Glyoxysomal malate dehydrogenase and malate synthase from soybean cotyledons (Glycine max L.): enzyme association, antibody production and cDNA cloning

Abstract

In order to investigate a possible association between soybean malate synthase (MS; L-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD+ oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionic-strength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (Mr) of 670,000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl2, with the elution of MS as an octamer of M(r) 510,000 and of gMDH as a dimer of M(r) 73,000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a lambda ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X6)HL motif also found in plant and mammalian thiolases.

Operation of glyoxylate cycle in halophilic archaea: presence of malate synthase and isocitrate lyase in Haloferax volcanii

“…The molecular mass of the subunit of malate synthase from Hf . volcanii (67±4 kDa) is similar to other eukaryal and bacterial subunits [24–26]. The molecular mass of the native enzyme (200±30 kDa) suggests that the enzyme is a trimer, like some malate synthases [27].…”

Marker-trait association study for protein content in chickpea (Cicer arietinum L.)

“…Of the 29 candidate genes identified based on GO annotation, gene-like protein-like malate synthase have been reported to be involved in carbohydrate metabolism pathway in soybean (Guex et al 1995); whereas 6-phosphogluconate dehydrogenase is involved in carbohydrate degradation and pentose phosphate pathway, while SufE-like protein are involved in cofactor and iron-sulphur cluster biosynthesis, and it has been reported that its deficiency results in abnormal seed development and arrested embryo development in Arabidopsis (Xu and Moller 2006). These genes need to be studied further for their direct or indirect involvement in regulation of seed protein.…”

Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes

“…Other PTS2-containing proteins dimerize or form higher multimers, suggesting that this may be a more general observation (Mathieu et al, 1994;Guex et al, 1995;Lee et al, 1997;Flynn et al, 1998;Chudzik et al, 2000). However, thiolase can be imported as a heterodimer when one subunit possesses a PTS2 and the other does not, which suggests that PTS2 dimerization is not required for import (Glover et al, 1994;Flynn et al, 1998).…”