NMR observation of hydrolysis of acetonitrile to acetamide catalyzed by binuclear silver cryptate.

NMR analyses (TOCSY, HMQC and DOSY) indicate that, in the presence of water, acetonitrile is exclusively hydrolyzed to acetamide catalyzed by bisilver cryptate complex, which contains coordinating acetonitrile molecule.

A Nedd8 conjugation pathway is essential for proteolytic targeting of p27Kip1 by ubiquitination.

Temporal control of p27(Kip1) (p27) degradation imposes periodicity in its activity during cell cycle progression and its accumulation during cell cycle exit. Degradation of p27 is initiated by phosphorylation of p27 at Thr-187, which marks the protein for ubiquitination by SCF(Skp2) and subsequent proteolysis by the 26S proteasome. Here we show that the p27 ubiquitination activity in cell extracts depends on the presence of the ubiquitin-like protein Nedd8 and enzymes that catalyze Nedd8 conjugation to proteins. Moreover, we show that reconstitution of the p27 ubiquitination activity of recombinant SCF(Skp2) also requires Nedd8 conjugation pathway components. Inactivation of the Nedd8 conjugation pathway by a dominant negative mutant of the Nedd8-conjugating enzyme Nce1/Ubc12 blocks the ubiquitination and degradation of p27 in cell extracts. Consistent with a role in cell-cycle progression, Nedd8 is expressed in proliferating cells and is itself down-regulated upon cellular differentiation. These results suggest that the Nedd8 conjugation pathway may regulate the turnover of p27(Kip1), independently of p27 phosphorylation, and further establishes the identity of protein components involved in p27 ubiquitination. Finally, these findings provide a direct demonstration of a function for Nedd8 in a biological process.

Nedd8 modification of cul-1 activates SCF(beta(TrCP))-dependent ubiquitination of IkappaBalpha.

Regulation of NF-kappaB occurs through phosphorylation-dependent ubiquitination of IkappaBalpha, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IkappaBalpha is carried out by a ubiquitin-ligase enzyme complex called SCF(beta(TrCP)). Here we show that Nedd8 modification of the Cul-1 component of SCF(beta(TrCP)) is important for function of SCF(beta(TrCP)) in ubiquitination of IkappaBalpha. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCF(beta(TrCP)), phosphorylated IkappaBalpha and beta-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCF(beta(TrCP)) in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed betaTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkappaBalpha required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCF(beta(TrCP)) containing a K720R mutant of Cul-1 only weakly supported IkappaBalpha ubiquitination compared to SCF(beta(TrCP)) containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCF(beta(TrCP)). These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkappaBalpha.

NMR diffusion and relaxation study of drug-protein interaction.

In this work, NMR diffusion and relaxation measurements are applied to the study of the interaction between the anti-inflammatory drug salicylate and the human serum albumin (HSA) in solutions. The self-diffusion coefficients and the spin-lattice relaxation rates of salicylate are measured as a function of the concentration. The dissociation constant, Kd, for drug/HSA complexes and the number of binding sites, n, are evaluated.

Primary structure and disulfide bridge location of arrowhead double-headed proteinase inhibitors.

Two arrowhead proteinase inhibitors (inhibitors A and B) were characterized and their primary structures were determined. Both inhibitors A and B are double-headed and multifunctional protease inhibitors. Inhibitor A inhibits an equimolar amount of trypsin and chymotrypsin simultaneously and weakly inhibits kallikrein. Inhibitor B inhibits two molecules of trypsin simultaneously and inhibits kallikrein more strongly than does inhibitor A. The amino acid sequences of inhibitors A and B were determined by sequencing the reduced and S-carboxamidomethylated proteins and their peptides produced by cyanogen bromide or proteolytic lysylendopeptidase or Staphylococcus aureus V8 protease cleavage. Inhibitors A and B consist of 150 amino acid residues with three disulfide bonds (Cys 43-Cys 89, Cys 110-Cys 119, and Cys 112-Cys 115) and share 90% sequence identity, with 13 different residues. Since the primary structures are totally different from those of all other serine protease inhibitors so far known, these inhibitors might be classified into a new protease inhibitor family.

Preferential binding of androgen to transcriptionally active chromatin in rat prostate.

Rat ventral prostate chromatin was fractionated by the Micrococcal nuclease procedure of Berkowitz and Riggs (Biochemistry 20, 7284-7290, 1981). Four chromatin fractions were obtained and analyzed by gel electrophoresis for their protein and DNA components, by hybridization of their DNAs with an androgen regulated cDNA for transcribed DNA sequence, and by [3H]dihydrotestosterone exchange assay and steroid competition experiment for specific androgen binding. All four chromatin fractions contained a complete complement of histones and DNA in multiples of about 200 base pairs, indicating repeating nucleosome structure. The active chromatin fraction, which has the shortest nucleosome repeats of 186 basepairs, was enriched in the androgen regulated sequence and bound androgen. These results correlate androgen binding sites with transcribed DNA sequence and structurally distinctive domain of chromatin in the rat prostate.

Acceptor proteins of rat prostate residual chromatin.

The androgen acceptor sites of the rat prostate residual chromatin (2 M NaCl insoluble fraction of chromatin) have been determined by steroid exchange assay, binding of translocated androgen-receptor complex in vitro, and solubilization of the acceptor protein(s) from the residual chromatin. Binding of [3H]dihydrotestosterone to the residual chromatin was saturable, displaying high affinity (Kd = 3.1 nM) and low capacity (6.3 nmol/mg of protein). The binding of [3H]dihydrotestosterone by the residual chromatin was androgen specific, as shown by steroid competition experiments. Intrachromatin binding study of translocated 5 alpha-[3H]dihydrotestosterone-receptor indicated that the residual chromatin contained 31% of the total chromatin-bound androgen, thus representing one of the major chromatin-androgen binding sites. The results suggested the presence of acceptor molecules in the residual chromatin with which the androgen-receptor interacted. To ascertain this, the residual chromatin was extracted with phenol, and the phenol-solubilized protein(s) was (were) assayed for acceptor activity by interaction with [3H]dihydrotestosterone-receptor complex. Comparison of phenol-solubilized residual proteins from rat prostate, spleen, and chicken erythrocyte indicated that [3H]dihydrotestosterone-receptor complex bound tissue specifically to the prostate residual protein and that the interaction required the presence of DNA. The possible importance of the residual DNA was examined by reannealing with cloned cDNAs coding for the subunit components of prostatic binding protein, an androgen-regulated oligomeric protein in rat prostate. The rates of reassociation kinetics of the residual DNA with the cDNAs were faster than with total DNA, equivalent to a 3-fold enrichment in prostatic binding protein coding sequences. The high salt resistant residual chromatin acceptor(s) thus appear(s) to be preferentially associated with androgen-activated genes.