Purification and characterization of 5'-deoxy-5'-methylthioadenosine phosphorylase from human placenta.
5'-Methylthioadenosine phosphorylase has been purified to homogeneity (30,000-fold) from human full-term placenta by a procedure involving covalent chromatography on organomercurial-agarose as the major step. The specific activity of the homogeneous enzyme is 10.2 mumol of 5'-methylthioadenosine cleaved per min per mg of protein, and the overall yield is about 20%. The enzyme has a molecular weight of 98,000, as determined by gel filtration on Sephacryl S-200 and Superose 6B, and is composed by three apparently identical subunits with a molecular weight of 32,500. The isoelectric point is 5.5, and the optimal pH ranges from 7.2 to 7.6. The resistance of the enzyme to thermal inactivation is increased remarkably by the addition of 5'-methylthioadenosine or phosphate. The homogeneous enzyme shows an absolute requirement for -SH-reducing agents and is specifically and rapidly inactivated by thiol-blocking compounds. The reaction catalyzed by the enzyme is fully reversible with a Keq of 1.39 X 10(-2) (in the direction of phosphorolysis) at 37 degrees C and pH 7.4. The Km values for 5'-methylthioadenosine, phosphate, adenine, and 5-methylthioribose 1-phosphate are 5, 320, 23, and 8 microM, respectively.
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D003839:Deoxyadenosines; D006358:Hot Temperature; D006801:Humans; D007526:Isoelectric Point; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000302:isolation & purification*; D010710:Phosphates / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D011683:Purine-Nucleoside Phosphorylase / Q000302:isolation & purification*; D013872:Thionucleosides / Q000378:metabolism
D003839:Deoxyadenosines; D046911:Macromolecular Substances; D010710:Phosphates; D013872:Thionucleosides; C008500:5'-methylthioadenosine; D010430:Pentosyltransferases; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase; D000241:Adenosine