Several properties of the cytosolic cholesterol ester hydrolase from bovine adrenal cortex were investigated and those properties were compared directly with those of the well-characterised hormone-sensitive lipase, the ratelimiting enzyme in adipose tissue lipolysis. Properties examined included : (a) activity against different substrates; (b) susceptibility to inhibition by NaF, Hg2+ ions and diisopropyl fluorophosphonate; (c) subunit molecular weight as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate ; (d) ability to serve as a substrate for cyclic AMP-dependent protein kinase; (e) effect of phosphorylation on enzyme activity: and (f) degradation pattern of polypeptides following limited proteolysis.In all respects the two enzymes exhibited essentially identical characteristics. It is therefore concluded that the same protein, or two very similar proteins, catalyses the hydrolysis of cholesterol esters in adrenal cortex and lipolysis in adipose tissue.The implication of this finding is discussed in relation to the hormonal control of steroidogenesis in adrenal cortex and of lipolysis in adipose tissue.Cholesterol ester hydrolase catalyses one of the ratelimiting steps in steroidogenesis in the adrenal cortex [I]. This enzyme catalyses the release of free cholesterol from stores of cholesterol esters present as lipid droplets in the cytoplasm. The free cholesterol is then transported into the mitochondria where it is converted to pregnenolone by the side-chain cleavage enzyme system. Following stress induced by ether anaesthesia, which is known to elevate the circulating levels of corticotrophin, the cholesterol ester stores are depleted and the activity of the cholesterol ester hydrolase is elevated [2]. Corticotrophin is thought to exert most of its effects via the actions of cyclic AMP, and it has been shown that, in the high-speed supernatant from adrenal cortex, the cholesterol ester hydrolase becomes activated following incubation with cyclic AMP and Mg-ATP [3], suggesting that activation results from phosphorylation of the enzyme.In addition to cholesterol esters, the lipid droplets contain significant levels of phospholipids and triacylglycerol, and adrenal cortex contains a triacylglycerol hydrolase which also becomes activated in response to corticotrophin [4]. This has led to the suggestion that one enzyme may catalyse both the cholesterol ester and triacylglycerol hydrolase activities and indeed the two activities co-purify through a limited purification procedure [ 5 ] . The ratio of the two activities does vary, however, in fractions obtained by precipitation at different concentrations of ammonium sulphate and the cholesterol ester hydrolase is more susceptible to inhibition by active Ahhrc.~'icrrions. DFP, diisopropylfluorophosphonate; corticotrophin, adrenocorticotrophic hormone; QAE-Sephadex, quaternary diethylL(2-hydroxypropy1)aminoethyl-Sephadex.En=~w?e.s. Cholesterol esterase or cholesterol ester hydrolase (EC 3.1.1.13): triacylglycerol lipase or...

Bovine kidney mitochondria1 branched-chain 2-oxoacid dehydrogenase complex is inactivated by covalent phosphorylation catalysed by a specific protein kinase intrinsic to the complex. It has been shown previously [Cook, K. C., Lawson, R. and Yeaman, S. J. (1983) FEBS Lett. 157,59-621 that tryptic digestion ofphosphorylated complex releases three phosphopeptides, indicative of multisite phosphorylation.In this communication we report several findings. The mitochondria1 branched-chain 2-oxoacid dehydrogenase complex catalyses a rate-limiting step in the oxidation of the essential branched-chain amino acids, namely leucine. isoleucine and valine [l]. This complex is analogous to the well-characterised pyruvate and 2-oxoglutarate component ( E2) around which are arranged the oxoacid dehydrogenase (E 1) and dihydrolipoyl dehydrogenase (E3) components. As with the mammalian pyruvate dehydrogenase complex the E l component of the branched-chain 2-oxoacid dehydrogenase consists of two types of subunit, termed x and /j [2]. The activity of the branched-chain 2-oxacid dehydrogenase complex is regulated by phosphorylation of the x subunit of the E l component by a protein kinase intrinsic to the complex [3 -51.I t has been shown previously by ourselves [6] and others [7] that more than one site on the s1 subunit can be phosphorylated by the kinase. The same sites are also phosphorylated within intact mitochondria 183. Inactivation of the complex in i9itr.o apparently correlates with phosphorylation of one site [6] and the amino acid sequence surrounding that site has been determined [9].Previously we have shown that tryptic digestion of phosphor-ylated complex yields three phosphopeptides termed T1, T3 and T3 [6], the major inactivating site being contained in peptitlc T1. In this communication we show that these three phosphopcptides contain only two sites of phosphorylation.The sequence around the second site has been determined. Both sites can be dephosphorylated in vitro by the cytosolic protein phosphatases 2A and 2C. Re-activation of the complex correlates with dephosphorylation of the site contained in tryptic phosphopeptide TI. MATEKlALS AND METHODSAll materials were of the highest available purity and were IS described in [5]. EnzymesBranched-chain 2-oxoacid dehydrogenase was purified from bovine kidney as described in [ 5 ] except that the final preparation was resuspended in 50 mM Tris/HCl pH 7.0 in place of 30 mM sodium phosphate. Phosphorylated branched-chain 2-oxoacid dehydrogenase was prepared as in [5]. The catalytic subunits of protein phosphatases 1A and 2A were purified to homogeneity from rabbit skeletal muscle by Mr H. Y. Lim-Tung [lo]. Protein phosphatase 2B was purified from rabbit skeletal muscle up to and including the affi-gel blue chromatography step by Dr A. A. Stewart [I I]. Protein phosphatase 2C was partially purified from rat liver by Professor Philip Cohen, using chromatography on DEAEcellulose and Sephadex GI00 superfine [12]. Trypsin (treated with L-I -tosyl-amido-2-phenylethyl chlorome...

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Branched-chain 2-oxo acid dehydrogenase complex was resolved into component E 1 and E2-kinase subcomplex by gel filtration in the presence of 1 M-NaCl. Essentially all the original activity of the complex can be regained after reconstitution of the component enzymes, reassociation being a rapid process. The specific activities of El and E2 were 25.1 and 19.0 units/mg respectively. Non-phosphorylated active El has an approx. 6-fold higher affinity for E2 than does phosphorylated El. The components of the branched-chain 2-oxo acid dehydrogenase complex do not crossreact with the respective components from the pyruvate dehydrogenase complex. The significance of these results and of the tight association of the kinase with E2 are discussed.Branched-chain 2-oxo acid dehydrogenase complex is a multienzyme complex catalysing the committed step in the breakdown of the essential amino acids leucine, isoleucine and valine. This complex is analogous to the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, and comprises an acyltransferase core (E2) around which are arranged the 2-oxo acid dehydrogenase (El) and dihydrolipoamide dehydrogenase (E3) components. In each complex the enzymes act in sequence to complete the oxidative decarboxylation of their respective substrates. As with the pyruvate dehydrogenase complex, the E I component of the branched-chain 2-oxo acid dehydrogenase complex consists of two polypeptides, termed a and fi. The activity of the complex can be controlled by phosphorylation/dephosphorylation of the a-subunit of the El component by a kinase intrinsic to the complex (Fatania et al., 1981;Odessey, 1982;Lawson et al., 1983). The kinase phosphorylates two sites closely grouped on the asubunit, and the amino acid sequence surrounding these sites has been determined, inactivation correlating closely with phosphorylation of one site (Cook et al., 1984). The amino acid sequence surrounding this site shows a distinct homology with Abbreviation used: SDS, sodium dodecyl sulphate.

Branched-chain 2-oxoacid dehydrogenase compIex has been purified to near homogeneity by a simple, rapid procedure. The final product contains endogenous kinase activity capable of phosphorylating and inactivating the compIex. Phosphorylation continues after complete inactivation, indicating the possibility of several phosphorylatabte sites.Branched-chain 2-oxoacid dehydrogenase complex Rapid purification Inactivation (Bovine kidney cortex) Phosphorylation