Purification and characterization of recombinant human 5'-methylthioadenosine phosphorylase: definite identification of coding cDNA.
5'-Methylthioadenosine phosphorylase gene maps on the 9p21 chromosome, strictly linked to the important tumor suppressor gene p16INK4A. Chromosomal deletions encompassing both the phosphorylase and p16INK4A genes cause the complete absence of the enzymatic activity in a large number of tumors, thus resulting in well-defined metabolic differences between malignant and normal cells. Recently, the cloning of the phosphorylase gene has been reported on the basis of indirect evidence. In order to demonstrate definitely the identification of 5'-methylthioadenosine phosphorylase gene, we have cloned the putative enzyme coding sequence in a prokaryotic expression vector and expressed the protein in bacteria. The recombinant phosphorylase has been purified to homogeneity and its physicochemical, immunological and kinetic features have been characterized. The results obtained allowed the conclusive demonstration of 5'-methylthioadenosine phosphorylase gene cloning and the use of recombinant protein for further characterization.
Della Ragione
F
F
Institute of Biochemistry of Macromolecules, Medical School, Second University of Naples, Italy.
Takabayashi
K
K
Mastropietro
S
S
Mercurio
C
C
Oliva
A
A
Russo
G L
GL
Della Pietra
V
V
Borriello
A
A
Nobori
T
T
Carson
D A
DA
Zappia
V
V
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002874:Chromosome Mapping; D002899:Chromosomes, Human, Pair 9; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D008040:Genetic Linkage; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008970:Molecular Weight; D010710:Phosphates / Q000494:pharmacology; D011683:Purine-Nucleoside Phosphorylase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D010710:Phosphates; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase