scholarly journals Highly diverged novel subunit composition of apicomplexan F-type ATP synthase identified from Toxoplasma gondii

2018 ◽  
Author(s):  
Rahul Salunke ◽  
Tobias Mourier ◽  
Manidipa Banerjee ◽  
Arnab Pain ◽  
Dhanasekaran Shanmugam
Keyword(s):  
Toxoplasma Gondii ◽  
Atp Synthase ◽  
Functional Characterization ◽  
Structural Features ◽  
Subunit Composition ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Apicomplexan Parasites ◽  
Fundamental Understanding

AbstractThe mitochondrial F-type ATP synthase, a multi-subunit nanomotor, is critical for maintaining cellular ATP levels. In Toxoplasma gondii and other apicomplexan parasites, many subunit components, necessary for proper assembly and functioning of this enzyme, appear to be missing. Here, we report the identification of 20 novel subunits of T. gondii F-type ATP synthase from mass spectrometry analysis of partially purified monomeric (~600 kDa) and dimeric (>1 MDa) forms of the enzyme. Despite extreme sequence diversification, key FO subunits, a, b and d, can be identified from conserved structural features. Orthologs for these proteins are restricted to apicomplexan, chromerid and dinoflagellate species. Interestingly, their absence in ciliates indicates a major diversion, with respect to subunit composition of this enzyme, within the alveolate clade. Discovery of these highly diversified novel components of the apicomplexan F-type ATP synthase complex could facilitate the development of novel anti-parasitic agents. Structural and functional characterization of this unusual enzyme complex will advance our fundamental understanding of energy metabolism in apicomplexan species.

scholarly journals GlcNAc De-N-Acetylase from the Hyperthermophilic ArchaeonSulfolobus solfataricus

2018 ◽  
Vol 85 (2) ◽  
Author(s):  
Roberta Iacono ◽  
Andrea Strazzulli ◽  
Luisa Maurelli ◽  
Nicola Curci ◽  
Angela Casillo ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Recombinant Enzyme ◽  
Mass Spectrometry Analysis ◽  
Genomic Context ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Additional Information ◽  
The One ◽  
Hydrolysis Of

ABSTRACTSulfolobus solfataricusis an aerobic crenarchaeal hyperthermophile with optimum growth at temperatures greater than 80°C and pH 2 to 4. Within the crenarchaeal group ofSulfolobales,N-acetylglucosamine (GlcNAc) has been shown to be a component of exopolysaccharides, forming their biofilms, and of theN-glycan decorating some proteins. The metabolism of GlcNAc is still poorly understood inArchaea, and one approach to gaining additional information is through the identification and functional characterization of carbohydrate active enzymes (CAZymes) involved in the modification of GlcNAc. The screening ofS. solfataricusextracts allowed the detection of a novel α-N-acetylglucosaminidase (α-GlcNAcase) activity, which has never been identified inArchaea. Mass spectrometry analysis of the purified activity showed a protein encoded by thesso2901gene. Interestingly, the purified recombinant enzyme, which was characterized in detail, revealed a novel de-N-acetylase activity specific for GlcNAc and derivatives. Thus, assays to identify an α-GlcNAcase found a GlcNAc de-N-acetylase instead. The α-GlcNAcase activity observed inS. solfataricusextracts did occur when SSO2901 was used in combination with an α-glucosidase. Furthermore, the inspection of the genomic context and the preliminary characterization of a putative glycosyltransferase immediately upstream ofsso2901(sso2900) suggest the involvement of these enzymes in the GlcNAc metabolism inS. solfataricus.IMPORTANCEIn this study, a preliminary screening of cellular extracts ofS. solfataricusallowed the identification of an α-N-acetylglucosaminidase activity. However, the characterization of the corresponding recombinant enzyme revealed a novel GlcNAc de-N-acetylase, which, in cooperation with the α-glucosidase, catalyzed the hydrolysis of O-α-GlcNAc glycosides. In addition, we show that the product of a gene flanking the one encoding the de-N-acetylase is a putative glycosyltransferase, suggesting the involvement of the two enzymes in the metabolism of GlcNAc. The discovery and functional analysis of novel enzymatic activities involved in the modification of this essential sugar represent a powerful strategy to shed light on the physiology and metabolism ofArchaea.

Abstract 546: Characterization of the I4399M Variant of Apolipoprotein(a): Implications for Altered Prothrombotic Properties of Lipoprotein(a)

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jackson McAiney ◽  
Corey Scipione ◽  
Daniel Simard ◽  
James Gauld ◽  
Michael Boffa ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Fibrin Clot ◽  
Mass Spectrometry Analysis ◽  
Clot Lysis ◽  
Elevated Plasma ◽  
Plasma Clot ◽  
Lipoprotein A ◽  
Spectrometry Analysis ◽  
Apolipoprotein A

Elevated plasma levels of lipoprotein(a) (Lp(a)) are a causal risk factor for CHD. Lp(a) closely resembles LDL, but contains an additional glycoprotein apolipoprotein(a) (apo(a)) that is structurally homologous to the fibrinolytic proenzyme plasminogen. Key sequence differences render the protease-like domain in apo(a) catalytically inactive. This has led to speculation that Lp(a) can oppose the fibrinolytic functions of plasminogen. A single nucleotide polymorphism (SNP) in the LPA gene encoding apo(a) results in an Ile to Met substitution at position 4399 in the protease-like domain. In population studies, this variant has been correlated with elevated plasma Lp(a) levels and with higher CHD risk, an effect that has been suggested to arise from effects of the variant on fibrin clot properties. We undertook a functional characterization of the effect of the I4399M substitution in apo(a). Molecular dynamics simulations of wild-type (wt) apo(a) and the Met variant revealed a shift from a buried (Ile) to slightly exposed (Met) environment, allowing for potential modification of the Met. Indeed, MALDI-TOF mass spectrometry analysis demonstrated the presence of a methionine sulfoxide moiety at this position in the Met variant. When 17-kringle recombinant forms of apo(a) were included in a plasma clot lysis assay, both the wt apo(a) and Met variant inhibited lysis, but the Met variant had a 50% greater effect. However, the Met variant was equally as efficient as wt apo(a) in inhibiting plasminogen activation on a fibrin surface. Apo(a) was also able to significantly shorten coagulation time for clots made from either purified fibrin or lipoprotein-deficient plasma, with the Met variant having twice as large an effect as wt apo(a). Morever, the Met variant resulted in greater turbidity of the clots whereas wt apo(a) had no effect. In agreement with these findings, SEM and confocal microscopy of fibrin clots showed that compared to wt apo(a), the I4399M variant resulted in significant alteration of the fibrin network, with a 4-fold increase in fibrin fiber width. Together, our data suggest that the Met4399 variant differs structurally from wt apo(a), which may underlie key differences related to its effects on fibrin clot architecture and fibrinolysis.

Identification and functional characterization of a sesquiterpene synthase gene from Eleutherococcus trifoliatus

Holzforschung ◽  
2012 ◽  
Vol 66 (2) ◽  
Author(s):  
Chi-Hsiang Wen ◽  
Yen-Hsueh Tseng ◽  
Fang-Hua Chu
Keyword(s):  
Functional Characterization ◽  
Mass Spectrometry Analysis ◽  
Terpene Synthase ◽  
Gas Chromatography Mass Spectrometry ◽  
Sesquiterpene Synthase ◽  
Reading Frame ◽  
Spectrometry Analysis ◽  
Authentic Standard ◽  
The Common

Abstract In the present study, one sesquiterpene synthase gene in Eleutherococcus trifoliatus was identified and characterized. Full-length cDNA was obtained from stems. It contained an open reading frame of 1671 bp (EtCop) with a predicted molecular mass of 64.5 kDa. The amino acid sequence of EtCop contained the common terpene synthase family motifs RR(x)8W, RxR and DDxxD. The recombinant protein from Escherichia coli was incubated with farnesyl diphosphate in order to identify the function of EtCop. The product of EtCop could be identified as an α-copaene by means of gas chromatography-mass spectrometry analysis and comparison with an authentic standard.

scholarly journals Effect of Ocular Hypertension on D-β-Aspartic Acid-Containing Proteins in the Retinas of Rats

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Takashi Kanamoto ◽  
Takashi Tachibana ◽  
Yasushi Kitaoka ◽  
Toshio Hisatomi ◽  
Yasuhiro Ikeda ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ocular Hypertension ◽  
Aspartic Acid ◽  
Atp Synthase ◽  
Wistar Rats ◽  
Protein Band ◽  
Mass Spectrometry Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Sds Page

Purpose. To investigate the effect of ocular hypertension-induced isomerization of aspartic acid in retinal proteins. Methods. Adult Wistar rats with ocular hypertension were used as an experimental model. D-β-aspartic acid-containing proteins were isolated by SDS-PAGE and western blot with an anti-D-β-aspartic acid antibody and identified by liquid chromatography-mass spectrometry analysis. The concentration of ATP was measured by ELISA. Results. D-β-aspartic acid was expressed in a protein band at around 44.5 kDa at much higher quantities in the retinas of rats with ocular hypertension than in those of normotensive rats. The 44.5 kDa protein band was mainly composed of α-enolase, S-arrestin, and ATP synthase subunits α and β, in both the ocular hypertensive and normotensive retinas. Moreover, increasing intraocular pressure was correlated with increasing ATP concentrations in the retinas of rats. Conclusion. Ocular hypertension affected the expression of proteins containing D-β-aspartic acid, including ATP synthase subunits, and up-regulation of ATP in the retinas of rats.

scholarly journals Identification of sugars and phenolic compounds in honey powders with the use of GC–MS, FTIR spectroscopy, and X-ray diffraction

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Katarzyna Kozłowicz ◽  
Renata Różyło ◽  
Bożena Gładyszewska ◽  
Arkadiusz Matwijczuk ◽  
Grzegorz Gładyszewski ◽  
...  
Keyword(s):  
Phenolic Compounds ◽  
Ftir Spectroscopy ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Coumaric Acid ◽  
X Ray Diffraction ◽  
Phenyllactic Acid ◽  
X Ray ◽  
Spectrometry Analysis

Abstract This work aimed at the chemical and structural characterization of powders obtained from chestnut flower honey (HFCh) and honey with Inca berry (HBlu). Honey powders were obtained by spray drying technique at low temperature (80/50 °C) with dehumidified air. Maltodextrin (DE 15) was used as a covering agent. The isolation and evaluation of phenolic compounds and sugars were done by gas chromatography–mass spectrometry analysis. Scanning electron microscopy, Fourier-transform infrared (FTIR) spectroscopy, and X-ray diffraction were performed to determine the morphology of the studied honey powders. The obtained results showed that the content of simple sugars amounted to 72.4 and 90.2 g × 100 g−1 in HFCh and HBlu, respectively. Glucose was found to be the dominant sugar with a concentration of 41.3 and 51.6 g × 100 g−1 in HFCh and HBlu, respectively. 3-Phenyllactic acid and ferulic acid were most frequently found in HFCh powder, whereas m-coumaric acid, benzoic acid, and cinnamic acid were the most common in HBlu powder. The largest changes in the FTIR spectra occurred in the following range of wavenumbers: 3335, 1640, and below 930 cm−1. The X-ray diffraction profiles revealed wide peaks, suggesting that both honey powders are amorphous and are characterized by a short-range order only.

Chemical and biological characterization of sclerosin, an antifungal lipopeptide

2012 ◽  
Vol 58 (8) ◽  
pp. 1027-1034 ◽  
Author(s):  
Chrystal L. Berry ◽  
Ann Karen C. Brassinga ◽  
Lynda J. Donald ◽  
W.G. Dilantha Fernando ◽  
Peter C. Loewen ◽  
...  
Keyword(s):  
Ring Opening ◽  
Chemical Structure ◽  
Cell Types ◽  
Mass Spectrometry Analysis ◽  
Bacillus Species ◽  
Biological Characterization ◽  
Spectrometry Analysis ◽  
C Elegans ◽  
Oomycete Pathogen

Pseudomonas sp. strain DF41 produces a lipopeptide, called sclerosin that inhibits the fungal pathogen Sclerotinia sclerotiorum . The aim of the current study was to deduce the chemical structure of this lipopeptide and further characterize its bioactivity. Mass spectrometry analysis determined the structure of sclerosin to be CH3-(CH2)6-CH(OH)-CH2-CO-Dhb-Pro-Ala-Leu/Ile-Ala-Val-Val-Dhb-Thr-Val-Leu/Ile-Dhp-Ala-Ala-Ala-Val-Dhb-Dhb-Ala-Dab-Ser-Val-OH, similar to corpeptins A and B of the tolaasin group, differing by only 3 amino acids in the peptide chain. Subjecting sclerosin to various ring opening procedures revealed no new ions, suggesting that this molecule is linear. As such, sclerosin represents a new member of the tolaasin lipopeptide group. Incubation of S. sclerotinia ascospores and sclerotia in the presence of sclerosin inhibited the germination of both cell types. Sclerosin also exhibited antimicrobial activity against Bacillus species. Conversely, this lipopeptide demonstrated no zoosporicidal activity against the oomycete pathogen Phytophthora infestans . Next, we assessed the effect of DF41 and a lipopeptide-deficient mutant on the growth and development of Caenorhabditis elegans larvae. We discovered that sclerosin did not protect DF41 from ingestion by and degradation in the C. elegans digestive tract. However, another metabolite produced by this bacterium appeared to shorten the life-span of the nematode compared to C. elegans growing on Escherichia coli OP50.

Topological and functional characterization of the F0I subunit of the membrane moiety of the mitochondrial H+-ATP synthase

1988 ◽  
Vol 173 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Josef HOUSTEK ◽  
Jan KOPECKY ◽  
Franco ZANOTTI ◽  
Ferruccio GUERRIERI ◽  
Emilio JIRILLO ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Functional Characterization
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