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for fixed-output experiments. The obligatory correlation between cardiac output and coronary flow in closed-aorta preparations makes it possible to isolate the metabolic effects of increased work from secondary effects due to alterations in substrate delivery or tissue O2 concentration. The pyruvate dehydrogenase assay system illustrated in Fig. 2 permits a rapid and quantitative transfer of l4COz from the reaction vessel to the scintillation vial. Initially the needle valve is open, the diaphragm valve is closed and there is no scintillation vial in place. The enzyme sample is incubated at atmospheric pressure for 2min at 35°C in 0.5ml of medium with the following final composition: 50m~-potassiumphosphate, pH7.5, 2rn~-MgCI,,0.5 mM-CoA, OS~M-NAD',0.1 mM-thiamin pyrophosphate and 0.5m~-[l-'~C]pyruvate (0.4pCi/pmol). The reaction is terminated by injecting 1ml of 1M-H,SO, through the Luer needle, and the needle valve is at once closed to prevent loss of co2. A new scintillation vial is screwed into position and evacuated by briefly opening the diaphragm valve. The valve contains 0.8 ml of C02-absorption fluid (phenethylamine/ ethanol/water, 5:2:13, by vol.) soaked into 25cm2 glass-fibre filter paper. A syringe is O attached ~ to the Luer needle, and the needle valve containing 1 ml of O . ~ M - N ~ H C is cautiously opened so that the NaHC03 solution is gradually sucked into the reaction vessel, and there is a gentle evolution of C02. The rolled tissue-paper filter prevents spray contamination of the scintillation vial. Evolution of CO, ceases after about 2min and the syringe is removed, allowing a gentle current of air to sweep the remaining COz into the scintillation vial. The vial is removed, and lOml of scintillation 'cocktail' added [Triton X-l14/xylene, 1:3, v/v containing 3 g of 2,5-diphenyloxazole/litreand 0.2g of 1,4-bis-(5-phenyloxazol-2-yl)benzene/litre].The filter paper disperses completely when shaken in this medium. When a poor batch of phenethylamine is used it may be necessary to wait a few minutes before scintillation counting for chemiluminescence to subside. Illingworth, J. A. & Mullings, R. (1976) Biochem. SOC.Trans. 4,291-292 Illingworth,J. A., Ford, W. C. L., Kobayashi,K. &Williamson,J. R. (1975) in Recent Advnnces in Studies of Cardiac Structure and Metabolism (Roy, P. E. & Harris, P., eds.), vol. 8, pp. 271290,University Park Press, Baltimore Neely, J. R., Liebermeister, H., Battersby, E. J. & Morgan, H. E. (1967) Am. J . Physiol. 212, 804-814

Improved Alkylation of Acetoacetyl-Coenzyme A Thiolase by Extension of Chain Length in Chloromethyl Ketone Fatty Acids R. ALAN CHALKLEY and DAVID P. BLOXHAM Department of Physiology and Biochemistry, University of Southatlipton, Southampton SO9 3TU, U.K.

We have initiated a programme to develop specific inhibitors for the enzymes involved in the early stages of cholesterol biosynthesis (Holland et a/., 1973; Bloxham, 1975). In this presentation, we describe studies with chloromethyl ketone fatty acids of the general formula H02C~CH2].-CO-CH2Cl(n = 2,4,6,8), and demonstrate that mitochondria1 acetoacetyl-CoA thiolase (acetyl-CoA-acetyl-CoA C-acetyltransferase, EC 2.3.1.9) has an increase in affinity for those compounds as their chain length increases. Mitochondria1 acetoacetyl-CoA thiolase possesses a cysteine at the active site, which is alkylated by highly rcactive reagents such as iodoacetamide (Gehring at al., 1968; Gehring &Harris, 1970). We were interested in examining the sensitivity of this enzyme to the less-reactive chloromethyl ketone group. For this purpose a series of chloromethyl ketone fatty acids was synthesized. These compounds were synthesized from dicarboxylic acids by a route involving the half-ester, the carboxyethyl acyl chloride, the VOl. 4

280

BIOCHEMICAL SOCIETY TRANSACTIONS

L

I 0

I

2

3

4

5

6

7

8

Time (mid Fig. 1. Improved alkylation of thiolase by extension of chain length in chloromethylketone fatty acids Thiolas was inactivated with chloromethyl ketone fatty acids C1CH2-CO-[CH2J,,-C02H of varying chain length as follows; 0 , n = 2 ( 5 m ~ ) 0; , n = 4 ( l m ~ ) A; , n = 6 ( 0 . 3 m ~ ) ; A , n = 8 (40,uM).

Table 1. Comparisonof alkylation by chlorornethylketone fatty acids and their CoA esters Thiolase was alkylated by either the free acid or the CoA ester. The approximate K , was evaluated for the free acid, but the kinetic pattern with the CoA esters was more complex and only the simplest data are presented. Methylenes in chloromethyl ketone fatty acid n=2 n=4 n=6 n=8

Ki Of free acid

01M) 20000 4300 450 33

Concn. of CoA ester giving 50 % in 5 min (C) @M) 15 2.0 1.4 2.5

CoA enhancement (KiIC)

1333 2150 321 13

carboxyethyl diazoketone and the carboxyethyl chloromethyl ketone. Structures were characterized by i.r. spectroscopy, mass spectrometry and nuclear magnetic resonance. Thiolase was purified from pig heart by a modification of the method of Stern (1955), and had a final specific activity of 0.66-1 pkatlmg when assayed by the method of Lynen et al. (1952). For inactivation experiments, thiolase with an active-site concentration of 6 , u (protomer ~ molecular weight 44000; Gehring & Riepertinger, 1968) was incubated at 20°C in 0.05~-potassiumphosphate, pH7.0, and the reaction was started by addition of a concentrated solution of inhibitor. Fig. 1 shows that, when theenzyme was incubated with the chloromethyl ketone fatty acids, then there was a progressive loss of enzyme activity, which followed pseudo-first-order kinetics for the shortchain compounds. It is also quite clear that, as the number of bridge methylenes increases, the time required for 1976

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inactivation decreases substantially. Further, the effective concentration of the inhibitor also decreases. This observation was unexpected, since, in its normal substrate, heart mitochondria1 8-oxoacyl-CoA thiolase has a marked preference for short-chain substrates, i.e. acetoacetyl-CoA (Stern & Ochoa, 1956).Therefore we decided to investigate whether converting the fatty acids into their CoA esters (Holland et al., 1973) had any differential effect on the ability to alkylate the enzyme. Table 1 compares the inhibitory properties of the chloromethyl ketone fatty acids with their corresponding CoA esters. The conversion into the CoA ester abolishes the clear relation between chain length and inhibitory potency, as all the CoA esters have roughly similar potency. It might have been anticipated, on the basis of theenzymes’ substrate preference, that, in the CoA series, inhibition would have decreased as chain length increased. This is not the case. The only effect consistent with the short-chain-substrate preference of the enzyme is the greater enhancement of inhibitory potency for the conversion of short-chain chloromethyl ketone fatty acids into their CoA esters. Protection experiments showed that the chloromethyl ketone fatty acids and their CoA esters were active-site-directed inhibitors. Either acetoacetyl-CoA ( 1 0 0 ~ or ~) acetyl-CoA ( 1 0 0 ~ provided ~) complete protection against inactivation by any of the inhibitors. At this stage it is assumed that the chloromethyl ketone group is susceptible to nucleophilic attack by the active-site cysteine (Gehring & Harris, 1970). The effect of exogenous thiol on this reaction was tested and showed that concentrations of CoA and dithiothreitol 50 times greater than the concentration of active-site thiol ( 6 , u ~ )only partially protected the enzyme against inactivation. The failure of exogenous thiol to protect against inactivation demonstrates that a non-covalent enzyme-inhibitor complex is strongly favoured, and indicates that the active-site thiol is more reactive than a simple thiol. The increased potency for inactivation of thiolase by extension of the methylene bridge of the chloromethyl ketone fatty acids may reflect a hydrophobic environment around the active-site thiol. Stronger apolar binding would be found where the methylene bridge has been extended. For the CoA esters tested here it is presumed that flexibility of the methylene bridge of these compounds allows folding, so that the chloromethyl ketone becomes close to the active-site thiol. We are grateful to the Medical Research Council for the award of a project grant to D. P. B. and a research studentship to R. A. C. Bloxham, D. P. (1975) Biochem. J. 147, 531-539 Gehring, U. & Harris, J. I. (1970) Eur. J. Biochem. 16, 492498 Gehring, U. & Riepertinger, C. (1968) Eur. J . Biochem. 6, 281-292 Gehring, U., Riepertinger, C. & Lynen, F. (1968) Eur. J. Biochem. 6, 264280 Holland. P. C., Clark, M. G. & Bloxham, D. P. (1973) Biochemistry 12, 3309-3315 Lynen, F., Wessely, 0. & Reuff, L. (1952) Angew. Chem. Znt. Ed. Engl. 64, 687 Stern, J. R. (1955) Methods Enzymol. 1, 581-585 Stern, J. R. & Ochoa, S . (1956) in BiochemicalProblems ofLipids (Popjak, G . & Le Breton, E., eds.), pp. 162-173, Butterworth, London

The Phospholipase Activity from Penicillium notaturn A. SHELTAWY, J. K. MARTIN and D. BORRILL Department of Biochemistry, University of Leeds, 9 Hyde Terrace, Leeds LS2 9LS, U.K.

Previous studies indicated that the lysophospholipase of the crude extract of Penici[liurn notatum can catalyse the hydrolysis of diacyl phospholipids only when the physical condition of their micelles is altered in one of two ways: (i) imparting a negative charge VOl. 4