ANALYTICAL

BIOCHEMISTRY

64, 606-608

SHORT

(

197.5)

COMMUNICATIONS

An Improved Method of Carboxylating

for Assay Enzymes

The fixation of ‘“CO, into an acid stable product is the most direct assay of carboxylase catalyzed reactions. The procedure employed includes termination of the enzymatic reaction with acid, which also liberates unreacted ‘“CO,. Subsequently, an aliquot of assay solution is either applied to filter paper and dried (l), or placed in a scintillation vial and dried at high temperature in a forced draft oven (2). These procedures entail time-consuming and error-prone handling as well as radioactive contamination of the atmosphere in the working area. We report a much improved method for determination of carboxylase activity, which avoids the shortcomings of the hitherto used methods. The new procedure has been used in this laboratory to assay phosphoenolpyruvate carboxylase, acetyl CoA carboxylase, malic enzyme (3) and ribulose 1, 5-diphosphate carboxylase (4) and is illustrated with the assay for acetyl CoA carboxylase. The assay mixture (final volume 0.25 ml, pH 7.5) contains in millimolars: ATP, 4.0: acetyl CoA, 0.24; imidazole. 100; MgCl,, 8.0; dithiothreitol, 2.0: and 25 (0.2 pCi/pM) of ‘“C-bicarbonate in a glass liquid scintillation counting vial. Vials are anchored and warmed in a 30°C circulating water bath, and the reaction is initiated by the addition of enzyme. The vials are then sealed with serum caps linked in a series through inlet and outlet 18 gauge needles interconnected with Tygon tubing (Fig. 1). After 15 min, reaction is terminated by injection of 0.1 ml of 3 N HCl with a Hamilton microliter syringe. Air is circulated through the vials, which remain anchored in the 30°C bath, for 15 min. Air from the outlet needles escapes through two 400 ml 40% NaOH traps connected in series, thus removing unfixed “CO,, and trapping it as sodium carbonate. The sealed assay vials are then moved to a 55°C water bath and taken to dryness under a current of air which has been passed through both Drierite and a Millipore filter holder containing Whatman no. 1 paper. Dried radioactive products are dissolved by sequential addition of 0.1 ml H,O, 3.0 ml ethanol and 15 ml toluene containing 15.1 g/gal 2,5diphenyloxazole. Vials are counted in a Packard Tri-Carb liquid scintillation counter (model 3320) and quenching is corrected by the channels ratio method. 606

SHORT

FIG. needles. lines. 6151.

I. Drawing The needles The letters (b) Tygon

of one \toppered in each vial denote tubing.

are

the following l/d in. o.d.

gauge x I in. td) Serum counting vial. for use with

607

COMMUNICATIONS

vial from connected, x

the

assay rig. respectively,

components: 1V4 in. (c)

vial stopper. 21 mm screw

Ih mm cap.

and of one of the component to the air inlet and air outlet

(a) T connectors. Hypodermic needle. o.d.

plug.

(e)

VIC in., Nalgene Luer-Lok hub.

(

An improved method for assay of carboxylating enzymes.

ANALYTICAL BIOCHEMISTRY 64, 606-608 SHORT ( 197.5) COMMUNICATIONS An Improved Method of Carboxylating for Assay Enzymes The fixation of ‘“CO,...
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