Author/Editor		Madrid-Marina, V; Lestan, B; Nowak, PJ; Fox, IH; Spychala, J
Title		Altered properties of human T-lymphoblast soluble low Km 5'-nucleotidase: comparison with B-lymphoblast enzyme
Type		članek
Source		Leuk Res
Vol. and No.		Letnik 17, št. 3
Publication year		1993
Volume		str. 231-40
Language		eng
Abstract		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+ more th. Mn2+ more th. Co2+, while the T-cell enzyme had maximum activity with Co2+ more th. Mn2+ more th. 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 more th. UMP more th. dUMP more th. dCMP more th. dAMP more th. IMP more th. GMP more th. dIMP more th. 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.
Descriptors		B-LYMPHOCYTES ISOENZYMES T-LYMPHOCYTES 5'-NUCLEOTIDASE BLOTTING, WESTERN CATIONS, DIVALENT CELLS, CULTURED CYTOPLASM HYDROGEN-ION CONCENTRATION ISOENZYMES KINETICS MACROMOLECULAR SYSTEMS MOLECULAR WEIGHT SUBSTRATE SPECIFICITY NUCLEOTIDASE