Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane

• Published in: The Journal of biological chemistry Vol. 266; no. 20; pp. 13185 - 13192
• Main Authors: KVAMME, E, INGEBORG, I. A, ROBERG, B
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15-07-1991
• Subjects: Alanine - pharmacology; Animals; Ethylmaleimide - pharmacology; Exact sciences and technology; Glutaminase - metabolism; Glutamine - metabolism; Hydroxybutyrate Dehydrogenase - metabolism; inner membranes; Intracellular Membranes - enzymology; kidney; Kidney Cortex - enzymology; Kinetics; Mathematical analysis; Mathematics; mitochondria; Mitochondria - enzymology; phosphate; Potential theory; Sciences and techniques of general use; Submitochondrial Particles - enzymology; Sulfhydryl Reagents - pharmacology; Swine; Enzyme; Enzyme inhibitor; Glutaminase; Kidney; Internal membrane; Pig; Vertebrata; Mitochondria; Mammalia; Enzymatic activity; Artiodactyla; Kinetics; Kinetic parameter; Localization; Ungulata
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)98822-8

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl
and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2
mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase
(a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This
indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase
have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could
not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine
transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable
effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated
from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which
has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine
is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when
the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate,
20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton
X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate
in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that
of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without
being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally
predominant external localization in the inner mitochondrial membrane.