Specific glutamic acid residues in targeted proteins induce exaggerated retardations in Phos-tag SDS-PAGE migration

2017 ◽  
Vol 38 (8) ◽  
pp. 1139-1146 ◽  
Author(s):  
Eiji Kinoshita ◽  
Emiko Kinoshita-Kikuta ◽  
Kiyonobu Karata ◽  
Toshiki Kawano ◽  
Atsuhiro Nishiyama ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Page Migration ◽  
Sds Page

Front Cover: Specific glutamic acid residues in targeted proteins induce exaggerated retardations in Phos-tag SDS-PAGE migration

2017 ◽  
Vol 38 (8) ◽  
pp. NA-NA
Author(s):  
Eiji Kinoshita ◽  
Emiko Kinoshita-Kikuta ◽  
Kiyonobu Karata ◽  
Toshiki Kawano ◽  
Atsuhiro Nishiyama ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Front Cover ◽  
Page Migration ◽  
Sds Page

scholarly journals Detergent binding explains anomalous SDS-PAGE migration of membrane proteins

2009 ◽  
Vol 106 (6) ◽  
pp. 1760-1765 ◽  
Author(s):  
Arianna Rath ◽  
Mira Glibowicka ◽  
Vincent G. Nadeau ◽  
Gong Chen ◽  
Charles M. Deber
Keyword(s):  
Membrane Proteins ◽  
Page Migration ◽  
Sds Page

Effects of Ultrasonic and Physical-Rays Treatment on the Molecular Structures of Poly(γ-Glutamic Acid)

2012 ◽  
Vol 518-523 ◽  
pp. 5550-5554
Author(s):  
Ming Hui ◽  
Chun Yuan Gao ◽  
Qing Tian ◽  
Xiao Bo Du ◽  
Xiang Long Yin
Keyword(s):  
Molecular Weight ◽  
Glutamic Acid ◽  
Molecular Weight Distribution ◽  
Weight Distribution ◽  
Molecular Structures ◽  
Low Molecular Weight ◽  
Chain Molecules ◽  
Sds Page ◽  
Γ Rays ◽  
Long Chain

Molecule structures of poly(γ-glutamic acid) (γ-PGA) were modified by ultrasonic, UV and 60Co γ-rays irradiation treatments, which might be used to prepare the polymer with low molecular weight. When 10 g/l γ-PGA solution was sonicated 60 times (400W, working time 3 s and interval 4 s) or that of 20 g/l solution was irradiated from 2 kGy to 10 kGy, the long-chain molecules were broken into smaller fragments. But, as the same solution of 10 g/l was irradiated by UV rays for 10 min, the molecular aggregates were observed in the solution so that the molecular weight distribution of γ-PGA became narrower compared with the control through the analysis of SDS-PAGE. These results would have a reference of the modification of γ-PGA molecules and the production of low molecular weight γ-PGA.

Isolation of tubulin polyglutamylase from Crithidia; binding to microtubules and tubulin, and glutamylation of mammalian brain alpha- and beta-tubulins

1999 ◽  
Vol 112 (13) ◽  
pp. 2185-2193 ◽  
Author(s):  
S. Westermann ◽  
A. Schneider ◽  
E.K. Horn ◽  
K. Weber
Keyword(s):  
Glutamic Acid ◽  
Gradient Centrifugation ◽  
Exchange Chromatography ◽  
Mammalian Brain ◽  
Side Chain ◽  
Dependent Manner ◽  
Sds Page ◽  
Microtubular Cytoskeleton ◽  
Low Efficiency ◽  
Carboxy Terminal

Trypanosomatids have a striking cage-like arrangement of submembraneous microtubules. We previously showed that alpha- and beta- tubulins of these stable microtubules are extensively modified by polyglutamylation. Cytoskeletal microtubular preparations obtained by Triton extraction of Leishmania tarentolae and Crithidia fasciculata retain an enzymatic activity that incorporates radioactive glutamic acid in a Mg2+-ATP-dependent manner into alpha- and beta-tubulins. The tubulin polyglutamylase is extracted by 0.25 M salt. The Crithidia enzyme can be purified by ATP-affinity chromatography, glycerol-gradient centrifugation and ion-exchange chromatography. After extraction from the microtubular cytoskeleton the glutamylase forms a complex with alphabeta tubulin, but behaves after removal of tubulin as a globular protein with a molecular mass of 38x10(3). In highly enriched fractions a corresponding band is the major polypeptide visible in SDS-PAGE. The enzyme from Crithidia recognises mammalian brain tubulin, where it incorporates glutamic acid preferentially into the more acidic variants of both alpha- and beta-tubulins. Synthetic peptides with an oligoglutamyl side chain, corresponding to the carboxy-terminal end of brain alpha- and beta-tubulins, are accepted by the enzyme, albeit at low efficiency. The polyglutamylase elongates the side chain by up to 3 and 5 residues, respectively. Other properties of the tubulin polyglutamylase are also discussed.

scholarly journals Use of Nonspecific, Glutamic Acid-Free, Media and High Glycerol or High Amylase as Inducing Parameters for Screening Bacillus Isolates Having High Yield of Polyglutamic Acid

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Nandita N. Baxi
Keyword(s):  
Glutamic Acid ◽  
Synthetic Medium ◽  
Maximum Yield ◽  
Proteinase K ◽  
High Yield ◽  
Polyglutamic Acid ◽  
Production Medium ◽  
Coomassie Blue ◽  
Sds Page ◽  
Free Media

Out of fifty-five Bacillus isolates obtained from ten different regional locations and sources, seven showed the ability to consistently produce specific extracellular polymeric substance (EPS) on rich as well as synthetic but nonspecific media which did not contain glutamic acid. The isolates were identified as either Bacillus licheniformis or Bacillus subtilis. The EPS from all isolates was resistant to alpha protease, proteinase K, and was thus of high molecular weight. Further it was detected after SDS-PAGE by methylene blue but not by coomassie blue R staining as in case of proteins with high proportion of acidic amino acids. Cell-free EPS, after acid hydrolysis, showed absence of carbohydrates and presence of only glutamic acid. Thus the native the EPS from all seven isolates was confirmed to be gamma polyglutamic acid (PGA) and not exopolysaccharide. The Bacillus isolate T which produced maximum polymer on all media tested had higher amylase: protease activity as compared to other strains. If inoculum was developed in rich medium as compared to synthetic medium, the PGA produced increased by twofold in the subsequent synthetic production medium. Similarly, use of inoculum consisting of young and vegetative cells also increased the PGA production by twofold though amount of inoculum did not affect yield of PGA. Though PGA was produced in even in the absence of glutamic acid supplementation in the production medium by all isolates, the yield of PGA increased by fourfold in the presence glutamic acid and the maximum yield was 30 g/l for isolate K. The supplementation of glutamine instead of glutamic acid into the medium caused an increase in the viscosity of the non-Newtonian solution of PGA.

scholarly journals Compositional changes of proteins and amino acids in germinating coffee seeds

2000 ◽  
Vol 43 (3) ◽  
pp. 259-265 ◽  
Author(s):  
Milton Massao Shimizu ◽  
Paulo Mazzafera
Keyword(s):  
Amino Acids ◽  
Glutamic Acid ◽  
Aspartic Acid ◽  
Free Amino Acids ◽  
Free Amino ◽  
Total Content ◽  
Total Soluble Protein ◽  
Sds Page ◽  
Compositional Changes ◽  
Quantitative Changes

Endosperm is the main reserve tissue in coffee seeds. Coffee (Coffea arabica L.) seeds were germinated for six weeks and qualitative and quantitative changes in amino acids and proteins were investigated. The total content of free amino acids were reduced during germination, however, protein content remained constant. SDS-PAGE profiles showed that legumin-like proteins became less stained in the last weeks. Asparagine, glutamic acid, aspartic acid, alanine and lysine were the major free amino acids, although serine and glutamine were also significant. Except for tyrosine, which increased with germination, all other amino acids were reduced. Analysis of the amino acid composition of the total soluble protein showed glutamic acid/glutamine and glycine as the main amino acids. However, other amino acids such as leucine, aspartic acid/asparagine, alanine, lysine, serine were also found in reasonable amounts.

scholarly journals Abnormal SDS-PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding

2012 ◽  
Vol 21 (8) ◽  
pp. 1197-1209 ◽  
Author(s):  
Yunhua Shi ◽  
Richard A. Mowery ◽  
Jonathan Ashley ◽  
Michelle Hentz ◽  
Alejandro J. Ramirez ◽  
...  
Keyword(s):  
Cytosolic Proteins ◽  
Page Migration ◽  
Sds Page

3-D organization of fibrinogen receptors using computer reconstruction and whole-mount microscopy

1988 ◽  
Vol 46 ◽  
pp. 30-31
Author(s):  
E. T. O'Toole ◽  
R. R. Hantgan ◽  
J. C. Lewis
Keyword(s):  
Whole Blood ◽  
Colloidal Gold ◽  
Blood Platelets ◽  
Cell Contact ◽  
Computer Assisted ◽  
Adherent Cells ◽  
Differential Centrifugation ◽  
Computer Reconstruction ◽  
Sds Page ◽  
Whole Mount

Thrombocytes (TC), the avian equivalent of blood platelets, support hemostasis by aggregating at sites of injury. Studies in our lab suggested that fibrinogen (fib) is a requisite cofactor for TC aggregation but operates by an undefined mechanism. To study the interaction of fib with TC and to identify fib receptors on cells, fib was purified from pigeon plasma, conjugated to colloidal gold and used both to facilitate aggregation and as a receptor probe. Described is the application of computer assisted reconstruction and stereo whole mount microscopy to visualize the 3-D organization of fib receptors at sites of cell contact in TC aggregates and on adherent cells.Pigeon TC were obtained from citrated whole blood by differential centrifugation, washed with Ca++ free Hank's balanced salts containing 0.3% EDTA (pH 6.5) and resuspended in Ca++ free Hank's. Pigeon fib was isolated by precipitation with PEG-1000 and the purity assessed by SDS-PAGE. Fib was conjugated to 25nm colloidal gold by vortexing and the conjugates used as the ligand to identify fib receptors.

Parallel Reduction of Plasma Levels of High and Low Molecular Weight Kininogen in Patients with Cirrhosis

1999 ◽  
Vol 82 (11) ◽  
pp. 1428-1432 ◽  
Author(s):  
Cheryl Scott ◽  
Francesco Salerno ◽  
Elettra Lorenzano ◽  
Werner Müller-Esterl ◽  
Angelo Agostoni ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Liver Disease ◽  
Liver Function ◽  
Plasma Levels ◽  
Plasma Concentrations ◽  
Normal Subjects ◽  
Low Molecular Weight ◽  
Major Band ◽  
Sds Page ◽  
Normal Controls

SummaryLittle is known about the regulation of high-molecular-weight-kininogen (HK) and low-molecular-weight-kininogen (LK) or the relationship of each to the degree of liver function impairment in patients with cirrhosis. In this study, we evaluated HK and LK quantitatively by a recently described particle concentration fluorescence immunoassay (PCFIA) and qualitatively by SDS PAGE and immunoblotting analyses in plasma from 33 patients with cirrhosis presenting various degrees of impairment of liver function. Thirty-three healthy subjects served as normal controls. Patients with cirrhosis had significantly lower plasma levels of HK (median 49 μg/ml [range 22-99 μg/ml]) and LK (58 μg/ml [15-100 μg/ml]) than normal subjects (HK 83 μg/ml [65-115 μg/ml]; LK 80 μg/ml [45-120 μg/ml]) (p < 0.0001). The plasma concentrations of HK and LK were directly related to plasma levels of cholinesterase (P < 0.0001) and albumin (P < 0.0001 and P < 0.001) and inversely to the Child-Pugh score (P < 0.0001) and to prothrombin time ratio (P < 0.0001) (reflecting the clinical and laboratory abnormalities in liver disease). Similar to normal individuals, in patients with cirrhosis, plasma HK and LK levels paralleled one another, suggesting that a coordinate regulation of those proteins persists in liver disease. SDS PAGE and immunoblotting analyses of kininogens in cirrhotic plasma showed a pattern similar to that observed in normal controls for LK (a single band at 66 kDa) with some lower molecular weight forms noted in cirrhotic plasma. A slight increase of cleavage of HK (a major band at 130 kDa and a faint but increased band at 107 kDa) was evident. The increased cleavage of HK was confirmed by the lower cleaved kininogen index (CKI), as compared to normal controls. These data suggest a defect in hepatic synthesis as well as increased destructive cleavage of both kininogens in plasma from patients with cirrhosis. The decrease of important regulatory proteins like kininogens may contribute to the imbalance in coagulation and fibrinolytic systems, which frequently occurs in cirrhotic patients.

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