Document Detail


Development of a peptidase-resistant substrate for single-cell measurement of protein kinase B activation.
MedLine Citation:
PMID:  22881604     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
An iterative design strategy using three criteria was utilized to develop a peptidase-resistant substrate peptide for protein kinase B. Libraries of peptides possessing non-native amino acids were screened for time to 50% phosphorylation, degradation half-life within a lysate, and appearance of a dominant fragment. The lead peptide possessed a half-life of 92 ± 7 and 16 ± 2 min in HeLa and LNCaP cytosolic lysates, respectively, representing a 4.6- and 2.7-fold lifetime improvement over that of the starting peptide. The redesigned peptide possessed a 4.5-fold improvement in phosphorylation efficiency compared to the starting peptide. The same peptide fragments were formed when the lead peptide was incubated in a lysate or loaded into single cells although the fragments formed in significantly different ratios suggesting that distinct peptidases metabolized the peptide in the two preparations. The rate of peptide degradation and phosphorylation was on average 0.1 ± 0.2 zmol pg(-1) s(-1) and 0.04 ± 0.08 zmol pg(-1) s(-1), respectively, for single LNCaP cells loaded with 4 ± 8 μM of peptide. Peptidase-resistant kinase substrates should find widespread utility in both lysate-based and single-cell assays of kinase activity.
Authors:
Angela Proctor; Qunzhao Wang; David S Lawrence; Nancy L Allbritton
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2012-08-09
Journal Detail:
Title:  Analytical chemistry     Volume:  84     ISSN:  1520-6882     ISO Abbreviation:  Anal. Chem.     Publication Date:  2012 Aug 
Date Detail:
Created Date:  2012-08-21     Completed Date:  2012-12-20     Revised Date:  2014-09-12    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  7195-202     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Amino Acid Substitution
Arginine
Cytosol / metabolism
Drug Design*
Enzyme Activation / drug effects
HeLa Cells
Humans
Oligopeptides / chemistry,  metabolism*,  pharmacology
Peptide Hydrolases / metabolism*
Phosphorylation
Protein Binding
Protein Kinase Inhibitors / chemistry,  metabolism*,  pharmacology
Proteolysis
Proto-Oncogene Proteins c-akt / antagonists & inhibitors,  metabolism*
Single-Cell Analysis*
Grant Support
ID/Acronym/Agency:
CA139599/CA/NCI NIH HHS; CA140173/CA/NCI NIH HHS; R01 CA139599/CA/NCI NIH HHS; R01 CA140173/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Oligopeptides; 0/Protein Kinase Inhibitors; 94ZLA3W45F/Arginine; EC 2.7.11.1/Proto-Oncogene Proteins c-akt; EC 3.4.-/Peptide Hydrolases
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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