Isolation of the myc transcription factor nucleoside diphosphate kinase and the multifunctional enzyme glyceraldehyde-3-phosphate dehydrogenase by cAMP affinity chromatography.
Cyclic AMP affinity chromatography applied to various mammalian tissue extracts yielded two proteins in addition to the regulatory subunits of protein kinase. This paper characterizes these proteins and provides a simple procedure for their preparation. The polypeptides (36 kDa and a 19 kDa/21 kDa doublet) were isolated from the cAMP matrix by sequential elution with cAMP solutions of increasing concentrations. Microsequencing was accomplished following chemical or enzymic degradation of isolated polypeptides. Partial amino acid sequences of the 36 kDa protein and analyses of its enzymic activity indicated identity with glyceraldehyde-3-phosphate dehydrogenase whilst the lower MW protein proved to be identical with mammalian nucleoside diphosphate kinase subunits. In both cases, binding to cAMP appeared to occur at the nucleotide (NAD and ATP, respectively) sites. In conclusion, we present a one step-procedure, applicable to tissue and cell extracts, which allows the simultaneous isolation of both glyceraldehyde-3-phosphate dehydrogenase and nucleoside diphosphate kinase. This procedure may help to elucidate the multiple functions of these two important enzymes.
Weber
B
B
Institut für Physiologische Chemie, Universitätskrankenhaus Eppendorf, Hamburg, Germany.
Weber
W
W
Buck
F
F
Hilz
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D000242:Cyclic AMP; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry / Q000302:isolation & purification*; D011817:Rabbits; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification*
D010446:Peptide Fragments; D016271:Proto-Oncogene Proteins c-myc; D014157:Transcription Factors; D000242:Cyclic AMP; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D009701:Nucleoside-Diphosphate Kinase