scholarly journals Site-specific modification of albumin by free radicals. Reaction with copper(II) and ascorbate

1986 ◽  
Vol 236 (2) ◽  
pp. 397-400 ◽  
Author(s):  
G Marx ◽  
M Chevion
Keyword(s):  
Gel Electrophoresis ◽  
Chelating Agents ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cross Linking ◽  
Oh Radicals ◽  
Site Specific ◽  
Metal Chelating ◽  
Specific Manner ◽  
A Site ◽  
New Protein

Exposure of albumin to Cu(II) (10-100 microM) and ascorbate (0.1-2 mM) results in extensive molecular modifications, indicated by decreased fluorescence and chain breaks. The rate of utilization of molecular oxygen and ascorbate as a function of Cu(II) concentration is non-linear at copper/albumin ratios of greater than 1. It appears that Cu(II) bound to the tightest albumin-binding site is less available to the ascorbate than the more loosely bound cation. SDS/polyacrylamide-gel electrophoresis reveals new protein bands corresponding to 50, 47, 22, 18 and 3 kDa. For such a cleavage pattern, relatively few (approximately 3) and rather specific chain breaks occurred. Repeated addition of portions of ascorbate to the albumin/Cu(II) mixture results in increased intensity of the new bands. The absence of Cu(II) or the presence of metal chelating agents is inhibitory. There was no evidence of intermolecular cross-linking or of the formation of insoluble, albumin-derived, material. A mechanism is proposed wherein the loosely bound Cu(II) participates in a Fenton-type reaction. This generates OH. radicals, which rapidly inter-react with the protein and modify it in a ‘site-specific’ manner.

scholarly journals Site Specific Genetic Incorporation of Azidophenylalanine in Schizosaccharomyces pombe

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Nan Shao ◽  
N. Sadananda Singh ◽  
Susan E. Slade ◽  
Alexandra M. E. Jones ◽  
Mohan K. Balasubramanian
Keyword(s):  
Amino Acids ◽  
Schizosaccharomyces Pombe ◽  
Unnatural Amino Acids ◽  
Building Blocks ◽  
Trna Synthetase ◽  
Cross Linking ◽  
Site Specific ◽  
Specific Manner ◽  
Protein Functions ◽  
A Site

Abstract The diversity of protein functions is impacted in significant part by the chemical properties of the twenty amino acids, which are used as building blocks for nearly all proteins. The ability to incorporate unnatural amino acids (UAA) into proteins in a site specific manner can vastly expand the repertoire of protein functions and also allows detailed analysis of protein function. In recent years UAAs have been incorporated in a site-specific manner into proteins in a number of organisms. In nearly all cases, the amber codon is used as a sense codon and an orthogonal tRNA/aminoacyl-tRNA synthetase (RS) pair is used to generate amber suppressing tRNAs charged with the UAA. In this work, we have developed tools to incorporate the cross-linking amino acid azido-phenylalanine (AzF) through the use of bacterial tRNATyr and a modified version of TyrRS, AzFRS, in Schizosaccharomyces pombe, which is an attractive model organism for the study of cell behavior and function. We have incorporated AzF into three different proteins. We show that the majority of AzF is modified to amino-phenyl alanine, but protein cross-linking was still observed. These studies set the stage for exploitation of this new technology for the analysis of S. pombe proteins.

Optimization of the posttranslational click modification of proteins

2011 ◽  
Vol 76 (9) ◽  
pp. 1089-1101
Author(s):  
Milan Vrabel ◽  
Emine Kaya ◽  
Stefan Prill ◽  
Veronika Ehmke ◽  
Thomas Carell
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Fluorescent Protein ◽  
Click Reaction ◽  
Yellow Fluorescent Protein ◽  
Trna Synthetase ◽  
Site Specific ◽  
Specific Manner ◽  
Modified Proteins ◽  
A Site

In order to develop efficient methods that would enable the synthesis of posttranslationaly modified proteins in a site-specific manner we have adopted the orthogonal pyrrolysyl-tRNA synthetase/tRNA pair to genetically encode various pyrrolysine analogs, which we were able to insert into the yellow fluorescent protein (YFP). These experiments showed that the alkene and alkyne containing amino acids 5 and 6 are superior substrates for the pyrrolysyl-tRNA synthetase and that they can be successfully incorporated into proteins. Using the Cu(I)-catalyzed Huisgen–Meldal–Sharpless click reaction, the alkyne containing YFP was finally glycosylated with various sugars. We confirmed the presence of the modified amino acids as well as the corresponding sugar modifications by HPLC-MS/MS mass spectrometry.

scholarly journals Preferential cross-linking of the small subunit of the electron-transfer flavoprotein to general acyl-CoA dehydrogenase

1987 ◽  
Vol 243 (2) ◽  
pp. 519-524 ◽  
Author(s):  
D J Steenkamp
Keyword(s):  
Electron Transfer ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Staining Intensity ◽  
Small Subunit ◽  
Cross Linking ◽  
Thiol Groups ◽  
Electron Transfer Flavoprotein ◽  
Acceptor Activity ◽  
Lysine Residues

The interaction between pig liver mitochondrial electron-transfer flavoprotein (ETF) and general acyl-CoA dehydrogenase (GAD) was investigated by means of the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Neither ETF or GAD contained reactive thiol groups. The substitution of 9.4 lysine residues/FAD group in GAD with pyridyl disulphide structures did not affect the catalytic activity of the enzyme. Thiol groups were introduced into ETF by thiolation with methyl 4-mercaptobutyrimidate. ETF containing 10.5 reactive thiol groups/FAD group showed undiminished electron-acceptor activity with respect to GAD. The reaction of thiolated ETF and GAD containing pyridyl disulphide structures resulted in a decreased staining intensity of the small subunit of ETF on SDS/polyacrylamide-gel electrophoresis. Preferential cross-linking of the smaller subunit of ETF to GAD did not take place when ETF was first treated with SDS, but was unaffected by reduction of GAD by octanoyl-CoA.

scholarly journals Osterix Regulates Tooth Root Formation in a Site-specific Manner

2015 ◽  
Vol 94 (3) ◽  
pp. 430-438 ◽  
Author(s):  
T.H. Kim ◽  
C.H. Bae ◽  
J.C. Lee ◽  
J.E. Kim ◽  
X. Yang ◽  
...  
Keyword(s):  
Root Formation ◽  
Tooth Root ◽  
Site Specific ◽  
Specific Manner ◽  
A Site

scholarly journals Hypoxia inducible factor signaling in breast tumors controls spontaneous tumor dissemination in a site-specific manner

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Vera M. Todd ◽  
Lawrence A. Vecchi ◽  
Miranda E. Clements ◽  
Katherine P. Snow ◽  
Cayla D. Ontko ◽  
...  
Keyword(s):  
Primary Tumor ◽  
Hypoxia Inducible Factor ◽  
Metastatic Tumor ◽  
Breast Tumors ◽  
Tumor Burden ◽  
Site Specific ◽  
Tumor Dissemination ◽  
Specific Manner ◽  
A Site ◽  
The Impact

AbstractHypoxia is a common feature in tumors and induces signaling that promotes tumor cell survival, invasion, and metastasis, but the impact of hypoxia inducible factor (HIF) signaling in the primary tumor on dissemination to bone in particular remains unclear. To better understand the contributions of hypoxia inducible factor 1 alpha (HIF1α), HIF2α, and general HIF pathway activation in metastasis, we employ a PyMT-driven spontaneous murine mammary carcinoma model with mammary specific deletion of Hif1α, Hif2α, or von Hippel-Lindau factor (Vhl) using the Cre-lox system. Here we show that Hif1α or Hif2α deletion in the primary tumor decreases metastatic tumor burden in the bone marrow, while Vhl deletion increases bone tumor burden, as hypothesized. Unexpectedly, Hif1α deletion increases metastatic tumor burden in the lung, while deletion of Hif2α or Vhl does not affect pulmonary metastasis. Mice with Hif1α deleted tumors also exhibit reduced bone volume as measured by micro computed tomography, suggesting that disruption of the osteogenic niche may be involved in the preference for lung dissemination observed in this group. Thus, we reveal that HIF signaling in breast tumors controls tumor dissemination in a site-specific manner.

scholarly journals Site-specific monoubiquitination downregulates Rab5 by disrupting effector binding and guanine nucleotide conversion

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Donghyuk Shin ◽  
Wooju Na ◽  
Ji-Hyung Lee ◽  
Gyuhee Kim ◽  
Jiseok Baek ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Regulatory Mechanism ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Guanine Nucleotide ◽  
Site Specific ◽  
Downstream Effectors ◽  
Specific Manner ◽  
A Site ◽  
Effector Binding

Rab GTPases, which are involved in intracellular trafficking pathways, have recently been reported to be ubiquitinated. However, the functions of ubiquitinated Rab proteins remain unexplored. Here we show that Rab5 is monoubiquitinated on K116, K140, and K165. Upon co-transfection with ubiquitin, Rab5 exhibited abnormalities in endosomal localization and EGF-induced EGF receptor degradation. Rab5 K140R and K165R mutants restored these abnormalities, whereas K116R did not. We derived structural models of individual monoubiquitinated Rab5 proteins (mUbRab5s) by solution scattering and observed different conformational flexibilities in a site-specific manner. Structural analysis combined with biochemical data revealed that interactions with downstream effectors were impeded in mUbRab5K140, whereas GDP release and GTP loading activities were altered in mUbRab5K165. By contrast, mUbRab5K116 apparently had no effect. We propose a regulatory mechanism of Rab5 where monoubiquitination downregulates effector recruitment and GDP/GTP conversion in a site-specific manner.

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