scholarly journals Dual Lipid Modification of the Yeast Gγ Subunit Ste18p Determines Membrane Localization of Gβγ

1999 ◽  
Vol 19 (11) ◽  
pp. 7705-7711 ◽  
Author(s):  
Jodi E. Hirschman ◽  
Duane D. Jenness
Keyword(s):  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cysteine Residue ◽  
Wild Type ◽  
Molecular Weights ◽  
Sds Page ◽  
Membrane Attachment ◽  
The Stability ◽  
Mutant Forms

ABSTRACT The pheromone response in the yeast Saccharomyces cerevisiae is mediated by a heterotrimeric G protein. The Gβγ subunit (a complex of Ste4p and Ste18p) is associated with both internal and plasma membranes, and a portion is not stably associated with either membrane fraction. Like Ras, Ste18p contains a farnesyl-directing CaaX box motif (C-terminal residues 107 to 110) and a cysteine residue (Cys 106) that is a potential site for palmitoylation. Mutant Ste18p containing serine at position 106 (mutation ste18-C106S) migrated more rapidly than wild-type Ste18p during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic mobility of wild-type Ste18p (but not the mutant Ste18p) was sensitive to hydroxylamine treatment, consistent with palmitoyl modification at Cys 106. Furthermore, immunoprecipitation of the Gβγ complex from cells cultured in the presence of [3H]palmitic acid resulted in two radioactive species on nonreducing SDS-PAGE gels, with molecular weights corresponding to Gγ and Gβγ. Substitution of serine for either Cys 107 or Cys 106 resulted in the failure of Gβγ to associate with membranes. The Cys 107 substitution also resulted in reduced steady-state accumulation of Ste18p, suggesting that the stability of Ste18p requires modification at Cys 107. All of the mutant forms of Ste18p formed complexes with Ste4p, as assessed by coimmunoprecipitation. We conclude that tight membrane attachment of the wild-type Gβγ depends on palmitoylation at Cys 106 and prenylation at Cys 107 of Ste18p.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

scholarly journals Effects of Wheat Bug (Eurygasterspp. andAeliaspp.) Infestation in Preharvest Period on Wheat Technological Quality and Gluten Composition

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Aleksandra M. Torbica ◽  
Jasna S. Mastilović ◽  
Milica M. Pojić ◽  
Žarko S. Kevrešan
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Wheat Gluten ◽  
Sodium Dodecyl ◽  
Deformation Energy ◽  
Wheat Varieties ◽  
Gluten Proteins ◽  
Molecular Weights ◽  
Sds Page ◽  
Technological Quality ◽  
Gluten Index

The effects of wheat bug infestation (Eurygasterspp. andAeliaspp.) on the composition of wheat gluten proteins and its influence on flour technological quality were investigated in the present study. Wheat samples of six wheat varieties, collected from two localities in northern Serbia, were characterized by significantly different level of wheat bug infestation. Composition of wheat gluten proteins was determined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE), while the selected parameters of technological quality were determined according to standard and modified empirical rheological methods (Farinograph, Extensograph, Alveograph, and Gluten Index). The surface morphology of the selected samples was viewed using scanning electron microscopy (SEM). Wheat from wheat bug-infested locality regardless of the variety had deteriorated technological quality expressed with higher Farinograph softening degree, lower or immeasurable Extensograph energy, and Alveograph deformation energy. The most important changes in the gluten proteins composition of bug-infested wheat were related to gliadin subunits with molecular weights below 75 kDa, which consequently caused deterioration of uniaxial and biaxial extensibility and dough softening during mixing.

scholarly journals Isolation of the intercellular glycoproteins of desmosomes.

1981 ◽  
Vol 90 (1) ◽  
pp. 243-248 ◽  
Author(s):  
G Gorbsky ◽  
M S Steinberg
Keyword(s):  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insoluble Residue ◽  
Differential Centrifugation ◽  
Electron Microscopic ◽  
Nonionic Detergent ◽  
Cell Layers ◽  
Membrane Attachment

To characterize the desmosome components that mediate intercellular adhesion and cytoskeletal-plasma membrane attachment, we prepared whole desmosomes and isolated desmosomal intercellular regions (desmosomal "cores") from the living cell layers of bovine muzzle epidermis. The tissue was disrupted in a nonionic detergent at low pH, sonicated, and the insoluble residue fractionated by differential centrifugation and metrizamide gradient centrifugation. Transmission electron microscopic analyses reveal that a fraction obtained after differential centrifugation is greatly enriched in whole desmosomes that possess intracellular plaques. Metrizamide gradient centrifugation removes most of the plaque material, leaving the intercellular components and the adjoining plasma membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with methods that reveal carbohydrate-containing moieties on gels demonstrate that certain proteins present in whole desmosomes are glycosylated. These glycoproteins are specifically and greatly enriched in the desmosome cores of which they are the principal protein constituents, and thus may function as the intercellular adhesive of the desmosome.

Acid and alkaline phosphatases of Capnocytophaga species. II. Isolation, purification, and characterization of the enzymes from Capnocytophaga ochracea

1983 ◽  
Vol 29 (10) ◽  
pp. 1361-1368 ◽  
Author(s):  
Thomas P. Poirier ◽  
Stanley C. Holt
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alkaline Phosphatases ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Sds Page ◽  
Kinetics Of

Capnocytophaga ochracea acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase was isolated by Ribi cell disruption and purified by sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE.) Both phosphatases eluted from Sephadex G-150 consistent with molecular weights (migration) of 140 000 and 110 000. SDS–PAGE demonstrated a 72 000 and 55 000 subunit molecular migration for AcP and AlP, respectively. The kinetics of activity of purified AcP and AIP on p-nitrophenol phosphate and phosphoseryl residues of the phosphoproteins are presented.

scholarly journals Synthesis of proteins from [35S]methionine by guinea pig megakaryocytes in vivo and time course of appearance of newly synthesized proteins in platelets

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 887-891 ◽  
Author(s):  
BP Schick
Keyword(s):  
Protein Synthesis ◽  
Guinea Pigs ◽  
Time Course ◽  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Molecular Weights ◽  
Sds Page ◽  
Relationship Of

The relationship of protein synthesis to megakaryocyte maturation has been studied in guinea pigs in vivo. Guinea pigs were injected with a single dose of [35S]methionine. Megakaryocytes and platelets were isolated daily for 4 days, and proteins from both cells were isolated by DEAE-Sephacel chromatography and analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. All proteins in megakaryocytes corresponding to stained bands on the SDS- PAGE gels were radiolabeled at 3 hours after injection. The greatest loss of radioactivity from the megakaryocytes occurred between 1 and 3 days after injection. Only trace labeling of platelet proteins was seen at 3 hours, representing almost entirely three bands at molecular weights 47,000, 52,000, and 66,000. At 24 hours only about 13% of the maximal labeling was present, but not all proteins were labeled. The maximal labeling was at 3 days. The pattern of labeling of platelets at 3 days was identical to that of megakaryocytes at 3 hours. The protein pattern of nonmegakaryocytic marrow cells was different from that of the platelets and megakaryocytes. Data presented here suggest that most protein synthesis in megakaryocytes is completed at least 24 hours before release of the platelets to the circulation, and suggest some specificity in the proteins that are synthesized at the terminal stages of maturation.

scholarly journals ANALYSIS OF PROTEIN CONCENTRATE FROM KAHAI (CARYODENDRON ORINOCENSE KARST) SEEDS CULTIVATED IN AMAZONIA OF ECUADOR

2018 ◽  
Vol 11 (6) ◽  
pp. 369
Author(s):  
Greffa J ◽  
Vilcacundo E ◽  
Altuna J ◽  
Carrillo W
Keyword(s):  
Extraction Method ◽  
Colorimetric Assay ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Heat Extraction ◽  
Precipitation Method ◽  
Protein Concentrate ◽  
Native Page ◽  
Molecular Weights ◽  
Sds Page

Objective: The aim of this study was to obtain Kahai protein concentrate from Caryodendron orinocense Karst cultivated in the Amazonic region of Ecuador, to characterize its proteins using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native electrophoresis methods, and to determine its content of total polyphenols.Methods: Kahai seeds (C. orinocense Karst) were utilized to obtain Kahai protein concentrate at pH 3.0, pH 4.0, pH 5.0, and pH 6.0 using the isoelectric precipitation method. The proteins were characterized using the SDS-PAGE and native-PAGE electrophoresis methods. Total polyphenols were determined using the colorimetric assay method.Results: The best treatment to obtain Kahai protein concentrate was at pH 5.0 with a 21.91% yield using the cold extraction method. The best treatment was at pH 6.0 with a 11.07% yield using the heat extraction method. Kahai concentrates presented a complex profile of proteins with molecular weights between 14 and 97 kDa. It was possible to obtain at the same time polyphenols at pH 5.0 with a value of 1028.58 mg gallic acid equivalents/100 g protein of sample.Conclusion: This study suggests that Kahai protein concentrates possess a high content of proteins and polyphenols that can be used to elaborate functional foods.

scholarly journals Synthesis of proteins from [35S]methionine by guinea pig megakaryocytes in vivo and time course of appearance of newly synthesized proteins in platelets

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 887-891 ◽  
Author(s):  
BP Schick
Keyword(s):  
Protein Synthesis ◽  
Guinea Pigs ◽  
Time Course ◽  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Molecular Weights ◽  
Sds Page ◽  
Relationship Of

Abstract The relationship of protein synthesis to megakaryocyte maturation has been studied in guinea pigs in vivo. Guinea pigs were injected with a single dose of [35S]methionine. Megakaryocytes and platelets were isolated daily for 4 days, and proteins from both cells were isolated by DEAE-Sephacel chromatography and analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. All proteins in megakaryocytes corresponding to stained bands on the SDS- PAGE gels were radiolabeled at 3 hours after injection. The greatest loss of radioactivity from the megakaryocytes occurred between 1 and 3 days after injection. Only trace labeling of platelet proteins was seen at 3 hours, representing almost entirely three bands at molecular weights 47,000, 52,000, and 66,000. At 24 hours only about 13% of the maximal labeling was present, but not all proteins were labeled. The maximal labeling was at 3 days. The pattern of labeling of platelets at 3 days was identical to that of megakaryocytes at 3 hours. The protein pattern of nonmegakaryocytic marrow cells was different from that of the platelets and megakaryocytes. Data presented here suggest that most protein synthesis in megakaryocytes is completed at least 24 hours before release of the platelets to the circulation, and suggest some specificity in the proteins that are synthesized at the terminal stages of maturation.

Characterization of the electrophoretic mobility mutation in the N protein of the tsD1 mutant of vesicular stomatitis virus New Jersey serotype

1982 ◽  
Vol 60 (11) ◽  
pp. 1065-1076 ◽  
Author(s):  
Earl Brown ◽  
Ludvik Prevec
Keyword(s):  
New Jersey ◽  
Electrophoretic Mobility ◽  
Vesicular Stomatitis Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Specific Chemical ◽  
N Protein ◽  
Sds Page ◽  
Vesicular Stomatitis

Some isolates of the temperature sensitive mutant tsD1 of complementation group D of vesicular stomatitis virus of New Jersey serotype have a nucleocapsid (N) protein which shows an increased electrophoretic mobility on sodium dodecyl sulfate –polyacrylamide gel electrophoresis (SDS–PAGE) when compared with wild type. Utilizing techniques involving specific chemical cleavage at tryptophan or methionine residues, as well as enzymatic cleavage with carboxypeptidases A and B, we have determined that residues near the carboxyterminus are responsible for the electrophoretic difference of the mutant protein. We have further shown that there are no differences in the tryptic peptides of the mutant compared with the wild type or a non-ts revertant in this region of the protein. We have identified a tryptic peptide located outside the relevant carboxyterminal region which is distinct in mutant and revertant. We conclude that the mutation producing the aberrant electrophoretic mobility of N protein of the tsD1 mutant is a missense point mutation located at least 40 amino acid residues from the carboxyterminus and which interacts with a more proximal carboxyregion so as to influence electrophoretic mobility on SDS–PAGE.

Glutathione synthetase from the fission yeast. Purification and its unique heteromeric subunit structure

1993 ◽  
Vol 71 (9-10) ◽  
pp. 447-453 ◽  
Author(s):  
Chiaki W. Nakagawa ◽  
Norihiro Mutoh ◽  
Yukimasa Hayashi
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Fission Yeast ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Glutathione Synthetase ◽  
Subunit Structure ◽  
Wild Type ◽  
Native Page ◽  
Sds Page

Glutathione (GSH) synthetase (EC 6.3.2.3) was purified from the fission yeast Schizosaccharomyces pombe L972h− and from the GSH synthetase deficient mutant MN101/pYS41, which harbors a plasmid containing the GSH synthetase gene of the fission yeast. GSH synthetase is expressed at 10 times higher the amount in MN101/pYS41 than in wild-type L972 h−. The purified enzyme gave a single band on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (native PAGE). The molecular weight of this enzyme was determined to be 1.2 × 105 by Sepharose CL-6B gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) revealed that this enzyme was composed of two kinds of subunits, A (Mr = 33 × 103) and B (Mr = 26 × 103), and existed as a heterotetramer (A2B2). The enzyme purified from the wild-type fission yeast, which did not harbor the plasmid, showed the same electrophoretic mobilities on both native PAGE and SDS–PAGE and similar catalytic properties under standard conditions. This enzyme is most active at 45 °C and pH 8.0–8.5 with 20 mM Mg2+ + 10 mM ATP and 50 mM K+. The strict requirement for the monovalent cation is rather specific for the enzymes from yeasts. The presence of sugar components in the enzyme is also observed, similar to that in the rat kidney enzyme.Key words: Schizosaccharomyces pombe, glutathione synthetase, heteromeric subunit structure.

scholarly journals Construction and Characterization of Haemophilus ducreyi Lipooligosaccharide (LOS) Mutants Defective in Expression of Heptosyltransferase III and β1,4-Glucosyltransferase: Identification of LOS Glycoforms Containing Lactosamine Repeats

2000 ◽  
Vol 68 (6) ◽  
pp. 3352-3361 ◽  
Author(s):  
Melanie J. Filiatrault ◽  
Bradford W. Gibson ◽  
Birgit Schilling ◽  
Shuhua Sun ◽  
Robert S. Munson ◽  
...  
Keyword(s):  
Lipid A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Haemophilus Ducreyi ◽  
Ionization Mass ◽  
Wild Type ◽  
Reading Frame ◽  
Plasmid Library ◽  
Sds Page ◽  
Isogenic Mutants

ABSTRACT To begin to understand the role of the lipooligosaccharide (LOS) molecule in chancroid infections, we constructed mutants defective in expression of glycosyltransferase genes. Pyocin lysis and immunoscreening was used to identify a LOS mutant of Haemophilus ducreyi 35000. This mutant, HD35000R, produced a LOS molecule that lacked the monoclonal antibody 3F11 epitope and migrated with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Structural studies indicated that the principal LOS glycoform contains lipid A, Kdo, and two of the three core heptose residues. HD35000R was transformed with a plasmid library of H. ducreyi 35000 DNA, and a clone producing the wild-type LOS was identified. Sequence analysis of the plasmid insert revealed one open reading frame (ORF) that encodes a protein with homology to the WaaQ (heptosyltransferase III) ofEscherichia coli. A second ORF had homology to the LgtF (glucosyltransferase) of Neisseria meningitidis. Individual isogenic mutants lacking expression of the putative H. ducreyi heptosyltransferase III, the putative glucosyltransferase, and both glycosyltransferases were constructed and characterized. Each mutant was complemented with the representative wild-type genes in trans to restore expression of parental LOS and confirm the function of each enzyme. Matrix-assisted laser desorption ionization mass spectrometry and SDS-PAGE analysis identified several unique LOS glycoforms containing di-, tri-, and poly-N-acetyllactosamine repeats added to the terminal region of the main LOS branch synthesized by the heptosyltransferase III mutant. These novel H. ducreyi mutants provide important tools for studying the regulation of LOS assembly and biosynthesis.

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