Document Detail


Purification and characterization of adenosine deaminase from a genetically enriched mouse cell line.
MedLine Citation:
PMID:  3902813     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Mammalian adenosine deaminase has been shown by genetic and biochemical evidence to be essential for the development of the immune system. For the purpose of studying the function and structure of this enzyme, we have isolated by genetic selection a mouse cell line, B-1/50, in which adenosine deaminase levels were increased 4,300-fold over the parent cell line. The enzyme was purified from these cells in large quantity and high yield by a simple two-step purification scheme. The enzyme derived from the B-1/50 cells was indistinguishable from that of the parental cells as judged by several biochemical criteria. The Km (30 microM) and Ki (4 nM) values using adenosine as substrate and 2'-deoxycoformycin as inhibitor, respectively, were identical for the enzyme derived from the parental cells as well as the adenosine deaminase gene amplification mutants. The enzyme from both cell types exhibited multiple isoelectric focusing forms which co-purified using our purification protocol. Electrophoretic analysis using sodium dodecyl sulfate-polyacrylamide gels showed that adenosine deaminase migrated with an apparent molecular weight of 41,000 or 36,000 depending on whether the enzyme was reduced or oxidized, respectively. This shift was reversible, indicating that proteolysis was not responsible for the faster migrating form. Monospecific antibodies raised against purified adenosine deaminase cross-reacted with the enzyme derived from the parental cells and precipitated 37% of the total soluble protein in the B-1/50 cells. Continued genetic selection resulted in the isolation of cells in which adenosine deaminase was overproduced by 11,400-fold and accounted for over 75% of the soluble protein.
Authors:
D E Ingolia; C Y Yeung; I F Orengo; M L Harrison; E G Frayne; F B Rudolph; R E Kellems
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  260     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1985 Oct 
Date Detail:
Created Date:  1985-11-29     Completed Date:  1985-11-29     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  13261-7     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Adenosine / metabolism
Adenosine Deaminase / antagonists & inhibitors,  isolation & purification,  metabolism*
Animals
Cell Line
Coformycin / analogs & derivatives,  pharmacology
Electrophoresis, Polyacrylamide Gel
Gene Amplification
Half-Life
Immunosorbent Techniques
Isoelectric Focusing
Kinetics
Mice
Molecular Weight
Mutation
Nucleoside Deaminases / metabolism*
Pentostatin
Grant Support
ID/Acronym/Agency:
AI20402/AI/NIAID NIH HHS; CA14030/CA/NCI NIH HHS; RR05425/RR/NCRR NIH HHS
Chemical
Reg. No./Substance:
11033-22-0/Coformycin; 53910-25-1/Pentostatin; 58-61-7/Adenosine; EC 3.5.4.-/Nucleoside Deaminases; EC 3.5.4.4/Adenosine Deaminase

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


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