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1.
Suspension-cultured cells and aseptically cultured roots ofintact plants of Atropa belladonna L. removed tropane alkaloidprecursors arginine (Arg) and ornithine (Orn) at nearly an equalrate from the feeding medium. A great part of Arg- and Orn-derived14C-label was found in ethanol-insoluble compounds, mostly inproteins already after 2 h feeding. Ethanol-soluble label inthe roots was found mainly in amino acids (e.g. glutamine, Gln)after 2 h feeding, and after 20 h also in some intermediatesof the urea cycle (e.g. argininosuccinate). In suspension cultures, subculturing of the initiation callusdecreased both the uptake of the basic amino acids tested andtheir binding on to the apoplastic space. After 20 h feedingwith Arg more label was found in organic acids in stationaryphase suspension cultures with repressed alkaloid synthesisthan in roots producing alkaloids. The growth phase and passagenumber also affected into which amino acids the label was incorporated.When the initiation callus was young (the 3rd passage), theintermediates of the urea cycle were actively labelled, butwhen the initiation callus was older (the 8th passage) and thesuspension formed roots, especially Gln was labelled. Only tracesof -N-methylornithine were detected in feeding experiments withOrn and Arg. Considerable arginase activity with a high pH optimumwas observed in cell suspensions and roots of A. belladonna. Key words: Atropa, arginine, ornithine, roots, suspension culture  相似文献   

2.
Summary The objective of these experiments was to examine short- and long-term (7 d) effects of arginine-deficient diets on free amino acid concentrations in hindlimb muscle of rats. In rats fed the control diet containing arginine (+Arg), muscle alanine and methionine concentrations were higher 1 and 2h after feeding compared to food-deprived rats, whereas branched-chain amino acids, arginine and asparagine concentrations were lower postprandially. In Experiment 1, rats were fed an arginine-deficient (–Arg) diet with glutamate (+Glu) substituted for arginine; alanine (+Ala), ornithine (+Orn) or citrulline (+Cit) were substituted for arginine in Experiment 2. In Experiment 1, arginine concentrations decreased in blood but not in muscle. This contrasts with rats fed –Arg/+Ala or –Arg/+Orn diets which had muscle arginine concentrations less than half the concentrations in controls or in rats fed the –Arg/+Cit diet. Muscle essential amino acids in Experiment 2 did not differ by diet, but muscle branched-chain amino acids were elevated relative to controls in the rats fed –Arg/+Ala or –Arg/+Orn diets; however, rats fed the –Arg/+Cit diet had levels similar to the controls. Also, muscle branched-chain amino acids were correlated with glutamine concentrations in both blood and muscle. The measurements in the post-meal period suggest that muscle amino acid concentrations may more closely reflect dietary amino acid patterns than do blood amino concentrations.Abbreviations BCAA branched-chain amino acids - BCKADH branched-chain ketoacid dehydrogenase - EAA essential amino acids - LNAA large neutral amino acids - NEAA nonessential amino acids - PDV portal-drained viscera - SELSM standard error of least squares means - SSA 5-sulfosalicylic acid - TAA total amino acids Mention of a trade name, proprietary product or specific equipment does not constitute a guarantee by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.  相似文献   

3.
Gabaculine, a potent suicide inhibitor of ornithine aminotransferase (OAT), at a dose of 50 mg/kg inhibited this enzyme in mouse tissues and dramatically increased tissue ornithine concentrations, whether or not arginine was present in the diet. Thus even under arginine deprivation there is catabolism of ornithine which involves OAT. This was confirmed by administration of [14C]ornithine to arginine-deprived mice. Gabaculine (3-amino-2,3-dihydrobenzoic acid) drastically decreased the release of 14CO2 and increased the radioactivity in the basic amino acids in the tissues. When [1-14C]glutamate was injected into mice deprived of arginine, a significant amount of radioactivity was recovered in tissue ornithine and arginine, and gabaculine decreased this labelling by about two-thirds, indicating that ornithine was synthesized in vivo from glutamate via OAT. In addition, we failed to detect in liver and small intestine alpha-N-acetylornithine, N-acetylglutamate kinase or N-acetylornithine aminotransferase, which are obligatory components of a potential route of ornithine synthesis from N-acetylglutamate. Our results indicate that at least 45 mumol of ornithine was synthesized and catabolized daily via OAT in the mouse deprived of arginine.  相似文献   

4.
The metabolism of polyamines (putrescine, spermidine, and spermine) has become the target of genetic manipulation because of their significance in plant development and possibly stress tolerance. We studied the polyamine metabolism in non-transgenic (NT) and transgenic cells of poplar (Populus nigra x maximowiczii) expressing a mouse Orn decarboxylase (odc) cDNA. The transgenic cells showed elevated levels of mouse ODC enzyme activity, severalfold higher amounts of putrescine, a small increase in spermidine, and a small reduction in spermine as compared with NT cells. The conversion of labeled ornithine (Orn) into putrescine was significantly higher in the transgenic than the NT cells. Whereas exogenously supplied Orn caused an increase in cellular putrescine in both cell lines, arginine at high concentrations was inhibitory to putrescine accumulation. The addition of urea and glutamine had no effect on polyamines in either of the cell lines. Inhibition of glutamine synthetase by methionine sulfoximine led to a substantial reduction in putrescine and spermidine in both cell lines. The results show that: (a) Transgenic expression of a heterologous odc gene can be used to modulate putrescine metabolism in plant cells, (b) accumulation of putrescine in high amounts does not affect the native arginine decarboxylase activity, (c) Orn biosynthesis occurs primarily from glutamine/glutamate and not from catabolic breakdown of arginine, (d) Orn biosynthesis may become a limiting factor for putrescine production in the odc transgenic cells, and (e) assimilation of nitrogen into glutamine keeps pace with an increased demand for its use for putrescine production.  相似文献   

5.
Polyamine levels of some helminth parasites were analyzed by reverse phase HPLC of benzoyl derivatives. Setaria cervi, Acanthocheilonema viteae, Hymenolepis nana, H. diminuta, and Ascaridia galli contained higher levels of spermine than spermidine while in Ancylostoma ceylanicum and Nippostrongylus brasiliensis the spermidine levels were higher than spermine; putrescine was either absent or present in minor quantities. The enzymes of polyamine biosynthesis viz., ornithine decarboxylase, S-adenosyl methionine (SAM)-decarboxylase, and arginine decarboxylase were present in very low to negligible amounts in all the parasites examined. A. ceylanicum exhibited high activity of ornithine amino transferase (OAT) and catalyzed appreciable decarboxylation of ornithine. The ornithine decarboxylating activity of A. ceylanicum was localized in the particulate fraction containing mitochondria, not inhibited by alpha-difluoromethyl ornithine, the specific inhibitor of ornithine decarboxylase (ODC), but inhibited in the presence of glutamate, suggesting the involvement of mitochondrial OAT rather than a true ODC in ornithine decarboxylation in this parasite. Significant activity of polyamine oxidase was also detected in helminth parasites. The absence of polyamine biosynthesizing enzymes in helminth parasites suggests their dependence on hosts for uptake and interconversion of polyamines, providing a potential target for chemotherapy.  相似文献   

6.
Ornithine aminotransferase (OAT) is a crucial enzyme in the synthesis of citrulline and arginine from glutamine/glutamate and proline by enterocytes of the small intestine. However, a role for OAT in intestinal polyamine synthesis and cell growth is not known. All-transretinoic acid (RA), an active metabolite of vitamin A, regulates the activity of several metabolic enzymes related to OAT, including ornithine decarboxylase and arginase, which may influence the function of OAT through effects on substrate (ornithine) availability. The objective of the present study was to test the hypothesis that RA regulates OAT mRNA expression and enzymatic activity in intestinal epithelial cells. Caco-2 cells were cultured for 12-72 h in the presence of 0, 0.01 and 1 microM RA and then used for measurements of OAT mRNA levels and enzyme activity as well as ornithine and polyamines. Treatment with RA induced increases in OAT gene expression and enzymatic activity, which resulted in decreased intracellular concentrations of ornithine and polyamines (putrescine, spermidine and spermine) in a dose-dependent manner. These changes occurred concomitantly with a decrease in the total number of cells, and the increase in OAT activity was due to increased OAT mRNA expression. In cells treated with 1 microM RA, addition of 10 microM putrescine to culture medium restored both cellular levels of polyamines and cell numbers to the values for the control group (without addition of RA). We conclude that exposure of Caco-2 cells to RA induces OAT expression for increasing ornithine catabolism. This leads to a reduced availability of intracellular ornithine for polyamine synthesis, thereby decreasing cell proliferation. These novel findings indicate a functional role for OAT in regulating intestinal polyamine synthesis and growth.  相似文献   

7.
Nitric oxide (NO) regulates numerous processes during endotoxemia and inflammation. However, the sequential changes in whole body (Wb) nitric oxide (NO) production during endotoxemia in vivo remain to be clarified. Male Swiss mice were injected intraperitoneally with saline (control group) or lipopolysaccharide (LPS group). After 0, 2, 4, 6, 9, 12, and 24 h, animals received a primed constant infusion of L-[guanidino-(15)N(2)-(2)H(2)]arginine, L-[ureido-(15)N]citrulline, L-[5-(15)N]glutamine, and L-[ring-(2)H(5)]phenylalanine in the jugular vein. Arterial blood was collected for plasma arginine (Arg), citrulline (Cit), glutamine (Gln), and phenylalanine (Phe) concentrations and tracer-to-tracee ratios. NO production was calculated as plasma Arg-to-Cit flux, Wb de novo Arg synthesis as plasma Cit-to-Arg flux, and Wb protein breakdown as plasma Phe flux. LPS reduced plasma Arg and Cit and increased Gln and Phe concentrations. Two peaks of NO production were observed at 4 and 12 h after LPS. Although LPS did not affect total Arg production, de novo Arg production decreased after 12 h. The second peak of NO production coincided with increased Wb Cit, Gln, and Phe production. In conclusion, the curve of NO production in both early and late phases of endotoxemia is not related to plasma Arg kinetics. However, because Wb Cit, Gln, and Phe fluxes increased concomitantly with the second peak of NO production, NO production is probably related to the catabolic phase of endotoxemia.  相似文献   

8.
The synthesis of citrulline from arginine in the small intestine depends on the provision of ornithine. To test the hypothesis that arginase II plays a central role in the supply of ornithine for citrulline synthesis, the contribution of dietary arginine, glutamine, and proline was determined by utilizing multitracer stable isotope protocols in arginase II knockout (AII(-/-)) and wild-type (WT) mice. The lack of arginase II resulted in a lower citrulline rate of appearance (121 vs. 137 μmol·kg(-1)·h(-1)) due to a reduced availability of ornithine; ornithine supplementation was able to restore the rate of citrulline production in AII(-/-) to levels comparable with WT mice. There were significant differences in the utilization of dietary citrulline precursors. The contribution of dietary arginine to the synthesis of citrulline was reduced from 45 to 10 μmol·kg(-1)·h(-1) due to the lack of arginase II. No enteral utilization of arginine was observed in AII(-/-) mice (WT = 25 μmol·kg(-1)·h(-1)), and the contribution of dietary arginine through plasma ornithine was reduced in the transgenic mice (20 vs. 13 μmol·kg(-1)·h(-1)). Dietary glutamine and proline utilization were greater in AII(-/-) than in WT mice (20 vs. 13 and 1.4 vs. 3.7 μmol·kg(-1)·h(-1), respectively). Most of the contribution of glutamine and proline was enteral rather than through plasma ornithine. The arginase isoform present in the small intestinal mucosa has the role of providing ornithine for citrulline synthesis. The lack of arginase II results in a greater contribution of plasma ornithine and dietary glutamine and proline to the synthesis of citrulline.  相似文献   

9.
10.
In this study, concentrations of free amino acids (FAA) and amino group containing compounds (AGCC) following graded diffuse traumatic brain injury (mild TBI, mTBI; severe TBI, sTBI) were evaluated. After 6, 12, 24, 48 and 120 hr aspartate (Asp), glutamate (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), histidine (His), glycine (Gly), threonine (Thr), citrulline (Cit), arginine (Arg), alanine (Ala), taurine (Tau), γ‐aminobutyrate (GABA), tyrosine (Tyr), S‐adenosylhomocysteine (SAH), l ‐cystathionine (l ‐Cystat), valine (Val), methionine (Met), tryptophane (Trp), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), ornithine (Orn), lysine (Lys), plus N‐acetylaspartate (NAA) were determined in whole brain extracts (n = 6 rats at each time for both TBI levels). Sham‐operated animals (n = 6) were used as controls. Results demonstrated that mTBI caused modest, transient changes in NAA, Asp, GABA, Gly, Arg. Following sTBI, animals showed profound, long‐lasting modifications of Glu, Gln, NAA, Asp, GABA, Ser, Gly, Ala, Arg, Citr, Tau, Met, SAH, l ‐Cystat, Tyr and Phe. Increase in Glu and Gln, depletion of NAA and Asp increase, suggested a link between NAA hydrolysis and excitotoxicity after sTBI. Additionally, sTBI rats showed net imbalances of the Glu‐Gln/GABA cycle between neurons and astrocytes, and of the methyl‐cycle (demonstrated by decrease in Met, and increase in SAH and l ‐Cystat), throughout the post‐injury period. Besides evidencing new potential targets for novel pharmacological treatments, these results suggest that the force acting on the brain tissue at the time of the impact is the main determinant of the reactions ignited and involving amino acid metabolism.  相似文献   

11.
In this work new methods for the determination of ornithine (Orn) and l-ornithine:2-oxoacid aminotransferase (OAT) activity are described. These methods were used to demonstrate linear interrelationships between brain GABA and Orn concentrations. Brain GABA levels were modulated by administration of vigabatrin (4-aminohex-5-enoic acid), a specific inactivator of GABA-T, which is not an inhibitor of OAT. The results suggest feed-back inhibition of OAT by GABA, a mechanism which is compatible with the assumption that Orn may serve in certain neurons as a precursor of glutamate and GABA.  相似文献   

12.
Zhou X  Wu X  Yin Y  Zhang C  He L 《Amino acids》2012,43(2):813-821
The objective of this study was to evaluate the effect of oral supplementation with a combination of arginine and glutamine on the intestinal mucosa and inflammatory cytokines of lipopolysaccharide (LPS)-induced adult rats. Fifty Sprague-Dawley rats (average weight of 185 ± 15 g) were randomly divided into five groups: control group A (CA) and control group B (CB), both orally supplemented with 0.9% saline; group Arg, supplemented with 300 mg/kg day(-1) arginine; group Gln, supplemented with 300 mg/kg day(-1) glutamine; group AG, supplemented with 150 mg/kg day(-1) arginine and 150 mg/kg day(-1) glutamine. The experiment lasted for 2 weeks. Food intake and body weight were measured during the experiment. At 10.00 h of day 15, animals were injected with 4 mg/kg LPS (group CB, Arg, Gln, and AG) or sterile saline (group CA) after supplementation. Then at 14.00 h, all animals were killed and blood and tissue collected. The results showed that compared with group CB, arginine concentration tended to be increased (P > 0.05) in group Arg and AG, while there was no significant difference in glutamine concentration among the groups challenged with LPS. Oral supplementation with arginine or/and glutamine mitigated morphology impairment (lower villus height, P < 0.05) in the jejunum and ileum induced by LPS challenge. LPS administration resulted in a significant increase in TNF-α, IL-1β, IL-6 and IL-10 mRNA abundance. Arginine only significantly decreased TNF-α mRNA abundance in the ileum, while glutamine significantly decreased both TNF-α and IL-10 mRNA in the ileum. A combination of arginine and glutamine significantly decreased TNF-α and IL-1β mRNA abundance in both the jejunum and ileum, while they also significantly decreased anti-inflammatory IL-10 in the ileum. These results revealed that an oral supply of combined arginine and glutamine had more favorable effects on the intestinal mucosa and inflammatory cytokines than a supply of arginine or glutamine alone.  相似文献   

13.
Sepsis is a severe catabolic condition. The loss of skeletal muscle protein mass is characterized by enhanced release of the amino acids glutamine and arginine, which (in)directly affects interorgan arginine and the related nitric oxide (NO) synthesis. To establish whether changes in muscle amino acid and protein kinetics are regulated by NO synthesized by nitric oxide synthase-2 or -3 (NOS2 or NOS3), we studied C57BL6/J wild-type (WT), NOS2-deficient (NOS2-/-), and NOS3-deficient (NOS3-/-) mice under control (unstimulated) and lipopolysaccharide (LPS)-treated conditions. Muscle amino acid metabolism was studied across the hindquarter by infusing the stable isotopes L-[ring-2H5]phenylalanine, L-[ring-2H2]tyrosine, L-[guanidino-15N2]arginine, and L-[ureido-13C,2H2]citrulline. Muscle blood flow was measured using radioactive p-aminohippuric acid dilution. Under baseline conditions, muscle blood flow was halved in NOS2-/- mice (P < 0.1), with simultaneous reductions in muscle glutamine, glycine, alanine, arginine release and glutamic acid, citrulline, valine, and leucine uptake (P < 0.1). After LPS treatment, (net) muscle protein synthesis increased in WT and NOS2-/- mice [LPS vs. control: 13 +/- 3 vs. 8 +/- 1 (SE) nmol.10 g(-1).min(-1) (WT), 18 +/- 5 vs. 7 +/- 2 nmol.10 g(-1).min(-1) (NOS2-/-); P < 0.05 for LPS vs. control]. This response was absent in NOS3-/- mice (LPS vs. control: 11 +/- 4 vs. 10 +/- 2 nmol.10 g(-1).min(-1)). In agreement, the increase in muscle arginine turnover after LPS was also absent in NOS3-/- mice. In conclusion, disruption of the NOS2 gene compromises muscle glutamine release and muscle blood flow in control mice, but had only minor effects after LPS. NOS3 activity is crucial for the increase in muscle arginine and protein turnover during early endotoxemia.  相似文献   

14.
The role of the δ-ornithine amino transferase (OAT) pathway in proline synthesis is still controversial and was assessed in leaves of cashew plants subjected to salinity. The activities of enzymes and the concentrations of metabolites involved in proline synthesis were examined in parallel with the capacity of exogenous ornithine and glutamate to induce proline accumulation. Proline accumulation was best correlated with OAT activity, which increased 4-fold and was paralleled by NADH oxidation coupled to the activities of OAT and Δ1-pyrroline-5-carboxylate reductase (P5CR), demonstrating the potential of proline synthesis via OAT/P5C. Overall, the activities of GS, GOGAT and aminating GDH remained practically unchanged under salinity. The activity of P5CR did not respond to NaCl whereas Δ1-pyrroline-5-carboxylate dehydrogenase was sharply repressed by salinity. We suggest that if the export of P5C from the mitochondria to the cytosol is possible, its subsequent conversion to proline by P5CR may be important. In a time-course experiment, proline accumulation was associated with disturbances in amino acid metabolism as indicated by large increases in the concentrations of ammonia, free amino acids, glutamine, arginine and ornithine. Conversely, glutamate concentrations increased moderately and only within the first 24 h. Exogenous feeding of ornithine as a precursor was very effective in inducing proline accumulation in intact plants and leaf discs, in which proline concentrations were several times higher than glutamate-fed or salt-treated plants. Our data suggest that proline accumulation might be a consequence of salt-induced increase in N recycling, resulting in increased levels of ornithine and other metabolites involved with proline synthesis and OAT activity. Under these metabolic circumstances the OAT pathway might contribute significantly to proline accumulation in salt-stressed cashew leaves.  相似文献   

15.
Ornithine decarboxylase (ODC) catalyzes the first step in the polyamine biosynthetic pathway, a highly regulated pathway in which activity increases during rapid growth. Other enzymes also metabolize ornithine, and in hepatomas, rate of growth correlates with decreased activity of these other enzymes, which thus channels more ornithine to polyamine biosynthesis. Ornithine is produced from arginase cleavage of arginine, which also serves as the precursor for nitric oxide production. To study whether short-term coordination of ornithine and arginine metabolism exists in rat colon, ODC, ornithine aminotransferase (OAT), arginase, ornithine, arginine, and polyamine levels were measured after two stimuli (refeeding and/or deoxycholate exposure) known to synergistically induce ODC activity. Increased ODC activity was accompanied by increased putrescine levels, whereas OAT and arginase activity were reduced by either treatment, accompanied by an increase in both arginine and ornithine levels. These results indicate a rapid reciprocal change in ODC, OAT, and arginase activity in response to refeeding or deoxycholate. The accompanying increases in ornithine and arginine concentration are likely to contribute to increased flux through the polyamine and nitric oxide biosynthetic pathways in vivo.  相似文献   

16.
The alarming increase in infections caused by multiple drug resistant bacteria including methicillin-resistant Staphylococcus aureus has prompted a desperate search for new antimicrobials. Augmenting the discoveries of completely new scaffolds with antimicrobial activity are efforts aimed at modifying existing molecules to optimize activity or reduce toxicity. We report herein the parallel solid-phase synthesis of analogues of the cationic antimicrobial peptide gramicidin S (GS) using amino acid side chain attachment strategy. The ornithine (Orn) residues were replaced by glutamine (Gln) and the aromatic d-phenylalanine (Phe) were replaced by different aromatic d-amino acids. Additional Gln containing GS analogues with all the possible combinations of the hydrophobic amino acids valine and leucine were also synthesized. In this work we also report the antibacterial activity of these analogs against several clinically-important drug-resistant Gram-positive and Gram-negative pathogens.  相似文献   

17.
We previously reported that intracellular proline (Pro) confers tolerance to ethanol on the yeast Saccharomyces cerevisiae. In this study, to improve the ethanol productivity of sake, a traditional Japanese alcoholic beverage, we successfully isolated several Pro-accumulating mutants derived from diploid sake yeast of S. cerevisiae by a conventional mutagenesis. Interestingly, one of them (strain A902-4) produced more than 10-fold greater amounts of ornithine (Orn) and Pro compared to the parent strain (K901). Orn is a non-proteinogenic amino acid and a precursor of both arginine (Arg) and Pro. It has some physiological functions, such as amelioration of negative states such as lassitude and improvement of sleep quality. We also identified a homo-allelic mutation in the ARG5,6 gene encoding the Thr340Ile variant N-acetylglutamate kinase (NAGK) in strain A902-4. The NAGK activity of the Thr340Ile variant was extremely insensitive to feedback inhibition by Arg, leading to intracellular Orn accumulation. This is the first report of the removal of feedback inhibition of NAGK activity in the industrial yeast, leading to high levels of intracellular Orn. Moreover, sake and sake cake brewed with strain A902-4 contained 4–5 times more Orn than those brewed with strain K901. The approach described here could be a practical method for the development of industrial yeast strains with overproduction of Orn.  相似文献   

18.
Restoration of the colonic epithelial barrier is an important response during colitis. L-arginine (L-Arg) is a semiessential amino acid that reduces murine colitis induced by Citrobacter rodentium. Cationic amino acid transporter (CAT) proteins increase L-Arg uptake into cells. L-Arg is utilized to produce nitric oxide (NO), by inducible NO synthase (iNOS), or L-ornithine (L-Orn) by arginase (Arg) enzymes. The latter is followed by generation of polyamines by ornithine decarboxylase (ODC) and L-proline (L-Pro) by ornithine aminotransferase (OAT). We show that L-Arg enhanced epithelial restitution in conditionally immortalized young adult mouse colon (YAMC) cells in a wound repair model, and in isolated mouse colonic epithelial cells (CECs), using a cell migration assay. Restitution was impaired by C. rodentium. Wounding induced CAT2, and inhibition of L-Arg uptake by the competitive inhibitor L-lysine (L-Lys) or by CAT2 shRNA, but not CAT1 shRNA, decreased restitution. Migration was impaired in CECs treated with L-Lys or from CAT2(-/-) mice. Wounding increased Arg1 expression, and inhibition of arginase with S-(2-boronoethyl)-L-cysteine (BEC) or Arg1 shRNA inhibited restitution in YAMC cells; cell migration in CECs was also impaired by BEC. Inhibition of ODC or iNOS did not alter restitution. L-Orn or L-Pro restored restitution in cells treated with BEC or Arg1 shRNA, whereas the polyamine putrescine had no benefit. Wounding increased OAT levels, OAT shRNA inhibited restitution, and L-Pro restored restitution in cells with OAT knockdown. Uptake of L-Arg, and its metabolism by Arg1 to L-Orn and conversion to L-Pro by OAT is essential for colonic epithelial wound repair.  相似文献   

19.
Human prorenin is an inactive zymogen comprising 43 amino acid residues at the amino terminus of human renin. The aim of this work was to determine why prorenin is inactive at neutral pH. Eighteen different mutant prorenins, in which positively charged residues in the propeptide were substituted with either glutamine (Gln) or lysine (Lys) residues by site-directed mutagenesis, were expressed in COS-7 cells and characterized. By replacing each of the three arginine (Arg) residues (Arg10P, Arg15P, and Arg20P) with Gln residues, partially active prorenins were produced, which exhibited significant but not full renin activity without trypsin activation. The effect of double or triple amino acid substitutions on the appearance of active prorenin was cumulative, the activity reaching about 80% in a mutant in which all the three Arg residues were replaced by Gln residues. In contrast, mutant prorenins with Lys residues substituted for the Arg residues were inactive. These results clearly indicate that the positive charges of the three Arg residues are essential for maintenance of the human prorenin in an inactive form.  相似文献   

20.
In sonicates of mouse brain synaptosomes, we demonstrated that gamma-aminobutyric acid (GABA) can be formed when L-ornithine (Orn) through L-glutamic acid (Glu), but not through putrescine (Put). Incubation of these sonicates with [3H]ORN yielded not only [3H]Glu and [3H]L-proline (Pro) but also produced [3H]GABA from the [3H]Glu. Formation of each of these three major amino acids from [3H]Orn was strongly inhibited by the addition of GABA (1-5 mM). The likely enzymatic site of this negative feedback inhibition by GABA appeared to be ornithine delta-aminotransferase (OAT). A radiometric procedure was employed to study the effects of the three amino acids cited above and of others found in the free form in brain on the activity of a 30-fold-purified OAT from rat brain. Enzyme activity was measured in the presence of low concentrations of Orn, such as might occur in vivo. OAT was inhibited by GABA to a considerably greater extent than by Glu, L-glutamine, or Put; no inhibition was found with Pro, glycine, aspartarte, taurine, or beta-alanine. The inhibition of GABA was competitive with Orn. These results clearly show that one of the molecular mechanisms underlying the negative feedback inhibition of synaptosomal GABA biosynthesis from Orn is a competitive inhibition by GABA of the brain OAT activity that is responsible for the formation of L-glutamic-gamma-semialdehyde in equilibrium with L-delta 1-pyrroline-5-carboxylic acid from Orn. Thus, the results suggest that GABA may play an important role in restricting the metabolic flow from Orn to Glu and thence to GABA. It is confirmed that L-canaline (delta-aminooxy-L-alpha-aminobutyric acid) is a potent and specific inhibitor of brain OAT whereas much weaker inhibition was observed with two other carbonyl-trapping agents, aminooxyacetic acid and hydrazine.  相似文献   

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