Isolation, cloning and characterization of a low-molecular-mass purine nucleoside- and nucleotide-binding protein.
A purine nucleoside- and nucleotide-binding protein has been isolated from extracts of rat and rabbit heart, calf aortic smooth muscle and rat liver using an affinity column containing adenosine bound through the N6-position. The protein, which was eluted by adenosine, was cloned and expressed in Escherichia coli. The deduced amino acid sequence has a calculated Mr of 13693 (p13.7). The expressed protein has properties identical with the protein isolated from heart and liver, including an anomalous, apparent Mr of 15300, observed on gel electrophoresis. Gel filtration shows it to be a dimer. p13.7 differs by only three amino acids out of 125 from protein kinase C inhibitor 1 [Pearson, DeWald, Mathews, Mozier, Zürcher-Neely, Heinrikson, Morris, McCubbin, McDonald, Fraser et al. (1990) J. Biol. Chem. 265, 4583-4591]. However, we have not been able to demonstrate inhibition of protein kinase C by physiological concentrations of p13.7, regardless of whether it was isolated from tissue extracts or expressed in E. coli. p13.7 is a member of the histidine triad motif family of proteins [Séraphin (1992) J. DNA Sequencing Mapping 3, 177-179]. The affinity of p13.7 for a number of different purine nucleosides and nucleotides, as measured by fluorescence titration and gel filtration, falls within the range 5-50 microM. On the basis of these properties and its crystal structure [Brenner, Garrison, Gilmour, Peisach, Ringe, Petsko and Lowenstein (1997) Nature Struct. Biol. 4, 231-238], we have coined the acronym HINT (histidine triad nucleotide-binding motif) to describe the family of proteins of which p13.7 is a member. Other proteins that bind to the affinity column have been identified as malate and lactate dehydrogenases, cAMP-binding proteins, adenosine kinase and S-adenosylhomocysteine hydrolase.
Gilmour
J
J
Biochemistry Department, Brandeis University, Waltham, MA 02254, USA.
Liang
N
N
Lowenstein
J M
JM
D000241:Adenosine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006867:Hydrolases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009928:Organ Specificity; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011684:Purine Nucleosides / Q000378:metabolism*; D011685:Purine Nucleotides / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D013050:Spectrometry, Fluorescence; D017186:Titrimetry
D011506:Proteins; D011684:Purine Nucleosides; D011685:Purine Nucleotides; C097552:histidine triad protein; D006867:Hydrolases; D000241:Adenosine