Cottonseed Malate Synthase

Abstract

Malate synthase (EC 4.13.2), an enzyme unique to the glyoxylate cycle, was purified to homogeneity from cotyledons of 72-hours, darkgrown cotton (Gossypium hirsutum L.) seedlings. Homogeneity of the enzyme was assessed by silver staining SDS-PAGE gels. Purification was accomplished by using a single buffer medium through six steps involving one ammonium sulfate fractionation and chromatography on three columns (Sephacryl S-300, DEAE element for studying certain aspects of protein trafficking is the availability of monospecific antibodies to specific organellar proteins. As part of our ongoing efforts to elucidate mechanisms of glyoxysomal enzyme acquisition in cotton seeds, we have purified and raised antibodies to isocitrate lyase (4) and catalase (13) and now report results for MS.MS has been partially purified from cottonseeds (20), purified to apparent homogeneity from only three oilseed species (cucumber 10, 22; castor bean 2, 5; corn 24), and purified to varying degrees in bacteria, yeast, and Euglena (Ref. 20 for references). In all procedures described for oilseeds, the enzyme was purified from glyoxysome fractions. In the procedure presented here the enzyme was extracted from homogenized cotyledons and purified using the same buffer medium for all steps. The final product was free of contaminating proteins as judged from silver-stained gels; this criterion for purity had not been applied previously to other purified MS preparations. New information is also presented on the immunological characteristics and cross-reactivity of the polyclonal antibodies raised in rabbits relative to different forms of MS in germinated and maturing seeds.MATERIALS AND METHODS Chemicals. PMSF, DTT, aprotinin, benzamidine-HCl, iodoacetamide, Sephacryl S-300, DEAE Sephacel, Phenyl Sepharose CL-4B, Protein-A Sepharose CL-4B, bovine thyroglobulin, BSA (98-99%), bovine heart L-lactic dehydrogenase, bovine liver catalase (C-I 0), glyoxylic acid (free acid and sodium salt), alkaline phosphatase conjugated goat anti-rabbit IgG, fast red violet LB salt, naphthol AS-BI phosphate (sodium salt), MOPS, Triton X-100, and PVP-10 were purchased from Sigma Chemical Co.

Relationship between Cottonseed Malate Synthase Aggregation Behavior and Suborganellar Location in Glyoxysomes and Endoplasmic Reticulum

“…Malate synthase (31), catalase (21), and ICL (3), CCR (12), and FCR (12) were assayed as previously described. Antimycin A (0.002 mM) and KCN (0.2 mM) were included in CCR assays to ensure ER specificity.…”

“…Rate-zonal centrifugation and calculations of S values were done essentially as in Trelease et al (31), except linear gradients were 17 to 45% w/w sucrose in the various buffers with or without KCI.…”

Purification and Characterization of Malate Synthase from the Glucose-grown Wood-rotting BasidiomyceteFomitopsis palustris

“…By gelˆltration on a TSK gel G3000SW XL column, the puriˆed enzyme was estimated to have a molecular mass of 520 kDa, consisting apparently of eight identical subunits (65 kDa) as an octamer, which is very close in molecular size to the enzymes from higher plants, such as Ricinus communis and Pinus taeda with the molecular masses of 575 kDa (64 kDa subunit) 25) and 520 kDa (62 kDa subunit), 16) respectively. However, the enzymes from Pinus densi‰ora and Gossypium hirsutum have slightly higher molecular masses of 630 kDa (62 kDa subunit) 15) and 750 kDa (63 kDa subunit), 26) respectively. Interestingly, there is no signiˆcant diŠerence in the subunit molecular mass, whereas the enzymes from Acinetobacter calcoaceticus and Corynebacterium glutamicum are in a monomeric form with a molecular mass of 75 18) and 82 kDa, 19) respectively.…”

Rapid purification of malate synthase from cotyledons of Brassica napus L

“…These data are in good agreement with previous reports on malate synthase purifications form Brassica napus L. [7] or Glycine max L. [8]; however, these reports describe procedures that involve at least three different chromatographic steps. The purification of malate synthase from homogenates of cotton seedlings involved even six steps with three different columns and resulted in a apparent homogeneity on silver stained SDS-PAGE [5] yielding 2.6% of the total enzyme activity and a specific activity of 133 nkat/mg protein for the purified malate synthase [5].…”

“…Malate synthase, a key enzyme for the glyoxylate cycle, catalyzes the condensation of acetyI-CoA and glyoxylate forming L-malate and CoA [2,3]. The enzyme was purified from several plant species [4][5][6][7][8]. Its activity is strictly dependent on divalent cations, such as Mg 2+ and it is comprised of subunits with a molecular weight of around 63 kDa which readily aggregate to oligomeric complexes [6][7][8][9].…”