An improved assay for long-chain acyl-CoA synthetase

ANALYTICAL

An

BIOCHEMISTRY

28, 59-64

Improved

Assay for Long-Chain Synthetase

D. J. GALTON Department

AND

(1969)

Acyl-CoA

T. RUSSELL FRASER

of Medicine, Royal Postgraduate London W.12, England

Medical

School,

Received May 13,1968

Fatty acyl-CoA synthetases (EC 6.2.1.3) are a group of enzymes which catalyse the following reaction: CH8(CH2),COOH

+ ATP

+ CoASH s

CH3(CH2),COSCoA

+ AMP

+ PPi

(1)

This reaction is of great importance for the activation of longfatty acids before their oxidation or incorporation into lipid. Current methods for assay of these synthetases are unsatisfactory. The method of Kornberg and Pricer (1) depends on the use of a hydroxylamine buffer which traps the acyl thioester as a hydroxyamic acid. This is then complexed with ferric ions and measured in a spectrophotometer. However, it has been shown that hydroxylamine inhibits the activity of acyl-synthetase (2), and the assay has to be conducted in the presence of unphysiological amounts of K+ ions, added as KOH, to neutralize the hydroxylamine hydrochloride. An alternative method is to measure the disappearance of sulfhydryl groups of coenzyme A either by the nitroprusside reaction or directly in a spectrophotometer (2). This method is relatively insensitive since it attempts to measure removal of coenzyme A in small amounts from a large pool in the incubation medium; and the sensitivity of this assay can be increased only by limiting the concentration of CoA, This paper describes a sensitive radiochemical assay for longchain fatty acyl-synthetases which measures the direct incorporation of 14C-palmitate into palmityl-CoA. Separation of W-labeled reactant from product is achieved on filter paper discs by washing in diethyl ether. chain

59

60

GALTON MATERIAL

AND FRASER AND

METHODS

Source of enzyme. Adipose tissue was obtained from the epididymal fat pads of rats (250300 gm) which were killed by cervical dislocation. Other specimens of adipose tissue were obtained from patients undergoing routine abdominal surgery and the tissue was treated in a manner similar to the rat. The tissue was homogenized (1:l) with KC1/EDTA/Mg2+/buffer (150 miW/5 W/5 miW) in a Potter-Elvehjem mortar. The homogenate was then centrifuged at 1,000~. for 10 minutes and the fat layer and nuclear pellet were discarded. The middle layer was kept for assay. Procedure. The reaction system contained in 1 ml: Tris buffer (pH 8) 50 mM; ATP 1OmiV; CoA 435 a; 2-mercaptoethanol 5 m&f; MgCl, 15mM; KF 12 m&f; and K+ palmitate 2 mM with 0.5 PC of W-1-palmitate. The fatty acid was added as the neutralized potassium salt and before addition it was heated until optically clear. Incubation was carried out under air at 37OC for periods up to 20 minutes. During incubation aliquots (25 ~1) of the reaction mixture were pipetted onto numbered filter paper discs (Whatman 1; 2.4 cm diameter (3) ) and placed in ice-cold trichloracetic acid (5% w/v). At the end of the experiment the filter discs were washed three times with acidified diethyl ether (approximately 10 ml per disc), and after the final wash the discs were dried in air. They were then placed in counting vials containing 10 ml of scintillation solution (POPOPl 0.05%, PPO 0.4 % in toluene) and counted in a Packard liquid scintillation spectrometer (model 314 EX) with an efficiency of 63 %. The efficiency of counting of 14C-standard on paper discs compared to scintillator solution alone was greater than 99%. The counts retained on the filter discs were taken to be a measure of the activity of acyl-CoA synthetase, and activities are expressed as cpm per filter disc. Protein was measured by the method of Lowry (4). RESULTS

Figure 1 demonstrates the adequacy of the three ether washes to remove almost all the 14C-palmitate pipetted onto the discs. No improvements in separation was obtained by prolonging contact of the discs with each ether wash beyond about 5 minutes. However the product of the reaction was removed only slightly from the discs by the ether washes (Fig. 1) and over 95% was retained after five washes for counting. 1 Abbreviations diphenyloxazole.

: POPOP

= 1,4-bis-2 (5-phenyloxazolyl)

benzene,

PPO = 2,5-

ASSAY FOR ACYL-COA

61

SYNTHETASE

ELUTION FROM FILTER DISCS

Number of washes.

FIG. 1. Effect of diethyl ether washes on retention by filter discs of 14Cpalmitate and 14C-pahnityl CoA. 14C-Labeled material was pipetted onto filter discs as described under “Methods” and washed in diethyl ether. Results are means of three experiments. (A) = palmityl CoA (B) = palmitate.

The number of retained counts on the filter discs was shown to be dependent on the presence of ATP, CoA, and Mg2+ in the reaction system (Table 1) , whereas omission of 2-mercaptoethanol and F had little effect. This would be expected from the properties of the reaction in equation 1. The dependence of the reaction on ATP Requirements system Complete -ATP -CoA -2-mercaptoethanol -KF -MgCln

for Activity

TABLE 1 of Acyl-CoA Synthetase in Adipose Tissue Counts

retained,

1832 185 164 1639 976 239

cpm

% activityo

100 1 0 90 49 4

Homogenates of adipose tissue (0.1 ml) were incubated for 10 minutes in the reaction system described under “Methods.” Counts retained on filter discs after three washes in acidified diethyl ether were measured in a Packard liquid scintillation spectrometer. Results are means of four experiments. 0 A zero time blank was subtracted from retained counts before calculating y0 activity.

GALTON

or

AND FRASER

,

,

, l/$Ar;M),

2

1

3

ATP

-

4

5

(mM)

FIG. 2. Dependence of activity of acyl-CoA synthetase on ATP. The reaction was carried out under conditions as described in “Methods” with increasing concentrations of ATP. Measurements of initial velocity were made over the first 10 minutes of incubation.

is shown in more detail in Figure 2 and gives an apparent K,, for of 1.3 X lo-‘M. The reaction was also shown to be linear with time up to 15

ATP

TIME-COURSE HOMOGENATE

ON

AND EFFECT OF ADDED THE SVNTHETASE REACTION

. 400 I/,), 1800

0 r

5

10 TIME

15 (min)

20

1000 kiLl. . 600 l-

200

A

l

,100 HOMOGENATE

,200 -

,300 (mgm

400 prokin)

FIG. 3. Dependence of activity of acyl-CoA synthetase on time and amount of added homogenate. The conditions of assay are described under “Methods” and each point is a mean of four experiments.

ASSAY

FOR

ACYL-COA

SYNTHETASE

63

minutes, and depended in a linear manner on the quantity of enzyme protein added to the system (Fig. 3). In four experiments the incubation was conducted in the presence of 1 M hydroxylamine buffer (pH 8) and after addition of 1 ml perchloric acid (3.5% w/v) the precipitate was collected by centrifugation. This precipitate reacted with ferric chloride Reagent A of Hill (diluted 1 :lOO in ethanol) to give a violet color (6) typical of an insoluble hydroxyamic acid, presumably palmithydroxamic acid. DISCUSSION

The validity of the assay described in this paper depends on the successful separation of 14C-palmitate from its CoA derivative by the washing procedure. Probably two factors contribute to the adequacy of the present separation. First, palmitate is very soluble in ether whereas palmityl-SCoA is not. Second, palmityl-SCoA adsorbs to filter paper more readily than palmitate due probably to ionic sites on the CoA residue. It is of relevance here that the slight polarity conferred by a double bond in oleate was sufficient to render it more difficult to elute from filter paper than palmitate. Evidence that in fact acyl-Co-A synthetase was being measured was provided by the dependence of the reaction on Co-A, ATP, and Mg2+ and a K, for ATP compatible with previous estimates for this enzyme (5). In order to confirm that palmityl-CoA was the product of the reaction, it was isolated as its water-insoluble hydroxamic acid, which then characteristically reacted with ferric ions to give a violet complex with absorption maximum at 520 rnp. The assay presented in this paper has many advantages over previous methods. It measures directly the incorporation of Wpalmitate into its thioester and the sensitivity of the assay can be altered, depending on the activity of the enzyme preparation, by varying the specific activity of the l”C-palmitate. The assay is very simple to perform and is particularly useful for kinetic studies in which the progress of the reaction can be followed continuously by sampling the incubation system. SUMMARY

A new and simple paper disc assay is described for measuring the activity of long-chain fatty acyl-CoA synthetases that depends on measuring “he direct incorporation of W-palmitate into its product. Oleate was found to be unsatisfactory for this procedure due to difficulty of elution from paper discs. The behavior of the assay system is shown to be consistent with the expected properties of the

64

GALTON

AND FRASER

enzyme, and this method has been found particularly kinetic studies in our laboratory.

useful for

REFERENCES 1. KOENBEXUI, A., PBIcm, W. E., JR., J. Biol. Chem. 204,329 (1953). 2. MAHLER, H. R., WAKIL, S. J., AND BOCK, R. M., J. Biol. Chem. 204, 453 (1953). 3. BOLLUM, F. J., J. Bid. Chem. 234,2733 (1959). 4. LGWRY, 0. H., ROSFXROUGH, N. J., Fm, A. L., AND RANDALL, R. J., J. Biof. C&m, 193,265 (1951). 5. bfAHLEE, H. R., in “Biochemists HandJmoK’ (C. Long, ed.), p. 418. Spon, London, 1961. 6. HILL, U. T., And. Chem. 19,932 (1947).