Purification and physical and kinetic characterization of an NAD+-dependent malate dehydrogenase from leaves of pineapple (Ananas comosus)

Ileana C. Cuevas, Florencio E. Podestá

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

An NAD+-dependent malate dehydrogenase (MDH, EC 1.1.1.37) was purified and characterized from leaves of pineapple (Ananas comosus), a plant with Crassulacean acid metabolism (CAM). The purified enzyme had a subunit molecular mass of 39.5 kDa. Its activity showed a maximum at pH 6.8-7.0 and decreased sharply towards pH 8.0. This activity profile coincided with a change in the aggregation state, as determined by gel filtration on high-performance liquid chromatography from a dimer at pH 7.0 to a tetramer at pH 8.0. This isozyme is one of at least 5 MDH in pineapple leaves distinguishable by non-denaturing isoelectric focusing and displayed an isoelectric point of 5.8. The ratio of oxaloacetate reduction versus malate oxidation rates varied between 431 and 52 at pH 6.8 and 7.5, respectively. Antibodies raised against the purified pineapple leaf MDH immunodecorated a single 39.5-kDa polypeptide in denatured crude leaf extracts, but did not cross-react with extracts from purified pineapple mitochondria possessing high MDH activity. The purified MDH was recognized by monoclonal antibodies raised against the cytosolic MDH from Echinococcus granulosus. These and other distinctive traits, such as its isoelectric point and its subunit mass, suggest that the purified isozyme is the cytosolic MDH. Its properties are consistent with an implied function in the night acidification typical of CAM plants, although it is less clear if it also has a role in the daytime decarboxylation of malate.

Original languageEnglish (US)
Pages (from-to)240-248
Number of pages9
JournalPhysiologia Plantarum
Volume108
Issue number3
DOIs
StatePublished - Mar 2000

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Ananas
Ananas comosus
Malate Dehydrogenase
malate dehydrogenase
pineapples
kinetics
Crassulacean acid metabolism
Isoelectric Point
leaves
malates
isoelectric point
Isoenzymes
isozymes
Echinococcus granulosus
Oxaloacetic Acid
Decarboxylation
Acids
decarboxylation
isoelectric focusing
Isoelectric Focusing

ASJC Scopus subject areas

  • Plant Science

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Purification and physical and kinetic characterization of an NAD+-dependent malate dehydrogenase from leaves of pineapple (Ananas comosus). / Cuevas, Ileana C.; Podestá, Florencio E.

In: Physiologia Plantarum, Vol. 108, No. 3, 03.2000, p. 240-248.

Research output: Contribution to journalArticle

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