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The selective retardation of NADP+-dependent dehydrogenases by immobilized procion red HE-3B.

ABSTRACT: The capacities of Procion Red HE-3B and Cibacron Blue F3G-A immobilized to Sepharose CL-4B and Matrex 201R for NAD+-, NADP+- and NAD(P)+-dependent dehydrogenases were measured. Procion Red HE-3B columns retarded NADP+-dependent dehydrogenases more effectively than NAD+-dependent dehydrogenases, whilst immobilized Cibacron Blue F3G-A retarded NAD+-dependent dehydrogenases more effectively than NADP+-dependent dehydrogenases. The capacity of procion Red HE-3B-Sepharose CL-4B for five dehydrogenases was highest in the region of 70nmol of immobilized ligand/ml of settled gel. The effects of using poly(ethyleneimine) as a spacer for both porous and pellicular supports were also examined. Four NADP+-dependent dehydrogenases were purified from yeast extract by using Procion Red HE-3B-Sepharose CL-4B. Two NAD+-dependent dehydrogenases were purified from the same source using Cibacron Blue F3G-A-Sepharose CL-4B. These results are discussed in relation to the use of immobilized Procion Red HE-3B to purify dehydrogenases. This immobilized dye's chromatograhic behaviour is compared with that of immobilized nucleotides. The most important feature of immobilized tirazine dyes seems to be their high operational capacities when compared with group-specific nucleotide adsorbents.

SUBMITTER: Watson DH

PROVIDER: S-EPMC1185813 | biostudies-other | 1978 Aug

REPOSITORIES: biostudies-other

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Project description:The formate dehydrogenases (Fdh) Fdh-O, Fdh-N, and Fdh-H, are the only proteins in Escherichia coli that incorporate selenocysteine at a specific position by decoding a UGA codon. However, an excess of selenium can lead to toxicity through misincorporation of selenocysteine into proteins. To determine whether selenocysteine substitutes for cysteine, we grew Escherichia coli in the presence of excess sodium selenite. The respiratory Fdh-N and Fdh-O enzymes, along with nitrate reductase (Nar) were co-purified from wild type strain MC4100 after anaerobic growth with nitrate and either 2 µM or 100 µM selenite. Mass spectrometric analysis of the catalytic subunits of both Fdhs identified the UGA-specified selenocysteine residue and revealed incorporation of additional, 'non-specific' selenocysteinyl residues, which always replaced particular cysteinyl residues. Although variable, their incorporation was not random and was independent of the selenite concentration used. Notably, these cysteines are likely to be non-essential for catalysis and they do not coordinate the iron-sulfur cluster. The remaining cysteinyl residues that could be identified were never substituted by selenocysteine. Selenomethionine was never observed in our analyses. Non-random substitution of particular cysteinyl residues was also noted in the electron-transferring subunit of both Fdhs as well as in the subunits of the Nar enzyme. Nar isolated from an E. coli selC mutant also showed a similar selenocysteine incorporation pattern to the wild-type indicating that non-specific selenocysteine incorporation was independent of the specific selenocysteine pathway. Thus, selenide replaces sulfide in the biosynthesis of cysteine and misacylated selenocysteyl-tRNA(Cys) decodes either UGU or UGC codons, which usually specify cysteine. Nevertheless, not every UGU or UGC codon was decoded as selenocysteine. Together, our results suggest that a degree of misincorporation of selenocysteine into enzymes through replacement of particular, non-essential cysteines, is tolerated and this might act as a buffering system to cope with excessive intracellular selenium.

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The selective retardation of NADP+-dependent dehydrogenases by immobilized procion red HE-3B.

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