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1.
The medicinal plants from the genus Coptis were widely applied in clinical treatment, but no stable and systemic methods have been established for species identification and quality evaluation. Although several analytical methods have been reported for the detection of alkaloids, so far no attempt has been made to analyze the interspecific relationships between molecular phylogeny and main alkaloids in Coptis. In this study, the phylogenetic relationships within the genus Coptis from China are resolved with high support. Coptis chinensis, Coptis deltoidea and Coptis omeiensis cluster together, and this clade (‘clade III’) is sister to Coptis teeta. Here, we provide evidences that the relationships inferred by chemical taxonomy are different to those of the present molecular phylogeny. Within clade III, C. deltoidea, C. omeiensis and C. chinensis are sister to C. teeta, while C. teeta shares more similar metabolic compounds with C. deltoidea, and C. omeiensis than with C. chinensis. Most likely, the metabolic components are mainly affected by environmental factors resulting in convergent evolution of alkaloid contents that do not reflect phylogenetic relationships. 相似文献
2.
Dried rhizomes of Coptis species are utilized as “Coptidis Rhizoma” (CR), an important herbal medicinal material in traditional Chinese medicine. Almost all CRs traded in the Korean herbal medicine market originate from Coptis chinensis (“Chun Hwang-Lyun” in Korean medical terminology). Other minor CRs originate from Coptis japonica (“Il Hwang-Lyun”). Although there is an obvious discrepancy in the price of traded CRs in the herbal market depending on the Coptis species, CRs originating from C. chinensis and C. japonica are often confused. Furthermore, the CR traded as “Chun Hwang-Lyun” is occasionally mixed with rhizomes of Coptis deltoidea and/or Coptis omeiensis. Therefore, we sought to discriminate C. chinensis from C. japonica, as well as C. deltoidea and C. omeiensis, by using nucleotide sequence differences in the partial trnL-F intergenic spacer. We developed an efficient real-time polymerase chain reaction (PCR)-based discrimination assay to separate samples of C. chinensis from those of C. japonica without the need to separate the DNA markers by using gel electrophoresis. In addition, we developed a multiplex PCR method with which we were able to discriminate samples of C. chinensis from those of C. deltoidea and C. omeiensis by amplifying the 153-bp DNA marker in C. chinensis in a single PCR process. 相似文献
3.
Kun-Li Xiang Sheng-Dan Wu Sheng-Xian Yu Yang Liu Florian Jabbour Andrey S. Erst Liang Zhao Wei Wang Zhi-Duan Chen 《PloS one》2016,11(4)
Coptis (Ranunculaceae) contains 15 species and is one of the pharmaceutically most important plant genera in eastern Asia. Understanding of the evolution of morphological characters and phylogenetic relationships within the genus is very limited. Here, we present the first comprehensive phylogenetic analysis of the genus based on two plastid and one nuclear markers. The phylogeny was reconstructed using Bayesian inference, as well as maximum parsimony and maximum likelihood methods. The Swofford-Olsen-Waddell-Hillis and Bayesian tests were used to assess the strength of the conflicts between traditional taxonomic units and those suggested by the phylogenetic inferences. Evolution of morphological characters was inferred using Bayesian method to identify synapomorphies for the infrageneric lineages. Our data recognize two strongly supported clades within Coptis. The first clade contains subgenus Coptis and section Japonocoptis of subgenus Metacoptis, supported by morphological characters, such as traits of the central leaflet base, petal color, and petal shape. The second clade consists of section Japonocoptis of subgenus Metacoptis. Coptis morii is not united with C. quinquefolia, in contrast with the view that C. morii is a synonym of C. quinquefolia. Two varieties of C. chinensis do not cluster together. Coptis groenlandica and C. lutescens are reduced to C. trifolia and C. japonica, respectively. Central leaflet base, sepal shape, and petal blade carry a strong phylogenetic signal in Coptis, while leaf type, sepal and petal color, and petal shape exhibit relatively higher levels of evolutionary flexibility. 相似文献
4.
中国黄连属6种1变种分属叶掌状三全裂和叶掌状五全裂2个类群,前者包括三角叶黄连、峨眉黄连、云南黄连、黄连和其变种短萼黄连;而分布于滇东南的五裂黄连和产于台湾的五叶黄连则归于后者。采用根尖压片法和卡宝品红染色法,对除五叶黄连外的中国黄连属5种1变种,以及日本黄连进行染色体核型比较分析,从细胞学角度为探讨中国黄连属植物的系统分类提供新的线索和证据。结果表明:(1)五裂黄连(2n=2x=18=2M+16m)、短萼黄连(2n=2x=18=8m+10sm)和日本黄连(2n=2x=18=12m+6sm)的染色体数目和核型均为首次报道。(2)7个材料的染色体基数均为9,除三角叶黄连为三倍体外,其余均为二倍体。(3)叶为掌状三全裂的二倍体种核型一致,为2A型,染色体类型以及不对称系数均很相似;叶为掌状五全裂的五裂黄连与五叶黄连的核型更接近,为1A型,核型特征的共性表明了这2个类群的自然属性。(4)三倍体三角叶黄连的不对称程度较高,核型为3A型,其染色体大小与峨眉黄连最接近。(5)根据核型不对称程度和染色体大小,结合地理分布,推测叶掌状五全裂种为本属的原始类群。 相似文献
5.
The theanine content of the leaves of 27 species or varieties of Theaceae plants was investigated. Theanine was present in 21 species or varieties, but in much lower amounts (<0.2 μmol/g fresh weight) than the quantity detected in Camellia sinensis var. sinensis. The major free amino acids in leaves of four species belonging to the genera Schima and Eurya, were glutamic acid, aspartic acid, glutamine, asparagine, alanine and proline and content of these amino acids is similar to or higher than theanine. Accumulation of free amino acids in these plants was generally lower than in C. sinensis var. sinensis. The biosynthetic activity of theanine, assessed by the incorporation of radioactivity from [14C]ethylamine, was detected in seedlings of two species of Schima. The theanine biosynthetic activity in roots was higher than that of leaves. 相似文献
6.
Adrienne Zaprasis Monika Bleisteiner Anne Kerres Tamara Hoffmann Erhard Bremer 《Applied and environmental microbiology》2015,81(1):250-259
The data presented here reveal a new facet of the physiological adjustment processes through which Bacillus subtilis can derive osmostress protection. We found that the import of proteogenic (Glu, Gln, Asp, Asn, and Arg) and of nonproteogenic (Orn and Cit) amino acids and their metabolic conversion into proline enhances growth under otherwise osmotically unfavorable conditions. Osmoprotection by amino acids depends on the functioning of the ProJ-ProA-ProH enzymes, but different entry points into this biosynthetic route are used by different amino acids to finally yield the compatible solute proline. Glu, Gln, Asp, and Asn are used to replenish the cellular pool of glutamate, the precursor for proline production, whereas Arg, Orn, and Cit are converted into γ-glutamic semialdehyde/Δ1-pyrroline-5-carboxylate, an intermediate in proline biosynthesis. The import of Glu, Gln, Asp, Asn, Arg, Orn, and Cit did not lead to a further increase in the size of the proline pool that is already present in osmotically stressed cells. Hence, our data suggest that osmoprotection of B. subtilis by this group of amino acids rests on the savings in biosynthetic building blocks and energy that would otherwise have to be devoted either to the synthesis of the proline precursor glutamate or of proline itself. Since glutamate is the direct biosynthetic precursor for proline, we studied its uptake and found that GltT, an Na+-coupled symporter, is the main uptake system for both glutamate and aspartate in B. subtilis. Collectively, our data show how effectively B. subtilis can exploit environmental resources to derive osmotic-stress protection through physiological means. 相似文献
7.
Storage forms of N were studied in below-ground structures of nine boreal forest understorey plants. The ericaceous shrubs
Vacciniumvitis-idaea and V.myrtillus, the fern Gymnocarpium dryopteris, the grass Deschampsia flexuosa, and the herbs Epilobium angustifolium, Maianthemum bifolium, Solidago virgaurea, Geranium sylvaticum and Trientalis europaea were sampled in early summer and late autumn from plots fertilised with a complete mixture of nutrients and from non-fertilised
control plots. Concentrations of total nitrogen, insoluble and soluble proteins, free amino acids and nitrate were measured,
and changes in absolute and relative concentrations of these N fractions between early summer and late autumn were used to
identify the forms in which the plants store N. In all species studied, the concentration of free amino acids increased both
between summer and autumn and in response to fertilisation, while the concentration of protein N increased only in response
to fertilisation. Thus, free amino acids appear to have a central role in N storage. In all of the species except G. dryopteris, D. flexuosa and S. virgaurea, arginine dominated the pool of free amino acids and thus arginine was the major form of stored N in most species. In D. flexuosa and S. virgurea, however, asparagine and arginine together were the major forms of stored N, while glutamine was the major free amino acid,
and N storage form, in G. dryopteris.
Received: 10 March 1996 / Accepted: 22 December 1996 相似文献
8.
Hiroyuki Yamaguchi Hitoshi Miura Kazuoki Ohsumi Takako Osaki Haruhiko Taguchi Tomoko Yamamoto Tomoko Hanawa Sachio Ogata Shigeru Kamiya 《Microbiology and immunology》1996,40(1):77-80
To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat-shock protein (HSP60), a dot blot analysis was perfomed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316-342, p327-359, p340-366, p316-326, p316-321, p319-323, and p321-326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316-342, p316-326 and p321-326, and 3C8 mAb p316-342 and p316-326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316-326. The sequence homology between p316-326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3. 相似文献
9.
We investigated the effects of injecting male-derived extracts on congeneric female receptivity in two species of Callosobruchus beetle, C. chinensis and C. maculatus. We also examined the influence of interspecific mating on female remating behaviour in these two species. Male-derived extracts reduced congeneric female receptivity in both species. As quick-acting components, extracts of C. chinensis male seminal vesicles reduced the receptivity of C. maculatus females, whereas extracts of C. maculatus male testes reduced the receptivity of C. chinensis females. As slow-acting components, extracts of male accessory glands of other species reduced the receptivity of both C. maculatus and chinensis females. After interspecific mating, the sperm of C. maculatus males were transferred to the reproductive organs of C. chinensis females, thereby reducing their receptivity. In contrast, no C. chinensis sperm were transferred to the reproductive organs of C. maculatus females; accordingly, the latter's receptivity was not reduced. Furthermore, the survival rate of C. chinensis females decreased markedly after interspecific mating. These results raise the possibility that under circumstances where populations of these two species share the same habitat, reproductive interference would occur only in the interactions between C. maculatus males and C. chinensis females. 相似文献
10.
Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen 总被引:4,自引:0,他引:4
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Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH2-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity. 相似文献
11.
《Harmful algae》2014
In order to develop an improved method of mitigating harmful algal blooms (HABs), we assessed the inhibitory effects of five Chinese traditional herbs, Andrographis paniculata (Burm.f.) Nees, Galla chinensis, Punica granatum L., Cortex phellodendri chinensis and Radix scutellariae, as well as clay modified with herb extract on the growth of two harmful algae, Phaeocystis globosa and Prorocentrum donghaiense. The results showed that the five Chinese herbs had varying effects on the target microalgae and inhibitory rates ranged from −35% to 100% at concentration of 0.3 g dry wt./L during 96 h treatments. Among the five herbs, G. chinensis exhibited the strongest inhibitory effect, almost 100% on P. globosa and P. donghaiense. The growth of P. globosa and P. donghaiense was completely inhibited by G. chinensis extracts (0.3 g dry wt./L) during all growth phases, and lag phase cultures were more sensitive than exponential phase and stationary phase cultures. The highest inhibitory rate (100% inhibition) on P. globosa was observed at lag phase, followed by exponential phase (73.1% inhibition), and stationary phase (57% inhibition). The highest inhibitory rate of 100% on P. donghaiense was also found at lag phase, 40.3% at exponential phase, and 23.4% at stationary phase. Furthermore, modified clay with G. chinensis extract significantly enhanced the inhibitory impact. Modified clay (0.3 g L−1) produced 95% of growth inhibition for both algae species at 24 h, and maintained the inhibition thereafter. Our study demonstrated that G. chinensis and clays modified with its extract significantly inhibited the growth of harmful species, therefore may provide ideas and another option for control of harmful algal blooms. 相似文献
12.
The hemibiotrophic soil-borne fungus Verticillium dahliae is a major pathogen of a number of economically important crop species. Here, the metabolic response of both tomato and Arabidopsis thaliana to V. dahliae infection was analysed by first using non-targeted GC-MS profiling. The leaf content of both major cell wall components glucuronic acid and xylose was reduced in the presence of the pathogen in tomato but enhanced in A. thaliana. The leaf content of the two tricarboxylic acid cycle intermediates fumaric acid and succinic acid was increased in the leaf of both species, reflecting a likely higher demand for reducing equivalents required for defence responses. A prominent group of affected compounds was amino acids and based on the targeted analysis in the root, it was shown that the level of 12 and four free amino acids was enhanced by the infection in, respectively, tomato and A. thaliana, with leucine and histidine being represented in both host species. The leaf content of six free amino acids was reduced in the leaf tissue of diseased A. thaliana plants, while that of two free amino acids was raised in the tomato plants. This study emphasizes the role of primary plant metabolites in adaptive responses when the fungus has colonized the plant. 相似文献
13.
Taxonomic classification of the important medicinal plant, Ficus deltoidea (Moraceae), is challenging because of the variability of its leaves and fig forms that occur within the species. We developed 16 nuclear Simple Sequence Repeat (nSSR) markers, and characterized them using 24 individuals from a natural population. We then studied the intraspecific variation of F. deltoidea subsp. deltoidea in Peninsular Malaysia using morphological and molecular approaches. Based on the morphological variations, we further determined the varieties that occur regionally under the above subspecies based on the leaf characteristics. As for molecular data sets, we used both chloroplast DNA (cpDNA) and nSSR markers to elucidate the phylogenetic relationship among the varieties. The cpDNA dendrogram yielded poorer resolution where most of the clades were forming paraphyletic complex. The cluster analysis based on nSSR is largely congruent with the morphological classification, with F. deltoidea subsp. deltoidea classified into four main varieties, namely var. deltoidea, var. angustifolia, var. kunstleri and var. lutescens. Our study demonstrates the applicability of molecular approach in complementing the conventional taxonomic classification. 相似文献
14.
Masayochi Iwaki Hiroyuki Kagamiyama Mitsuhiro Nozaki 《Archives of biochemistry and biophysics》1981,210(1):210-223
The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper. 相似文献
15.
Yuko Ogawa Atsushi Ohnishi Yoshikuni Goto Yoshiki Sakuma Jobu Watanabe Akira Hattori Masafumi Tsujimoto 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
Aminopeptidase B (EC 3.4.11.6, APB) preferentially hydrolyzes N-terminal basic amino acids of synthetic and peptide substrates. APB is involved in the production and maturation of peptide hormones and neurotransmitters such as miniglucagon, cholecystokinin and enkephalin by cleaving N-terminal basic amino acids in extended precursor proteins. Therefore, the specificity for basic amino acids is crucial for the biological function of APB.Methods
Site-directed mutagenesis and molecular modeling of the S1 site were used to identify amino acid residues of the human APB responsible for the basic amino acid preference and enzymatic efficiency.Results
Substitution of Gln169 with Asn caused a significant decrease in hydrolytic activity toward the fluorescent substrate Lys-4-methylcoumaryl-7-amide (MCA). Substantial retardation of enzyme activity was observed toward Arg-MCA and substitution with Glu caused complete loss of enzymatic activity of APB. Substitution with Asn led to an increase in IC50 values of inhibitors that interact with the catalytic pocket of APB. The EC50 value of chloride ion binding was also found to increase with the Asn mutant. Gln169 was required for maximal cleavage of the peptide substrates. Molecular modeling suggested that interaction of Gln169 with the N-terminal Arg residue of the substrate could be bridged by a chloride anion.Conclusion
Gln169 is crucial for obtaining optimal enzymatic activity and the unique basic amino acid preference of APB via maintaining the appropriate catalytic pocket structure and thus for its function as a processing enzyme of peptide hormones and neurotransmitters. 相似文献16.
《Insect Biochemistry》1984,14(3):353-358
Free amino acid concentrations in plasma of larval and adult Hydropsyche betteni and Cheumatopsyche pettiti were determined. Free amino acids in whole-body homogenates of these two species were also investigated.Larval haemoplasm of both species contained high concentrations of serine, proline, alanine, lysine, valine, threonine and arginine. Total free amino acid concentrations were consistently higher in C. pettiti larvae (33.1 mM) than in H. betteni larvae (24.6 mM).Haemoplasm from adult specimens of H. betteni contained high concentrations of serine and proline, followed by alanine, valine, lysine and histidine. Serine was found in very high concentrations in plasma from C. pettiti adults, followed by proline, glytamic acid, histidine and arginine. The mean total concentration of free amino acids was higher in C. pettiti adults (18.6 mM) than in H. betteni adults (13.3 mM).Comparison of free amino acids from haemoplasm and whole-body homogenates suggests that concentrations of free amino acids in body tissues differ from those present in plasma for these two insects. 相似文献
17.
The mass spectra of the O-trimethylsilylated trifluoro-dideuteroethyl polyamino alcohols, produced by LiAlD4-reduction and O-trimethylsilylation of N-trifluororacetyl oligopeptide methyl esters, are evaluated. Characteristic mass spectra of derivatives are shown which are derived from peptides containing all protein amino acids including Arg, His, Trp, Gln, Asn and carboxyl terminal amides as well as modified Cys-residues. The mass spectra of these derivatives can be easily interpreted in terms of the amino acid sequence of the original peptides since they contain abundant and intensity-balanced sequence-determining ions. 相似文献
18.
A revision of the dioecious genus Chersodoma (Senecioneae, Asteraceae) recognizes nine species, including a new species, C. deltoidea Sagást. & M. O. Dillon, from northern Peru and a status change for a northwestern Argentine endemic, C. glabriuscula (Cabrera) M. O. Dillon & Sagást. Chersodoma is confined to the Andean Cordillera and is the only dioecious member of the Senecioneae in continental South America. A cladistic analysis of a morphological data set supports the monophyly of the genus and the recognition of two previously established subgenera. Subgenus Chersodoma contains three species (C. argentina, C. candida, C. jodopappa) with greatest diversity in the semi-arid puna of northwestern Argentina and adjacent Bolivia and Chile. Subgenus Diclinanthus B. Nord., contains six species (C. antennaria, C. arequipensis, C. deltoidea, C. juanisernii, C. glabriuscula, and C. ovopedata) with greatest diversity in Peru. A key to Andean genera of Senecioneae is provided. A discussion of species relationships and historical biogeography is presented as suggested by morphological parsimony analysis. 相似文献
19.
《Biochemical Systematics and Ecology》1987,15(4):427-432
Interspecific variation of protein amino acids between peanut, horse bean, french bean, lentil, cowpea and chick-pea was undertaken in relation to larval development of C. maculatus. Twenty-one groups of amino acids were identified and quantified. In all seeds, free amino acids exist in low quantities; but they are more numerous than bound amino acids. Essential amino acids for man are sufficient in the six species, except for the sulphur amino acids in all seeds, and for lysine in peanut. Utilization of those pulses, after cooking, in human food complements cereals. Larval development possibilities of C. maculatus upon the used seeds shows up that: (a) with chick-pea and cowpea, the bruchid thrives normally. However, although amino acids are higher in cowpea than in chick-pea, the better performances were observed on the latter; (b) peanut, french bean, horse bean and lentil are unsuitable for larval development of the insect. Although they are rich in amino acids, all larvae die at first in star within these pulses. We conclude that larval development of C. maculatus does not depend only on the amounts of amino acids in the seeds but also on other trophic factors. These could consist of the balance between different nutritive compounds and the presence of allelochemical substances in the tested seeds which bruchid larvae are unable to utilize. 相似文献
20.
Matthew R. Sapio Monique Vessaz Pierre Thomas Pierre Genton Lloyd D. Fricker Annick Salzmann 《PloS one》2015,10(4)
Carboxypeptidase A6 (CPA6) is a peptidase that removes C-terminal hydrophobic amino acids from peptides and proteins. The CPA6 gene is expressed in the brains of humans and animals, with high levels of expression during development. It is translated with a prodomain (as proCPA6), which is removed before secretion. The active form of CPA6 binds tightly to the extracellular matrix (ECM) where it is thought to function in the processing of peptides and proteins. Mutations in the CPA6 gene have been identified in patients with temporal lobe epilepsy and febrile seizures. In the present study, we screened for CPA6 mutations in patients with juvenile myoclonic epilepsy and identified two novel missense mutations: Arg36His and Asn271Ser. Patients harboring these mutations also presented with generalized epilepsy. Neither of the novel mutations was found in a control population. Asn271 is highly conserved in CPA6 and other related metallocarboxypeptidases. Arg36 is present in the prodomain and is not highly conserved. To assess structural consequences of the amino acid substitutions, both mutants were modeled within the predicted structure of the enzyme. To examine the effects of these mutations on enzyme expression and activity, we expressed the mutated enzymes in human embryonic kidney 293T cells. These analyses revealed that Asn271Ser abolished enzymatic activity, while Arg36His led to a ~50% reduction in CPA6 levels in the ECM. Pulse-chase using radio-labeled amino acids was performed to follow secretion. Newly-synthesized CPA6 appeared in the ECM with peak levels between 2-8 hours. There was no major difference in time course between wild-type and mutant forms, although the amount of radiolabeled CPA6 in the ECM was lower for the mutants. Our experiments demonstrate that these mutations in CPA6 are deleterious and provide further evidence for the involvement of CPA6 mutations in the predisposition for several types of epilepsy. 相似文献