Regulation of oxidative degradation of L-lysine in rat liver mitochondria.
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ABSTRACT:
SUBMITTER: Scislowski PW
PROVIDER: S-EPMC1138248 | biostudies-other | 1994 Jun
REPOSITORIES: biostudies-other
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Project description:1. The catabolism of glycine was studied in isolated rat liver mitochondria by measuring release of 14CO2 from [1-14C]-glycine. Incubation of mitochondria in a medium containing 0.5 microM free Ca2+ resulted in an 8-fold increase in the rate of degradation of glycine. Intraperitoneal injection of glucagon (33 or 100 micrograms/100 g body wt.) 25 min before killing of rats also resulted in a 3-fold or 10-fold (depending on dosage) increase in the rate of catabolism of glycine. 2. Both the stimulation by free Ca2+ and that by injection of glucagon in vivo were dependent on phosphate in the incubation medium. This requirement for phosphate was specific, as replacement of phosphate by other permeant anions such as thiocyanate and acetate did not permit the stimulation. The phosphate-dependent stimulation of glycine catabolism by Ca2+ was also evident when mitochondria were incubated in the absence of K+. 3. Mitochondria isolated from rats previously injected with glucagon showed elevated rates of degradation of glycine even in the presence of rotenone, provided that regeneration of NAD+ was affected by providing acetoacetate. 4. Hypo-osmolarity of the medium markedly stimulated the rate of degradation of glycine by mitochondria. Although hypo-osmolarity-induced stimulation of glycine degradation was accompanied by parallel changes in mitochondrial matrix volume, no measurable changes in matrix volume were observed in mitochondria stimulated either by free Ca2+ (0.5 microM) or by injection of glucagon in vivo. Furthermore, Ca2+ stimulated glycine decarboxylation in mitochondria exposed to either hyper-osmolar (410 mosmol) or hypo-osmolar (210 mosmol) conditions. Although hyper-osmolarity decreased and hypo-osmolarity increased matrix volume, stimulation of glycine degradation by Ca2+ was not associated with any further changes in matrix volume. 5. These data demonstrate that the regulation of hepatic glycine oxidation by glucagon and by free Ca2+ is largely independent of changes in mitochondrial matrix volume.
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Project description:Data, both for and against the presence of a mitochondrial nitric-oxide synthase (NOS) isoform, is in the refereed literature. However, irrefutable evidence has not been forthcoming. In light of this controversy, we designed studies to investigate the existence of the putative mitochondrial NOS. Using repeated differential centrifugation followed by Percoll gradient fractionation, ultrapure, never frozen rat liver mitochondria and submitochondrial particles were obtained. Following trypsin digestion and desalting, the mitochondrial samples were analyzed by nano-HPLC-coupled linear ion trap-mass spectrometry. Linear ion trap-mass spectrometry analyses of rat liver mitochondria as well as submitochondrial particles were negative for any peptide from any NOS isoform. However, recombinant neuronal NOS-derived peptides from spiked mitochondrial samples were easily detected, down to 50 fmol on column. The protein calmodulin (CaM), absolutely required for NOS activity, was absent, whereas peptides from CaM-spiked samples were detected. Also, l-[(14)C]arginine to l-[(14)C]citrulline conversion assays were negative for NOS activity. Finally, Western blot analyses of rat liver mitochondria, using NOS (neuronal or endothelial) and CaM antibodies, were negative for any NOS isoform or CaM. In conclusion, and in light of our present limits of detection, data from carefully conducted, properly controlled experiments for NOS detection, utilizing three independent yet complementary methodologies, independently as well as collectively, refute the claim that a NOS isoform exists within rat liver mitochondria.
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