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1.
Streptococcus bovis JB1 cells energized with glucose transported glutamine at a rate of 7 nmol/mg of protein per min at a pH of 5.0 to 7.5; sodium had little effect on the transport rate. Because valinomycin-treated cells loaded with K and diluted into Na (pH 6.5) to create an artificial delta psi took up little glutamine, it appeared that transport was driven by phosphate-bond energy rather than proton motive force. The kinetics of glutamine transport by glucose-energized cells were biphasic, and it appeared that facilitated diffusion was also involved, particularly at high glutamine concentrations. Glucose-depleted cultures took up glutamine and produced ammonia, but the rate of transport per unit of glutamine (V/S) by nonenergized cells was at least 1,000-fold less than the V/S by glucose-energized cells. Glutamine was converted to pyroglutamate and ammonia by a pathway that did not involve a glutaminase reaction or glutamate production. No ammonia production from pyroglutamate was detected. S. bovis was unable to take up glutamate, but intracellular glutamate concentrations were as high as 7 mM. Glutamate was produced from ammonia via a glutamate dehydrogenase reaction. Cells contained high concentrations of 2-oxoglutarate and NADPH that inhibited glutamate deamination and favored glutamate formation. Since the carbon skeleton of glutamine was lost as pyroglutamate, glutamate formation occurred at the expense of glucose. Arginine deamination is often used as a taxonomic tool in classifying streptococci, and it had generally been assumed that other amino acids could not be fermented. To our knowledge, this is the first report of glutamine conversion to pyroglutamate and ammonia in streptococci.  相似文献   

2.
Formation of N-terminal pyroglutamate (pGlu or pE) from glutaminyl or glutamyl precursors is catalyzed by glutaminyl cyclases (QC). As the formation of pGlu-amyloid has been linked with Alzheimer's disease, inhibitors of QCs are currently the subject of intense development. Here, we report three crystal structures of N-glycosylated mammalian QC from humans (hQC) and mice (mQC). Whereas the overall structures of the enzymes are similar to those reported previously, two surface loops in the neighborhood of the active center exhibit conformational variability. Furthermore, two conserved cysteine residues form a disulfide bond at the base of the active center that was not present in previous reports of hQC structure. Site-directed mutagenesis suggests a structure-stabilizing role of the disulfide bond. At the entrance to the active center, the conserved tryptophan residue, W(207), which displayed multiple orientations in previous structure, shows a single conformation in both glycosylated human and murine QCs. Although mutagenesis of W(207) into leucine or glutamine altered substrate conversion significantly, the binding constants of inhibitors such as the highly potent PQ50 (PBD150) were minimally affected. The crystal structure of PQ50 bound to the active center of murine QC reveals principal binding determinants provided by the catalytic zinc ion and a hydrophobic funnel. This study presents a first comparison of two mammalian QCs containing typical, conserved post-translational modifications.  相似文献   

3.
4.
The mechanism for the post-translational conversion of glutamine to pyroglutamic acid on the N terminus of newly synthesized peptides and proteins is unknown. An assay is reported that permits measurement of the rate of conversion of Gln-His-Pro-NH2 to pyroGlu-His-Pro-NH2 (TRH). Using this assay, we demonstrate that the spontaneous cyclization of the N-terminal glutamine of this peptide occurs only slowly under physiological conditions. Furthermore, we describe the presence in rat brain, porcine pituitary, and human B lymphocytes of an enzyme(s) which converts Gln-His-Pro-NH2 into pyroGlu-His-Pro-NH2. The enzyme(s) appears to be a glycoprotein, is maximally active at neutral pH, has a Mr of 55,000, and contains catalytically significant sulfhydryl groups. The product of the enzymatic reaction was confirmed by high resolution fast atom bombardment-mass spectrometry. In preliminary studies, we find that over 90% of the enzyme in bovine adrenal medulla is contained in the soluble chromaffin vesicle fraction. These findings indicate that in vivo the post-translational conversion of a glutaminyl-peptide into a pyroglutamyl-peptide is neither spontaneous nor abiotic as has been previously proposed.  相似文献   

5.
The biosynthetic activities of the polypeptide subunits alpha and beta of glutamine synthetase (GS) were inhibited in vitro by glycine and serine. These amino acids inhibited the growth of a mutant strain with partial GS activity when grown on glutamate as the nitrogen source and also blocked the synthesis of the glutamine in vivo, thus demonstrating the inhibitory effect on GS activity in vivo. Glycine and serine lowered the intracellular glutamine pool and regulated GS beta synthesis. A preferential induction of synthesis of the GS beta polypeptide was observed when either of these amino acids was present in the medium. On this basis, we obtained a glycine-sensitive mutant which showed a structural alteration of the GS beta polypeptide. The double regulatory effect of either glycine or serine on glutamine synthesis may be considered an example of the regulation of glutamine synthesis by alpha-amino nitrogen. It may be a mechanism that regulates the assimilation of ammonium into glutamate versus glutamine.  相似文献   

6.
7.
Ammonia assimilation in Bacillus polymyxa. 15N NMR and enzymatic studies   总被引:4,自引:0,他引:4  
Pathways of ammonia assimilation into glutamic acid and alanine in Bacillus polymyxa were investigated by 15N NMR spectroscopy in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthetase, alanine dehydrogenase, and glutamic-alanine transaminase. Ammonia was found to be assimilated into glutamic acid predominantly by NADPH-dependent glutamate dehydrogenase with a Km of 2.9 mM for NH4+ not only in ammonia-grown cells but also in nitrate-grown and nitrogen-fixing cells in which the intracellular NH4+ concentrations were 11.2, 1.04, and 1.5 mM, respectively. In ammonia-grown cells, the specific activity of alanine dehydrogenase was higher than that of glutamic-alanine transaminase, but the glutamate dehydrogenase/glutamic-alanine transaminase pathway was found to be the major pathway of 15NH4+ assimilation into [15N]alanine. The in vitro specific activities of glutamate dehydrogenase and glutamine synthetase, which represent the rates of synthesis of glutamic acid and glutamine, respectively, in the presence of enzyme-saturating concentrations of substrates and coenzymes are compared with the in vivo rates of biosynthesis of [15N]glutamic acid and [alpha,gamma-15N]glutamine observed by NMR, and implications of the results for factors limiting the rates of their biosynthesis in ammonia- and nitrate-grown cells are discussed.  相似文献   

8.
Physiology of ammonium assimilation in Neurospora crassa.   总被引:12,自引:6,他引:6       下载免费PDF全文
In Neurospora crassa the assimilation of high and low concentrations of ammonium occurs by two different pathways. When the fungi are growing exponentially on ammonium excess, this compound is fixed by a glutamic dehydrogenase and an octameric glutamine synthetase (GS). The synthesis of this GS polypeptide (beta) is regulated by the nitrogen source present in excess; being higher on glutamate, intermediate on ammonium, and lower on glutamine. When N. crassa is growing in fed-batch ammonium-limited cultures a different polypeptide of GS (alpha), arranged as a tetramer, is synthesized. In both conditions synthesis in vivo correlates with the data obtained with an in vitro translation system primed with N. crassa RNA. This different expression of alpha and beta GS polypeptides was also observed when the cultures were shifted from excess to low nitrogen, and vice versa. By agarose gel electrophoresis in the presence of methylmercury hydroxide, some separation of different mRNAs that direct the in vitro synthesis of alpha and beta GS polypeptides has been accomplished. Data are presented that establish the operation of the tetrameric alpha GS and of glutamate synthase in the assimilation of ammonium in low concentration.  相似文献   

9.
Limited proteolysis of glutamine synthetase from Escherichia coli has been studied under nondenaturing conditions (pH 7.6, 20 degrees C). Trypsin cleaves the polypeptide chain of glutamine synthetase into two principal fragments, Mr = about 32,000 and 18,000. The covalently bound AMP group is attached to the larger fragment and its presence does not affect cleavage. Although the cleaved polypeptide chain does not dissociate under nondenaturing conditions, catalytic activity is lost. Chymotrypsin and Staphylococcus aureus protease produce similar cleavages in glutamine synthetase. The substrate L-glutamate retards tryptic as well as chymotryptic digestion. Tryptic digestion is also retarded by some of the feedback inhibitors of glutamine synthetase including CTP, L-alanine, L-serine, L-histidine, and glucosamine 6-phosphate. An implication of these findings is that there is a region of the glutamine synthetase polypeptide chain that is particularly susceptible to proteolysis. Either the glutamate and inhibitor sites are formed partly by this suceptible peptide or the binding of glutamate and some inhibitors induces conformational changes within the E. coli glutamine synthetase molecule in the region of the susceptible peptide.  相似文献   

10.
Transformation of glutamine of mixed population of microorganisms-symbionts is demonstrated. Protection of glutamine and its intermediates (glutamate and pyroglutamate) under the influence of ionophore (monensin) is found. It is accompanied by the reduced (by 30-70 per cent and more) levels of ammonia formation, total VFA, acetate, butyrate, pH, alpha-ketoglutarate dehydrogenase and glutamine synthetase at parallel increase of L-aspartate and L-alanine: 2-oxoglutarate, aminotransferase activity, redox potential of stability of protein content. It is suggested that the rapid change of Eh level on the 24th hour of incubation reflects the main stage of pyroglutamate formation being one of the final products of glutamine degradation when monensin is used. Ionophore affects first of all the inhibition of metabolic processes in gram-positive rumen microorganisms.  相似文献   

11.
The IgG1 has N-terminal glutamic acid (E) and glutamine (Q). Conversion of E to pyroglutamate (pE) results in no charge change while that of glutamine (Q) to pE results in a loss of amine group. C-terminal lysine is often cleaved off in the bioreactor culture. The terminal heterogeneities can be detected by many analytical methods. A simple CEX profile was shown.
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12.
An N-terminal truncated isoform of the amyloid beta-peptide (A beta) that begins with a pyroglutamate (pE) residue at position 3 [A beta3(pE)-42] is the predominant isoform found in senile plaques. Based upon previous in vitro studies regarding A beta N-terminal truncated isoforms, it has been hypothesized that A beta3(pE)-x isoforms may aggregate more rapidly and become more toxic than corresponding Abeta1-x peptides. However, the toxicity and aggregation properties of A beta3(pE)-42 and A beta3(pE)-40 have not previously been examined. After initial solubilization and 1-week preaggregation of each peptide at 37 degrees C and pH 7.4, the toxicity of 5-50 microM A beta3(pE)-42 was similar to that of A beta1-42. Moreover, the toxicity of A beta3(pE)-40 paralleled that induced by A beta1-40 in both 1 day in vitro (DIV) cortical and 7 DIV hippocampal cells. Circular dichroism spectra did not reveal major differences in secondary structure between aged A beta1-42, A beta3(pE)-42, A beta3(pE)-40, and A beta1-40 or freshly solubilized forms of these peptides. Overall, the data indicate that the loss of the two N-terminal amino acids and the cyclization of glutamate at position 3 do not alter the extracellular toxicity of A beta.  相似文献   

13.
The photosynthetic bacterium Rhodospirillum rubrum regulates the activity of its nitrogenase (N2ase) by interconverting the enzyme into three distinct enzymatic species: N2ase A (a fully active form) and two regulatory forms, N2ase Ractive and N2ase Rinactive. N2ase R is distinguished from N2ase A in vitro by the requirement of its Fe protein for activation by a Mn2+-dependent activating factor. N2ase is converted from the A to the R form in response to certain environmental factors such as carbon starvation, depletion of intracellular adenosine triphosphate, or the addition of NH4+ (or glutamate) to a culture of N-starved cells. The rapid inhibition of R. rubrum N2ase in vivo by NH4+ was shown to result from the conversion of N2ase A to N2ase Rinactive. On depletion of NH4+ from the culture, whole-cell N2ase activity returned; however, the enzyme remained in the R form. Unlike the effect of NH4+, adding glutamate to cells containing N2ase A did not inhibit in vivo activity, but converted the enzyme to the R form (N2ase Ractive). Although glutamate-induced N2ase R formation was much slower than the NH4+-induced reaction, it occurred in the presence of rifampin, indicating that de novo protein synthesis was not involved. This suggested that N2ase R was formed by a modification of N2ase A. Although glutamine synthetase in involved in the conversion of N2ase A to R, the adenylylation state of glutamine synthetase appears not to be involved in regulating this nitrogenase reaction.  相似文献   

14.
During either production or storage, the LC214-HC220 disulfide in therapeutic antibodies can convert to a thioether bond. Here we report that a thioether forms at the same position on antibodies in vivo. An IgG1κ therapeutic antibody dosed in humans formed a thioether at this position at a rate of about 0.1%/day while circulating in blood. Thioether modifications were also found at this position in endogenous antibodies isolated from healthy human subjects, at levels consistent with this conversion rate. For both endogenous antibodies and recombinant antibodies studied in vivo, thioether conversion rates were faster for IgG1 antibodies containing λ light chains than those containing κ light chains. These light chain reaction rate differences were replicated in vitro. Additional mechanistic studies showed that base-catalyzed thioether formation through the light chain dehydrogenation was more preferred on antibodies with λ light chains, which may help explain the observed reaction rate differences.  相似文献   

15.
Acute hyperammonemia was induced by 15NH4+ infusion in portacaval-shunted (PCS) and control rats to investigate its effects on cerebral metabolism of glutamine, glutamate and gamma-aminobutyrate. Cerebral 15N-metabolites were observed by 15N-NMR spectroscopy in the ex vivo brain, removed in toto at the end of infusion. Key 15N-metabolites in the brain and liver were quantitated and their specific activities measured by NMR and biochemical assays in perchloric acid extracts of the freeze-clamped organs. In the ex vivo brain, [gamma-15N]glutamine, present at tissue concentrations of 3-5 mumol/g with 15N enrichment of 36-48%, was observable within 6-13 min of data acquisition. [alpha-15N]glutamine/glutamate, each present at 0.5-1 mumol/g (approx. 10% enrichment), were observed in 27 min. The results demonstrate the feasibility of observing these cerebral metabolites by 15N-NMR within a physiological time scale. In a rat pretreated with glutamine synthetase inhibitor, L-methionine DL-sulfoximine, cerebral [15N]gamma-aminobutyrate was observed after 910 min. In PCS rats, decreased 15NH4+ removal in the liver was accompanied by formation of approx. 2-fold higher concentration of cerebral [gamma-15N]glutamine relative to that in weight-matched controls. The result suggests that increased diffusion of blood-borne 15NH3 into the brain led to increased [gamma-15N]glutamine synthesis in astrocytes as well as ammonia-mediated inhibition of glutaminase.  相似文献   

16.
Two DNA vaccines were constructed encoding the ectodomain (domains I, II and III) of the DENV2 envelope protein (pE1D2) or only its domain III (pE2D2), fused to the human tissue plasminogen activator signal peptide (t-PA). The expression and secretion of recombinant proteins was confirmed in vitro in BHK cells transfected with the two plasmids, detected by immunofluorescence or immunoprecipitation of metabolically labeled gene products, using polyclonal and monoclonal antibodies against DENV2. Besides, results reveal that the ectodomain of the E protein can be efficiently expressed in vivo, in a mammalian system, without the prM protein that is hypothesized to act as a chaperonin during dengue infection. Balb/c mice were immunized with the DNA vaccines and challenged with a lethal dose of DENV2. All pE1D2-vaccinated mice survived challenge, while 45% of animals immunized with the pE2D2 died after infection. Furthermore, only 10% of pE1D2-immunized mice presented some clinical signs of infection after challenge, whereas most of animals inoculated with the pE2D2 showed effects of the disease with high morbidity degrees. Levels of neutralizing antibodies were significantly higher in pE1D2-vaccinated mice than in pE2D2-immunized animals, also suggesting that the pE1D2 vaccine was more protective than the pE2D2.  相似文献   

17.
In the present review, we summarize the current achievements of modelling early intraneuronal Aβ (amyloid β-peptide) accumulation in transgenic mice with the resulting pathological consequences. Of special importance will be to discuss recent developments and the translation of the results to AD (Alzheimer's disease). N-terminally truncated AβpE3 (Aβ starting with pyroglutamate at position 3) represents a major fraction of all Aβ peptides in the brain of AD patients. Recently, we generated a novel mAb (monoclonal antibody), 9D5, that selectively recognizes oligomeric assemblies of AβpE3 and demonstrated the potential involvement of oligomeric AβpE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost non-detectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal staining was observed. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall Aβ levels and stabilized behavioural deficits. In summary, we have demonstrated that intraneuronal Aβ is a valid risk factor in model systems and AD patients. This feature of AD pathology was successful in identifying novel low-molecular-mass oligomeric Aβ-specific antibodies for diagnosis and therapy.  相似文献   

18.
The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2-15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mM KCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na(+)-amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine-induced increase in intrasynaptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.  相似文献   

19.
We studied the effects of sodium valproate, a widely used antiepileptic drug and a hyperammonemic agent, on L-[1-14C]glutamine and L-[1-14C]glutamate metabolism in isolated human kidney-cortex tubules. Valproate markedly stimulated glutamine removal as well as the formation of ammonia, 14CO2, pyruvate, lactate and alanine, but it inhibited glucose synthesis; the increase in ammonia formation was explained by a stimulation by valproate mainly of flux through glutaminase (EC 3.5.1.2) and to a much lesser extent of flux through glutamate dehydrogenase (EC 1.4.1.3). By contrast, valproate did not stimulate glutamate removal or ammonia formation, suggesting that the increase in flux through glutamate dehydrogenase observed with glutamine as substrate was secondary to the increase in flux through glutaminase. Accumulation of pyruvate, alanine and lactate in the presence of valproate was less from glutamate than from glutamine. Inhibition by aminooxyacetate of accumulation of alanine from glutamine caused by valproate did not prevent the acceleration of glutamine utilization and the subsequent stimulation of ammonia formation. It is concluded from these data, which are the first concerning the in vitro metabolism of glutamine and glutamate in human kidney-cortex tubules, that the stimulatory effect of valproate is primarily exerted at the level of glutaminase in human renal cortex.  相似文献   

20.
To test the significance of the purine nucleotide cycle in renal ammoniagenesis, studies were conducted with rat kidney cortical slices using glutamate or glutamine labelled in the alpha-amino group with 15N. Glucose production by normal kidney slices with 2 mM-glutamine was equal to that with 3 mM-glutamate. With L-[15N]glutamate as sole substrate, one-third of the total ammonia produced by kidney slices was labelled, indicating significant deamination of glutamate or other amino acids from the cellular pool. Ammonia produced from the amino group of L-[alpha-15N]glutamine was 4-fold higher than from glutamate at similar glucose production rates. Glucose and ammonia formation from glutamine by kidney slices obtained from rats with chronic metabolic acidosis was found to be 70% higher than by normal kidney slices. The contribution of the amino group of glutamine to total ammonia production was similar in both types of kidneys. No 15N was found in the amino group of adenine nucleotides after incubation of kidney slices from normal or chronically acidotic rats with labelled glutamine. Addition of Pi, a strong inhibitor of AMP deaminase, had no effect on ammonia formation from glutamine. Likewise, fructose, which may induce a decrease in endogenous Pi, had no effect on ammonia formation. The data obtained suggest that the contribution of the purine nucleotide cycle to ammonia formation from glutamine in rat renal tissue is insignificant.  相似文献   

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