H2-M3 is an MHC class Ib molecule of the mouse with a unique preference for N-formylated peptides, which may come from the N-termini of endogenous, mitochondrial proteins or foreign, bacterial proteins. The crystal structure of M3 revealed a hydrophobic peptide-binding groove with an occluded A pocket and the peptide shifted one residue relative to class Ia structures. The formyl group is held by a novel hydrogen bonding network, involving His9 on the bottom of the groove, and the side chain of the P1 methionine is lodged in the B pocket. M3 is a full-service histocompatibility (H) antigen, i.e. self-M3 can present endogenous peptides as minor H antigens and foreign, bacterial antigens in a defensive immune response to infection; and foreign M3 complexed with endogenous self-peptides.

Purified Drosophila lebanonensis alcohol dehydrogenase (Adh) revealed one enzymically active zone in starch gel electrophoresis at pH 8.5. This zone was located on the cathode side of the origin. Incubation of D. lebanonensis Adh with NAD+ and acetone altered the electrophoretic pattern to more anodal migrating zones. D. lebanonensis Adh has an Mr of 56,000, a subunit of Mr of 28 000 and is a dimer with two active sites per enzyme molecule. This agrees with a polypeptide chain of 247 residues. Metal analysis by plasma emission spectroscopy indicated that this insect alcohol dehydrogenase is not a metalloenzyme. In studies of the substrate specificity and stereospecificity, D. lebanonensis Adh was more active with secondary than with primary alcohols. Both alkyl groups in the secondary alcohols interacted hydrophobically with the alcohol binding region of the active site. The catalytic centre activity for propan-2-ol was 7.4 s-1 and the maximum velocity of most secondary alcohols was approximately the same and indicative of rate-limiting enzyme-coenzyme dissociation. For primary alcohols the maximum velocity varied and was much lower than for secondary alcohols. The catalytic centre activity for ethanol was 2.4 s-1. With [2H6]ethanol a primary kinetic 2H isotope effect of 2.8 indicated that the interconversion of the ternary complexes was rate-limiting. Pyrazole was an ethanol-competitive inhibitor of the enzyme. The difference spectra of the enzyme-NAD+-pyrazole complex gave an absorption peak at 305 nm with epsilon 305 14.5 X 10(3) M-1 X cm-1. Concentrations and amounts of active enzyme can thus be determined. A kinetic rate assay to determine the concentration of enzyme active sites is also presented. This has been developed from active site concentrations established by titration at 305 nm of the enzyme and pyrazole with NAD+. In contrast with the amino acid composition, which indicated that D. lebanonensis Adh and the D. melanogaster alleloenzymes were not closely related, the enzymological studies showed that their active sites were similar although differing markedly from those of zinc alcohol dehydrogenases.

Rates of peroxisomal beta-oxidation were measured as fatty acyl-CoA-dependent NAD+ reduction, by using solubilized peroxisomal fractions isolated from livers of rats treated with clofibrate. Medium- to long-chain saturated fatty acyl-CoA esters as well as long-chain polyunsaturated fatty acyl-CoA esters were used. Peroxisomal beta-oxidation shows optimal specificity towards long-chain polyunsaturated acyl-CoA esters. Eicosa-8,11,14-trienoyl-CoA, eicosa-11,14,17-trienoyl-CoA and docosa-7,10,13,16-tetraenoyl-CoA all gave Vmax. values of about 150% of that obtained with palmitoyl-CoA. The Km values obtained with these fatty acyl-CoA esters were 17 +/- 6, 13 +/- 4 and 22 +/- 3 microM respectively, which are in the same range as the value for palmitoyl-CoA (13.8 +/- 1 microM). Myristoyl-CoA gave the higher Vmax. (110% of the palmitoyl-CoA value) of the saturated fatty acyl-CoAs tested. Substrate inhibition was mostly observed with acyl-CoA esters giving Vmax. values higher than 50% of that given by palmitoyl-CoA.

1. 14C-labelled fatty acyl-CoA esters resulting from beta-oxidation of [U-14C]hexadecanoate by peroxisomal fractions isolated from rats treated with clofibrate showed the presence of the full range of saturated intermediates down to acetyl-CoA. 2. The pattern of intermediates generated was fairly constant. At low concentrations of [U-14C]hexadecanoate (50 microM), decanoyl-CoA was present in lowest amounts. At higher concentrations of [U-14C]hexadecanoate (greater than 100 microM), all intermediates of chain length shorter than 12 carbon atoms (except acetyl-CoA) were present at similar low concentrations; the process of beta-oxidation now resembling chain-shortening of hexadecanoate by two cycles of beta-oxidation. 3. In the absence of an NAD(+)-regenerating system [pyruvate and lactate dehydrogenase (EC 1.1.1.28)] 2-enoyl- and 3-hydroxyacyl-CoA esters were generated, suggesting that re-oxidation of NADH is essential for optimal rates of peroxisomal beta-oxidation in vitro. 4. At high concentrations of [U-14C]hexadecanoate (greater than 100 microM), 3-oxohexadecanoyl-CoA was produced, suggesting that thiolase (acetyl-CoA acetyltransferase; EC 2.3.1.9) can become rate-limiting for peroxisomal beta-oxidation.