Kinetic superiority of intra- vs. extracellular pentose pathway flux: studies in porous adipocytes

Abstract

Considering how the cytosol is typically prepared, to wit, by cell disruption and ultracentrifugation, historically this compartment has been deemed unstructured and kinetically analogous to a solubilized system. By prudently permeabilizing rat adipocytes so that there is scant enzyme egress, intermediary metabolism can be explored intracellularly. Herein, cytoplasmic flux vs. a soluble reference system was compared. The three-enzyme oxidative portion of the pentose pathway was examined using [1-14C]glucose 6-phosphate (G-6-P); both the steady-state velocities (nu o) and transient times (tau ss) were compared in each system. At low G-6-P levels (much less than km), nu o is dependent solely on the activity of the first enzyme, and tau ss is determined by the distal two enzymes. In our experiments, the need also arose to compare tau ss between preparations, wherein the enzyme concentrations were unequal. It is shown that tau ss.nu o/G-6-P serves as an index of kinetic efficiency. Over various dilutions, the kinetic value (nu o/G-6-P) for the porous cells ranged from 2.0 to 23.9 x 10(-6) l/min, with corresponding tau ss values of 18-1.0 min. The respective values for the solubilized enzyme system were from 4.9 to 42.2 x 10(-6) l/min and from 15.2 to 1.1 min. This abbreviated pathway occurring in porous cells was nearly twice as fast at reaching steady state than the corresponding solubilized system. We conclude that cytoplasmic flux is kinetically efficient and that metabolic studies conducted only under Vmax conditions and ignoring tau ss could overlook the cellular effects of hormones or pathological states.