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Biochimico et Biophysics Acta, 573 (1979) 197-200 0 Elsevier/North-Holland Biomedical Press

BBA Report

BBA 51239

THE INHIBITION OF STEAROYL-COENZYME A DESATURASE BY PHENYLLACTATE AND PHENYLPYRUVATE

WALTER SCOTT and J. LINDSLEY FOOTE

Department of Chemistry, Western Michigan University, Kalamazoo, MI 49008 (U.S.A.)

(Received November 28th, 1978)

Key words: Stearoyl-CoA desaturase; Phenyllactate; Phenylpyruvate

Summary

The inhibition of oleic acid from stearic acid was studied using a rat liver microsomal preparation. Phenyllactic acid and phenylpyruvic acid are partial- ly inhibitory. This may be related to the low oleic acid to stearic acid ratios found in brains of phenylketonuric persons and experimentally phenylketo- nuric rats.

The ratio of oleic acid to stearic acid has been found to be low in glycero- phospholipids from brains of phenylketonuric persons [l] and rats [2]. Since there are increased concentrations of metabolites such as phenylpyruvic acid and phenyllactic acid [ 31, as a result of the abnormal phenylalanine meta- bolism in phenyl ketonuria, it seemed reasonable that these metabolites might inhibit the conversion of stearic acid to oleic acid thus bringing about the ob- served low ratio. The results are reported here.

The labeled stearic acid and stearoyl-CoA were purchased from New Eng- land Nuclear Corp., Boston, MA. The nonlabeled fatty acids used for diluents and standards were obtained from Applied Science Laboratories, Inc., State College, PA. The metabolites tested and the unlabeled stearoyl-CoA were pur- chased from Sigma Chemical Co., St. Louis, MO.

Rat liver microsomes were isolated as described by Paulsrud et al. [5]. Rats (Charles River Laboratory) were deprived of food for 24 h. Following food deprivation, the rats were refed for 21 h and killed. The livers were homo- genized in two volumes of 0.25 M sucrose, 0.005 M MgC12 at 0°C. The homo- genates were centrifuged to obtain the microsomal fraction. Microsomal sus- pensions were kept frozen until used. Protein levels were determined by the Lowry method as described by Chaykin [6].

The stearoyl-CoA desaturase complex was assayed with two different sets of incubation conditions. One set of conditions using ammonium stearate

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as the substrate was based on Paulsrud et al. [ 51, while the other using stea- royl-CoA was done according to Holloway [ 71. In all cases, a frozen micro- somal suspension was thawed as needed in an ice-water bath, Incubation mix- tures (1.5 ml total volume in 25-ml culture tubes) were placed in a water bath shaker at 39°C for 15 min. At the end of each incubation, the tubes were re- moved from the water bath and immersed in ice-water.

With ammonium stearate the incubation mixture was 67 mM potassium phosphate buffer, pH 7.35, 2.5 mM MgC12, 125 mM sucrose, 2 mM NADH, 100 mM ATP, 215 mM Co A, 0.1 mM ~monium stearate ( [1-14C] stearate, specific activity 0.32 Ci/mol), 3.2 mg microsomal protein (except as indicated). Potential inhibitors were added at concentrations given in the tables. Assays were terminated by the addition of 1.0 ml 10% methanolic HCl. 0.25 mg each of stearic and oleic acid were added in 2.0 ml chloroform/methanol (l:l, v/v). The total lipids were then extracted using the Folch method 181. The addition and mixing of 1 ml chloroform and 1 ml water followed by centri- fugation produced two layers separated by a protein pellicle. 1 ml of the organic layer was removed and dried under a stream of nitrogen. The lipids were transesterified, according to Glass and Christopherson [9]. Unesterified lipids were then esterified [9]. The mixture was again dried under nitrogen and the residue redissolved in 0.10 ml hexane and spotted on a 5% silver ni- trate TLC plate. The plate was developed twice in the same direction using hexane/diethyl ether/acetic acid (94:4:2, v/v/v). The fatty acid methyl esters were visualized by spraying the plates with a 0.2% solution of 2,7-dichloro- fluorescein in methanol. The methyl oleate and methyl stearate spots, as iden- tified by comparison with standards, were scraped off the plate and collected in scintillation vials. The r~ioacti~ty was determined, using a toluene-based scintillation counting solution, in a liquid scintillation spectrophotometer.

With stearoyl-CoA the incubation mixture was 67 mM potassium phos- phate buffer, pH 7.35, 2.5 mM MgClz , 125 mM sucrose, 2 mM NADH, 0.067 mM stearoyl-CoA ([ 1-i4C] stearoyl-CoA, specific activity 2.1 Cifmol), and 6.3 mg microsomal protein (except as indicated). After the incubation, 1.0 ml 8.5% KOH in 95% ethanol was added to each tube. The tubes were then capped with glass marbles and placed in a boiling water bath for 20 min. Following saponification, the tubes were cooled to room temperature and then 2.0 ml of a chloroform/methanol/hexane (1 :1:78, v/v/v) solution containing 0.25 mg each of stearic acid and oleic acid were added. After acidification with 0.1 ml concen~ated HCl, 1.0 ml from the upper (hexane) layer was re- moved. The remaining solution was extracted 4 times by adding 1.2 ml hexane, vortexing, and withdrawing 1.Q ml of the hexane layer. The combined hexane layers were evaporated to dryness under a stream of nitrogen. Methyl- ation was then carried out as above [9]. The samples were dried again under nitrogen and treated the same as the methyl esters of the ammonium stearate assay.

The activity of the desaturase complex as a function of the quantity of microsomal protein present is given in Table I. The amount of stearate desatu- rated was a linear function of the amount of protein present.

Table II presents results showing the activity of the desaturase in the presence of pheny~ac~te or phenylpy~vate. Both showed partial ~hibition.

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TABLE I

OLEIC ACID FORMED WITH VARYING MICROSOMAL PROTEIN

Enzyme preparations varied considerably in activity. It should not be concluded that ammonium stearate is a better substrate than stearoyl-CoA. Percent average deviations were 8.7 and 7.0. respec- tively for the two substrates.

Substrate Protein (mg) Oleic acid formed (nmol)

nmol/mg protein

Ammonium 1.6 1.44 0.90 stearate 2.4 1.83 0.76

3.2 2.66 0.80 4.0 3.86 0.96

Stearoyl coenzyme A

t: 0.96 0.30 1.76 0.37

6.3 2.08 0.33

TABLE II

INHIBITION OF OLEIC ACID FORMATION BY 100 @I PHENYLLACTATE OR PHENYLPYRWATE*

Added inhibitor Oleic acid formed (nmol)

Expt. 1 Expt. 2 Expt. 3 Expt. 4

None 7.46 4.89 2.78 7.92 Phenyllactate 3.40 3.82 Phenylpyruvate 2.26 5.82

Percent inhibition 64 22 19 27

*Ammonium stearate substrate.

Several other substances were tested for inhibition and had no effect. These included phenylalanine, indolepyruvic acid, and Z-amino-1-phenylethanol. Since phenyllactate appeared to be the most promising inhibitor of the com- pounds tested, it was further tested at varying concentrations using stearoyl- CoA as substrate. These data are shown in Table III. It can be seen that, phenyllactate is a partial inhibitor of the desaturase complex over the concen- tration range of 55-165 PM with maximum effect near 80 PM.

TABLE III

INHIBITION OF OLEIC ACID FORMATION BY VARYING CONCENTRATIONS OF PHENYG LACTATE*

Phenyllactate (j.&f) Oleic acid formed Inhibition (nmol) (96)

0 6.76 27 6.86 64 4.24 26 81 2.08 64

167 2.91 49 333 6.82 -18

- *Stearoyl-CoA substrate.

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The inhibition of the desaturase by phenyllactate and phenylpyruvate is consistent with the initial hypothesis. Furthermore, the brains from rats having experiment~ly induced phenylketon~ia had low oleicjstearic acid ratios following the period of rapid myelination, during which the conversion of phenylalanine to phenyllactate is rapid [lo]. Therefore, during the period of rapid brain development in an experimentally phenylketonuric rat, the phenylalanine concentration is high and is at the same time converted to phenylla~ti~ acid at a rapid rate. The phenyllactate may then inhibit the conversion of stearate to oleate and hence the observed abnormal ratios be- tween these two acids. As discussed before [ 11, since the 24 carbon fatty acids are formed by elongation of the respective 18 carbon acids the same rational could account for the low 24:1/24:0 ratio found in phenylketonuric brain.

References

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J.B. and Frederickson, D.S., eds.), pp. 266-296, McGraw-Hill, New York 4 Strittmatter, P., Spats, L., Corcoran, D., Rogers, M.J., Setlow. B. and Redline, R. (1974) hoc. NatI.

Aead. Sci. U.S. 71.4666-4669 5 Paulsrud, J.R., Stewart, SE., Graff, G. and Holman, R,T. (1970) Lipids 6, 611-616 6 Chaykin, S. (1966) Biochemistry Laboratory Techniques, p. 20, John Wiley, New York 7 Holloway. P.W. (1976) in Methods in Enzymology. (Lowenstein, J.M., ed.), Vol. 35, pp. 263-262,

Academic Press, New York 8 Folch, J., Lees, M. and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509 9 Qass, R.L. and Christopherson, S.W. (1969) Chem. Phys. Lipids 3. 405-408

10 Edwards, D.J. and Blau, K. (1972) Biochem. J. 130, 495-503