosphorylation of protein components of isolated zymogen granule membranes from the rat pancreas

Subcellular Distribution and Response to Gastrointestinal Hormones of Adenylate Cyclase in the Rat Pancreas Partial Purification of a Stable Plasma Membrane Preparation

Studies on the Orientation of Cyclic AMP-Dependent Protein Kinase in Human Erythrocyte Membranes

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