Copurification of vimentin, energy metabolism enzymes, and a MER5 homolog with nucleoside diphosphate kinase. Identification of tissue-specific interactions.
Chromatography on immobilized antibodies specific to nucleoside diphosphate (NDP) kinase was utilized for affinity purification of this enzyme from detergent extracts of frog heart post-mitochondrial fractions. SDS-polyacrylamide gel electrophoresis analysis of eluates from these supports shows that five polypeptides co-purify with nucleoside diphosphate (NDP) kinase. Tryptic digests of each band were analyzed by mass spectrometric microsequencing. Data base searches by peptide mass matching and sequence homology led to the identification of these proteins as glyceraldehyde-3-phosphate dehydrogenase (40 kDa), creatine kinase (45 kDa), vimentin (55 kDa), pyruvate kinase (60 kDa), and a putative member of the antioxidant protein family (28 kDa). Distinct protein compositions were found in eluates of lung and liver extracts processed in a like manner. The 28-kDa band and vimentin were associated with NDP kinase from all tissues, but co-purification of pyruvate kinase was seen only in liver, while creatine kinase and glyceraldehyde-3-phosphate dehydrogenase were absent from eluates from lung and liver. The results suggest that while NDP kinase is associated with vimentin intermediate filaments and an antioxidant protein in most tissues, it interacts with energy metabolism enzymes in a tissue-specific manner.
Otero
A S
AS
Department of Molecular Physiology and Biological Physics, University of Virginia Medical School, Charlottesville, Virginia 22906, USA. ado2@virginia.edu
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D003402:Creatine Kinase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004734:Energy Metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007256:Information Systems; D007381:Intermediate Filament Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology*; D009363:Neoplasm Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011770:Pyruvate Kinase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011892:Rana catesbeiana; D014746:Vimentin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014982:Xenopus laevis
D007381:Intermediate Filament Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D014746:Vimentin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011770:Pyruvate Kinase; D003402:Creatine Kinase; D009701:Nucleoside-Diphosphate Kinase