AJP - Cell Physiology, Vol 261, Issue 3 C476-C481, Copyright © 1991 by American Physiological Society
Kinetic superiority of intra- vs. extracellular pentose pathway flux: studies in porous adipocytes
K. L. McCormick and G. J. Mick
Department of Pediatric Endocrinology, State University of New York Health Science Center, Syracuse 13210.
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.
