Argininosuccinate synthetase and argininosuccinate lyase are localized around mitochondria: An immunocytochemical study

Argininosuccinate synthetase and argininosuccinate lyase are soluble cytoplasmic enzymes of the urea cycle. Previous biochemical studies using permeabilized hepatocytes showed that these enzymes are organized in situ, and function as if they are located next to the outer membrane of mitochondria. We have now confirmed and extended those observations in intact liver by means of immunocytochemistry at the electron microscope level. Morphometric analysis of the electron micrographs shows that argininosuccinate synthetase and argininosuccinate lyase are located in the immediate vicinity of the mitochondria, predominantly next to the cytoplasmic surface of the outer membrane. Some immuno-specific protein is also observed in the endoplasmic reticulum in the immediate vicinity of the mitochondria. These results support our previous biochemical findings, and additionally suggest that virtually all of the argininosuccinate synthetase and argininosuccinate lyase of the liver parenchymal cell are located just outside the mitochondria. © 1996 Wiley-Liss, Inc.     

Induction of astrocyte argininosuccinate synthetase and argininosuccinate lyase by dibutyryl cyclic AMP and dexamethasone

Arginine is an intermediate in the elimination of excess nitrogen and is the substrate for nitric oxide synthesis. Arginine synthesis has been reported in brain tissue. We have studied the activity of the arginine biosynthetic enzymes argininosuccinate synthetase and argininosuccinate lyase in dexamethasone and/or dibutyryl cyclic AMP treated rat astrocyte cultures. Argininosuccinate lyase activity was stimulated by treatment with either effector and an additive effect was obtained when both agents were added simultaneously. Argininosuccinate synthetase was also increased in dexamethasone treated astrocytes. The effect of dibutyryl cyclic AMP on argininosuccinate synthetase was variable, suggesting a role for additional factors in its regulation as compared to argininosuccinate lyase. Regulation of arginine synthesis in astrocytes may be important to insure that arginine is not limiting for nitric oxide synthesis in neural tissue.     

Regulation of messenger ribonucleic acid levels for five urea cycle enzymes in cultured rat hepatocytes. Requirements for cyclic adenosine monophosphate, glucocorticoids, and ongoing protein synthesis

In adult rat liver, amounts of the urea cycle enzymes are regulated by diet, glucocorticoids, and cAMP. Rat hepatocytes cultured in chemically defined medium were used to precisely define the roles of glucocorticoids and cAMP in regulation of these enzymes at the pretranslational level. With the exception of ornithine transcarbamylase mRNA, cultured rat hepatocytes retain the capacity to express mRNAs for the urea cycle enzymes at the same level observed for liver of intact rats. In the absence of added hormones, mRNAs for argininosuccinate synthetase and argininosuccinate lyase remained at or above normal in vivo levels, while mRNAs for the other three enzymes declined to very low levels. Messenger RNAs for carbamyl phosphate synthetase I, argininosuccinate synthetase, argininosuccinate lyase, and arginase increased in response to either dexamethasone or 8-(4-chlorophenylthio) cAMP (CPT-cAMP). Half-maximal responses occurred at 2-3 nM dexamethasone and at 2-7 microM CPT-cAMP. Cycloheximide abolished the response to dexamethasone but not to CPT-cAMP, suggesting that dexamethasone induced expression of an intermediate gene product required for induction of these mRNAs. The effects of a combination of both hormones were additive for argininosuccinate lyase mRNA and synergistic for carbamyl phosphate synthetase I, argininosuccinate synthetase, and arginase mRNAs. Messenger RNA for ornithine transcarbamylase showed little or no response to any condition tested. Depending on the particular mRNA and hormonal condition tested, increases in mRNA levels ranged from 1.4- to 70-fold above control values.(ABSTRACT TRUNCATED AT 250 WORDS)     

The localization within plant cells of enzymes involved in arginine biosynthesis

Studies were carried out to determine the distribution of the following: (1) carbamoyl phosphate synthetase (EC 2.7.2.9), (2) ornithine carbamoyltransferase (EC 2.1.3.3), (3) argininosuccinate synthetase (EC 6.3.4.5), and (4) argininosuccinate lyase (EC 4.3.2.1) in soybean cells grown in suspension culture. Protoplasts were produced from the soybean cells by treatment with cellulase (EC 3.2.1.4) and pectinase (EC 3.2.1.15); the protoplasts were then ruptured by osmotic shock with distilled water. This treatment was followed by differential centrifugation and sucrose density gradient centrifugation to isolate various organelle fractions including mitochondria and plastids. Examination of these fractions using specific enzyme assays showed that carbamoylphosphate synthetase and ornithine carbamoyltransferase were localized in a fraction found to be composed primarily of plastids. Argininosuccinate synthetase and argininosuccinate lyase appeared to be associated with either the cytosol or a membrane fraction in close association with the cytosol such as the endoplasmic reticulum or protoplast membrane.     

Relative argininosuccinate synthetase mRNA levels and gene copy number in canavanine-resistant lymphoblasts

Mutants resistant to the arginine analogue, canavanine, have been isolated from two normal lymphoblast lines, MGL8B2 and MGL33. These mutants constitutively express up to 200-fold higher amounts of structurally normal argininosuccinate synthetase, the urea cycle enzyme that converts citrulline to argininosuccinate. Relative levels of argininosuccinate synthetase mRNA were compared among normal and canavanine-resistant lines using in vitro translation of poly(adenylic acid) RNA and blot hybridization of total cytoplasmic RNA to an argininosuccinate synthetase cDNA. Both of these approaches indicated that the canavanine-resistant lines contain increased steady-state levels of synthetase-specifc mRNA relative to their sensitive parents and that these were roughly correlated with levels of enzyme activity. Blot hybridization of Eco RI-digested genomic DNA preparations revealed no detectable differences in argininosuccinate synthetase structural gene copy number between normal and canavanine-resistant lymphoblasts, demonstrating that the canavanine-resistant phenotype is not caused by gene amplification.     

Ureogenesis in a freshwater teleost: an unusual sub-cellular localization of ornithine-urea cycle enzymes in the freshwater air-breathing teleost Heteropneustes fossilis.

Sub-cellular localization of different ornithine-urea cycle enzymes was studied in the liver and kidney of a freshwater air-breathing teleost. Carbamyl phosphate synthetase, ornithine transcarbamylase, and arginase were found to be localized inside the mitochondria, and argininosuccinate synthetase and argininosuccinate lyase were found in the soluble fraction. Mitochondrial localization of arginase, a feature known in marine elasmobranchs and toadfishes, indicates the evolutionary position of H. fossilis to be different from that of present day freshwater teleosts.     

Arginine metabolism in Saccharomyces cerevisiae: subcellular localization of the enzymes.

Subcellular localization of enzymes of arginine metabolism in Saccharomyces cerevisiae was studied by partial fractionation and stepwise homogenization of spheroplast lysates. These enzymes could clearly be divided into two groups. The first group comprised the five enzymes of the acetylated compound cycle, i.e., acetylglutamate synthase, acetylglutamate kinase, acetylglutamyl-phosphate reductase, acetylornithine aminotransferase, and acetylornithine-glutamate acetyltransferase. These enzymes were exclusively particulate. Comparison with citrate synthase and cytochrome oxidase, and results from isopycnic gradient analysis, suggested that these enzymes were associated with the mitochondria. By contrast, enzymatic activities going from ornithine to arginine, i.e., arginine pathway-specific carbamoylphosphate synthetase, ornithine carbamoyltransferase, argininosuccinate synthetase, and argininosuccinate lyase, and the two first catabolic enzymes, arginase and ornithine aminotransferase, were in the "soluble" fraction of the cell.     

Control of argininosuccinate synthetase by arginine in human lymphoblasts

Human lymphoblasts in long-term culture have the enzyme activities necessary to convert citrulline to arginine: argininosuccinate synthetase and argininosuccinate lyase. Upon transfer from arginine-supplemented to citrulline-supplemented medium, lymphoblasts exhibit a lag period before resuming exponential growth. During this lag the specific activity of argininosuccinate synthetase increases an average of 60-fold. Argininosuccinate lyase activity remains unchanged. If normal lymphoblasts are starved in arginine-deficient medium without citrulline or if argininosuccinate lyase--deficient lymphoblasts are transferred to citrulline-containing medium, argininosuccinate synthetase activity increases linearly for several days and reaches even higher levels. Cycloheximide blocks the increase in enzyme activity. Cells grown in citrulline medium and pulse labeled with 35S-methionine incorporate more 35S-methionine into argininosuccinate synthetase protein than cells grown in arginine; the rate of disappearance of radioactively labeled enzyme is the same in citrulline- and arginine-grown cells. Arginine or a closely related metabolite thus appears to repress the synthesis of argininosuccinate synthetase of human lymphoblasts in culture.     

Nutritional and hormonal regulation of mRNA abundance for arginine biosynthetic enzymes in kidney

Argininosuccinate synthetase and argininosuccinate lyase catalyze the synthesis of arginine from citrulline in kidney and also serve as components of the urea cycle in liver of ureotelic animals. Dietary and hormonal regulation of mRNAs encoding these enzymes have been well studied in liver but not in kidney. Messenger RNAs for these enzymes are localized within the renal cortex. Starvation and extreme variations in dietary protein content (0% vs 60% casein) produced 2.6- to 3.5-fold increases in mRNA abundance for these two enzymes in rat kidney. Argininosuccinate lyase mRNA was not induced by dibutyryl cAMP, dexamethasone, or a combination of the two agents. In contrast, argininosuccinate synthetase mRNA was induced 2-fold by dibutyryl cAMP but was unresponsive to dexamethasone. Thus, diet and hormones regulate levels of these mRNAs in rat kidney, but the responses are both qualitatively and quantitatively distinct from the responses previously reported for rat liver.     

Nitro analogs of substrates for argininosuccinate synthetase and argininosuccinate lyase

The nitro analogs of aspartate and argininosuccinate were synthesized and tested as substrates and inhibitors of argininosuccinate synthetase and argininosuccinate lyase, respectively. The Vmax for 3-nitro-2-aminopropionic acid was found to be 60% of the maximal rate of aspartate utilization in the reaction catalyzed by argininosuccinate synthetase. Only the nitronate form of this substrate, in which the C-3 hydrogen is ionized, was substrate active, indicating a requirement for a negatively charged group at the beta carbon. The V/K of the nitro analog of aspartate was 85% of the value of aspartate after correcting for the percentage of the active nitronate species. The nitro analog of argininosuccinate, N3-(L-1-carboxy-2-nitroethyl)-L-arginine, was a strong competitive inhibitor of argininosuccinate lyase but was not a substrate. The pH dependence of the observed pKi was consistent with the ionized carbon acid (pK = 8.2) in the nitronate configuration as the inhibitory material. The pH-independent pKi of 2.7 microM is 20 times smaller than the Km of argininosuccinate at pH 7.5. These results suggest that the tighter binding of the nitro analog relative to the substrate is due to the similarity in structure to a carbanionic intermediate in the reaction pathway.     

Channeling of urea cycle intermediates in situ in permeabilized hepatocytes

Preferential use of endogenously generated intermediates by the enzymes of the urea cycle was observed using isolated rat hepatocytes made permeable to low molecular weight compounds with alpha-toxin. The permeabilized cells synthesized [14C]urea from added NH4Cl, [14C]HCO3-, ornithine, and aspartate, using succinate as a respiratory substrate; with all substrates saturating, about 4 nmol of urea were formed per min/mg dry weight of cells. Urea usually accounted for about 40-50% of the total (NH3 + ornithine)-dependent counts, arginine for less than 10%, and citrulline for about 30%. Very tight channeling of arginine between argininosuccinate lyase and arginase was shown by the fact that the addition of a 200-fold excess of unlabeled arginine to the incubations did not decrease the percentage of counts found in urea or increase that found in arginine, even though a substantial amount of the added arginine was hydrolyzed inside the cells. The channeling of argininosuccinate between its synthetase and lyase was demonstrated by similar observations; unlabeled argininosuccinate added in 200-fold excess decreased the percentage of counts in urea by only 25%. Channeling of citrulline from its site of synthesis by ornithine transcarbamylase in the mitochondrial matrix to argininosuccinate synthetase in the cytoplasmic space was also shown. These results strongly suggest that the three "soluble" cytoplasmic enzymes of the urea cycle are grouped around the mitochondria and are spatially organized within the cell in such a way that intermediates can be efficiently transferred between them.     

Immunohistochemical Localization of Arginase II and Other Enzymes of Arginine Metabolism in Rat Kidney and Liver

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were collocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.     

Regulation of genes for inducible nitric oxide synthase and urea cycle enzymes in rat liver in endotoxin shock

Arginine is an intermediate of the urea cycle in the liver. It is synthesized by the first four enzymes of the cycle, carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, and argininosuccinate lyase, and is hydrolyzed to urea and ornithine by arginase I, forming the cycle. In endotoxemia shock, inducible nitric oxide (NO) synthase (iNOS) is induced in hepatocytes and arginine is utilized for NO production. Regulation of the genes for iNOS and the urea cycle enzymes was studied using lipopolysaccharide (LPS)-treated rat livers. When rats were injected intraperitoneally with LPS, iNOS mRNA was markedly induced. Cationic amino acid transporter-2 and C/EBPbeta mRNAs were also highly increased. In contrast, mRNAs for all the urea cycle enzymes except ornithine transcarbamylase were gradually decreased and reached 16-28% of controls at 12 h. However, all these enzymes remained unchanged at protein level up to 24 h. In light of these results, we suggest that synthesis of urea cycle enzymes is downregulated and that the protein synthetic capacity is directed to synthesis of proteins required for defense against endotoxemia.     

Subcellular location of glutamine synthetase and urea cycle enzymes in liver of spiny dogfish (Squalus acanthias)

Glutamine synthetase and glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, both of which are present in high concentrations in liver of urea-retaining elasmobranchs, have been found to be located exclusively in the mitochondria in liver from the representative elasmobranch Squalus acanthias. This observation is consistent with the view that the function of this unique carbamoyl-phosphate synthetase is related to urea synthesis, and that the initial nitrogen-donating substrate for urea synthesis in these species is glutamine rather than ammonia. The urea cycle enzymes, ornithine carbamoyltransferase and arginase, are also located in the mitochondria, whereas argininosuccinate synthetase and argininosuccinate lyase are located in the cytosol. Glutamine synthetase and arginase are mitochondrial enzymes in uricotelic species, but are normally found in the cytoplasm in ureotelic species. the properties of the elasmobranch arginase, however, are characteristic of arginases from ureotelic species (e.g. the Km for arginine is 1.2 mM, and the enzyme has an Mr congruent to 100,000).     

Fine-structural localization of proenkephalin mRNAs in the hypothalamic magnocellular dorsal nucleus of the guinea pig: a comparison of radioisotopic and enzymatic in situ hybridization methods at the light- and electron-microscopic levels

With the aim of localizing proenkephalin mRNAs in neurons of the hypothalamic magnocellular dorsal nucleus of the guinea pig, we compared the in situ hybridization signals obtained on Vibratome sections with a method employing either a biotinylated or a digoxigenin-labeled oligonucleotide detected by means of the alkaline phosphatase reaction. Since the hybridization approach using the biotinylated probe was more sensitive than the digoxigenin method, the ultrastructural localization of hybrids in neurons of the magnocellular dorsal nucleus was studied by the use of the former procedure, and was further compared with results of in situ hybridization using a ³⁵S-labeled probe. Biotin was detected via an amplified avidin-biotin-peroxidase complex. Radioactive hybrids were localized over extended cytoplasmic compartments rich in rough endopoasmic reticulum and also in nuclear indentations. The method based on biotinylated probe proved to be sensitive and provided high-resolution labeling in well-preserved specimens. Proenkephalin mRNAs were clearly localized within circumscribed cytoplasmic compartments. The immunoprecipitates were mainly observed within the rough endoplasmic reticulum, especially at the periphery of the cell. The reticulum was dominated by elongated parallel cisternae. The labeling also appeared in a paranuclear position, mainly in nuclear indentations. The labeling was found on the outer surface of the endoplasmic lamellae. The remainder of the reticulum was unlabeled. Neuronal processes were free of labeling.     

Messenger RNA sorting in enterocytes. Co-localization with encoded proteins.

This study describes the intracellular compartmentalization of three different mRNAs in the polarized rat fetal enterocyte. They encode proteins that are known to be localized within different regions of the epithelial cell namely (i) the apical, membrane-bound glycoprotein, lactase-phlorizin hydrolase (lactase), (ii) the mitochondrially localized enzyme, carbamoylphosphate synthetase (CPS), and (iii) the cytoplasmically localized enzyme, phosphoenolpyruvate carboxykinase (PEPCK). These mRNAs are found in close proximity to their respective protein products, i.e. the apical membrane, mitochondria and cytoplasm, respectively. The significance of these observations is twofold; (i) they indicate that mRNAs are sorted into specific domains of the cytosol of intestinal epithelial cells; and (ii) they imply the presence of two distinct pathways of mRNA targeting one that allows transport of mRNAs that are translated on ribosomes associated with the rough endoplasmic reticulum (lactase mRNA), and the other that allows sorting of mRNAs that are translated on free polysomes (CPS and PEPCK mRNA).     

Protein synthesis in the livers of aging mice studied by electron microscopic radioautography.

The application of 3H-leucine results in labeling of the liver cells of mice in which protein is synthesized at various ages of the animals. Quantitative changes of protein synthesis in the hepatocytes of aging mice were studied by electron microscopic radioautography. The silver grains in the hepatocytes were mainly located over the rough surfaced endoplasmic reticulum, mitochondria, Golgi apparatus, cytoplasmic matrix, and a few over the nuclei. The number of silver grains in the cytoplasm and nuclei of the hepatocytes gradually increased after birth, reached the maximum at 1 month after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the hepatocyte cytoplasm was more than that in nuclei at various ages. The number of silver grains in the rough surfaced endoplasmic reticulum and mitochondria gradually increased from embryo to 1 month after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the Golgi apparatus showed almost no change from fetal stage to 6 months after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the cytoplasmic matrix gradually increased from fetal stage to 2 months after birth, then decreased with aging until the 24th month. These changes reflect the quantity of protein synthesized in each cell organelle at various ages of animals.     

Complementary distribution of carbamoylphosphate synthetase (ammonia) and glutamine synthetase in rat liver acinus is regulated at a pretranslational level

We studied the distribution of the mRNAs for carbamoylphosphate synthetase (ammonia) and glutamine synthetase in frozen sections of adult rat liver by in situ hybridization to [35S]-labeled cDNA probes. The density of silver grains resulting from hybridization to the labeled cDNA probe for carbamoylphosphate synthetase is highest around the portal venules, decreases towards the central venule, and is virtually absent from an area two to three cells wide that lines the central venules in which mRNA for glutamine synthetase is predominantly localized. Therefore, both mRNAs show the same complementary distribution within the liver acinus that was found for the proteins they encode, demonstrating that compartmentalization of the expression of these enzymes is controlled at a pretranslational level. In addition, we found that carbamoylphosphate synthetase mRNA is present mainly in the epithelium of the crypts of the proximal part of the small intestine, whereas carbamoylphosphate synthetase protein is present in the epithelium of both crypts and villi.     

Cytoplasmic organelles on the road to mRNA decay

Localization of both mRNAs and mRNA decay factors to internal membranes of eukaryotic cells provides a means of coordinately regulating mRNAs with common functions as well as coupling organelle function to mRNA turnover. The classic mechanism of mRNA localization to membranes is the signal sequence-dependent targeting of mRNAs encoding membrane and secreted proteins to the cytoplasmic surface of the endoplasmic reticulum. More recently, however, mRNAs encoding proteins with cytosolic or nuclear functions have been found associated with various organelles, in many cases through unknown mechanisms. Furthermore, there are several types of RNA granules, many of which are sites of mRNA degradation; these are frequently found associated with membrane-bound organelles such as endosomes and mitochondria. In this review we summarize recent findings that link organelle function and mRNA localization to mRNA decay. This article is part of a Special Issue entitled: RNA Decay mechanisms.