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1.
Pyroglutamate aminopeptidase II is a highly specific membrane-bound ectopeptidase proposed to inactivate thyrotropin releasing hormone (TRH) in brain extracellular space. Its activity was measured in primary cell cultures of fetal brain in an attempt to define its cellular localization. Enzyme activity was detected in hypothalamic or cortical cell membrane fractions from 4- to 12-day-old cultures. When proliferation of nonneuronal cells was abolished by cytosine arabinoside treatment, pyroglutamate aminopeptidase II specific activity was increased as compared to untreated cultures, the opposite was observed for pyroglutamate amino-peptidase I activity. Treatment of cortical cells with the neurotoxic agent glutamate reduced simultaneously pyroglutamate aminopeptidase II and glutamate decarboxylase activities. Glial cell cultures expressed pyroglutamate aminopeptidase I or glutamate synthase activities but not pyroglutamate aminopeptidase II. The data suggest that pyroglutamate aminopeptidase II is predominantly localized in neuronal cells. This is consistent with a role for pyroglutamate aminopeptidase II in TRH-ergic synaptic transmission.  相似文献   

2.
The effect of protein-modifying reagents on the activity of a purified preparation of a thyroliberin-hydrolysing pyroglutamate aminopeptidase, solubilised from synaptosomal membranes of guinea-pig brain by treatment with papain, was investigated. The results indicated that tyrosine, histidine, arginine, and possibly lysine residues were necessary for expression of catalytic activity and that these tyrosine, histidine, and arginine residues were probably located at the active site of the enzyme. Cysteine, serine, glutamate, and aspartate residues were not involved in the expression of catalytic activity.  相似文献   

3.
A comparison was made of the distribution of amino terminal end groups in the cellular proteins of a number of microbes. Among the procaryotes, methionine is a highly variable but virtually ubiquitous major protein end group. This is consistent with its possible role as a general amino acid initiator of protein biosynthesis in the procaryotes. Generally, however, alanine is the most abundant of the major end groups, followed in decreasing order by serine, threonine, the acidic amino acids, and occasionally lysine. No other new major end-groups were found. Among 15 representatives of the Enterobacteriaceae, retention of the initiating methionine terminus of the cellular protein varies considerably at a tribal level and is randomized at a familial level. The profiles of the five remaining end groups, however, are strikingly uniform, and are, for example, close to but significantly different from those of the Erwineae. Among the taxonomically more heterogeneous Bacillaceae, end-group profiles vary more and are sometimes unrelated. End-group analysis is thus particularly useful as a molecular criterion of taxonomy in assessing familial homogeneity. Free NH(2) termini in eucaryote cell proteins are fewer, and they have increased acidic amino acid components and no methionine; they are otherwise similar to those of the procaryotes.  相似文献   

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Kohorn BD  Tobin EM 《Plant physiology》1986,82(4):1172-1174
We have previously isolated and sequenced two genes encoding light-harvesting chlorophyll a/b-proteins (LHCP) from Lemna gibba. One of these, AB30, encodes a protein that is highly homologous to LHCP sequences reported from other species, but the second, AB19, encodes a protein that has a transit peptide and first 12 amino-terminal residues of the mature protein that are substantially different. Despite these differences, we can demonstrate that AB19 encoded protein synthesized in vitro can be imported into isolated chloroplasts, and we provide evidence that at least some of the imported molecules are assembled into the light-harvesting complex of photosystem II. Thus, our results are consistent with the possibility that there are two functional forms of LHCP.  相似文献   

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Synthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacterium Escherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-l-tyrosine incorporation system in E. coli. A translational switch was turned on after addition of 3-iodo-l-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants.  相似文献   

8.
Amino acid residues in the metal-binding and putative substrate-binding sites of Escherichia coli methionine aminopeptidase (MAP) were mutated, and their effects on the function of the enzyme were investigated. Substitution of any amino acid residue at the metal-binding site resulted in complete loss of the two cobalt ions bound to the protein and diminished the enzyme activity. However, only Cys70 and Trp221 at the putative substrate-binding site are involved in the catalytic activity of MAP. Changing either of them caused partial loss of enzyme activity, while mutations at both positions abolished MAP function. Both residues are found to be conserved in type I but not type II MAPs.  相似文献   

9.
The functional significance of amino acid residues Lys-265, Asp-270, Lys-277, Asp-288, Asp-347, Glu-349, and Arg-351 of Bacillus kaustophilus leucine aminopeptidase was explored by site-directed mutagenesis. Variants with an apparent molecular mass of approximately 54 kDa were overexpressed in Escherichia coli and purified to homogeneity by nickel-chelate chromatography. The purified mutant enzymes had no LAP activity, implying that these residues are important for the catalytic reaction of the enzyme.  相似文献   

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Russian Journal of Bioorganic Chemistry - Recombinant major allergens Phl p 1 from meadow timothy (Phleum pratense) and Amb a 1 from ragweed (Ambrosia artemisiifolia) were obtained in E. coli...  相似文献   

12.
In the prion diseases, a prolonged, asymptomatic incubation period precedes the onset of neurologic dysfunction. At present, a noninvasive test is not available for the presymptomatic diagnosis of prion disease, and thus the report of a test for prions using urine has been of great interest (Shaked, G. M., Shaked, Y., Kariv-Inbal, Z., Halimi, M., Avraham, I., and Gabizon, R. (2001) J. Biol. Chem. 276, 31479-31482). Using Western immunoblots with the anti-prion protein (PrP) 3F4 monoclonal antibody and an anti-mouse IgG secondary antibody, a protease-resistant PrP was reported in the urine of Syrian hamsters and humans with prion disease. Here we have demonstrated that this purportedly "protease-resistant PrP" band in the urine of diseased hamsters is detectable using the anti-mouse IgG secondary antibody in the absence of the 3F4 monoclonal antibody. Mass spectrometric analysis identified an immunoglobulin light chain in the band but found no PrP peptides. No similar band was found in the urine of uninfected hamsters or in brain homogenates from normal or prion-infected hamsters. Moreover, the band in the urine of infected hamsters was not detected using two chimeric human-mouse recombinant anti-PrP antibody fragments followed by an anti-human IgG secondary antibody. Our results indicate that the band detected under previously published conditions is due to the cross-reactivity of the anti-mouse IgG antibody with IgG light chains and possibly heavy chain fragments in urine, but not with PrP.  相似文献   

13.
Using a commercial protein expression system, we sought the crucial elements and conditions for the expression of proteins with genetically encoded unnatural amino acids. By identifying the most important translational components, we were able to increase suppression efficiency to 55% and to increase mutant protein yields to levels higher than achieved with wild type expression (120%), reaching over 500 µg/mL of translated protein (comprising 25 µg in 50 µL of reaction mixture). To our knowledge, these results are the highest obtained for both in vivo and in vitro systems. We also demonstrated that efficiency of nonsense suppression depends greatly on the nucleotide following the stop codon. Insights gained in this thorough analysis could prove useful for augmenting in vivo expression levels as well.  相似文献   

14.
一株高产纤维素酶真菌的选育与鉴定   总被引:1,自引:0,他引:1  
采用微生物常规分离筛选方法,以蘑菇培养基质、牛瘤胃内容物、落叶堆和腐木等为材料,从中分离筛选得到一株高产纤维素酶的真菌Z90;进一步对该菌株进行紫外诱变育种,获得产酶能力更强且遗传稳定的突变菌株Z90-19,突变菌株Z90-19产CMCase和FPA的活性分别达到11.61±0.17U·mL-1和2.15±0.14U·mL-1;对该菌株进行鉴定,确定菌株Z90为核茎点霉(Phoma putaminum)。  相似文献   

15.
Abstract: Molecular cloning has revealed that there are six classes of subunits capable of forming GABA-gated chloride channel receptors. GABAA receptors are composed of α, β, γ, δ, and ε/χ subunits, whereas GABAC receptors appear to contain ρ subunits. However, retinal cells exhibiting GABAC responses express α, β, and ρ subunits, raising the possibility that GABAC receptors may be a mixture of subunit classes. Using in vitro translated protein, we determined that human GABAA receptor subunits α1, α5, and β1 did not coimmunoprecipitate with full-length ρ1, ρ2, or the N-terminal domain of ρ1 that contains signals for ρ-subunit interaction. To explore the molecular mechanism underlying these apparently exclusive combinations, chimeric subunits were created and tested for interaction with the wild-type subunits. Transfer of the N terminus of β1 to ρ1 created a β1ρ1 chimera that coimmunoprecipitated with the α1 subunit but not with the ρ2 subunit. Furthermore, exchanging the N terminus of the ρ1 subunit with the corresponding region of β1 produced a ρ1β1 chimera that interfered with ρ1 receptor expression in Xenopus oocytes, whereas the full-length β1 subunit had no effect. Together, these results indicate that sequences in the N termini direct assembly of ρ subunits and GABAA subunits into GABAC and GABAA receptors, respectively.  相似文献   

16.
After modification with monomethoxyl-poly(ethylene glycol)-5000, a recombinant intracellular uricase from Bacillus fastidiosus ATCC 29604 showed residual activity of about 65%, a thermo-inactivation half-life >85 h, a circulating half-life about 20 h in rats in vivo, consistent effects of common cations, and consistent optima for reaction temperature and pH. Thus, this uricase can be formulated via modification with monomethoxyl-poly(ethylene glycol).  相似文献   

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对表达双功能谷胱甘肽合成酶的重组大肠杆菌发酵生产谷胱甘肽(Glutathione,GSH)进行氨基酸添加策略优化,结果表明:基本培养基中未添加氨基酸时GSH产量为0.81 g/L;诱导2 h后添加17 mmol/L半胱氨酸GSH产量为1.16 g/L,比不加氨基酸提高43%;添加17 mmol/L的3种前体氨基酸,GSH产量达到3.86 g/L,比只添加半胱氨酸提高2.33倍;进一步提高3种氨基酸添加量至25 mmol/L,GSH产量可达4.64 g/L,比不添加氨基酸提高4.73倍,总生产强度高达317.8 mg/(L·h),半胱氨酸转化为谷胱甘肽达到0.60 mol/mol;考察氨基酸添加模式发现一次性添加25 mmol/L氨基酸较恒速流加模式生产速率提高了29.8%。后续在50 L罐放大生产GSH,产量为4.31 g/L,总生产强度达到310.1 mg/(L·h),为工业化放大生产GSH奠定了基础。  相似文献   

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Membrane-bound aminopeptidase activities in livers of rats with experimental renal failure were assayed. Only aminopeptidase A activity was decreased with the reduction in renal function, but aminopeptidase B and Leu-aminopeptidase activity did not change. The liver membrane-bound aminopeptidase A activity was inhibited by the addition of angiotensin I or -II in the enzyme assay system. From these results, it is expected that a decrease in liver membrane-bound aminopeptidase A activity may play a role in increasing angiotensin II during renal failure.  相似文献   

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