Altered properties of human T-lymphoblast soluble low Km 5'-nucleotidase: comparison with B-lymphoblast enzyme.
Soluble low Km 5'-nucleotidases have been purified from human cultured T- and B-lymphoblasts to compare their properties and to examine the mechanism of different rates of nucleotide dephosphorylation. The enzyme from B-lymphoblasts (MGL-8) was 4385-fold purified with a specific activity of 114 mumol/min/mg, while the enzyme from T-lymphoblasts (CEM, MOLT-4) was 4355-fold purified with a specific activity of 35 mumol/min/mg. The activity of both enzymes have an absolute requirement for Mg++. The B-cell enzyme has maximum activity with Mg2+ > Mn2+ > Co2+, while the T-cell enzyme had maximum activity with Co2+ > Mn2+ > Mg2+. The optimum activity was at pH 7.4-9.0 for the B-cell enzyme and pH 9.0 for the T-cell enzyme. Substrate specificity was the same for both enzymes with the following relative Vmax values: CMP > UMP > dUMP > dCMP > dAMP > IMP > GMP > dIMP > dGMP. The Km values for AMP and IMP were 12 and 25 microM for the B-cell enzyme, and 7.0 and 12 microM for the T-cell enzyme. ATP and ADP are competitive inhibitors of these enzymes with apparent Ki values of 100 and 20 microM for the B-cell enzyme, and 44 microM and 8 microM for the T-cell enzyme, respectively. The apparent molecular mass by gel filtration column chromatography is 145 kD for the B-cell enzyme and 72 kDa for the T-cell enzyme. The subunit molecular masses by Western blots are 69.2 kD for both enzymes. These properties suggest that the B-lymphoblast enzyme is identical or similar to the enzyme from human placenta. However, the T-cell enzyme has some different properties. We conclude that these differences plus a lower content of low Km 5'-nucleotidase in T-cells may account for the decreased ability of T-lymphoblasts to dephosphorylate nucleotides and may contribute to the selective cytotoxicity of deoxyribonucleosides for T-lymphoblasts as compared to B-lymphoblasts.
Madrid-Marina
V
V
University of Michigan, Department of Internal Medicine, Ann Arbor 48109.
Lestan
B
B
Nowak
P J
PJ
Fox
I H
IH
Spychala
J
J
D015720:5'-Nucleotidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001402:B-Lymphocytes / Q000201:enzymology*; D015153:Blotting, Western; D002413:Cations, Divalent / Q000494:pharmacology; D002478:Cells, Cultured; D003593:Cytoplasm / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000201:enzymology*
D002413:Cations, Divalent; D007527:Isoenzymes; D046911:Macromolecular Substances; D015720:5'-Nucleotidase