The lipoate acetyltransferase (E2, Mr 70 000) and protein X (Mr 51 000) subunits of the bovine pyruvate dehydrogenase multienzyme complex (PDC) core assembly are antigenically distinct polypeptides. However comparison of the N-terminal amino acid sequence of the E2 and X polypeptides reveals significant homology between the two components. Selective tryptic release of the 14C-labelled acetylated lipoyl domains of E2 and protein X from native PDC generates stable, radiolabelled 34 and 15 kDa fragments, respectively. Thus, in contrast to E2 which contains two tandemly-arranged lipoyl domains, protein X appears to contain only a single lipoyl domain located at its N-terminus.Pyruvate dehydrogenase complex; Component X; Lipoyl domain; Homology; Component E2; (Bovine heart)

Selective proteolysis of the protein X subunit of native bovine heart pyruvate dehydrogenase complex may be accomplished without loss of overall complex activity. Partial loss of function occurs if Mg2+ and thiamin pyrophosphate are not present during proteinase arg C treatment as these cofactors are necessary to prevent cleavage of the E1 alpha subunit. Specific degradation of component X leads to marked alterations in the general enzymic properties of the complex. Lipoamide dehydrogenase (E3) exhibits a decreased affinity for the core assembly and the complex is much more susceptible to inactivation at high ionic strength. The inactive form of the complex is not readily re-activated by removal of salt. It appears that intact protein X and specifically the presence of its cleaved lipoyl domain is not essential for maintenance of an enzymically active pyruvate dehydrogenase complex. However, this protein has an important structural role in promoting the correct association of E3 with the E2 core assembly, an interaction that is required for optimal catalytic efficiency of the complex.

The arrangement of the large (70,000-Mr) and small (30,000-Mr) subunits of succinate dehydrogenase in the mitochondrial inner membrane was investigated by immunoblot analysis of bovine heart mitochondria (right-side-out, outer membrane disrupted) or submitochondrial particles (inside-out) that had been subjected to surface-specific proteolysis. Both subunits were resistant to proteinase treatment provided that the integrity of the inner membrane was preserved, suggesting that neither subunit is exposed at the cytoplasmic surface of the membrane. The bulk of the small subunit appears to protrude into the matrix compartment, since the 30,000-Mr polypeptide is degraded extensively during limited proteolysis of submitochondrial particles without the appearance of an immunologically reactive membrane-associated fragment: moreover, a soluble 27,000-Mr peptide derived from this subunit is observed transiently on incubation with trypsin. Similar data obtained from the large subunit suggest that this polypeptide interacts with the matrix side of the inner membrane via two distinct domains; these are detected as stable membrane-associated fragments of 32,000 Mr and 27,000 Mr after treatment of submitochondrial particles with papain or proteinase K, although the 27,000-Mr fragment can be degraded further to low-Mr peptides with trypsin or alpha-chymotrypsin. A stable 32,000-34,000-Mr fragment is generated by a variety of specific and non-specific proteinases, indicating that it may be embedded largely within the lipid bilayer, or is inaccessible to proteolytic attack owing to its proximity to the surface of the intact membrane, possibly interacting with the hydrophobic membrane anchoring polypeptides of the succinate: ubiquinone reductase complex.

626th MEETING. SHEFFIELD 77s 4.6, respectively) and insensitivity to typical phosphodicstcrase inhibitors such as methylxanthines. Both enzymes were active in the absence of a metal cofactor but while the cyclic CMP phosphodiesterase was stimulated by Fe3+, Mg?+ and C 0 2 + and inhibited by Zn'+, Mn?+ and Ca?', the multifunctional enzyme differed in that it was stimulated by Mg'+, Ca2+ and Mn" and inhibited by Co?+ and Z n 2 + . Both enzymes were inhibited by phosphate and stimulated by cytosine and cytidine; the multifunctional enzyme, however. was insensitive to organic antioxidants, while the cyclic-CMP-specific phosphodiesterase was stimulated by cysteinc, glutathione and mercaptoethanol. The cyclic-CMP-specific enzyme was stimulated by testosterone and an endogenous protein and inhibited by calmodulin and a second endogenous protein; the multifunctional enzyme was insensitive to each of these factors.Of major significance is the response of thcse enzymes to the presence of other cyclic nucleotides during the hydrolysis of cyclic CMP. The multifunctional phosphodiesterase was competitively inhibited by 2', 3'and 3', 5'-cyclic nucleotides and deoxycyclic nucleotides; the cyclic-CMP-specific enzyme was also inhibited by these compounds, but it was less sensitive and inhibition varied from competitive, noncompetitive t o mixed inhibition dependent upon the nature of the nitrogenous base and the position of the cyclic phosphate ring. The K , of the cyclic-CMP-specific phosphodiesterase was an order of magnitude greater than that of the multifunctional phosphodiesterase, which is present at a higher concentration in most mammalian tissues. As V,,;,, for each enzyme is of the same order of magnitude, in thc absence of compartmentation effects and o f inhibition by other cyclic nucleotides, it can be deduced that the hydrolysis of cyclic CMP to CMP would be catalysed predominantly by the multifunctional phosphodiesterase. In the presence of high cellular concentrations of cyclic AMP and cyclic GMP, cyclic CMP hydrolysis would on the other hand be predominantly catalysed by the cyclic-CMP-specific phosphodiesterase, and sensitize the cyclic CMP turnover rate to changes in concentration of testosterone, calmodulin and the two endogenous proteinaceous effectors of cyclic CMP phosphodiesterase. The relative concentrations of cyclic AMP and cyclic GMP would thus appear t o be crucial factors in determination of the rate of breakdown of cyclic CMP.Although the cellular concentration of cyclic CMP is significantly lower than those of cyclic AMP and cyclic GMP, it is also feasible that cyclic CMP may exert an effect o n purine cyclic nucleotide hydrolysis by competing for the active site of the multifunctional phosphodiesterase. The effect of cyclic CMP on cyclic AMP and cyclic GMP hydrolysis may not be only confined to the activity of the multifunctional phosphodiesterase; analysis of a number of other phosphodiesterase activities from a variety of mammalian tissues has shown that cyclic CMP can act as a competitive, non-c...