A simple procedure is described for the purification of the pyruvate dehydrogenase complex and dihydrolipoamide dehydrogenase from Bacillus subtilis. The method is rapid and applicable to small quantities of bacterial cells. The purified pyruvate dehydrogenase complex (s0(20),w = 73S) comprises multiple copies of four different types of polypeptide chain, with apparent Mr values of 59 500, 55 000, 42 500 and 36 000: these were identified as the polypeptide chains of the lipoate acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and the two types of subunit of the pyruvate decarboxylase (E1) components respectively. Pyruvate dehydrogenase complexes were also purified from two ace (acetate-requiring) mutants of B. subtilis. That from mutant 61142 was found to be inactive, owing to an inactive E1 component, which was bound less tightly than wild-type E1 and was gradually lost from the E2E3 subcomplex during purification. Subunit-exchange experiments demonstrated that the E2E3 subcomplex retained full enzymic activity, suggesting that the lesion was limited to the E1 component. Mutant 61141R elaborated a functional pyruvate dehydrogenase complex, but this also contained a defective E1 component, the Km for pyruvate being raised from 0.4 mM to 4.3 mM. The E1 component rapidly dissociated from the E2E3 subcomplex at low temperature (0-4 degrees C), leaving an E2E3 subcomplex which by subunit-exchange experiments was judged to retain full enzymic activity. These ace mutants provide interesting opportunities to analyse defects in the self-assembly and catalytic activity of the pyruvate dehydrogenase complex.

The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase activities of Bacillus subtilis were found to co-purify as a single multienzyme complex. Mutants of B. subtilis with defects in the pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase complex were correspondingly affected in branched-chain 2-oxo acid dehydrogenase complex activity. Selective inhibition of the E1 or lipoate acetyltransferase (E2) components in vitro led to parallel losses in pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex activity. The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes of B. subtilis at the very least share many structural components, and are probably one and the same. The E3 component appeared to be identical for the pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes in this organism and to be the product of a single structural gene. Long-chain branched fatty acids are thought to be essential for maintaining membrane fluidity in B. subtilis, and it was observed that the ace (pyruvate dehydrogenase complex) mutant 61142 was unable rapidly to take up acetoacetate, unlike the wild-type, indicative of a defect in membrane permeability. A single pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex can be seen as an economical means of supplying two different sets of essential metabolites.

The recently characterized M,-50000 polypeptide associated with mammalian pyruvate dehydrogenase complex, referred to as component or protein X, was shown to incorporate N-ethylmaleimide only in the presence of pyruvate or NADH. Component X, modified with N-ethy1[2,3-'4C]maleimide in the presence of pyruvate. was isolated and subjected to acid hydrolysis. The radioactive products were resolved on an amino acid analyser and these coeluted with products from similarly modified and hydrolysed lipoate acetyltransferase. Preincubation of pyruvate dehydrogenase complex with pyruvate or NADH and acetyl-CoA resulted in a time-dependent diminution of incorporation of radiolabelled N-ethylmaleimide into component X and lipoate acetyltransferase and, correspondingly, in the extent of inhibition of overall complex activity by N-ethylmaleimide.Pyruvate dehydrogenase complex (PDC) from mammalian sources comprises a structural core formed by 60 E2 subunits ( M , 52000) organised into a pentagonal dodecahedron (532 symmetry), to which El( a2p2 = M , 154000) and E3 ( a homodimer of M , 110000) subunits are bound to give a large array of 8.5 x lo6 [2]. Most evidence suggests that only a single lipoyl moiety is present on each E2 polypeptide [2, 31, although there have been some contrary indications [4]. Lipoic acid is covalently attached to E2 polypeptides by means of an amide bond to the Nb-amino group of lysine residues [5]. Studies employing NMR or electron microscopy [6, 71 have shown that the lipoyl groups of E2 subunits are sited on extended regions of polypeptide, which are highly mobile relative to other parts of the multienzyme complex. This would enable any one lipoyl group to interact over more of the complex surface than the length of the lysine-lipoate arm, 1.4 nm, would at first suggest. Limited proteolysis of ox kidney PDC results in the release of a domain bearing lipoic acid from the E2 subunit leaving a structural domain which remains as a high-M, assemblage [8]. Substrate-induced S-acetylation of lipoyl groups on the E2 subunits generates an extra thiol on the adjacent sulphur atom in the dithiolane ring, which may thus be specifically modified with N-ethylmaleimide [9].It has become clear recently that mammalian PDC contains a previously unrecognised M , 50000 polypeptide, which forms a close physical and functional union with the E2 assembly [lo]. In addition the E2 assembly, isolated from the PDC of Saccharomyces cerevisiae, shows a tightly bound polypeptide of approximate M , 50000, which is in similar

The effects of elevated temperature and of digestion with a variety of proteinases on the flocforming ability of flocculent strains of Saccharomyces cerevisiae, both genetically defined (FLO1 and FLO5) laboratory and genetically undefined brewing strains, have been determined. This has permitted classification of the flocculent phenotypes of these strains according to criteria other than quantitative grading of flocculence. The flocculent phenotypes conferred by both the FLO1 and the FLO5 gene were irreversibly lost upon treatment with pronase, proteinase K, trypsin or 2-mercaptoethanol treatments. However, the floc-forming ability of cells of the FLO1 strain ABXL-1D was destroyed by chymotrypsin digestion and was stable to incubation at 70 degrees C, whereas the floc-forming ability of cells of the FLO5 strain ABXR-11A was resistant to the action of chymotrypsin and was heat labile. Tetrad analysis of a cross of these FLO1 and FLO5 strains indicated that the chymotrypsin and heat sensitivity phenotypes were FLO-gene determined. It appears that expression of the FLO1 and FLO5 genes leads to the production of different and characteristic cell-wall proteins underlying their respective flocculent phenotypes.

Component X, the recently recognised subunit of mammalian pyruvate dehydrogenase complex, was shown by immune blotting to be present in all of nine tissues dissected from rat. This finding indicated that component X was not an isoenzyme of the lipoate acetyltransferase (E2) associated with one or a limited number of tissues.Native pyruvate dehydrogenase complex was shown to bind IgG raised to isolated component X, indicating that there were at least some regions of the X subunit exposed at the periphery of the complex.Lipoyl groups of ox heart pyruvate dehydrogenase complex were specifically cross-linked by reaction with phenylene-o-bismaleimide in the presence of pyruvate and the subunits contributing to the products of crosslinking were identified by immune blotting. Species with very high MI containing both E2 and component X, were formed in high yield, as well as apparent E2/E2 and E2/X dimers and trimers and an X/X dimer. These results showed that acetylated lipoyl groups of different E2 and X subunits were able to interact in all possible combinations.The types of cross-linked E2 products formed suggested that two thiols, reactible with phenylene-o-bismaleimide, were rapidly generated in the presence of pyruvate. The results were most easily explained by the presence of two acetylatable lipoyl groups on each E2 polypeptide.Pyruvate dehydrogenase complex (PDC) is responsible for the conversion of pyruvate to acetyl-CoA with the overall reactionIn mammalian systems the enzyme is located in the mitochondrion and is a large multienzyme complex of approximate M I = 8.5 x lo6 formed by an assembly of 60 lipoate acetyltransferase subunits (E2) arranged as a pentagonal dodecahedron, bound to which are multiple copies of pyruvate dehydrogenase (El ) and lipoamide dehydrogenase (E3) subunits [l]. Also bound to the pyruvate dehydrogenase complex, although in lower proportions, is a specific kinase involved in regulation of the complex activity by means of covalent modification. The complex is inactivated by the intrinsic kinase, which phosphorylates the Ela subunit and reactivation is achieved by the action of pyruvate dehydrogenase phosphatase [2]. The phosphatase activity does not copurify with the complex. An additional subunit of MI 50000 has recently been identified and has been designated protein or component X [3,4]. Similarly to E2, component X contains covalently bound lipoic acid [5] which is acetylated in the presence of pyruvate or acetyl-CoA and may be reduced by NADH [6]. Immunological characterisation and comparison of peptides produced by limited proteolysis has indicated that component X is unlikely to be derived from E2 by proteolytic cleavage of the E2 polypeptide [3, 61.Studies employing electron microscopy have shown that the lipoyl groups on E2 subunits of PDC are located on peripherally extended regions of polypeptide [7] and analysis with proton NMR show these to be highly mobile relative to other parts of the complex [S]. This mobility would allow interaction of the lipoic acid at spatially...