Here, we show that three enzymes belonging to the 1B group of the aldo-keto reductase (AKR) superfamily, i.e., human placental aldose reductase (AKR1B1), mouse kidney aldose reductase (AKR1B3) and mouse vas deferens protein (AKR1B7), catalyse the reduction of prostaglandin (PG) H(2), a common intermediate of various prostanoids, to form PGF(2alpha) in the presence of NADPH. AKR1B1, AKR1B3 and AKR1B7 displayed higher affinities for PGH(2) (K(m) = 1.9, 9.3 and 3.8 microM, respectively) and V(max) values (26, 53 and 44 nmol/min/mg protein, respectively) than did the human lung PGF(2alpha) synthase (AKR1C3; 18 microM and 4 nmol/min/mg protein, respectively). The PGF(2alpha) synthase activity of AKR1B1 and AKR1B3 was efficiently inhibited by two AKR inhibitors, tolrestat (K(i) = 3.6 and 0.26 microM, respectively) and sorbinil (K(i) = 21.7 and 0.89 microM, respectively), in a non-competitive or mixed-type manner, whereas that of AKR1B7 was not sensitive to these inhibitors (K(i) = 9.2 and 18 mM, respectively). These data provide a molecular basis for investigating novel functional roles for AKR1B members and PGF(2alpha) as mediators of physiological and pathological processes in mammalian organisms.

The DNA bound on the surface of the histone octamer in the nucleosome core particle partially protects the &-amino side-chains of a subset of the lysine residues from reductive methylation. Most of the strongly protected lysines, which probably define the path of the DNA on the octamer surface, are in the globular ('structured') regions of the core histones rather than in the N-terminal or C-terminal 'tails'. Analysis of the protected peptides shows that the three strongest lysine-containing DNA-binding sites in the core histones contain the sequence -Lys/ Arg-Lys-Thr/Ser-. On the assumption that the lysine-containing regions protected from chemical modification are also those found in lysine-DNA cross-links in another study [Mirzabekov et al. (1978) Proc. Nut1 Acad. Sci. USA 75, 41 84 -41 881, particular DNA-protected peptides may be tentatively assigned to particular DNA contact points. T h s leads to a more detailed description of the DNA-binding regions on the octamer surface in the nucleosome core particle. Strong contacts, reflected in strongly protected lysines, may well contribute to the distortion of the DNA from smooth bending [Richmond et al. (1984) Nature (Lond.) 311, 532-5371.A low-resolution (2.0 nm) structure, deduced for the nucleosome core particle, defined in outline the region within the structure occupied by particular histones, and the path of the DNA [1]. Subsequently these features were determined directly from X-ray diffraction analysis of nucleosome core particle crystals at 0.7 nm resolution [2]. However, the details of histone chain folding and of histone-DNA interactions remain at present unresolved. Some interactions may be particularly strong and may be responsible for distortion of the DNA from otherwise smooth bending [2]. They seem likely to involve electrostatic linkages between lysine and arginine side-chains on the surface of the histone octamer and DNA phosphates.We describe here a procedure for specifically radiolabelling lysine residues that are afforded protection against chemical modification in the native structure by the DNA. These are presumed to be engaged in electrostatic interaction with DNA phosphates and to direct the path of the DNA on the octarner surface. MATERIALS AND METHODSPreparation of nucleosome core particles and 'tail-less' core particles Chromatin was prepared from chicken erythrocyte nuclei [4] at AZ60 = 100 (read in 1 M NaOH) by digestion with micrococcal nuclease (5 units/ml nuclei for 4.25 min at 37°C) NazEDTA, pH 7.5. H1 and H5 were removed by treatment with the cation-exchange resin, AG5OW-X2 [6] and the stripped chromatin was dialysed into 10 mM Tris/HCl, 0.1 mM Na2EDTA, 0.25 mM phenylmethylsulphonyl fluoride (PMSF), pH 7.5, concentrated against solid sucrose and redialysed against the same buffer. It was digested to 146-basepair nucleosome core particles with micrococcal nuclease, under conditions determined from an analytical time course of digestion and analysis of the extracted DNA in a 6% polyacrylamide/7 M urea gel. The bulk digest wa...

The bifunctional enzyme CoA synthase can be nicked by trypsin without loss of its activities. The original dimer of subunit M r approx. 61000 yields fragments of M r 41000 and 22000 as seen on gel electrophoresis in the presence of SDS, but the nicked enzyme retains the native M r of 118 000. Further proteolysis occurs rapidly in the absence of protecting substrates. The N‐terminal of native CoA synthase is proline, and proteolysis exposes glycine as a second N‐terminal. This evidence strongly suggests that the subunits are identical.