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PMID: 3462724 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

The association of NADPH with the guanine nucleotide exchange factor from rabbit reticulocytes: a role of pyridine dinucleotides in eukaryotic polypeptide chain initiation.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 83 ·No. 18 ·1986-09-00 ·Pages 6746-50

Dholakia JN, Mueser TC, Woodley CL, Parkhurst LJ, Wahba AJ

Abstract

The guanine nucleotide exchange factor (GEF) was purified to apparent homogeneity from postribosomal supernatants of rabbit reticulocytes by chromatography on DEAE-cellulose and phosphocellulose, fractionation by glycerol gradients, and chromatography on Mono S and Mono Q (Pharmacia). At the Mono S step GEF is isolated as a complex with the eukaryotic polypeptide chain initiation factor 2 (eIF-2) and is separated from this factor by column chromatography on Mono Q. An emission spectrum characteristic of a reduced pyridine dinucleotide was observed when GEF was subjected to fluorescence analysis. By both coupled enzymatic analysis and chromatography on reverse-phase or Mono Q columns, the bound dinucleotide associated with GEF was determined to be NADPH. The GEF-catalyzed exchange of eIF-2-bound GDP for GTP was markedly inhibited by NAD+ and NADP+. This inhibition was not observed in the presence of equimolar concentrations of NADPH. Similarly, the stimulation of ternary complex (eIF-2 X GTP X Met-tRNAf) formation by GEF in the presence of 1 mM Mg2+ was abolished in the presence of oxidized pyridine dinucleotide. These results demonstrate that pyridine dinucleotides may be directly involved in the regulation of polypeptide chain initiation by acting as allosteric regulators of GEF activity.

MeSH Terms
Animals Eukaryotic Initiation Factor-2 Guanine Nucleotide Exchange Factors Guanosine Diphosphate/metabolism Light NAD/physiology NADP/analysis,physiology Peptide Chain Initiation, Translational Peptide Initiation Factors/physiology Protein Biosynthesis Proteins/analysis,physiology Rabbits Reticulocytes/metabolism Scattering, Radiation Spectrometry, Fluorescence
Chemicals
Eukaryotic Initiation Factor-2 Guanine Nucleotide Exchange Factors Peptide Initiation Factors Proteins NAD Guanosine Diphosphate NADP
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Dholakia J N
Mueser T C
Woodley C L
Parkhurst L J
Wahba A J
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35 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.83.18.6746
Published
1986-09-00
Pages
6746-50
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC386586
Subset
IM
Grants
NIADDK NIH HHS · AM 26288 · United States
NIGMS NIH HHS · GM 25451 · United States
NHLBI NIH HHS · HL 15,284 · United States
External Links
PubMed source DOI: 10.1073/pnas.83.18.6746 PMC Full Text View journal details
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