Subcellular localization of fumarase in mammalian cells and tissues

Fumarase, a mitochondrial matrix protein, is previously indicated to be present in substantial amounts in the cytosol as well. However, recent studies show that newly synthesized human fumarase is efficiently imported into mitochondria with no detectable amount in the cytosol. To clarify its subcellular localization, the subcellular distribution of fumarase in mammalian cells/tissues was examined by a number of different methods. Cell fractionation using either a mitochondria fraction kit or extraction with low concentrations of digitonin, detected no fumarase in a 100,000 g supernatant fraction. Immunoflourescence labeling with an affinity-purified antibody to fumarase and an antibody to the mitochondrial Hsp60 protein showed identical labeling pattern with labeling seen mainly in mitochondria. Detailed studies were performed using high-resolution immunogold electron microscopy to determine the subcellular localization of fumarase in rat tissues, embedded in LR White resin. In thin sections from kidney, liver, heart, adrenal gland and anterior pituitary, strong and specific labeling due to fumarase antibody was only detected in mitochondria. However, in the pancreatic acinar cells, in addition to mitochondria, highly significant labeling was also observed in the zymogen granules and endoplasmic reticulum. The observed labeling in all cases was completely abolished upon omission of the primary antibody indicating that it was specific. In a western blot of purified zymogen granules, a fumarase-antibody cross-reactive protein of the same molecular mass as seen in the mitochondria was present. These results provide evidence that fumarase in mammalian cells/tissues is mainly localized in mitochondria and significant amounts of this protein are not present in the cytosol. However, these studies also reveal that in certain tissues, in addition to mitochondria, this protein is also present at specific extramitochondrial sites. Although the cellular function of fumarase at these extramitochondrial locations is not known, the appearance/localization of fumarase outside mitochondria may help explain how mutations in this mitochondrial protein can give rise to a number of different types of cancers.     

Ferrochelatase and δ-aminolaevulate synthetase in brain, heart, kidney and liver of normal and porphyric rats. The induction of δ-aminolaevulate synthetase in kidney cytosol and mitochondria by allylisopropylacetamide

1. delta-Aminolaevulate synthetase was detected in liver and kidney mitochondria prepared from normal rats. 2. The administration of allylisopropylacetamide induced an increase in delta-aminolaevulate synthetase in both liver and kidney mitochondria and the enzyme also appeared in the cytosol fraction of both tissues. Comparison with the distribution of glutamate dehydrogenase indicated that this soluble kidney delta-aminolaevulate synthetase was truly of cytosol origin and did not arise from disrupted mitochondria. The kidney cytosol enzyme was inhibited by 50% by 50mum-protohaem. 3. delta-Aminolaevulate synthetase could not be detected in mitochondria or cytosol from heart or brain from normal or porphyric rats. 4. The administration of allylisopropylacetamide caused little or no increase in ferrochelatase or cytochrome content of liver, kidney, heart or brain mitochondria.     

Role of structural and functional elements of mouse methionine-S-sulfoxide reductase in its subcellular distribution

Oxidized forms of methionine residues in proteins can be repaired by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). In mammals, three MsrBs are present, which are targeted to various subcellular compartments. In contrast, only a single mammalian MsrA gene is known whose products have been detected in both cytosol and mitochondria. Factors that determine the location of the protein in these compartments are not known. Here, we found that MsrA was present in cytosol, nucleus, and mitochondria in mouse cells and tissues and that the major enzyme forms detected in various compartments were generated from a single-translation product rather than by alternative translation initiation. Both cytosolic and mitochondrial forms were processed with respect to the N-terminal signal peptide, and the distribution of the protein occurred post-translationally. Deletion of amino acids 69-108, 69-83, 84-108, or 217-233, which contained elements important for MsrA structure and function, led to exclusive mitochondrial location of MsrA, whereas a region that affected substrate binding but was not part of the overall fold had no influence on the subcellular distribution. The data suggested that proper structure-function organization of MsrA played a role in subcellular distribution of this protein in mouse cells. These findings were recapitulated by expressing various forms of mouse MsrA in Saccharomyces cerevisiae, suggesting conservation of the mechanisms responsible for distribution of the mammalian enzyme among different cellular compartments.     

Branched-chain amino acid aminotransferase in mouse testicular tissue

Branched-chain amino acid aminotransferase (L-leucine:2-oxoglutarate aminotransferase, EC 2.6.1.6) activity was determined in several tissues of the mouse. Testis homogenates presented a specific activity very close to that of heart extracts which were the most active. Enzyme activity was detectable in testes from 5-day-old mice and increased steadily during development to reach a maximum at the 20th day of life. The transaminase was present in the cytosol of testicular homogenates and also associated, probably in the matrix, with a special type of mitochondria present in spermatozoa and gametogenic cells. The enzyme from testis is active against the three branched-chain amino acids and catalyses the reaction in both directions. Highest activity and lowest Km were obtained with L-leucine. Activity with L-valine was the lowest. The enzyme from the mitochondrial fraction showed identical properties to that from the soluble phase. The possible participation of this aminotransferase in a shuttle system transferring reducing equivalents from cytoplasm to mitochondria is postulated.     

Subcellular localization of the leucine biosynthetic enzymes in yeast

When baker's yeast spheroplasts were lysed by mild osmotic shock, practically all of the isopropylmalate isomerase and the beta-isopropylmalate dehydrogenase was released into the 30,000 x g supernatant fraction, as was the cytosol marker enzyme, glucose-6-phosphate dehydrogenase. alpha-Isopropylmalate synthase, however, was not detected in the initial supernatant, but could be progressively solubilized by homogenization, appearing more slowly than citrate synthase but faster than cytochrome oxidase. Of the total glutamate-alpha-ketoisocaproate transaminase activity, approximately 20% was in the initial soluble fraction, whereas solubilization of the remainder again required homogenization of the spheroplast lysate. Results from sucrose density gradient centrifugation of a cell-free particulate fraction and comparison with marker enzymes suggested that alpha-isopropylmalate synthase was located in the mitochondria. It thus appears that, in yeast, the first specific enzyme in the leucine biosynthetic pathway (alpha-isopropylmalate synthase) is particulate, whereas the next two enzymes in the pathway (isopropylmalate isomerase and beta-isopropylmalate dehydrogenase) are "soluble," with glutamate-alpha-ketoisocaproate transaminase activity being located in both the cytosol and particulate cell fractions.     

Ultrastructural localization of PGP 9.5 and ubiquitin immunoreactivities in rat ductus epididymidis epithelium

Summary The distribution of protein gene product 9.5 (PGP) and ubiquitin in the spermatozoa and epithelial cells in the different regions of the rat duetus epididymidis (proximal caput, distal caput, corpus and cauda) was studied by Western blotting analyses and electron microscopical immunogold labelling. Western blotting analyses showed that the PGP immunoreactive band was very intense in the caput and cauda epididymidis and almost irrelevant in the corpus, while the ubiquitin immunoreactive band was intense in the distal caput and cauda. No ubiquitin immunoreactive band was observed in the proximal caput and only a very weak band was seen in the corpus. The results of electron microscopical immunogold labelling varied from one epididymal region to another. The proximal caput epididymidis presented immunoreaction to PGP in the rough endoplasmic reticulum, cytosol, mitochondria and microvilli of most principal cells, and in the cytosol, rough endoplasmic reticulum and mitochondria of most basal cells. No ubiquitin immunoreaction was observed in this epididymal region. In the distal caput epididymidis, PGP immunoreactivity was detected in some principal and basal cells in the same intracellular locations as described in the proximal caput. In this region, ubiquitin immunoreactivity appears in the apical cytosol and mitochondria of principal cells. The corpus epididymidis showed no immunoreaction to PGP or ubiquitin. In the cauda epididymidis, immunostaining to PGP was observed in most clear cells and in isolated principal cells. The intracellular location of PGP in both cell types was the cytosol, mitochondria and microvilli. Ubiquitin immunoreactivity was detected in the perinuclear cytosol and mitochondria — but not in the digestive vacuoles — of some clear cells. Scanty ubiquitin immunolabelling was also found in the microvilli, cytosol and mitochondria of some principal cells. The head of the spermatozoa present in the ductal lumen in all epididymal regions immunoreacted intensely to PGP. Ubiquitin was detected in the intermediate piece and residual cytoplasm of intraluminal spermatozoa present in the corpus and cauda epididymidis. These findings suggest that a non-ubiquitinated PGP irnrnunoreactive protein is secreted by the principal cells in caput epididymidis and binds the spermatozoon heads. It is possible that the clear cells of the cauda epididymidis secrete the ubiquitin that binds to spermatozoon tail.     

Stereoselective arylpropionyl-CoA thioester formation in vitro

The inversion from R- to S-enantiomer that occurs for some arylpropionic acids may have both toxicological and therapeutic implications. To characterize some properties of this inversion, arylpropionyl-CoA thioester formation was studied in rat tissue homogenates and subcellular fractions for the enantiomers of fenoprofen, ibuprofen, and flurbiprofen. Thioesters were formed from (R)-fenoprofen (64%) and (R)-ibuprofen (33%) but not from the corresponding S-enantiomers or the enantiomers of flurbiprofen. This correlates with the extensive inversion of fenoprofen and ibuprofen and lack of inversion of flurbiprofen in vivo. Subcellular fractions from rat liver showed thioester formation to occur in mitochondria and microsomes but not cytosol. Once formed, the thioesters were readily racemized by whole rat liver homogenate, mitochondria, and cytosol, but only partially inverted (S:R = 0.3) in microsomes. Thioester formation from fenoprofen and ibuprofen was studied in tissue homogenate obtained from liver, diaphragm, kidney, lung, skeletal muscle, smooth muscle, fat, caecum, and intestines. The liver was at least 50-fold more efficient than the other tissues studied and would be expected to be a major organ of enantiomeric inversion. Our data support the hypothesis that R- to S-enantiomeric inversion of arylpropionic acids proceeds via the stereoselective formation of CoA thioesters followed by enzymatic racemization and hydrolysis of the thioesters to regenerate free acid.     

Role of the mitochondrial anion transporters in the regulation of ammoniagenesis in renal cortex mitochondria of the rabbit and rat.

The purpose of this study was to investigate factors which may regulate ammoniagenesis in the kidney cortex. Emphasis was placed on the segment of the pathway by which the carbons derived from glutamine must exit from the mitochondrion. These pathways were compared in the rat with high rates of ammoniagenesis and the rabbit which has a low rate of ammoniagenesis. The dicarboxylate transporter, which is essential for ammoniagenesis, has a maximum velocity which was much lower in the rabbit. The malate concentration required for half-maximal rates of transport was 14 nmol/mg mitochondrial protein and similar in both species. There was no effect of chronic metabolic acidosis on dicarboxylate transporter activity. The tricarboxylate transporter activity with phosphoenol pyruvate as substrate also had a low activity in the rabbit kidney-cortex mitochondria. The maximum velocity of phosphate dependent glutaminase, glutamate dehydrogenase and phosphoenolpyruvate carboxykinase were all much greater than the maximal rate of ammoniagenesis observed in vivo in the rabbit. Therefore, the low rates of ammoniagenesis and the failure to adapt to acidosis in the rabbit are best explained by factors influencing the dicarboxylate transporter.     

Microsomal and cytosolic epoxide hydrolases in rhesus monkey liver, and in normal and neoplastic human liver

The cytosolic epoxide hydrolase (EH-LC) was observed in rhesus monkey liver cytosol, and in both normal and neoplastic human liver. Microsomal epoxide hydrolase (EH-LM) was detected not only in the microsomes of normal and neoplastic human liver and normal rhesus monkey liver, but also in the cytosol of these tissues. No apparent differences were observed between the EH-LM in liver cytosol and that in microsomes. No major differences were observed between the levels of EH-LM in the cytosol of normal and that in neoplastic human liver.     

Co-distribution of annexin VI and actin in secretory ameloblasts and odontoblasts of rat incisor

Summary Annexin VI and actin were detected by immunoblot analysis in the enamel- and dentin-related portions of dental tissues. Annexin VI was found mainly in the particulate fraction whereas actin was detected in both the soluble and particulate fractions. By immunoelectron microscopy, annexin VI antibodies conjugated with colloidal gold were seen to label the mitochondria, the cytosol and the nucleus of secretory ameloblasts and odontoblasts of rat incisor. In the processes of these cell, the plasmalemmal undercoat was labeled. Antiactin antibodies labeled the desmosome-like junctions, the cytosol, and the mitochondria of the cell bodies. Extensive labeling was seen at the periphery of the Tomes' processes and odontoblast processes. These results suggest that annexin VI may play a role in Ca²⁺-regulation in the cell bodies, especially as a calcium receptor protein in the mitochondria. Moreover, annexin VI and actin seem to be co-distributed in secretory processes. Thus, these proteins might be both involved in exocytotic and endocytotic events.     

Analysis of peptides secreted from cultured mouse brain tissue

Peptides represent a major class of cell–cell signaling molecules. Most peptidomic studies have focused on peptides present in brain or other tissues. For a peptide to function in intercellular signaling, it must be secreted. The present study was undertaken to identify the major peptides secreted from mouse brain slices that were cultured in oxygenated buffer for 3–4 h. Approximately 75% of the peptides identified in extracts of cultured slices matched the previously reported peptide content of heat-inactivated mouse brain tissue, whereas only 2% matched the peptide content of unheated brain tissue; the latter showed a large number of postmortem changes. As found with extracts of heat-inactivated mouse brain, the extracts of cultured brain slices represented secretory pathway peptides as well as peptides derived from intracellular proteins such as those present in the cytosol and mitochondria. A subset of the peptides detected in the extracts of the cultured slices was detected in the culture media. The vast majority of secreted peptides arose from intracellular proteins and not secretory pathway proteins. The peptide RVD-hemopressin, a CB1 cannabinoid receptor agonist, was detected in culture media, which is consistent with a role for RVD-hemopressin as a non-classical neuropeptide. Taken together with previous studies, the present results show that short-term culture of mouse brain slices is an appropriate system to study peptide secretion, especially the non-conventional pathway(s) by which peptides produced from intracellular proteins are secreted. This article is part of a Special Issue entitled: An Updated Secretome.     

Relationships between carnitine and coenzyme A esters in tissues of normal and alloxan-diabetic sheep

1. The total acid-soluble carnitine concentrations of four tissues from Merino sheep showed a wide variation not reported for other species. The concentrations were 134, 538, 3510 and 12900nmol/g wet wt. for liver, kidney cortex, heart and skeletal muscle (M. biceps femoris) respectively. 2. The concentration of acetyl-CoA was approximately equal to the concentration of free CoA in all four tissues and the concentration of acid-soluble CoA (free CoA plus acetyl-CoA) decreased in the order liver>kidney cortex>heart>skeletal muscle. 3. The total amount of acid-soluble carnitine in skeletal muscle of lambs was 40% of that in the adult sheep, whereas the concentration of acid-soluble CoA was 2.5 times as much. A similar inverse relationship between carnitine and CoA concentrations was observed when different muscles in the adult sheep were compared. 4. Carnitine was confined to the cytosol in all four tissues examined, whereas CoA was equally distributed between the mitochondria and cytosol in liver, approx. 25% was present in the cytosol in kidney cortex and virtually none in this fraction in heart and skeletal muscle. 5. Carnitine acetyltransferase (EC 2.3.1.7) was confined to the mitochondria in all four tissues and at least 90% of the activity was latent. 6. Acetate thiokinase (EC 6.2.1.1) was predominantly (90%) present in the cytosol in liver, but less than 10% was present in this fraction in heart and skeletal muscle. 7. In alloxan-diabetes, the concentration of acetylcarnitine was increased in all four tissues examined, but the total acid-soluble carnitine concentration was increased sevenfold in the liver and twofold in kidney cortex. 8. The concentration of acetyl-CoA was approximately equal to that of free CoA in the four tissues of the alloxan diabetic sheep, but the concentration of acid-soluble CoA in liver increased approximately twofold in alloxan-diabetes. 9. The relationship between CoA and carnitine and the role of carnitine acetyltransferase in the various tissues is discussed. The quantitative importance of carnitine in ruminant metabolism is also emphasized.     

Subcellular Localization and Developmental Changes of Aspartate-alpha-Ketoglutarate Transaminase Isozymes in the Cotyledons of Cucumber Seedlings

The total activity of aspartate-α-ketoglutarate transaminase in the cotyledons of cucumber (Cucumis sativus L.) seeds increased 17-fold during the first 2 days of germination in darkness and then declined gradually to 20% of the peak activity after 10 days. Exposure of the seedlings to light at day 3 accelerated the decline. The enzyme in the cotyledon extracts from seedlings at various ages was resolved into six distinct isozymes by starch gel electrophoresis. Isozymes 1 and 2 were glyoxysomal isozymes with different developmental patterns. Isozyme 1 followed the developmental pattern of the total enzyme activity in darkness, and was rapidly eliminated upon illumination. Isozyme 2 increased in activity to a peak at day 2 and declined rapidly thereafter, and disappeared completely at day 6; this developmental pattern was independent of light. No major difference in the optimal pH for activity, substrate specificity, and reversibility was observed between isozymes 1 and 2. The combined developmental pattern of isozymes 1 and 2 during germination correlated with that of the glyoxysomes. Isozyme 3 was located in the cytosol and its developmental pattern followed that of the total activity. Isozymes 4,5, and 6 were plastid isozymes and appeared only after 2 days of germination. Unlike many other chloroplast enzymes, the appearance of the chloroplast transaminase isozymes was under temporal control and was independent of illumination. No enzyme activity was detected in isolated mitochondria. The findings illustrate a complicated cellular control system for the appearance of various organelle-specific transaminase isozymes and thus the amino acid metabolism during germination.     

Regional and subcellular distribution of enzymes of branched-chain amino acid metabolism in brains of normal and diabetic rats

Branched-chain-amino-acid:alpha-ketoglutarate transaminase and branched-chain alpha-ketoacid dehydrogenase have been assayed in brains of control and of streptozotocin-induced diabetic rats. Enzyme activities were measured in five distinct regions of the brain: cerebellum, pons + medulla, midbrain, thalamus + hypothalamus, and telencephalon. Subcellular distribution of these enzymes in whole brain was assessed by fractionating brain homogenate into cytoplasm, free mitochondria, and synaptosomes. The following enzymes were used as markers: lactate dehydrogenase for cytoplasm, glutamate dehydrogenase for mitochondria, and glutamate decarboxylase for synaptosomes. The activity of the branched-chain amino acid transaminase in all brain regions was considerably higher than that of the branched-chain alpha-ketoacid dehydrogenase. While the highest activity of the transaminase occurred in brain-stem regions, the highest activity of the dehydrogenase was present in cerebellum and telencephalon. Diabetes did not affect the activity of the transaminase, but it caused a decrease in the total activity of the dehydrogenase in midbrain and in thalamus + hypothalamus. The transaminase was localized in the cytoplasmic fraction of whole brain, while the dehydrogenase was enriched in the free mitochondria.     

Enzymes of glycerol metabolism in the storage tissues of Fatty seedlings

Various enzymes of glycerol metabolism in the extracts of 5-day-old eastor bean (Ricinus communis L. var. Hale) endosperm and 4-day-old peanut (Archis hypogaea L.) cotyledon were studied. NAD-glycerol dehydrogenase and NAD-α-glycerolphosphate dehydrogenase were not detected. Glycerol kinase was detected in the soluble fractions and an α-glycerolphosphate oxidoreductase was found in the particulate fractions. The particulate fractions were separated into various organelle fractions by sucrose gradient centrifugation and the α-glycerolphosphate oxidoreductase was shown to be present in the mitochondria. The properties of the castor bean mitochondrial α-glycerolphosphate oxidoreductase resembled those of a similar enzyme present in the mitochondria of many animal tissues. A survey showed that the α-glycerolphosphate oxidoreductase was present in great amount only in the storage tissues of fatty seedlings but not in other nonfatty plant tissues. It is concluded that in the storage tissues of fatty seedlings, the soluble glycerol kinase and the mitochondrial cytochrome-linked α-glycerolphosphate oxidoreductase are the two enzymes responsible for the initial conversion of glycerol to hexose.     

Development of substrate cycle enzymes in the hamster small intestine

A system of enzymes is required for the transport of reducing equivalents from reduced nicotinamide adenine dinucleotide (NADH) generated in the cytosol into the mitochondria by the substrate cycles. These substrate cycle enzymes are necessary for the flow of pyruvate derived from glucose into the mitochondria for oxidative decarboxylation and for the efficient production of adenosine 5′-triphosphate (ATP) for the unique intestinal nutrient transport functions. The enzymes of the l-glycerol 3-phosphate and the l-malate/l-aspartate substrate cycles are present before birth and increase significantly at the 7-day postnatal period of development. The key enzymes monitored in the intestinal subcellular fractions were NAD-linked l-glycerol-3-phosphate dehydrogenase, flavoprotein-linked l-glycerol-3-phosphate dehydrogenase, l-malate dehydrogenase, and l-glutamate-oxaloacetate transaminase.     

Mitochondrial glutathione uptake: characterization in isolated brain mitochondria and astrocytes in culture

Glutathione in the mitochondria is an important determinant of cellular responses to oxidative stress. Mitochondrial glutathione is maintained by uptake from the cytosol, a process that has been little studied in brain cells. In the present study, measurements using isolated rat brain mitochondria showed a rapid uptake of [³H]-glutathione that was strongly influenced by the mitochondrial glutathione content. [³H]-glutathione incorporated into the mitochondria was not rapidly released. Uptake was inhibited by substrates and inhibitors for several known mitochondrial anion transporters. Citrate, isocitrate and benzene-1,2,3-tricarboxylate were particularly effective inhibitors, suggesting a possible role for a tricarboxylate carrier in the glutathione transport. The properties of uptake differed greatly from those reported previously for mitochondria from kidney and liver. In astrocytes in primary culture, diethylmaleate or hydrogen peroxide treatment resulted in depletion of cytosolic and mitochondrial glutathione. The pattern of restoration of glutathione content in the presence of glutathione precursors following treatment with diethylmaleate was consistent with uptake into mitochondria being controlled primarily by the glutathione gradient between the cytosol and mitochondria. However, following hydrogen peroxide treatment, recovery of glutathione in the mitochondria initially preceded comparable proportional restoration in the cytosol, suggesting the possibility of additional controls on glutathione uptake in some conditions.     

Structuro-kinetic organization of the tricarboxylic acid cycle in the active functioning of mitochondria

Taking into account structural and functional organization of mitochondrial processes it has been shown that at active work there functions in mitochondria an accelerated mechanism of succinic acid formation via coupling of glutamate-oxalacetate transaminase and alpha-ketoglutaratdehydrogenase. This way is closed up into a cycle with the participation of cytosol transaminases which support influx of glutamate, pyruvate and malic acid into mitochondria. When provision of the mitochondria with the substrate proceeds along the transaminase pathway the initial slow region of the tricarboxylic acid cycle is omitted. Thus at active work a faster course is selected. It permits realization of the advantages of succinate dehydrogenase high activity and of oxidation efficiency of succinic acid generated in mitochondria which is essentially higher than that under oxidation of succinic acid and even more of other substrates of the tricarboxylic acid cycle.     

Organelle-specific Isozymes of Aspartate-alpha-Ketoglutarate Transaminase in Spinach Leaves

Four distinct isozymes of aspartate-α-ketoglutarate transaminase in a spinach (Spinacia oleracea L.) leaf extract were separated by starch gel electrophoresis. Of the total aspartate-α-ketoglutarate transaminase activity, approximately 45% was represented by the chloroplast isozyme, 26% by the cytosol isozyme, 19% by the mitochondrial isozyme, and 3 to 10% by the peroxisomal isozyme. The aspartate-α-ketoglutarate transamination activity in the four subcellular compartments behaved similarly. It was freely reversible and α-ketoglutarate was preferred to pyruvate or glyoxylate as the amino group acceptor. With glutamate as the amino group donor, oxaloacetate was superior to pyruvate or glyoxylate as the acceptor in chloroplasts, mitochondria, and cytosol, while pyruvate or glyoxylate was preferred to oxaloacetate as the acceptor in peroxisomes.