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

Dimroth, Peter

doi:10.1016/0014-5793(82)80016-1

Cited by 24 publications

(15 citation statements)

References 19 publications

(17 reference statements)

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“…After disrupting the cells with a French pressure chamber, the membranes were isolated as described [2, 31. Oxaloacetate decarboxylase was solubilized from these membranes with 1.5% Triton X-100 and purified on a monomeric avidinSepharose CL-4B column [8]. The column was prepared as described [lo].…”

Section: Methodsmentioning

confidence: 99%

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Subunit composition of oxaloacetate decarboxylase and characterization of the α chain as carboxyltransferase

Dimroth¹,

Thomer²

1983

European Journal of Biochemistry

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Oxaloacetate decarboxylase from Klebsiella aerogenes was shown to be composed of three different subunits a, b, y with M , 65 000, 34000 and 12000, respectively. On dodecylsulfate/polyacrylamide gels the smallest of these subunits was heavily stained with silver but poorly with Coomassie brilliant blue. All three subunits were resolved and clearly detectable by high-performance liquid chromatography in a dodecylsulfate-containing buffer. Biotin was localized exclusively in the a chain.Freezing and thawing of the isolated membranes in the presence of 1 M LiCl released the a chain which was subsequently purified to near homogeniety by affinity chromatography on monomeric avidin-Sepharose. No p or y chain were detectable in this a chain preparation and no oxaloacetate decarboxylation was catalyzed. The isolated a chain, however, was a catalytically active carboxyltransferase as evidenced from the isotopic exchange between [l-'4C]pyruvate and oxaloacetate. The rate of this exchange reaction was about 9 U/mg protein and was completely independent of the presence of N a + ions. The ease with which the a chain was released from the membrane characterize this subunit as a peripheral membrane protein. The p and y chain, on the other hand, stick so firmlyin the membrane that they are only released by detergents, thus indicating that these are integral membrane proteins. Limited tryptic digestion of oxaloacetate decarboxylase led to a rapid cleavage of the a chain, yielding a polypeptide of M , 52 000 which was devoid of biotin. Degradation of the p chain required prolonged incubation periods and was markedly influenced by Na' ions which had a protective effect against proteolysis. A proton is required in the decarboxylation of oxaloacetate and C 0 2 arises as primary product. The other alternative, i. e. generation of HCO; with H 2 0 as substrate, has been excluded.The sodium transport decarboxylases constitute a unique group of vectorial catalysts converting the chemical energy of decarboxylation reactions into electrochemical gradients of Na [6] were also shown to be sodiumtransport decarboxylases. These three enzymes are remarkably similar in many properties, e. g. in their localization in the cell membrane, the specific activation by Na' ions and in containing biotin as a prosthetic group. Carboxylation of this prosthetic group by carboxyl transfer is the first step in the overall reaction and has been demonstrated for oxaloacetate decarboxylase to be independent of Na' ions [7]. The N1-carboxybiotin enzyme intermediate is subsequently decarboxylated in an Naf-dependent reaction and this drives the uphill transport of Na' ions through the membrane.The biotin prosthetic group of the sodium-transport decarboxylases has tremendously facilitated the purification of these enzymes which is achieved by affinity chromatography on monomeric avidin-Sepharose columns [5, 81. Dodecylsulfate

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Section: Methodsmentioning

confidence: 99%

“…Biotin analyses of the individual subunits had indicated that the prosthetic group was localized in the a chain [8]. Due to the limited sensitivity of this procedure, low amounts of biotin in the other subunits would not be detectable.…”

Section: Subunit Composition Of Oxaloacetate Decarboxylasementioning

confidence: 99%

Subunit composition of oxaloacetate decarboxylase and characterization of the α chain as carboxyltransferase

Dimroth¹,

Thomer²

1983

European Journal of Biochemistry

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“…No influence was observed by this agent on the coupling ratio since the decarboxylation rate also increased. The organisms were grown for about 15 h at 37 "C as described in Materials and Methods after inoculation with 5 % of a freshly grown culture on a medium containing 2 Fig. 7.…”

Section: Reconstitution Of the Sodium Pumpmentioning

confidence: 99%

Purification, characterisation and reconstitution of glutaconyl‐CoA decarboxylase, a biotin‐dependent sodium pump from anaerobic bacteria

Semmler

1983

European Journal of Biochemistry

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Glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans is a biotin enzyme, which is integrated into membranes. It is activated by Triton X‐100 and inhibited by avidin. The results obtained by a combination of both agents indicate that biotin and the substrate‐binding site are located on the same side of the membrane. The decarboxylase was solubilized with Triton X‐100 and purified by affinity chromatography on monomeric avidin‐Sepharose. The enzyme is composed of three types of polypeptides: the group of α chains (Mr 120000–140000) containing the biotin, the β chain (60000) and an apparently hydrophobic γ chain (35000). Sodium ions specifically protected the latter chain from tryptic digestion. It was supposed, therefore, that this chain might function as the Na+ channel. The β and γ chains but not the α chain could be labelled by N‐ethyl‐[14C]maleimide. Similar decarboxylases but with much smaller biotin peptides (Mr 15000–20000) were isolated from Peptococcus aerogenes and Clostridium symbiosum. The decarboxylases from all three organisms could be reconstituted to active sodium pumps by incubation with phospholipid vesicles and octylglucoside followed by dilution. The Na+ uptake catalysed by the enzyme from A. fermentans was completely inhibited by monensin and activated twofold by valinomycin/K+ indicating an electrogenic Na+ pump. The coupling between Na+ transport and decarboxylation was not tight. During the reaction the ratio decreased from initially 1 to 0.2. The three organisms mentioned above and Clostridium tetanomorphum without glutaconyl‐CoA decarboxylase are able to ferment glutamate and require 10 mM Na+ for rapid growth. There is no correlation between the concentration of monensin necessary to inhibit growth and the presence of decarboxylase in these organisms.

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“…Oxaloacetate decarboxylase of Klebsiella aerogenes was prepared as described [4] by chromatography of a solubilized membrane extract on monomeric avidin-Sepharose. The specific activity of enzyme specimens ranged over 20 -47 U/ mg protein.…”

Section: Methodsmentioning

confidence: 99%

“…Oxaloacetate decarboxylase has been highly purified by affinity chromatography on monomeric avidin-Sepharose [4]. The enzyme is composed of three different polypeptide chains 01, j?, y with M, of 65000,34000 and 12000, respectively [5].…”

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confidence: 99%

Kinetic analysis of the reaction mechanism of oxaloacetate decarboxylase from Klebsiella aerogenes

Dimroth¹,

Thomer²

1986

Eur J Biochem

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The mechanism of oxaloacetate decarboxylase of Klebsiella aerogenes was investigated by enzyme kinetic methods. The activity of the decarboxylase was strictly dependent on the presence of Na' or Li' ions. For Li' the K,,, was about 17 times higher and the Vmx about 4 times lower than for Na'. No activity was detectable at Naf concentrations < 5 pM. The curve for initial velocity versus Na+ concentration was hyperbolic.Initial velocity patterns with oxaloacetate or Na' as the varied substrate at various fixed concentrations of the cosubstrate produced a pattern of parallel lines which is characteristic for a ping-pong mechanism. Product inhibition by pyruvate was competitive versus oxaloacetate and noncompetitive versus Na' . Oxalate, a dead-end inhibitor, was competitive versus oxaloacetate and uncompetitive versus Na' . The inhibition patterns are not consistent with a ping-pong mechanism comprising a single catalytic site but are analogous to kinetic patterns observed with the related biotin enzyme transcarboxylase, for which a catalytic mechanism at two different and independent sites has been demonstrated.The kinetic and other data support an oxaloacetate decarboxylase mechanism at two different sites of the enzyme with the intermediate formation of a carboxybiotin-enzyme complex. The first site is the carboxyltransferase which is localized on the a chain and the second site is the carboxybiotin-enzyme decarboxylase which is probably localized on the fl and/or y subunit. Binding studies with oxalate indicated that this is bound with high affinity to the CI chain. The affinity was not affected by Na' or by complex formation with the fi and y subunits. Oxalate protected the decarboxylase from heat inactivation but not from tryptic hydrolysis.The carboxybiotin-enzyme intermediate prepared from oxaloacetate decarboxylase with high specific activity was rapidly decarboxylated in the presence of Na' ions alone. The effect of pyruvate on this reaction, noted previously, probably results from inhomogeneity of the enzyme preparation used which contained a considerable amount of free a subunits.

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

Cited by 24 publications

References 19 publications

Subunit composition of oxaloacetate decarboxylase and characterization of the α chain as carboxyltransferase

Subunit composition of oxaloacetate decarboxylase and characterization of the α chain as carboxyltransferase

Purification, characterisation and reconstitution of glutaconyl‐CoA decarboxylase, a biotin‐dependent sodium pump from anaerobic bacteria

Kinetic analysis of the reaction mechanism of oxaloacetate decarboxylase from Klebsiella aerogenes

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