scholarly journals A preliminary study of Dihydroorotase from Methanococcus jannaschii

2014 ◽  
Vol 70 (a1) ◽  
pp. C481-C481
Author(s):  
Aditya Singh ◽  
Michael Colaneri ◽  
Jacqueline Vitali
Keyword(s):  
Mass Spectrometry ◽  
De Novo ◽  
Hydrophobic Interaction Chromatography ◽  
Size Exclusion ◽  
Mass Spectrometry Analysis ◽  
Yale University ◽  
Spectrometry Analysis ◽  
Bacteria And Fungi ◽  
Physics Department ◽  
Research Institute

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamoyl-L-aspartate to form L-dihydroorotate in the third step of de novo pyrimidine biosynthesis. It is a Zinc metalloenzyme and a member of the aminohydrolase superfamily. There are two classes of the enzyme. Class I, typically ~45 kDa, is found in higher organisms, bacteria and yeast. Class II, typically ~38 kDa, is found in bacteria and fungi. Some organisms have multiple DHOase sequences. The M. jannaschii pyrC gene coding for DHOase was subcloned and expressed in E. coli. Protein purification consisted of ammonium sulfate precipitation, heat treatment at 850C, and phenyl-sepharose hydrophobic interaction chromatography. The protein was confirmed in the SDS gel using Liquid Chromatography-Mass Spectrometry (Proteomics Laboratory, Lerner Research Institute, Cleveland, OH). Size Exclusion Chromatography-Laser Light Scattering (Keck Biotechnology Laboratory, Yale University, New Haven, CT) indicated that the protein is a monomer in solution with a molecular weight of 47 kDa. A model constructed with the I-TASSER server (Zhang, 2008) suggested that the binding site contains only one Zn ion per monomer coordinated by the conserved His56, His58 and Asp302. Asp146 is further away and does not coordinate with the Zn ion. According to the mass spectrometry analysis, the protein does not contain a carboxylated lysine. Our progress on this study will be presented. Acknowledgements: We thank Dr. Belinda Willard (Lerner Research Institute) for the LC-MS and Dr. Ewa Folta-Stogniew (Yale University) for the SEC- LS analysis. The presentation was supported in part by a graduate faculty travel award and by a contribution from the Physics Department at Cleveland State University.

scholarly journals Algorithm of Protein Sequence Determination by Combination of the Edman Degradation Method and Shotgun Mass Spectrometry

2018 ◽  
Vol 1 (4) ◽  
pp. e00087
Author(s):  
V.S. Skvortsov ◽  
A.V. Mikurova ◽  
N.E. Vavilov ◽  
V.G. Zgoda
Keyword(s):  
Mass Spectrometry ◽  
Protein Sequence ◽  
De Novo ◽  
Mass Spectrometry Analysis ◽  
Protein Hydrolysis ◽  
Edman Degradation ◽  
Terminal Part ◽  
Sequence Determination ◽  
Spectrometry Analysis ◽  
Shotgun Mass Spectrometry

An algorithm combining advantages of the Edman degradation method and de novo mass-spectrometric sequencing was developed. The protein from the “Diaskintest” diagnostic test was used for analysis. The protein was digested with trypsin and 5 steps of Edman degradation were carried out sequentially for the mixture of peptides. At each stage, the resulting mixture was analyzed by shotgun mass spectrometry analysis. The results of mass-spectrometry were analyzed both by the well-known de novo sequencing programs Novor and PepNovo+, and by own program that clustered individual spectra with a C-terminal signature formed by Y-ions. This approach allows us to determine confidently the amino acid sequence of the N-terminal part of the peptides obtained after the protein hydrolysis by trypsin.

Study of ketone body kinetics in children by a combined perfusion of 13C and 2H3 tracers

1987 ◽  
Vol 253 (5) ◽  
pp. E496-E502 ◽  
Author(s):  
P. F. Bougneres ◽  
P. Ferre
Keyword(s):  
Mass Spectrometry ◽  
Ketone Body ◽  
Turnover Rate ◽  
De Novo ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Turnover Rates ◽  
Short Term ◽  
Spectrometry Analysis ◽  
Insight Into

Ketone body kinetics were quantified in six children (3-5 yr old), who were fasted for 13-22 h, by a combined perfusion of [3-13C]acetoacetate ([13C]AcAc) and D-(-)-beta-[4,4,4-2H3]hydroxybutyrate (beta-[2H3]OHB) and gas chromatography-mass spectrometry analysis. Results were analyzed according to the "single-pool" (combined enrichments) or the "two-accessible pools" models. After 20-22 h of fasting, ketone body turnover rate was 30-50 mumol.kg-1.min-1, a rate achieved after several days of fasting in adults. At low ketosis, acetoacetate was the ketone body preferentially synthesized de novo and utilized irreversibly. When ketosis increased, acetoacetate irreversible disposal was not enhanced, since it was largely converted into beta-OHB, whereas beta-OHB irreversible disposal was very much increased. The single-pool and two-pool models gave similar ketone body turnover rates when [13C]AcAc was the tracer, whereas the use of beta-[2H3]OHB gave some more divergent results, especially at low ketosis. These studies demonstrate that ketogenesis is very active in short-term fasted children and that the use of a combined infusion of [13C]AcAc and beta-[2H3]OHB is a convenient way to give insight into individual ketone body kinetics.

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.

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