Selective permeability of rat liver mitochondria to purified aspartate aminotransferases in vitro.
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ABSTRACT:
SUBMITTER: Marra E
PROVIDER: S-EPMC1164847 | biostudies-other | 1977 Jun
REPOSITORIES: biostudies-other
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Project description:1. The mitochondrial malate dehydrogenase from rat liver has been purified to a state of homogeneity as judged by starch-gel electrophoresis and the cytoplasmic isoenzyme has been obtained in a partically purified state. 2. Inhibition of the isoenzymes by sulphite has been studied. 3. In mitochondria loaded with sulphite, the catalytic activity of the (partially inhibited) internal malate dehydrogenase has been measured by addition of oxaloacetate to the suspension medium and observation of the consequent decrease in fluorescence of NADH. 4. Addition of mitochondrial malate dehydrogenase to suspensions of mitochondria loaded with sulphite resulted in an increase in the level of intramitochondrial enzymic activity as measured by the above technique. Addition of the cytoplasmic isoenzyme did not result in such an increase. 5. These results show that mitochondria in suspension are permeable to the mitochondrial malate dehydrogenase but not to the cytoplasmic isoenzyme. 6. This conclusion has been confirmed by direct measurement of a decrease of enzyme activity in solution and an increase inside the mitochondria after incubation of organelles in solutions containing mitochondrial malate dehydrogenase. No such effect was observed with the cytoplasmic isoenzyme. 7. Some features of the permeation process have been studied.
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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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Project description:NO and cGMP administered at reperfusion after ischaemia prevent injury to hepatocytes mediated by the MPT (mitochondrial permeability transition). To characterize further the mechanism of protection, the ability of hepatic cytosol in combination with cyclic nucleotides to delay onset of the calcium-induced MPT was evaluated in isolated rat liver mitochondria. Liver cytosol plus cGMP or cAMP dose-dependently inhibited the MPT, required ATP hydrolysis for inhibition and did not inhibit mitochondrial calcium uptake. Specific peptide inhibitors for PKA (protein kinase A), but not PKG (protein kinase G), abolished cytosol-induced inhibition of MPT onset. Activity assays showed a cGMP- and cAMP-stimulated protein kinase activity in liver cytosol that was completely inhibited by PKI, a PKA peptide inhibitor. Size-exclusion chromatography of liver cytosol produced a single peak of cGMP/cAMP-stimulated kinase activity with an estimated protein size of 180-220 kDa. This fraction was PKI-sensitive and delayed onset of the MPT. Incubation of active catalytic PKA subunit directly with mitochondria in the absence of cytosol and cyclic nucleotide also delayed MPT onset, and incubation with purified outer membranes led to phosphorylation of a major 31 kDa band. After ischaemia, administration at reperfusion of membrane-permeant cAMPs and cAMP-mobilizing glucagon prevented reperfusion injury to hepatocytes. In conclusion, PKA in liver cytosol activated by cGMP or cAMP acts directly on mitochondria to delay onset of the MPT and protect hepatocytes from cell death after ischaemia/reperfusion.
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Project description:1. Butan-1-ol solubilizes that portion of rat liver mitochondrial aspartate aminotransferase (EC 2.6.1.1) that cannot be solubilized by ultrasonics and other treatments. 2. A difference in electrophoretic mobilities, chromatographic behaviour and solubility characteristics between the enzymes solubilized by ultrasonic treatment and by butan-1-ol was observed, suggesting the occurrence of two forms of this enzyme in rat liver mitochondria. 3. Half the aspartate aminotransferase activity of rat kidney homogenate was present in a high-speed supernatant fraction, the remainder being in the mitochondria. 4. A considerable increase in aspartate aminotransferase activity was observed when kidney mitochondrial suspensions were treated with ultrasonics or detergents. 5. All the activity after maximum activation was recoverable in the supernatant after centrifugation at 105000g for 1hr. 6. The electrophoretic mobility of the kidney mitochondrial enzyme was cathodic and that of the supernatant enzyme anodic. 7. Cortisone administration increased the activities of both mitochondrial and supernatant aspartate aminotransferases of liver, but only that of the supernatant enzyme of kidney.
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Project description:The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active site via a Schiff base to a conserved lysine. An active-site mutant showed that Trypanosoma brucei AAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the N-terminal arm.
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Project description:The distribution of pyruvate (glyoxylate) aminotransferases in the particulate fraction of rat liver homogenates was examined by centrifugation in a sucrose density graident. Aminotransferase activities towards serine, phenylalanine and histidine with pyruvate and those towards phenylalanine and histidine with glyoxylate were nearly identically distributed. Some 50-55% of the particulate activity was localized in the peroxisomes and the remainder in the mitochondria. Most of alanine-glyoxylate aminotransferase activity was localized in the mitochondria, with some activity in the peroxisomes. Glucagon injection resulted in increases of these enzyme activities in the mitochondria, but not in the peroxisomes.
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Project description:MicroRNAs (miRNAs) are a class of small approximately 22 nt noncoding (nc) RNAs that regulate gene expression post-transcriptionally by direct binding to target sites on mRNAs. They comprise more than 1,000 novel species in mammalian cells and exert their function by modulating gene expression through several different mechanisms, including translational inhibition, and/or degradation of target mRNAs. Mitochondria maintain and express their own genome, which is distinct from the nuclear transcriptional and translational apparatus. Thus, they provide a potential site for miRNA mediated post-transcriptional regulation. To determine whether they maintain a unique miRNA population, we examined the miRNA profile from highly purified and RNase treated mitochondria from adult rat liver. Fifteen miRNAs were identified by microarray analysis of which, five were confirmed by TaqMan 5'nuclease assays using rat specific probes. Functional analysis of the miRNAs indicated that they were not targeted to the mitochondrial genome nor were they complementary to nuclear RNAs encoding mitochondrial proteins. Rather, the mitochondria-associated miRNAs appear to be involved in the expression of genes associated with apoptosis, cell proliferation, and differentiation. Given the central role that mitochondria play in apoptosis, the results suggest that they might serve as reservoirs of select miRNAs that may modulate these processes in a coordinate fashion.
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Project description:The opening of the permeability transition pore (PTP) in mitochondria is a key event in the initiation of cell death in various pathologic states, including ischemia/reperfusion. The activation of K+ transport into mitochondria protects cells from ischemia/reperfusion. However, the role of K+ transport in PTP regulation is unclear. Here, we studied the role of K+ and other monovalent cations in the regulation of the PTP opening in an in vitro model. The registration of the PTP opening, membrane potential, Ca2+-retention capacity, matrix pH, and K+ transport was performed using standard spectral and electrode techniques. We found that the presence of all cations tested in the medium (K+, Na+, choline+, and Li+) strongly stimulated the PTP opening compared with sucrose. Several possible reasons for this were examined: the effect of ionic strength, the influx of cations through selective and non-selective channels and exchangers, the suppression of Ca2+/H+ exchange, and the influx of anions. The data obtained indicate that the mechanism of PTP stimulation by cations includes the suppression of K+/H+ exchange and acidification of the matrix, which facilitates the influx of phosphate. Thus, the K+/H+ exchanger and the phosphate carrier together with selective K+ channels compose a PTP regulatory triad, which might operate in vivo.
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Project description:1. Aspartate-carbamoyltransferase activity was concentrated from rat-liver preparations. Only l-aspartate, beta-benzyl-l-aspartate and beta-erythro-hydroxy-dl-aspartate were carbamoylated enzymically. The K(m) for l-aspartate and carbamoyl phosphate have been determined by three methods: colorimetric procedure, radioactive assay with [(14)C]aspartate and an assay with [(14)C]carbamoyl phosphate. 2. The K(m) for aspartate has been determined as a function of the pH; the pK of the functional group at the active site of the enzyme, pK(e), was at pH9.0. Enzymic activity was diminished in the presence of N-ethylmaleimide, p-hydroxymercuribenzoate and the heavy metals Ag(+), Hg(2+), or Zn(2+). The inhibitions could be prevented by mercaptoethanol. These findings suggested the association of a thiol group with the enzymic activity. 3. Enzymic activity was also decreased by sodium lauryl sulphate, urea and dioxan. Competitive inhibition (with l-aspartate) was manifested by maleate, succinate, oxaloacetate, beta-erythro-hydroxy-dl-aspartate and beta-benzyl-l-aspartate. The K(i) for most of these inhibitions has been determined. 4. The properties of the liver enzyme are compared with those of Escherichia coli aspartate carbamoyltransferase and the implications of the findings are discussed.
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Project description:The sequences of the coenzyme-binding peptide of both cytoplasmic and mitochondrial aspartate aminotransferases from sheep liver were determined. The holoenzymes were treated with NaBH4 and digested with chymotrypsin; peptides containing bound pyridoxal phosphate were then isolated. One phosphopyridoxyl peptide was obtained from sheep liver cytoplasmic aspartate aminotransferase. Its sequence was Ser-Ne-(phosphopyridoxyl)-Lys-Asn-Phe. This sequence is identical with that reported for the homologous peptide from pig heart cytoplasmic aspartate aminotransferase. Two phosphopyridoxyl peptides with different RF values were isolated from the sheep liver mitochondrial isoenzyme. They had the same N-terminal amino acid and similar amino acid composition. The mitochondrial phosphopyridoxyl peptide of highest yield and purity had the sequence Ala-Ne-(phosphopyridoxyl)-Lys-Asx-Met-Gly-Leu-Tyr. The sequence of the first four amino acids is identical with that already reported for the phosphopyridoxyl tetrapeptide from the pig heart mitochondrial isoenzyme. The heptapeptide found for the sheep liver mitochondrial isoenzyme closely resembles the corresponding sequence taken from the primary structure of the pig heart cytoplasmic aspartate aminotransferase.
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