scholarly journals Identification of membrane dipeptidase as a major glycosyl-phosphatidylinositol-anchored protein of the pancreatic zymogen granule membrane, and evidence for its release by phospholipase A

1997 ◽  
Vol 324 (1) ◽  
pp. 151-157 ◽  
Author(s):  
Nigel M. HOOPER ◽  
Sarah COOK ◽  
Jean LAINÉ ◽  
Denis LeBEL
Keyword(s):  
Enzyme Activity ◽  
Phospholipase A ◽  
Zymogen Granule ◽  
Binding Properties ◽  
Glycosyl Phosphatidylinositol ◽  
Reducing Conditions ◽  
Sds Page ◽  
Granule Membrane ◽  
Membrane Bound ◽  
Polyclonal Antisera

Membrane dipeptidase (EC 3.4.13.19) enzyme activity that is inhibited by cilastatin has been detected in pancreatic zymogen granule membranes of human, porcine and rat origin. Immunoelectrophoretic blot analysis of human and porcine pancreatic zymogen granule membranes with polyclonal antisera raised against the corresponding kidney membrane dipeptidase revealed that the enzyme is a disulphide-linked homodimer of subunit mass 61 kDa in the human and 45 kDa in the pig. Although membrane dipeptidase was, along with glycoprotein-2, one of the only two major components of carbonate high pH-washed membranes, no enzyme activity or immunoreactivity was detected in the zymogen granule contents. Digestion with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and subsequent recognition by antibodies specific for the cross-reacting determinant, revealed that membrane dipeptidase in human and porcine pancreatic zymogen granule membranes is glycosyl-phosphatidylinositol-anchored. Membrane dipeptidase was released from the pancreatic zymogen granule membranes by an endogenous hydrolase, and the released form migrated as a disulphide-linked dimer on SDS/PAGE under non-reducing conditions. Under reducing conditions it migrated with the same apparent molecular mass as the membrane-bound form, and was still a substrate for bacterial PI-PLC. Treatment of kidney microvillar membranes with phospholipase A2 resulted in the release of membrane dipeptidase in a form that demonstrated electrophoretic and cilastatin–Sepharose binding properties identical to those of the endogenously released form of the enzyme from zymogen granule membranes. These results indicate that the glycosyl-phosphatidylinositol anchor on the pancreatic membrane dipeptidase is cleaved by an endogenous hydrolase, probably a phospholipase A, and that this cleavage may promote the release of the protein from the membrane.

Reducing conditions induce a total degradation of the major zymogen granule membrane protein in both its membranous and its soluble form. Immunochemical quantitation of the two forms

1986 ◽  
Vol 64 (5) ◽  
pp. 456-462 ◽  
Author(s):  
Jean Paquette ◽  
François A. Leblond ◽  
Marlyne Beattie ◽  
Denis LeBel
Keyword(s):  
Membrane Protein ◽  
Gel Filtration ◽  
Soluble Form ◽  
Major Protein ◽  
Zymogen Granule ◽  
Chloromethyl Ketone ◽  
Reducing Conditions ◽  
Immunochemical Quantitation ◽  
Granule Membrane ◽  
Different Levels

The major protein of the pig pancreatic zymogen granule membrane is an integral glycoprotein of 92 × 103 daltons (Da) which amounts to 25% of the total proteins of this membrane. When zymogen granule membranes were prepared in presence of 5 mM dithiothreitol (DTT), this glycoprotein specifically vanished from the membrane preparation. During membrane purification two other fractions were produced out of the purified granules: a soluble fraction of zymogens referred to as granule content and a dense pellet. The possibility that DTT could release the 92-kDa protein from the membrane to these other fractions has been rejected. Altogether, addition of DTT during the lysis of the granules induced a total degradation of the 92-kDa protein. This hydrolysis could be inhibited by phenylmethylsulfonyl fluoride but not by N-α-p-tosyl-L-lysine chloromethyl ketone or L-1-tosylamide-2-phenylethylchloromethyl ketone. In the course of these experiments, using gel filtration of the granule content, it was found that the 92-kDa protein was also present in the granule content in the form of an aggregate of 300 kDa. A protease was present in this aggregate and could hydrolyse the 92-kDa protein upon addition of DTT. From immunoblotting studies and rocket immunoelectrophoresis, it was found that the soluble 92-kDa protein was antigenically similar to the membrane protein and that 44% of the immunoreactive glycoprotein of the granule was soluble in the content. A cross-reacting fragment of 65 kDa has been observed in all the fractions, yet at different levels. It is concluded that as much of the 92-kDa protein is soluble in the content as it is anchored in the membrane. The protease responsible for its degradation upon addition of DTT seems to be closely associated with the protein and could be involved in its posttranslational solubilization leading to its secretion.

Two polyclonal antisera to rat luteal LH/CG receptor with different ligand binding inhibition and immunohistochemical receptor detection capabilities

1991 ◽  
Vol 125 (3) ◽  
pp. 305-312 ◽  
Author(s):  
Jouni T. Lakkakorpi ◽  
Kari P. Keinänen ◽  
Hannu J. Rajaniemi
Keyword(s):  
Luteal Cell ◽  
Complex Method ◽  
Dependent Manner ◽  
Hormone Binding ◽  
Sds Page ◽  
Antibody Titres ◽  
Membrane Bound ◽  
Polyclonal Antisera ◽  
Detection Capabilities ◽  
Reduced Forms

Abstract. Polyclonal antisera to a SDS-denatured and partially renatured rat luteal 90 K LH/CG receptor were raised in rabbits, characterized, and their applicability for immunohistochemical location of the receptor examined. The LH/CG receptor was purified by hCG-affinity chromatography and subjected either to a preparative SDS-PAGE or Western blotting. Gel slices containing the SDS-denatured or nitrocellulose strips containing the renatured 90 K LH/CG receptor were used for immunization. The antisera, termed ARS-2 and ARS-3, respectively, possessed similar antibody titres. Both antisera were able to recognize the native, SDS-denatured, and SDS-denatured and reduced forms of the LH/CG receptor on dot blots, but only ARS-3 contained antibodies to the hormone binding site or a region near to it, as it was able to inhibit the hCG binding to the membrane-bound LH/CG receptor in a dilution-dependent manner. Both antisera recognized the receptor-hCG complex, but ARS-2 stained the complex with about 50% less intensity than the free receptor. ARS-3 located the LH/CG receptor distinctly on the luteal cell surfaces in immunohistochemical staining with peroxidase antiperoxidase complex method, but ARS-2, although it possessed similar antibody titre, revealed negligible staining. Thus, the antisera readily recognize the native receptor, but differ in their capability for inhibiting hormone binding. Only ARS-3, produced against the renatured receptor, contains sufficient amounts of antibodies capable of recognizing free and occupied receptors in immunohistochemistry.

Structure of the pig pancreatic GP-2: role of intramolecular disulfides in the resistance to proteolysis

1989 ◽  
Vol 67 (6) ◽  
pp. 281-287 ◽  
Author(s):  
Denis Lebel ◽  
Jean Paquette
Keyword(s):  
Disulfide Bonds ◽  
Hydrophobic Interaction Chromatography ◽  
Zymogen Granule ◽  
Proteolytic Digestion ◽  
Tryptic Fragment ◽  
Reducing Conditions ◽  
Granule Membrane ◽  
Reconstituted System ◽  
Specific Degradation

GP-2 is the major membrane glycoprotein characteristic of the pancreatic zymogen granule membrane. When granules are lysed in the presence of DTT, GP-2 becomes completely and specifically degraded. This proteolysis was reproducible with the same characteristics in the purified granule membrane. The protease was purified from this source using hydrophobic interaction chromatography. The proteolytic activity was identified as a 29-kDa protein because, in a reconstituted system containing both the purified GP-2 and the 29-kDa protein, the proteolytic degradation of GP-2 was sensitive to the same spectrum and concentrations of inhibitors or reducing agents as in the membrane. The activity was characteristic of a serine protease. It was also shown that GP-2 only becomes sensitive to proteolytic digestion when its disulfide bonds are reduced, and that DTT does not activate the protease. Seven intramolecular disulfide bonds were identified on GP-2. All of them are located in a 65-kDa tryptic fragment that is very resistant to exogenous proteases under nonreducing conditions. Because of the quite specific degradation of GP-2 under reducing conditions, we believe that the 29-kDa protease must be closely associated with GP-2 on the membrane. This protease could be responsible, in part, for the solubilization of the GP-2 from the membrane into the zymogen granule content and its resulting secretion by the pancreas.Key words: GP-2, zymogen granule, disulfide, exocrine, pancreas, secretion.

scholarly journals Evidence for an apical, nonregulated protein secretion in pig exocrine pancreas

1994 ◽  
Vol 267 (5) ◽  
pp. G764-G771
Author(s):  
G. Viau ◽  
J. Laine ◽  
F. Levenez ◽  
A. M. Gueugneau ◽  
T. Corring ◽  
...  
Keyword(s):  
Protein Secretion ◽  
Exocrine Pancreas ◽  
Specific Activity ◽  
Secretory Cells ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Secretory Product ◽  
Glycosyl Phosphatidylinositol ◽  
Granule Membrane ◽  
The One

Secretory proteins are segregated into two pathways out of the trans-Golgi network of regulated secretory cells. To identify proteins specifically secreted by pathways other than the one leading to zymogen granule exocytosis in the exocrine pancreas, conscious permanently cannulated pigs were perfused with atropine to inhibit the regulated fusion of granules. Atropine almost totally inhibited the protein secretion after 1 h of perfusion. The secretion of GP-2, a glycosyl phosphatidylinositol-anchored protein of the zymogen granule membrane, was partially inhibited but was never totally abolished by atropine perfusion. The pattern of proteins secreted under atropine was almost totally different. Soluble GP-2 was the major secretory product. Its specific activity increased 60 times over its normal level in all other conditions. This secretion clearly originated from nonregulated pathways. Results suggest that during the atropine block the apical plasmalemma could be the source of the released GP-2 and that the sustained nature of this release is compatible with a replenishment of the plasmalemma with GP-2 by the continuous exocytosis of vesicles from the nonregulated pathways.

Cholesterol-dependent interaction of syncollin with the membrane of the pancreatic zymogen granule

2001 ◽  
Vol 356 (3) ◽  
pp. 843-850 ◽  
Author(s):  
Alois HODEL ◽  
Seong J. AN ◽  
Neal J. HANSEN ◽  
Jared LAWRENCE ◽  
Barbara WÄSLE ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Membrane Lipids ◽  
Zymogen Granule ◽  
Dependent Manner ◽  
Phosphatidylcholine Liposomes ◽  
Sds Page ◽  
Luminal Side ◽  
Granule Membrane ◽  
In Vitro Interaction

Syncollin is a protein of the pancreatic zymogen granule that was isolated through its ability to bind to syntaxin. Despite this in vitro interaction, it is now clear that syncollin is present on the luminal side of the zymogen granule membrane. Here we show that there are two pools of syncollin within the zymogen granule: one free in the lumen and the other tightly associated with the granule membrane. When unheated or cross-linked samples of membrane-derived syncollin are analysed by SDS/PAGE, higher-order forms are seen in addition to the monomer, which has an apparent molecular mass of 16kDa. Extraction of cholesterol from the granule membrane by treatment with methyl-β-cyclodextrin causes the detachment of syncollin, and this effect is enhanced at a high salt concentration. Purified syncollin is able to bind to brain liposomes at pH5.0, but not at pH11.0, a condition that also causes its extraction from granule membranes. Syncollin binds only poorly to dioleoyl phosphatidylcholine liposomes, but binding is dramatically enhanced by the inclusion of cholesterol. Finally, cholesterol can be co-immunoprecipitated with syncollin. We conclude that syncollin is able to interact directly with membrane lipids, and to insert into the granule membrane in a cholesterol-dependent manner. Membrane-associated syncollin apparently exists as a homo-oligomer, possibly consisting of six subunits, and its association with the membrane may be stabilized by electrostatic interactions with either other proteins or phospholipids.

Exterior and Interior Events in Early Stage of Thrombin-induced Platelet Aggregation

1977 ◽  
Vol 38 (03) ◽  
pp. 0630-0639 ◽  
Author(s):  
Shuichi Hashimoto ◽  
Sachiko Shibata ◽  
Bonro Kobayashi
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Early Stage ◽  
Adenyl Cyclase ◽  
Cellular Component ◽  
Primary Event ◽  
Rabbit Platelets ◽  
Membrane Bound

SummaryTreatment of washed rabbit platelets with 1 u/ml of thrombin at 37° C resulted in a disappearance from platelets of a protein with 250,000 dalton molecular weight which was shown to be originated from plasma membrane. Parallel loss of adenyl cyclase was noted, and both reactions were complete within 30 sec. From the patterns of disc electrophoretograms, the importance of quick suppression of thrombin action in demonstrating the primary event was stressed.Thrombin induced an apparent activation of membrane bound phosphodiesterase. This reaction was also complete within 30 sec. The cellular component which contained the enzyme activity was distinct from plasma membrane. Soluble phosphodiesterase was not influenced by thrombin at all.These reactions required intact platelet cells to react with thrombin, and no reaction was detected when subcellular preparations were treated with thrombin.Possibility of collaboration of changes in externally located synthetic enzyme with those in internally located degrading enzyme in the early phase of thrombin action on platelets was suggested.

scholarly journals Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

2014 ◽  
Vol 81 (3) ◽  
pp. 1024-1031 ◽  
Author(s):  
Bhagyalakshmi Kalidass ◽  
Muhammad Farhan Ul-Haque ◽  
Bipin S. Baral ◽  
Alan A. DiSpirito ◽  
Jeremy D. Semrau
Keyword(s):  
Methane Monooxygenase ◽  
High Specificity ◽  
Copper Uptake ◽  
Content Type ◽  
Soluble Methane Monooxygenase ◽  
Sds Page ◽  
Genes Encoding ◽  
Key Factor ◽  
Membrane Bound

ABSTRACTIt is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that inMethylosinus trichosporiumOB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced byM. trichosporiumOB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and activein situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin.

Characterization of the Metamitron Deaminating Enzyme Activity from Sugar Beet ( Beta vulgaris L.) Leaves

1979 ◽  
Vol 34 (11) ◽  
pp. 948-950 ◽  
Author(s):  
Carl Fedtke ◽  
Robert R. Schmidt
Keyword(s):  
Cytochrome C ◽  
Sugar Beet ◽  
Electron Donor ◽  
Nitrogen Atmosphere ◽  
Enzyme Preparations ◽  
Reducing Conditions ◽  
Trade Name ◽  
Membrane Bound

Abstract The enzymatic activity from sugar beet leaves which is responsible for the detoxification of the herbicide metamitron (4-amino-4,5-dihydro-3-methyl-6-phenyl-1, 2, 4-triazin-5-one, trade name Goltix®) has been characterized in vitro. The detoxification occurs by rapid deamination in vivo as well as in vitro. However, the deamination in vitro is only maximal under reducing conditions, i. e. with an electron donor and in a nitrogen atmosphere. The electron donor may be cystein, glutathione, dithionite or ascorbate. The enzymatic deamination further requires the addition of cytochrome c and a “supernatant factor”, which may be replaced by FMN, FAD or DCPIP. However, in the presence of FMN or DCPIP cytochrome c is not essential but only stimulatory. The partic­ulate as well as the soluble metamitron deaminating enzyme preparations obtained take up oxygen when supplied with cysteine and FMN. The particulate enzyme appears in the peroxysome-fraction. It is therefore suggested, that the enzymatic deamination of metamitron in sugar beet leaves is mediated by a proxisomal membrane bound electron transport system which alternatively may reduce oxygen or metamitron (deaminating).

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