Approaches for submicrosequencing

Tsugita, Akira; Ataka, Tatsuaki; Uchida, Toyoaki

doi:10.1007/bf00247761

Cited by 26 publications

(12 citation statements)

References 17 publications

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“…Samples containing the FomA protein were subjected to SDS-PAGE, and the Coomassie-stained FomA bands were excised. The protein was extracted from the gel by incubation in 70 % formic acid at room temperature overnight (Tsugita et al, 1987), and submitted to the protein sequencing facilities at the University of Oslo for N-terminal sequencing.…”

Section: Trypsin Treatment Of Intact Cells and Cell Envelopes Con-mentioning

confidence: 99%

Structural characterization of the fusobacterial non-specific porin FomA suggests a 14-stranded topology, unlike the classical porins

et al. 2002

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Section: Trypsin Treatment Of Intact Cells and Cell Envelopes Con-mentioning

confidence: 99%

Structural characterization of the fusobacterial non-specific porin FomA suggests a 14-stranded topology, unlike the classical porins

et al. 2002

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“…Protein samples were hydrolyzed in the gas phase according to procedures described in [19]. Amino-acid analyses were performed on a Beckman 6300 amino-acid analyser according to the instructions of the manufacturer.…”

Section: Amino-acid Composition and Nh-terminal Sequence Determinationmentioning

confidence: 99%

Subunit composition, crystallization and preliminary crystallographic studies of the Desulfovibrio gigas aldehyde oxidoreductase containing molybdenum and [2Fe‐2S] centers

Romão

Barata

Archer

et al. 1993

European Journal of Biochemistry

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The Desulfovibrio gigas aldehyde oxidoreductase contains molybdenum bound to a pterin cofactor and [2Fe-2S] centers. The enzyme was characterized by SDSPAGE, gel-filtration and analytical ultracentrifugation experiments. It was crystallized at !"C, pH 7.2, using isopropanol and MgC1, as precipitants. The crystals diffract beyond 0.3-nm (3.0-A) resolution and belong to space group P6,22 or its enantiomorph, with cell dimensions u = b = 14.45 nm and c = 16.32 nm. There is one subunit/asymmetric unit which gives a packing density of 2.5 X nm'/Da (2.5 A3/Da), consistent with the experimental crystal density, p = 1.14 g/cm3. One dimer (approximately 2 X 100 kDa) is located on a crystallographic twofold axis.The detailed knowledge of the structure and the catalytic mechanism of molybdenum-containing enzymes has considerably increased during the last decade [I -61 and, in particular, the biochemistry of aldehyde oxidoreductases has been extensively studied [4, 51. Molybdenum is a relevant transition metal in biological systems. Two groups of molybdo-proteins have been described. In the first group Mo is associated with iron in a complex cluster type structure (FeMocofactor) in the nitrogenase enzyme. Recently, a structural model of the FeMoco was proposed based on crystallographic analysis, consisting of [4Fe,3S] and [lMo,3Fe,3S] clusters bridged by three nonprotein ligands [7, 81. In the second group Mo is contained in an organic structural component (pterin), designated as Mocofactor (molybdopterin), generally associated with ironsulfur centers and/or a flavin or heme center, as in the case of molybdenum oxotransferases [6].From sulfate-reducing bacteria (strict anaerobes) enzymes related to the second group have been isolated and characterized [9-171. They represent unique situations for the presence of such a group of enzymes in the prokaryotic world, since most often these proteins are studied in eukaryotes (e.g. plants, insects and higher animals).The molybdenum iron-sulfur protein (MOP) isolated from D. gigas has analogies with the molybdenum hydrox- An important advance on these studies was the possibility to isolate the enzyme from 57Fe-enriched media with obvious interest for an iron-sulfur-center site labelling, enhanced sensitivity of the Mossbauer studies (an advantage with respect to mammalian systems) and the possibility of a direct measurement of substrate binding [14]. Previously described molybdenum (V) resting-type and slow-type EPR signals and the extended-X-ray-absorption fine-structure (EXAFS) spectrum of the molybdenum center indicated close similarities to desulfo-xanthine oxidase (inactive) [lo-121. In addition, it was later demonstrated for MOP, a new Mo(V) EPR signal (rapid type 2) centered at EPR average g-value (gav) of 1.9742, similar to those observed for xanthine and aldehyde oxidases [12]. These rapid EPR signals have been shown, in these proteins, to be physiologically significant, as they develop within the enzymeturnover time scale. A molybdenum cofactor, liberated from the protei...

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“…After staining with Coomassie blue R250, each polypeptide band was excised and extracted with 70% formic acid. Subunit A was isolated by Bio-Gel P-10 (Bio-Rad) column chromatography according to [9] and directly used for the following sequencing work. …”

Section: Methodsmentioning

confidence: 99%

Amino acid sequence of spinach ferredoxin:thioredoxin reductase variable subunit

Iwadate

Yano

Kamo

et al. 1994

European Journal of Biochemistry

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Ferredoxin:thioredoxin reductase (FTR) is an iron‐sulfur protein, which, in the presence of ferredoxin and thioredoxin, catalyses the light‐dependent activation of several photosynthetic enzymes. Spinach FTR consists of two dissimilar polypeptide chains, A and B, present in equal amounts. Whereas subunit B seems to be responsible for the catalytic activity, subunit A has no known catalytic function. We found earlier that the N‐terminus of subunit A, also called the variable subunit, shows terminal redundancy and that 2–3 of its serine residues are phosphorylated [Tsugita, A. Yano, K., Gardet‐Salvi, L. & Schürmann, P. (1991) Protein Sequence Data Anal. 4, 9–13]. We now report the complete amino acid sequence of subunit A, determined by conventional protein sequencing methods. The polypeptide chain with a calculated molecular mass of 12 669 Da consists of 112 amino acids and has a calculated isoelectric point of 5.4. The analysis of the sequence supports the idea that this subunit has no catalytic function. The comparison with a known cyano‐bacterial FTR reveals about 58% similarity and the striking presence of a N‐terminal extension in the spinach protein. This extension may be responsible for the reported size variability of this subunit.

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Approaches for submicrosequencing

Cited by 26 publications

References 17 publications

Structural characterization of the fusobacterial non-specific porin FomA suggests a 14-stranded topology, unlike the classical porins

Structural characterization of the fusobacterial non-specific porin FomA suggests a 14-stranded topology, unlike the classical porins

Subunit composition, crystallization and preliminary crystallographic studies of the Desulfovibrio gigas aldehyde oxidoreductase containing molybdenum and [2Fe‐2S] centers

Amino acid sequence of spinach ferredoxin:thioredoxin reductase variable subunit

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