Physicochemical and immunological studies on mammalian 5'-deoxy-5'-methylthioadenosine phosphorylase.
5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAase) was purified to homogeneity (10,000-fold) from bovine liver with a recovery of 12%. The pure protein shows a molecular weight of about 98,000 +/- 3,000 and is composed of three apparently identical subunits. Several physicochemical features have been investigated including hydrodynamic properties, amino acid composition, and secondary structure. In particular, the CD spectrum of the protein indicates a very low alpha-helical content and a large percent of beta-structure and random coil. The pure protein was used to raise specific rabbit antisera but, because of the scarce antigenic properties of the native enzyme, different chemically modified forms were prepared and employed as immunogens. Among the antibodies obtained, those to keyhole limpet hemocyanin-MTAase recognize both the native and the denatured enzyme and are also active against the human protein. Therefore, they were employed as a tool to investigate the occurrence of inactive forms of MTAase in two human malignant cell lines lacking this enzymatic activity. The results obtained with K562 and Jurkat cells indicate that the protein is absent in these phosphorylase-deficient cell lines.
Della Ragione
F
F
Institute of Biochemistry of Macromolecules, First Medical School, University of Naples, Italy.
Oliva
A
A
Gragnaniello
V
V
Russo
G L
GL
Palumbo
R
R
Zappia
V
V
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000936:Antigen-Antibody Complex; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D007106:Immune Sera; D015151:Immunoblotting; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000302:isolation & purification*; D011487:Protein Conformation; D011683:Purine-Nucleoside Phosphorylase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D014461:Ultracentrifugation
D000596:Amino Acids; D000936:Antigen-Antibody Complex; D007106:Immune Sera; D046911:Macromolecular Substances; D010430:Pentosyltransferases; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase