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Uridine Diphosphate Glucose Pyrophosphorylase of Acanthamoeba castellanii

PURIFICATION, KINETIC, AND DEVELOPMENTAL STUDIES

From the ¹ From the Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78284

When cells of Acanthamoeba castellanii (Neff) are placed on a salts medium the amoebas encyst with the formation of a cellulose-containing cyst wall and an in vitro system synthesizing cellulose from UDP-glucose becomes detectable after about 12 hours of encystment. In the present study cellular levels of UDP-glucose were observed to increase 5-fold within 5 hours of the induction of encystment and to remain at elevated levels for 5 to 10 hours, apparently making UDP-glucose available to the cells at the time of cellulose synthesis. The increased level of UDP-glucose during encystment is not the reflection of an increase in uridine diphosphate glucose pyrophosphorylase activity detectable in vitro. The specific activity and the activity per cell were examined as a function of development in cell extracts. The specific activity was constant for the first 20 hours of encystment while the activity per cell decreased almost from the beginning of encystment.

The uridine diphosphate glucose pyrophosphorylase (EC 2.7.7.9) from trophozoites was purified to homogeneity as determined by polyacrylamide gel electrophoresis. The purified enzyme (final specific activity 15,833 units per mg) was highly specific for both UTP and UDP-glucose. The divalent cation requirement was most readily satisfied by magnesium and the pH optimum was 8 to 9 in the direction of UDP-glucose synthesis and 7.5 to 7.6 in the direction of pyrophosphorolysis.

The enzyme was characterized kinetically as having an ordered mechanism of catalysis. Pyrophosphorylase was subject to product inhibition by UTP and pyrophosphate. The following K_m values were obtained: UTP, 5.8 x 10^-5 m; glucose 1-phosphate, 1.4 x 10^-4 m; UDP-glucose, 5.0 x 10^-5 m; pyrophosphate, 2.4 x 10^-3 m.

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