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. 1984 Apr;74(4):834–840. doi: 10.1104/pp.74.4.834

Purification and Comparative Characterization of an Enolase from Spinach 1

Sukanto Sinha 1, John M Brewer 1
PMCID: PMC1066777  PMID: 16663518

Abstract

An enolase has been purified to apparent homogeneity, as measured by gel electrophoresis, some 400-fold from spinach (Spinacia oleracea). This is the first plant enolase that has been purified to homogeneity. At moderate ionic strengths, the 5,5-dithio-bis-2-(nitrobenzoate) (DTNB)-or parachloromercuribenzoate-reacted enzyme elutes from a Bio-Gel P-200 column with somewhat greater volumes than the yeast enzyme (Mr = 93,000) indicating a greater size. Its elution volume from Ultrogel in 50% ammonium sulfate, however, suggests it exists as an active monomer (Mr = 47,000). Sodium dodecyl sulfate-gel electrophoresis indicates the subunit molecular weight is 50,000 ± 3,000, like that of yeast enolase.

The enzyme contains 23 ± 4 half-cystines per mole of subunit. Titrations with DTNB in guanidine hydrochloride or nondenaturing media indicate that most of these, if not all, are in the reduced state. Reaction of one or more of the sulfhydryls with DTNB or parachloromercuribenzoate stabilizes the enzyme.

The kinetic parameters of the reaction catalyzed by spinach enolase, as well as the inhibitions by transition metal ions and fluoride, are similar to those properties of the yeast and rabbit muscle enzymes.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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