.~,Y,,LI ,JLAL BIOCHEMISTRY 71, 209-213 (1976)

An Automated Continuous Assay of Membrane-Bound and Soluble ATPases and Related Enzymes

ALFRED ARNOLD,' H. UWEWOLF,~ BERND P. ACKERMANN,~,~ AND HERMANN BADER'

‘Abteilung Pharmakologie der Unirzersittit Ulm, D-7900 Ulm, Oberer Eselsberg, and Vnstitut ,fitr Biochemie der Universitiit Main;, D-6500 Mainz,

Job.Joachim-Becher-Weg

Received July 3, 1975; accepted October 17, 1975

A simple, rapid, and precise method is described for the continuous automated determination of the activity of membrane-bound enzymes which deliberate inor- ganic phosphate, e.g.. ATPases and phosphatases. The characteristics of this method, which is based on the determination of liberated phosphate in the pres- ence of nucleotides, are: (A) the enzyme reaction can be followed continuously during a certain period, thus providing a higher precision, as compared to other methods in which the enzyme reaction is measured by few distinct deter- minations: (B) the enzyme protein and other (membrane) proteins of the enzyme preparation have not to be removed during the continuous determination of enzyme activity because they remain solubilized after denaturation; and (C) low or moderate concentration of nonionic detergents do not disturb the reading of the absorbancy.

The measurement of ATPase activity by direct determination of re- leased phosphate is, on account of its simplicity, the method of choice as compared to others, e.g., the optical method using an ATP-regenerating system (1) or the radiochemical assay (2). Inorganic phosphate can be measured simply by the method of Fiske and Subbarow (3), as modified by Lacy (4).

Usually, the enzyme reaction is stopped by the addition of a suitable acid, and the precipitated protein is removed by centrifugation (5) or filtration (6). Berenblum and Chain (7) separated the inorganic phosphate from ATP and protein by extracting the phosphomolybdate complex into an organic phase. Kline et al. (8) use the dialysis of the inorganic phosphate into the reducing reagent to separate the released inorganic phosphate from the ATP and the Na, K-ATPase. An automated assay for sonicated Na,K-ATPase activity was described by Josephson et al. (9). Another single-point assay uses the solubilisation of the enzyme pro- tein prior to the phosphate determination (lo- 13).

We now present an automated and continuous method for the direct

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Copyright 0 1976 by Academic Presr. Inc. All rights of reproduction !n any form reserved

210 ARNOLD ET AL.

determination of the inorganic phosphate released from ATP by soluble and membrane-bound ATPases avoiding filtration, centrifugation, extrac- tion, or dialysis. This method combines the method of Schwartz and Bodansky (14) and the methods which solubilize the enzyme protein prior to the phosphate determination.

MATERIALS AND METHODS

Chemicals

Oxidized glutathione was purchased from Serva, Heidelberg, and all other chemicals were from E. Merck, Darmstadt. In all cases, the metal ions were used as chlorides. The water was twice distilled in a quartz apparatus.

Reagents

Acid molybdate. 7 mM ammoniummolybdate ((NHJGMo,0Z4*24 H,O); 0.9 M H&SO,; 0.3 mM potassium-antimonyl-tartrate (KSbO(OOC)z- (CHOH),.1/2 H,O).

Ascorbic acid. 0.114 M ascorbic acid; 10 PM KH,PO,. The phosphate was added in order to decrease adsorption and desorption of Pi in the tube system.

Trichloroacetic acid. 25 mM CC&. COOH, 28 mM sodium dodecyl- sulfate (SDS).

Cleaning solution. After each series of experiments, the tube system was rinsed with a solution containing 0.1 mM EDTA and 1 mM NaOH.

Buffer solution for enzyme test media. 8 mM Tris-maleate, pH 7.1; 80 mM Na’; 3.2 mM reduced glutathione; 0.16 mM oxidized glutathione; 1.6 mM Mg2+; 32 PM Ca2+ (as Ca2+-Mg2+-EDTA-buffer (15) consisting of 0.32 mM EDTA and 0.32 mM (Ca2+ + Mg2+)) or as indicated in Results.

Enzyme. High-affinity Ca2+-dependent ATPase of human erythrocyte membranes was prepared by the method of Wolf (5). In 8 ml buffer solu- tion, the enzyme protein (0.05 mg protein/ml or as indicated in Results) was incubated for 10 min in a water bath at 30°C before starting the reac- tion with 2 ml of substrate. The test volume was 10 ml throughout. The protein concentration of the enzyme suspension was measured by the method of Lowry et al. (16).

Equipment

The manifold for assaying ATPase activity is shown in Fig. 1. An Is- matec 13-canal proportioning pump contains six tubes for the transport of the following solutions (the recommended inner diameters in inches are given in parentheses): ATPase reaction medium (0.056); trichloroacetic acid (0.056); air (0.04); acid molybdate (0.035); ascorbic acid (0.035); and waste (0.081).

CONTINUOUS ASSAY OF ATPases 211

FIG. 1. Flow diagram of the automated ATPase assay. The diameters of pump tubings are shown in inches.

After addition of the substrate to the enzyme reaction medium, the tube is shaken vigorously without delay and a suction pipe, connected with the proportioning pump, is placed into the probe for 7 min. The suction pipe is a glass capillary tube with an inner diameter of 1 mm. After 7 min, the reaction tube is rinsed with water for an additional 3 min.

Immediately behind the pump, the enzyme reaction is stopped by addi- tion of trichloroacetic acid, and the enzyme protein is solubilized by SDS. Then, air, acid molybdate, and ascorbic acid is added consecutively. After passing a mixing coil, the reduction of phosphomolybdate to molybdenum blue takes place within 3 min during the passage of a second coil placed in a water bath of 40°C.

After removal of the air in an air debubbler, the mixture passes an Eppendorf photometer via a flow cell with a 15mm light path. The increase of optical density at 578 nm is recorded by an Eppendorf linear recorder (Kompensationsschreiber 6511).

RESULTS AND DISCUSSION

Schwartz and Bodansky (14) have utilized a continuous flow system to study the time course of alkaline phosphatase. This method cannot be used for membrane-bound enzymes. In order to circumvent this difficulty, we developed a new concept of an automated continuous assay in which the protein of the enzyme preparation is solubilized by SDS as already described by single point assays (lo- 13).

SDS at concentrations of 0.1 to 1.0% has no effect on the color develop- ment (see also 13). Due to the addition of 0.8% (28 mM) SDS, an enzyme suspension containing about 1.3 mg protein/ml remains solubilized and therefore does not disturb the reading of the optical density of the phospho- molybdate complex. Fig. 2 shows the effect of increasing concentrations of Ca2+-dependent ATPase on the liberation of inorganic phosphate.

Since Triton X-100 is a useful tool to solubilize ATPases (17,18), this nonionic detergent is often present during the determination of the ATPase activity. However, this substance reacts quantitatively with ammonium-

212 ARNOLD ET AL.

mg Prote,n/m/

FIG. 2. The effect of increasing concentrations of enzyme on the liberation of inorganic phosphate. Buffer solution as indicated in Reagents. Final substrate concentration was 1 mM MgATP*-. Each point is the mean of eight values. The standard deviation is shown in the graph.

phosphomolybdate yielding a precipitate (19), thus interfering with the reading of the optical density of the phosphomolybdate complex. Due to the addition of SDS to the trichloroacetic acid solution, the presence of membrane solubilizing materials such as Triton X-100 in the enzyme test solution at concentrations up to 0.4% does not disturb the reading of the optical density (see also 13).

For a period of about 7 min, which can be extended easily by increasing the volume of the test medium, the turnover of substrate can be followed directly by recording the complete curve, thus including a very easy control, whether or not the enzyme reaction is in the steady-state phase. This is one of the most important criteria for kinetic investigations of

FIG. 3. Recording of a typical experiment in which the Ca*+ sensitivity of the Ca*+- dependent ATPase was determined. Buffer solution as indicated in Reagents. The final substrate concentration was 1 mM MgATPZ-. Curves: (1) 1 ELM Ca2+; (2) 2 PM Ca2+; (3) blank: (4) 10 pM Ca2+; (5) 40 pM Ca*+; (6) blank. The distance from one vertical line to the next is equivalent to 2 min reaction time. The sensitivity was increased twofold at the recorder.

CONTINUOUS ASSAY OF ATPases 213

enzymes which cannot be seen directly with any other method described. A typical record of the Ca2+-dependent ATPase activity at different Ca2+ concentrations is shown in Fig. 3. The production of inorganic phos- phate is linear with time both in cases of enzyme reaction and of blanks. With this method, it is possible to determine low ATPase activities of less than 10 nmol Pi produced/mg proteinsminute.

Inorganic phosphorus standards were treated in a fashion similar to the enzyme reaction mixture showing linearity of the optical density on the Pi concentration.

This method can also be used for other insoluble phosphohydrolizing enzymes like (Na+ + K+)-ATPase or phosphatases in the presence of proteins.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Barbara Bramesfeld, Deutsches Rotes Kreuz, Bad Kreuznach, for the supply of fresh human erythrocytes. This work was supported by a grant of the Deutsche Forschungsgemeinschaft.

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