Oxalacetate Decarboxylase of Aerobacter aerogenes. I. Inhibition by Avidin and Requirement for Sodium Ion^*

Abstract

The oxalacetate (OAA) decarboxylase present in extracts of Aerobacter aerogenes did not require added divalent cation for its activity and was not inhibited by 0.1 µ ethylenediaminetetracetate (EDTA). The decarboxylase was inhibited 95% by avidin (0.5 unit), but not by avidin pretreated with biotin, indicating that it is a biotinoprotein. After dialysis or salt fractionation, the enzyme was largely (90%) inactivated and could be reactivated by the T A he anaerobic dissimilation of citric acid by cell suspensions of Aerobacter indologenes (Brewer and Werkman, 1939) and by cell-free extracts of Aerobacter aerogenes (Dagley and Dawes, 1935a) involves the cleavage of citrate to oxalacetate (OAA)* 1 *and acetate followed by the decarboxylation of OAA to pyruvate. This paper demonstrates that the OAA decarboxylase (OAA 4-carboxy-lyase) of A. aerogenes is a novel inducible enzyme of this type which is sensitive to avidin but not to EDTA. Moreover, it specifically requires sodium ion for activity. Experimental SectionCell Growth and Extraction. A. aerogenes NCTC 418 was grown without aeration at 37°in 20-1. bottles filled to the neck with medium above which a 1-in. layer of sterile mineral oil was placed. The medium used contained per liter of distilled water: monobasic potassium phosphate, 2 g; ammonium sulfate, 1 g; magnesium sulfate-7H20, 0.4 g; and 21 g of trisodium citrate-2H20 (Merck, reagent grade). It was adjusted to pH 7.0 with 7 n sodium hydroxide. A 1 % inoculum of similarly grown cells was added and after 24 hr the newly grown cells were cooled to stop gas production and harvested with a refrigerated Sharpies centrifuge. The harvested unwashed cells were stored at -20°for up to 8 months before extraction without

Metabolic Activities in Extracts of Mesophyll and Bundle Sheath Cells of Panicum miliaceum (L.) in Relation to the C4 Dicarboxylic Acid Pathway of Photosynthesis

“…Malic enzyme has been reported to be low in leaf extracts of P. miliaceumn (18 Reference 9. acetate decarboxylase by the method of Stern (37) and Kosicki (26); and PEP carboxytransphosphorylase by an exchange reaction using NaH`4CO3 as described by Wood, Davis, and Willard (40), without finding measurable activity.…”

Purification of the sodium transport enzyme oxaloacetate decarboxylase by affinity chromatography on avidin sepharose

“…Upon decarboxylation of oxaloacetate an electrochemical gradient of Na' is established and in this way part of the energy of the highly exergonic decarboxylation reaction is conserved. In accordance with this biological function, oxaloacetate decarboxylase is specifically activated by Na' [l] and bound to the cytoplasmic membrane [ 1,6]. The enzyme has been solubilized from the isolated membranes with non-ionic detergents and purified -4.5.fold over the crude membrane extract [6].…”

A mutant phosphoenolpyruvate carboxykinase in Escherichia coli conferring oxaloacetate decarboxylase activity

“…To confirm these results biochemically, we used an NADHlinked enzyme assay, which was modified from a published Oad assay, to detect both Pck activity and Oad activity (17). Briefly, cultures of JCL1305 harboring pCK601, pCK601m1, pCK601m3, or pJF118EH (the cloning vector) were harvested at early stationary phase, spun for 15 min at 12,000 ϫ g, and resuspended in a buffer containing 0.1 M potassium phosphate (pH 8.0) and 4 mM MgCl 2 .…”

“…Samples were taken at 0, 40, and 60 min and assayed for OAA and pyruvate levels in the reaction mixture. OAA levels were determined in a solution containing 0.1 M potassium phosphate buffer pH 7.6 and 0.5 M NADH by the decrease in A 340 due to a stoichiometric decrease in NADH after the addition of malate dehydrogenase to this assay mixture (17). After A 340 stabilized, lactate dehydrogenase was added, and the second decrease in A 340 was used to quantitate the amount of pyruvate.…”