Development of Glutaminase Along the Villus–Crypt Axis in the Jejunum of Rat

Abstract

The activity of glutaminase (E.C. 3.5.1.2), the entry enzyme for oxidation of glutamine, was measured in enterocytes isolated along the villus-crypt axis from rat jejunum. Specific activity of glutaminase was 5.05 +/- 0.24 mumol glutamate/mg protein/h in villus cells (fully differentiated cells) and 4.16 +/- 0.30 in the deep crypt (undifferentiated cells). Activity of glutaminase was significantly (p less than 0.05) increased in cells isolated from the villus-crypt junction (differentiating cells) compared to the activity of the enzyme in both the villus and crypt at 6.21 +/- 0.45. A similar pattern of activity of glutaminase was observed when the cells of the villus-crypt gradient were separated by sequential horizontal sectioning with a cryostat. Oxidation of L-[U-14C]glutamine to 14CO2 was also significantly (p less than 0.01) higher in cells isolated from the villus-crypt junction compared to both villus or deep crypt cells. The quantity of glutaminase protein was determined by a dot immunobinding assay using an antibody to purified glutaminase. Immunoreactive glutaminase protein relative to total cellular protein was 6.06 +/- 0.40 cpm/microgram homogenate protein in the villus cells, 3.01 +/- 0.24 (p less than 0.05) at the villus-crypt junction, and 4.49 +/- 0.57 (p less than 0.05) in the deep crypt. Thus, the highest activity of glutaminase present in the villus-crypt junction is the result of an increase in activity of the enzyme rather than an increase in the enzyme protein.