Glucose 6-phosphate dehydrogenase from larval Taenia crassiceps (cysticerci): purification and properties.
Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) was purified to homogeneity from the soluble fraction of larval Taenia crassiceps (Eucestoda: Cyclophyllidea) by a three-step protocol. Specific activity of the pure enzyme was 33.8 +/- 2.1 U mg(-1) at 25 degrees C and pH 7.8 with D: -glucose 6-phosphate and NADP+ as substrates. The activity increases to 67.6 +/- 3.9 U mg(-1) at 39 degrees C, a more physiological temperature in the intermediary host. Enzyme activity was maximal between pH 6.7 and 7.8. Km values were 14 +/- 1.7 microM and 1.3 +/- 0.4 microM for glucose 6-phosphate and NADP+, respectively. The enzyme showed absolute specificity for its sugar substrate. NAD+ was also a substrate but with a low catalytic efficiency (207 M(-1) s(-1)). No essential requirement for Mg++ or Ca++ was observed. Relative molecular mass of the native enzyme was 134,000 +/- 17,200, while a value of 61,000 +/- 1,700 was obtained for the enzyme subunit. Thus, glucose 6-phosphate dehydrogenase from T. crassiceps exists as a dimeric protein. The enzyme's isoelectric point was 4.5. The enzyme's activity dependence on temperature was complex, resulting in a biphasic Arrhenius plot. Activation energies of 9.91 +/- 0.51 and 7.94 +/- 0.45 kcal mol(-1) were obtained. Initial velocity patterns complemented with inhibition studies by product and substrate's analogues support a random bi bi sequential mechanism in rapid equilibrium. The low Ki value of 1.95 microM found for NADPH suggests a potential regulatory role for this nucleotide.
Rendón
Juan L
JL
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apartado Postal No. 70-159, 04510 Mexico City, Mexico. jrendon@bq.unam.mx
Del Arenal
Irene P
IP
Guevara-Flores
Alberto
A
Mendoza-Hernández
Guillermo
G
Pardo
Juan Pablo
JP
D000818:Animals; D003067:Coenzymes / Q000494:pharmacology; D019281:Dimerization; D005260:Female; D019298:Glucose-6-Phosphate / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D007814:Larva / Q000201:enzymology; D008670:Metals / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D013379:Substrate Specificity; D013621:Taenia / Q000201:enzymology*; D013696:Temperature
D003067:Coenzymes; D008670:Metals; D009243:NAD; D009249:NADP; D019298:Glucose-6-Phosphate; D005954:Glucosephosphate Dehydrogenase