Effects of mitochondrial swelling and calcium on phosphate‐activated glutaminase in pig renal mitochondria

Abstract

The effects of mitochondrial swelling and calcium have been used to study the possible function of the glutamine transporter in regulating glutamine hydrolysis. Salt-induced swelling of pig renal mitochondria and an iso-osmotic mixed salt solution and swelling caused by reducing the osmolarity of the incubation medium, are accompanied by activation of glutamine hydrolysis. Regulation of the glutaminase activity by salt-induced mitochondrial swelling is likely to have physiological importance, similar to the regulation of hepatic glutaminase by changing the matrix volume, that has been described by others.0.1 -1 .0 mM calcium stimulates glutamine hydrolysis and the calcium activation curve follows MichaelisMenten kinetics. The calcium activation is reversible, it is unaffected by phosphate, high glutamine and mitochondrial calcium uptake, as well as by sonication and the activation is calmodulin independent. The calcium activation is additive to that of swelling.Similar to calcium, hypo-osmotic swelling mainly increases the apparent V,,, for glutamine, whereas the apparent K, is little changed, indicating that the effects are primarily on the phosphate-activated glutaminase itself rather than on the glutamine transporter. Furthermore, calcium which activates glutamine hydrolysis, inhibits glutamine uptake into the mitochondria and so does alanine having no effect on glutamine hydrolysis. Therefore, it is indicative that glutamine transport is not rate limiting for glutamine hydrolysis.Phosphate-activated glutaminase (PAG) has an important metabolic function in the kidney as a key enzyme in ammonia production and most workers agree that the enzyme is localized to the inner mitochondrial membrane [l]. The enzyme has been purified to homogeneity from various sources; the first being from pig renal cortex [2]. Phosphate is a main activator although the enzyme is regulated by a variety of compounds among which the inhibitory reaction products glutamate and ammonia are assumed to play an essential role. In addition, acetyl-CoA activates the purified enzyme [3], whereas certain acyl-CoA derivatives activate at low concentrations and inhibit at higher ones [4]. Several authors have provided evidence for a glutamine transporter in the inner mitochondrial membrane of rat kidney and liver [S -81.In this paper we report a study of the pronounced stimulatory effect on glutamine hydrolysis by salt-induced and hypo-osmotic swelling of pig renal mitochondria. In addition, we have investigated the calcium activation of glutamine hydrolysis in renal mitochondria which previously has been found in rat brain synaptosomes [9-111. The effects of calcium and swelling have been used as tools to study the possible rate limitation of glutamine hydrolysis by the glutamine transporter.

Properties and submitochondrial localization of pig and rat renal phosphate-activated glutaminase

“…This is at variance with previous findings for pig kidney and rat brain mitochondria using Triton X-100 (12,23), and may be caused by loss of activity in the earlier work due to instability of the enzyme. However, the pig renal mitochondrial PAG activity is also increased by swelling the mitochondria in a hypotonic medium whereby the membrane may be damaged and the matrix volume greatly increased (10). This is also supported by the finding that the sum of the enzyme activities of the isolated Mb-PAG and Sol-PAG exceeds that of intact mitochondria.…”

“…However, we assume that due to the high concentration of glutamate inside the inner mitochondrial membrane and matrix, only PAG molecules that have their active site accessible to the outer face of this membrane are enzymically active. Factors that lower the internal Glu concentration or increase the activation by phosphate, such as certain fatty-acyl CoA de-rivatives and fatty acids in low concentrations and calcium (10,11), may convert the dormant PAG into a functionally active one. Hence, dormant PAG may constitute a reserve that can be drawn on when needed.…”