Phos-tag SDS-PAGE systems for phosphorylation profiling of proteins with a wide range of molecular masses under neutral pH conditions

PROTEOMICS ◽  
2012 ◽  
Vol 12 (2) ◽  
pp. 192-202 ◽  
Author(s):  
Eiji Kinoshita ◽  
Emiko Kinoshita-Kikuta ◽  
Tohru Koike
Keyword(s):  
Neutral Ph ◽  
Sds Page ◽  
Wide Range ◽  
Molecular Masses ◽  
Ph Conditions

O-linked but not N-linked glycosylation is necessary for the secretion of endoglucanases I and II by Trichoderma reesei

1987 ◽  
Vol 33 (8) ◽  
pp. 698-703 ◽  
Author(s):  
C. P. Kubicek ◽  
T. Panda ◽  
G. Schreferl-kunar ◽  
F. Gruber ◽  
R. Messner
Keyword(s):  
Trichoderma Reesei ◽  
Protein Glycosylation ◽  
The Other ◽  
Detectable Effect ◽  
Secreted Protein ◽  
Intracellular Protein ◽  
Molecular Weights ◽  
Sds Page ◽  
Wide Range ◽  
Molecular Masses

The effect of inhibiting protein glycosylation was studied in nongrowing mycelia and protoplasts of Trichoderma reesei which secreted two endoglucanases (I and II) upon addition of sophorose. Tunicamycin (40 μg∙mL−1) inhibited incorporation of N-acetylglucosamine into secreted protein, but had no effect on secretion of total protein or endoglucanases. The secreted endoglucanases I and II exhibited relative molecular masses of 58 and 45 kilodaltons, respectively, irrespective of the presence of tunicamycin. On the other hand 2-deoxy-D-glucose inhibited the biosynthesis of extracellular as well as intracellular protein over a wide range of concentrations; at 50 μg∙mL−1, however, it inhibited the synthesis of extracellular protein more strongly. The synthesis of endoglucanases I and II was decreased accordingly under these conditions. SDS–PAGE did not reveal the secretion of endoglucanases with smaller molecular weights. When the two endoglucanases were purified and subjected to Endo H treatment or β-elimination, the former had no detectable effect, whereas the latter released all carbohydrate from the protein. Nevertheless, endoglucanases I and II contained 1.3 and 0.5 mol of glucosamine per mol enzyme, respectively. It is concluded that endoglucanases I and II from T. reesei contain mainly O-linked neutral carbohydrate, which is required for their secretion.

scholarly journals Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced profiling of protein phosphorylation

2012 ◽  
Vol 56 (Suppl_1) ◽  
pp. s41-s44
Author(s):  
Emiko Kinoshita-Kikuta ◽  
Eiji Kinoshita ◽  
Tohru Koike
Keyword(s):  
Protein Phosphorylation ◽  
Neutral Ph ◽  
Sds Page ◽  
Ph Conditions

scholarly journals Purification and characterization of cytosolic and microsomal cyclophilins from maize (Zea mays)

1996 ◽  
Vol 315 (3) ◽  
pp. 965-970 ◽  
Author(s):  
Philip S. SHELDON ◽  
Michael A. VENIS
Keyword(s):  
Cyclosporin A ◽  
Sequence Similarity ◽  
Terminal Sequence ◽  
Purification And Characterization ◽  
Cyclophilin B ◽  
Sds Page ◽  
Wide Range ◽  
Molecular Masses ◽  
High Level

Methods for the purification and separation of peptidyl prolyl cis–trans isomerase (PPI) from cytosolic and microsomal fractions of etiolated maize are described. On SDS/PAGE, the purified preparations appear as single polypeptides with molecular masses of 17.5 kDa and 17.7 kDa respectively. Instead of using immobilized cyclosporin A derivatives as affinity adsorbents, our methods employ conventional techniques enabling purification of the proteins on a much larger scale than previously described. An antiserum raised against the cytosolic PPI recognizes polypeptides of similar molecular mass from a wide range of plant species on an immunoblot. There is virtually no recognition of the microsomal PPI. The cytosolic and microsomal PPIs are inhibited by cyclosporin A (Ki = 6 nM in both cases), indicating that they are cyclophilins. The cytosolic enzyme is inactivated by 5 mM N-ethylmaleimide and 2 mM phenylglyoxal. N-terminal sequencing of the microsomal PPI indicates a high level of sequence similarity with the N-terminal sequence of mature animal s-cyclophilin (cyclophilin B).

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

Influence of bend geometry on flow accelerated corrosion under neutral pH conditions

2021 ◽  
Vol 122 ◽  
pp. 105127
Author(s):  
P. Madasamy ◽  
M. Mukunthan ◽  
P. Chandramohan ◽  
T.V. Krishna Mohan ◽  
Andrews Sylvanus ◽  
...  
Keyword(s):  
Accelerated Corrosion ◽  
Neutral Ph ◽  
Ph Conditions ◽  
Flow Accelerated Corrosion

scholarly journals Identification of phosphatidylserine-binding proteins in human white blood cells

1990 ◽  
Vol 269 (3) ◽  
pp. 723-728 ◽  
Author(s):  
M Wolf ◽  
M Baggiolini
Keyword(s):  
Protein Kinase C ◽  
Blood Cells ◽  
Binding Proteins ◽  
White Blood Cells ◽  
Human Neutrophils ◽  
Kinase C ◽  
Sds Page ◽  
Molecular Masses ◽  
Human White Blood Cells ◽  
Monocytes And Lymphocytes

Cytosol and membrane fractions from human neutrophils, monocytes, lymphocytes and platelets were separated by SDS/PAGE, blotted on to nitrocellulose and assayed for selective binding of phosphatidylserine (PS). Two PS-binding proteins with apparent molecular masses of 115 kDa and 100 kDa were identified in the cytosol of neutrophils, monocytes and lymphocytes. Corresponding bands along with other PS-binding proteins were detected in platelets in both cytosol and membrane fractions. These proteins were also found to bind protein kinase C (PKC) provided that PS was present. The 115 kDa and 100 kDa proteins (PS-p115/110) were partially purified from neutrophils and were used for the study of PS and PKC binding. The binding of PS did not require Ca2+ or Mg2+ and was inhibited by phosphatidic acid, by 1-alkyl-2-acetylphosphocholine and, to a lesser extent, by other lipids. The binding of PKC, however, was strictly PS- and Ca2(+)-dependent and seems to occur secondarily to PS binding.

scholarly journals Role of maltase in the utilization of sucrose by Candida albicans

1993 ◽  
Vol 291 (3) ◽  
pp. 765-771 ◽  
Author(s):  
P R Williamson ◽  
M A Huber ◽  
J E Bennett
Keyword(s):  
Candida Albicans ◽  
Intracellular Localization ◽  
Cross Reactivity ◽  
Neutral Sugar ◽  
Sequence Specificity ◽  
Western Blots ◽  
Sds Page ◽  
Molecular Masses ◽  
Kinetics Of

Two isoenzymes of maltase (EC 3.2.1.20) were purified to homogeneity from Candida albicans. Isoenzymes I and II were found to have apparent molecular masses of 63 and 66 kDa on SDS/PAGE with isoelectric points of 5.0 and 4.6 respectively. Both isoenzymes resembled each other in similar N-terminal sequence, specificity for the alpha(1-−>4) glycosidic linkage and immune cross-reactivity on Western blots using a maltase II antigen-purified rabbit antibody. Maltase was induced by growth on sucrose whereas beta-fructofuranosidase activity could not be detected under similar conditions. Maltase I and II were shown to be unglycosylated enzymes by neutral sugar assay, and more than 90% of alpha-glucosidase activity was recoverable from spheroplasts. These data, in combination with other results from this laboratory [Geber, Williamson, Rex, Sweeney and Bennett (1992) J. Bacteriol. 174, 6992-6996] showing lack of a plausible leader sequence in genomic or mRNA transcripts, suggest an intracellular localization of the enzyme. To establish further the mechanism of sucrose assimilation by maltase, the existence of a sucrose-inducible H+/sucrose syn-transporter was demonstrated by (1) the kinetics of sucrose-induced [14C]sucrose uptake, (2) recovery of intact [14C]sucrose from ground cells by t.l.c. and (3) transport of 0.83 mol of H+/mol of [14C]sucrose. In total, the above is consistent with a mechanism whereby sucrose is transported into C. albicans to be hydrolysed by an intracellular maltase.

scholarly journals Heat shock induces protein tyrosine phosphorylation in cultured cells.

1989 ◽  
Vol 108 (6) ◽  
pp. 2029-2035 ◽  
Author(s):  
P A Maher ◽  
E B Pasquale
Keyword(s):  
Heat Shock ◽  
Tyrosine Phosphorylation ◽  
Cultured Cells ◽  
Protein Tyrosine Phosphorylation ◽  
Animal Cells ◽  
Protein Tyrosine ◽  
Rous Sarcoma ◽  
Wide Range ◽  
Sarcoma Virus ◽  
Molecular Masses

We examined the effect of heat shock on protein tyrosine phosphorylation in cultured animal cells using antiphosphotyrosine antibodies in immunoblotting and immunofluorescence microscopy experiments. Heat shock significantly elevated the level of phosphotyrosine in proteins in most of the cultured cells examined, including fibroblasts, epithelial cells, nerve cells, and muscle cells, but not in Rous sarcoma virus-transformed fibroblasts. The increase in protein tyrosine phosphorylation induced by heat shock occurred in proteins with a wide range of molecular masses and was dependent on the temperature and duration of the heat shock.

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