Identification of human calphoglin-induced phosphoglucomutase phosphorylation in Escherichia coli.
Orthologous proteomes, universal protein networks conserved from bacteria to mammals, dictate the core functions of cells. To isolate mammalian protein sequences that interact with bacterial signaling proteins, a BLASTP genome search was performed using catalytic domains of bacterial phosphoryl-transfer enzymes as probes. A [32P]phosphoryl-transfer assay of these mammalian cDNA-expressing Escherichia coli cells was used to screen proteins retrieved from the database. Here we report that the expression of a human protein, named calphoglin, resulted in a significant increase in the phosphorylation of a 55-kDa protein in E. coli. The phosphorylation of the 55-kDa protein was acid-stable and its isoelectric point was determined to be 5.4. The 55-kDa protein was sequentially purified from an E. coli extract using three chromatography and two-dimensional polyacrylamide gel electrophoresis. Finally, the 55-kDa protein was purified 830-fold to homogeneity and the N-terminal amino acid sequence was analyzed. The sequence obtained, AIHNRAGQPAQQ, was identical to the N-terminal amino acids of E. coli phosphoglucomutase (PGM). This method may be applicable to the detection and analysis of other orthologous proteomes.
Ingi
Tatsuya
T
Department of Neurophysiology, Brain Research Institute, Niigata University, 1-757 Asahi-machi, Niigata 951-8585, Japan. tingi@bri.niigata-u.ac.jp
Mori
Hirotada
H
Inuzuka
Madoka
M
D000595:Amino Acid Sequence; D000837:Anion Exchange Resins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D002852:Chromatography, Ion Exchange / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010733:Phosphoglucomutase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D020539:Sequence Analysis, Protein; D014157:Transcription Factors; D014162:Transfection
D000837:Anion Exchange Resins; C495392:CALCOCO1 protein, human; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D014157:Transcription Factors; D010733:Phosphoglucomutase