Document Detail


Effect of proteolytic enzymes, storage and reduction on the structure and biological activity of pertussigen, a toxin from Bordetella pertussis.
MedLine Citation:
PMID:  3914965     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Pertussigen (Ptx), referred to by many different names, including pertussis toxin, was separated into five polypeptide subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a discontinuous Tris-glycine buffer system. Under non-reducing conditions, the apparent molecular weights of the polypeptides (mean 10(-3)) were: S1 (26.3), S2 (24.4), S3 (22.7), S4 (12.2), and S5 (11.3). Under reducing conditions, the apparent molecular weights (mean 10(-3)) were: S1 (28.2), S2 (24.8), S3 (24.3), S4 (12.2) and S5 (13.9). The identity of the individual polypeptide subunits was further confirmed by their unique two-dimensional peptide maps. The polypeptides which showed an apparent increase in molecular weight under reducing conditions were those previously found to contain at least two cysteine residues. Reducing conditions also altered the reactivity of S3 and S2 to polyclonal rabbit antibody in electrophoretic transfer (Western) blot analysis. When Ptx was stored in solution at 4 degrees C, S1 and S5 underwent a gradual decrease in apparent molecular weight, as judged by SDS-PAGE. This decrease occurred in three different buffer systems, and was similar to a decrease in apparent molecular weight of S1 and S5 after treatment with the proteolytic enzymes subtilisin or proteinase K. Neither the changes due to storage nor proteolysis affected the activity of Ptx in regard to hemagglutination, lymphocytosis promotion or histamine sensitization. These changes did, however appear to modify the reactivity of S5 in the Western blot. Both the "endogenous" and enzyme-induced changes in S1 and S5 could be stopped by phenylmethanesulfonyl fluoride. These data suggest that S1 and S5 have exposed determinants in the intact Ptx molecule which are readily cleaved by proteases, but have little bearing on the biological activity of the intact molecule. Resistance to inactivation by proteolytic cleavage may help explain the long duration of Ptx activity within in vivo biological systems.
Authors:
M S Peppler; R C Judd; J J Munoz
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Publication Detail:
Type:  In Vitro; Journal Article    
Journal Detail:
Title:  Developments in biological standardization     Volume:  61     ISSN:  0301-5149     ISO Abbreviation:  Dev. Biol. Stand.     Publication Date:  1985  
Date Detail:
Created Date:  1986-07-18     Completed Date:  1986-07-18     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0427140     Medline TA:  Dev Biol Stand     Country:  SWITZERLAND    
Other Details:
Languages:  eng     Pagination:  75-87     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Animals
Hemagglutination / drug effects
Histamine / pharmacology
Lymphocytosis / chemically induced
Mice
Molecular Weight
Oxidation-Reduction
Peptide Hydrolases / pharmacology
Pertussis Toxin*
Protein Conformation
Rabbits
Structure-Activity Relationship
Virulence Factors, Bordetella / immunology,  isolation & purification,  pharmacology*
Chemical
Reg. No./Substance:
0/Virulence Factors, Bordetella; 51-45-6/Histamine; EC 2.4.2.31/Pertussis Toxin; EC 3.4.-/Peptide Hydrolases

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


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