Reversal by CoA of palmityl-CoA inhibition of long chain ACYL-CoaA synthetase activity

Biochimictr 11:

rt Biophqsicn

Acttr, 306 (1973)

Elsevier Scientific Publishing Company.

15-20

Amsterdam

- Printed in The Netherlands

BBA 56237

REVERSAL OF LONG

BY CoA OF PALMITYL-CoA CHAIN

ACYL-CoA

TNHIBITION

SYNTHETASE

ACTIVITY

SHRI V. PANDE Clinictrl Rcsetrrch

Institute

of Montrenl,

Motltrecrl

130, Queht~

iCatmiuJ

(Received October 23rd, I 972)

SUMMARY

Low concentrations of palmityl-CoA inhibited the activity of long chain acylCoA synthetase in a reversible manner. The inhibition was competitive with respect to CoA and the apparent K, for palmityl-CoA (4 I’M) was close to the apparent K,, for CoA (3.3 HIM). This effect appears capable of influencing the rate of fatty acid activation in 11iro.

INTRODUCTION

Long chain acyl-CoA esters inhibit the activities of several enzymes in vitro and there has been considerable speculation as to whether these may have a physiological role’-“. There is little doubt that because of their detergent properties long chain acylCoA esters can unspecifically and irreversibly inhibit various enzyme activities probably by causing enzyme inactivation. Accordingly such effects appear unsuitable for being considered for any metabolic role in viva”.“. It is to be anticipated on the other hand, that the probability of those effects being functional in viva would be greater which become manifested at relatively lower levels of long chain acyl-CoA esters, involve reversible effects and result from a relatively specific interaction between the acyl-CoA esters and enzymes. As shown by us’ and subsequently by other investiof mitochondrial adenine nucleotide translocase by low gators8-‘O the inhibition levels of long chain acyl-CoA esters appears to be an effect of this kind. Experiments described below show that at lower levels of palmityl-CoA, the inhibition of microsomal long chain acyl-CoA synthetase (acid: CoA ligase (AMP), EC 6.2. I .2) is also a fully reversible effect and that it is competitive with respect to CoA. This effect is unlike that described previously” in which an irreversible inhibition was seen because of the higher concentration of long chain acyl-CoA ester employed. MATERIALS

Dithiothreitol, CoA, palmityl-CoA, D-pantothenic acid (hemicalcium salt), and n-pantethine were from Sigma; atractyloside from Calbiochem; ATP from P.L.

s. \;. PANDE

16

Biochemicais: [I-‘“C]palmitic acid from Radiochemical Center, other chemicals of high purity from Fisher or from Baker.

Amersham,

and

METHODS

Methods for the preparation of microsomal fraction from rat liver, of protein estimation and of radioactive assay procedure for long acyl-CoA synthetase were as previously described’ I. Exact details of the rea~tioji system employed are given in legends. RESULTS

AND

DISCUSSION

In a previous paper it was noted that when the palnlityl-CoA synthetase was assayed by following the conversion of [I-14C]palmitate to [I-“C]palmityl-CoA only a limited linearity was seen with time of assay” unlike that observed in the hydroxThe possibility that non-linearity in the radioactive assay amate assay procedure’2. was a consequence of product il~llibitioil was investigated and considered unlikely because under the conditions of radioactive assay, addition of palmityl-CoA, AMP and pyrophosphate in amounts expected to accumulate during the course of usual assay were found to be without effect on linearity. However, during the course of these experiments, it was realized that acided palmityl-CoA inhibited the acyl-CoA

0

4

2

I C0ApM

6

x IO

Fig. I. Effect of CoA concentration on the microsomal activation of palmitate in rhc absence 01 presence of added paimityf-CoA. The assay system in a final volume of 4oo jtl contained: 30 mM potassium phosphate (pH 7.41, 5 mM dithiothreitol, 2 mM ATP, 2 mM M&12, different concentrrttions of CoA, IOO /tM [I-‘%‘]pafmitate (2.8 to+ cpminmole) and where shown 2.4 #rM palmityl-CoA, Assays were initiated by the addition of microsomes (2 pg protein). lncub~tf(~ns were for 5 min at 37 ‘C. Y is expressed as nmole [r-“C]palmitrl-CoA formed.

synthetase activity when the concentration of CoA present was low. Subsequent experiments showed that palmityl-CoA behaved as a competitive inhibitor with respect to CoA (Fig. 1). Presence of 2.4 ~ttM palmityl-CoA caused the apparent I(,, for CoA to increase from 3.3 to 8 ;tM without changing the V. The Ki for palmityl-CoA de-

REVERSAL

OF PALMITYL-CoA

INHIBITION

pM PALMIlYL-&A

Fig. 2. Effect of palmityl-COA concentration on the microsomal activation of palmitate at different levels of CoA. Reaction conditions were as described in the legend for Fig. I except that the concentrations of CoA and of palm~tyl-CoA were as shown. V is expressed as nmole ~I-‘~C]palm~t~l-CoA formed.

termined by the method of Dixon13, was 4 ,LIM(Fig. 2). Thus the affinity of the enzyme for CoA and palmityl-CoA was nearly alike. Although above experiments suggested that the inhibition was reversible, the possibility (see ref. 6) that simultaneous presence of CoA offered protection to the enzyme against irreversibie inactivation by palmityl-CoA required investigation. if the latter were true then the preliminary exposure of enzyme to inhibitory concentration of palmityl-CoA in the absence of CoA should result in a loss of activity that TABLE

I

REVERSIBILITY ACTIVITY

OF THE

PALMITYL-CoA

INHIBITION

OF ACYL-CoA

SYNTHETASE

Reaction mixture in a ftnal volume of 325 1’1 contained: 12 gmoles potassium phosphate (pH 7.4), 2 @moles dithiothr~itol, 0.8 gmole ATP, 0.8 Jtmole MgCl,, and vvhere indicated 1.6 nmoles palmitylCoA. Following temperature equ~lib~tion at 37 C, 25 ,~l of microsomes (Z ,~g protein) were added. 5 and 5.5 min later, respectively, were added 0.96 Lmole CoA with 20 Bmoles of potassium phosphate (pH 7.4) and 60 nmoles of [I-‘QZ]paImitate (2.8 IO’ cpm/nmole). Final volume was 400 //I. 5 min after the addition of palmitate, reactions were arrested by the addition of 60 ,uI of 0.5 M H,SO, and chilling tuhes in ice. Following components were then added with continuous mixing in the order given: 20 ~1 of I M Tris-HCI (pH 7.4); 60 /ll of I M KOH; and to serve as carriers .s /cl of I mM palmityCCoA and 30 ~tl of 40 mM potassium palmitate. After keeping tubes at 37 ‘C for _s min the contents were acidified by the addition of IOO ,uI of 0.5 M HzSO,. Free fatty acids were removed by extractions with ether and radioactivity remaining in the aqueous phase due to [i-‘4C]palmityl-CoA formation was deternlilled as described previously”. Pc~fmiryi-CoA prior

inrvbcrtion

4.57* 0.0

_.-..

presenf i~hri)

during

Pafmitrrte ~nmofes 0.33

rrcrivnted

i

1 n.o18*”

0.32 - 0.014 ~ ..-._____ .* Final concentration of added palmityl-CoA during ** Average of four separate analyses with S.E.M.

subsequent

assay

was 4 pM.

I8

S. V. PANDE

would not be reversed by subsequently added CoA. Table B shows, however, that this was not so because even after iI~cLibati(~n of the enzyme with palm~tyl-boa in the absence of CoA. palmityl-CoA inhibition was not evident when the subsequent assay was carried out in the presence of a large excess of CoA. Other experiments showed (data not elaborated) that the extent of inhibition by palmityl-CoA was not enhanced by prior incubation of microsomes with the inhibitory amounts of palmityl-CoA. Clearly in the present case the inhibition of long chain acyl-CoA synthetase by palmityl-CoA was a fully reversible effect. Recently atractyloside has been reported to inhibit ~~litochondrial ATPdependent long chain fatty acid activating enzyme’” and Skrede and Bremer” have observed that increase in CoA concentration counteracted such inhibition. Atractyloside inhibited the microsomal fatty acid activating enzyme also (Table If, Expt I) but unlike that of palmityl-CoA. relatively higher levels of atractyloside were required for the inhibition to be evident. Further. in contrast to that reported for the mitochondrial enzyme”, for the microsomal enzyme and under the assay conditions employed, the inhibitory effectiveness of atractyloside was independent of CoA concentratio (Expt II, Table II).

TABLE

II

INHIUITION

OF

ATRACTY Reaction

system in a final volume

dithiotbrcitol, Co.A

and

incubations

c\-/)I

MICROSOMAL

PALMITYL-CoA

SYNTHETASE

ACTIVITY

BY

LOS1 DE

z mM

ATP,

of 1:tractylosidc. wxc

z mM

of400

Reactions

for 5 min at 37

111contained:

MgClz.

IOO pM

were starlcd

30 mM

potassium

[t-‘JC]palmitatc, by the addition

phosphate

and indicated

(pH 7.4), 5 mM concentrations

of 7 jry of microsomal

of

protein.

C.

I 0.20

50 250 2500

0.19

5

0.17

‘3 60

0.08 O.fO

4o

0.14 0.42

40

0.3

4oo 400

0.43 0.33

I

24 26 -33

Under the conditions (as in Fig. zf where palmityl-CoA was inhibitory simu!taneous inclusion of AMP and of pyrophosphate in amounts twice as much as that of palmityl-CoA did not increase the inhibitory effect any further as compared to that seen with palmityl-CoA alone. Variations in the concentrations of palmitate present were also likewise without effect on the inhibitory effectiveness of palmityl-CoA. It appears therefore, that if paimityl-CoA and CoA compete for a common binding

REVERSAL

OF PALMITYL-CoA

INHIBITION

19

site* on enzyme then competition occurs at a stage where palmityl-CoA is bound to the enzyme largely through the hydrophilic end. However, unlike palmityl-boa. Dpantothenic acid in up to 500 FM and n-pantetheine in up to 1600 PM did not affect the palmityl-CoA synthetase activity when tested in the presence of 5 /IM CoA. These results are in line with the finding of Bar-Tana et n/.” that pantetheine does not substitute for CoA in the palmityl-CoA synthetase reaction. The presently observed effect appears capable of being functional in ~ivo to keep the activation of long chain fatty acids within a check specially under conditions of uniimiting free fatty acid availability and hence it could be specially important for adipose tissue. Unlike in other tissues where generally availability of free fatty acids appears to be an important factor that determines the rate of fatty acid activation”, the situation in adipose tissue is quite different frequently. It is known that due to active lipolysis, the intracellular levels of free fatty acids become so elevated in adipose tissue that this tissue releases free fatty acids into the circulation**. Thus if utilization of long chain acyl-CoA esters were to become slower relative to that of fatty acid activation, then as the concentration of long chain acyl-CoA esters would go up that of CoA would decrease. As a consequence, further activation of fatty acids would slow down. This woutd be appropriate for the occasion as under such conditions continued unchecked activation of fatty acids may not only be meaningless but perhaps undesirable because of possible deleterious effects of increasing levels of long chain acyl-CoA esters in riro. However, if the situation were to change so that utilization of long chain acyl-CoA ester became faster, then as the level of long chain acyl-CoA would decrease that of CoA would increase and this would in turn accelerate the rate of fatty acid activation. Clearly this mechanism could function in viva to control the levels of long chain acyl-CoA esters within desirable limits while allowing rates of fatty acid activation to be influenced by the momentary needs of a cell. Takeda et af.2’ have reported that the ATP-citrate lyase activity is reversibfy inhibited by palmityl-CoA and that CoA counteracts this inhibition. The inhibition of pyruvate kinase activity by palmityl-CoA is also decreased by the presence of CoA (unpublished data). Experiments are underway to determine whether these represent part of a general regulatory mechanism functional in viro and whether a common reaction mechanism, such as acylation of enzymic thiol groups by long chain acylCoA esters, may be involved in some of these cases. ACKNOWLEDGMENTS

Council

of

* The possibility that palmityCCoA and CoA may bind at different sites on enzyme and these bindings cause reversible and opposing coniormational change in the structure of enzyme ref. 6) can not be eliminated at present.

that (see

Canada

This work was supported by grants from the Medical Research (MA-&J) and the Quebec Heart Foundation. I thank Miss Lise Gendron for excellent technical assistance.

** It has been observed in experiments in taitro that an appreciable buildup of free fatty acids can occur in heart 18-20 and a release of free fatty acids from heart has also been noted but it is not known up to what extent such situations may at times be attained in vivn.

20

S. V. PANDE

REFERENCES I Wieland. 0. (1968) Adr. M~‘mb. Disorti. 7, 1-47 2 Srere, P. A. (I 968) in The Mcfaholic Roles 01 Cirnrrc (Goodwin, T. W.. cd.), pp. I 1-2I, Academic Press. New York 3 Garland. I’. B. (1968) in Tiw Alrtcholic Roles of Citrrrr~(Goodwin,T. W., ed.). pp. 41~60. Academic Press. New York 4 Lynen, F. (I 970) in Control Processes irr Multicc~llr~lcrr Orgtrnirnrs (Wolstenholme, G. E. W. and Knight, J., eds), pp. 28-47, J. and A. Churchill. London 5 Taketa, K. and Pogell. B. M. ! 1966) J. Bi:i/. Che/u. 241, 720~726 6 Pande. S. V. and Mead, J. F. ( 1968) J. Biol. C~PIII. 243. 6180~ 6185 7 Pande. S. V. and Blanchaer. M. C. (1971) J. Biol. Chew. 246. 402241 I 8 Harris, R. A., Farmer, E. and Ozawa, T. (1972) Arch. RIOC/IPI)I. Bioph,~~s. 150, ~y,-zog y Lerner, E.. Shug. A. L.., Elson, C. and Shrago. E. (1972) J. Biol. C’hm. 247, r5 I ~-I~Ic) IO Stucki, J. W., Brawand, F. and Walter, P. (1972) O/r. J. Bioclreu~. 27. 181~19t I t Pande, S. V. ( 1972) Biochim. Biophj,s. .drfir 270. 197 -208 I2 Pande, S. V. and Mead, J. F. (I 968) J. Biol. C/wn~. 243. 352236 I I 3 Dixon, M. (1953) Biochrm. J. 55. 170 14 Alexandre, A., Rossi, C. R., Sartorelli. L. and Siliprandi, N.. (1969) FEBS Left. 3, 279-282 t5 Skrede. S. and Bremer. J. (1970) Ew. J. Bioclwur. 14. 465-472 16 Bar-Tana, J., Rose, G. and Shapiro. B. t 1971) Biochem. J. I 22, 3533362 I 7 Pande, S. V. ( I 97 I) J. Bid. Chrnt. 246, 5384-5390 t8 Olson, R. E. (1962) Natwe 195, 5977599 D. (1966) AI):. J. PIIysiol. 2 IO. 280-286 19 Challoner. D. R. and Steinberg, 20 Willenbrands, A. F. (1964) Biochiw. Biop/r),s. Acfrr 84, 607-610 2 I Takeda. Y.. Suzuki, F., Adachi. K. and Tanioka, H. (I 970) 81h N/,/r. Proc. IfIt. Congr. 66-69