Colorimetric and Fluorometric Dual-Readout Protein Kinase Assay by Tuning the Active Surface of Nanoceria

2021 ◽  
Author(s):  
Sujuan Sun ◽  
Lijun Zhang ◽  
Xiaohui Lu ◽  
Wei Ren ◽  
Chenghui Liu
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Active Surface ◽  
Kinase Assay ◽  
Protein Kinase Activity ◽  
Phosphorylated Peptides

Herein, we demonstrate that the active surface of nanoceria can be fine-tuned by phosphorylated peptides. Accordingly, a colorimetric and fluorometric dual-readout strategy is rationally developed for assaying protein kinase activity....

The effect of methacholine and histamine on cyclic AMP-dependent protein kinase activity in the guinea-pig isolated trachea

1992 ◽  
Vol 70 (3) ◽  
pp. 344-348 ◽  
Author(s):  
J. M. Langlands ◽  
I. W. Rodger
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Guinea Pig ◽  
Kinase Activity ◽  
Activity Ratio ◽  
Kinase Assay ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Isolated Trachea ◽  
Dependent Protein

The effects of methacholine and histamine were examined on cyclic AMP-dependent protein kinase (A-kinase) activity in guinea-pig isolated trachea, using kemptide as a substrate for phosphorylation during the determination of the enzyme activity. Methacholine (EC90, 10 μM) induced a rapid reduction in the basal A-kinase activity ratio, which was maximal after 30 s. This initial reduction coincided with the early phase of isometric tension development, and returned to control levels 4 min after the addition of methacholine. Pretreatment with atropine inhibited the methacholine response. In contrast, histamine (EQ90, 30 μM) was without effect upon A-kinase activity ratio. The results establish the sensitivity of the A-kinase assay using kemptide and demonstrate that not all contractile agonists have the capacity to inhibit basal activity of A-kinase in airway smooth muscle.Key words: A-kinase, cholinomimetics, guinea-pig trachealis, smooth muscle contraction.

Phosphorylation-regulated crosslinking of gold nanoparticles: a new strategy for colorimetric detection of protein kinase activity

The Analyst ◽  
2015 ◽  
Vol 140 (16) ◽  
pp. 5685-5691 ◽  
Author(s):  
Sujuan Sun ◽  
Haixia Shen ◽  
Chenghui Liu ◽  
Zhengping Li
Keyword(s):  
Gold Nanoparticles ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Colorimetric Detection ◽  
Kinase Assay ◽  
Protein Kinase Activity ◽  
New Strategy ◽  
Peptide Phosphorylation

A facile colorimetric protein kinase assay has been developed based on the peptide phosphorylation-tuned crosslinking and aggregation of gold nanoparticles.

scholarly journals A Protein Kinase Activity Associated with Epstein-Barr Virus BGLF4 Phosphorylates the Viral Early Antigen EA-D In Vitro

2000 ◽  
Vol 74 (7) ◽  
pp. 3093-3104 ◽  
Author(s):  
Mei-Ru Chen ◽  
Shin-Jye Chang ◽  
Hsiaowen Huang ◽  
Jen-Yang Chen
Keyword(s):  
Protein Kinase ◽  
Epstein Barr Virus ◽  
Kinase Activity ◽  
Kinase Assay ◽  
Specific Antiserum ◽  
Protein Kinase Activity ◽  
Early Antigen ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) open reading frame BGLF4 was identified as a potential Ser/Thr protein kinase gene through the recognition of amino acid sequence motifs characteristic of conserved regions within the catalytic domains of protein kinases. In order to investigate this potential kinase activity, BGLF4 was expressed inEscherichia coli and the purified protein was used to generate a specific antiserum. Recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase, was used to infect 293 and 293T cells after transient transfection with a plasmid containing BGLF4 under the control of the T7 promoter. Autophosphorylation of the BGLF4 protein was demonstrated using the specific antiserum in an immune complex kinase assay. In addition, EBNA-1-tagged BGLF4 and EBNA-1 monoclonal antibody 5C11 were used to demonstrate the specificity of the kinase activity and to locate BGLF4 in the cytoplasm of transfected cells. Manganese ions were found to be essential for autophosphorylation of BGLF4, and magnesium can stimulate the activity. BGLF4 can utilize GTP, in addition to ATP, as a phosphate donor in this assay. BGLF4 can phosphorylate histone and casein in vitro. Among the potential viral protein substrates we examined, the EBV early antigen (EA-D, BMRF1), a DNA polymerase accessory factor and an important transactivator during lytic infection, was found to be phosphorylated by BGLF4 in vitro. Amino acids 1 to 26 of BGLF4, but not the predicted conserved catalytic domain, were found to be essential for autophosphorylation of BGLF4.

scholarly journals A Scintillating Microplate Assay for the Assessment of Protein Kinase Activity

1998 ◽  
Vol 3 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Grace R. Nakayama ◽  
Michael P. Nova ◽  
Zahra Parandoosh
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinases ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Kinase Assay ◽  
Protein Kinase Activity ◽  
Discovery Research ◽  
Cell Extracts ◽  
Drug Discovery Research

Protein kinases, a class of enzymes that phosphorylate certain tyrosine, serine, and threonine residues, play an important role in cellular functions and are important targets in drug discovery research. Thus, it is of interest to develop a simple assay that can be used to measure protein kinase activity toward specific substrates and is suitable for the high throughput screening (HTS) of potential kinase inhibitors. The scintillation proximity concept has been successfully applied for measuring specific kinase activity using surfaces passively coated with a peptide substrate. In this study, we evaluated kinase assay performance on three ScintiStrip platforms: unmodified surface, streptavidin-coated surface, and streptavidin covalently attached to surface. The high affinity of streptavidin toward biotin-linked peptide substrates makes it a unique platform for measuring specific incorporation of radiolabeled phosphate into selected substrates of specific enzymes in the presence of others. Therefore, this assay may be used with cell extracts containing impure kinases as well as with purified enzymes. The scope of this assay was demonstrated with purified tyrosine kinases (e.g., p60c-src kinase) and A431 cell extracts. This scintillation proximity assay is universal, simple, rapid, accurate, and can be adapted for use with robotics for HTS.

Effect of Thrombin on Phosphorylation of Platelet Membrane Proteins

1976 ◽  
Vol 35 (03) ◽  
pp. 635-642 ◽  
Author(s):  
M Steiner
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Qualitative Change ◽  
Protein Kinase Activity ◽  
Protein Substrates ◽  
Phosphoprotein Phosphatase ◽  
Progressive Loss ◽  
Platelet Membranes ◽  
Endogenous Protein ◽  
Considerable Loss

SummaryThe effect of thrombin on the phosphorylating activity of platelet membranes was compared to that of trypsin. Preincubation of non-32P phosphorylated platelet membranes with or without either of these two enzymes resulted in a considerable loss of membrane protein kinase activity which was most severe when trypsin was used. Protein kinase activity and endogenous protein acceptors decreased in parallel. 32P-phosphorylated membranes showed a slow but progressive loss of label which was accelerated by trypsin. Thrombin under these conditions prevented the loss of 32P-phosphate. These results are interpreted to indicate a thrombin-induced destruction of a phosphoprotein phosphatase. The protein kinase activity of phosphorylated platelet membranes using endogenous or exogenous protein substrates showed a significant reduction compared to non-phosphorylated membranes suggesting a deactivation of protein kinase by phosphorylation of platelet membranes. Neither thrombin nor trypsin caused a qualitative change in the membrane polypeptides accepting 32P-phosphate but resulted in quantitative alterations of their ability to become phosphorylated.

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