Active site labeling of dopamine beta-hydroxylase by two mechanism-based inhibitors: 6-hydroxybenzofuran and phenylhydrazine

6-Hydroxybenzofuran and phenylhydrazine are mechanism-based inhibitors of dopamine beta-hydroxylase (D beta H; EC 1.14.17.1). We report here the isolation and characterization of radiolabeled peptides obtained after inactivation of D beta H with [3H]6-hydroxybenzofuran and [14C]phenylhydrazine followed by digestion with Staphylococcus aureus V8 protease. Inactivation of D beta H with [3H]6-hydroxybenzofuran gave only one labeled peptide, whereas inactivation with [14C]phenylhydrazine gave several labeled peptides. Each inhibitor labeled a unique tyrosine in the enzyme corresponding to Tyr477 in the primary sequence of the bovine enzyme (Robertson, J. G., Desai, P. R., Kumar, A., Farrington, G. K., Fitzpatrick, P. F., and Villafranca, J. J. (1990) J. Biol. Chem. 265, 1029-1035). In addition, [14C]phenylhydrazine also labeled a unique histidine (His249) as well as several other peptides. Examination of the complete peptide profile obtained by high pressure liquid chromatography analysis also revealed the presence of a modified but nonradioactive peptide. This peptide was isolated and sequenced and was identical whether the enzyme was inactivated by 6-hydroxybenzofuran or phenylhydrazine. An arginine at position 503 was missing from the sequence cycle performed by Edman degradation of the modified peptide, but arginine was present in the identical peptide isolated from native dopamine beta-hydroxylase. These data are analyzed based on an inactivation mechanism involving formation of enzyme bound radicals (Fitzpatrick, P. F., and Villafranca, J. J. (1986) J. Biol. Chem. 261, 4510-4518) interacting with active site amino acids that may have a role in substrate binding and binding of the copper ions at the active site.     

Ability of laboratory methods to predict in-use efficacy of antimicrobial preservatives in an experimental cosmetic

Volume 60, no. 12, p. 4553: a present address for S. J. Wells should be given, as follows: (dag) Present address: Cadbury Beverages North America, Trumbull, CT 06611. [This corrects the article on p. 4553 in vol. 60.].     

Impact of Ramadan intermittent fasting on cognitive function in trained cyclists: a pilot study

This study assessed selected measures of cognitive function in trained cyclists who observed daylight fasting during Ramadan. Eleven cyclists volunteered to participate (age: 21.6±4.8 years, VO₂max: 57.7±5.6 ml kg⁻¹·min⁻¹) and were followed for 2 months. Cognitive function (Cambridge Neuropsychological Test Automated Battery (CANTAB), Reaction Time index (RTI) and Rapid Visual Information Processing (RVP) tests) and sleep architecture (ambulatory EEG) were assessed: before Ramadan (BR), in the 1st week (RA1) and 4th week of Ramadan (RA4), and 2 weeks post-Ramadan (PR). Both cognitive tests were performed twice per day: before and after Ramadan at 8-10 a.m. and 4-6 p.m., and during Ramadan at 4-6 p.m. and 0-2 a.m., respectively. Training load (TL) by the rating of perceived exertion (RPE) method and wellness (Hooper index) were measured daily. If the TL increased over the study period, this variable was stable during Ramadan. The perceived fatigue and delayed onset muscle soreness (DOMS) increased at RA4. Sleep patterns and architecture showed clear disturbances, with significant increases in the number of awakenings and light sleep durations during Ramadan (RA1 and RA4), together with decreased durations of deep and REM sleep stages at PR. RTI (simple and multiple reaction index) reaction and movement times did not vary over the study period. The RVP test showed reduced false alarms during Ramadan, suggesting reduced impulsivity. Overall accuracy significantly increased at RA1, RA4 and PR compared to baseline. At RA4, the accuracy was higher at 0-2 a.m. compared to 4-6 p.m. Despite the observed disturbances in sleep architecture, Ramadan fasting did not negatively impact the cognitive performance of trained cyclists from the Middle East.     

Development of system B0,+ and a broad-scope Na(+)-dependent transporter of zwitterionic amino acids in preimplantation mouse conceptuses

The nature and ontogeny of Na(+)-dependent L-alanine transport was examined in mouse eggs and preimplantation conceptuses. Mediated L-alanine uptake was not detected in fertilized or unfertilized eggs, but a small amount of Na(+)-dependent L-alanine transport was detected in 2-cell conceptuses. Na(+)-dependent alanine transport was more rapid at the 8-cell stage of development, and more than 10-fold faster in blastocysts than in 8-cell conceptuses. Analog inhibition analyses were consistent with the interpretation that L-lysine-sensitive and L-lysine-resistant components of transport were present at the 2-cell, 8-cell and blastocyst stages of development. The range of amino acids and their analogs that inhibited the most conspicuous component of alanine transport in blastocysts was consistent with the conclusion that system B0,+ is largely responsible for L-alanine uptake in these conceptuses. Moreover, system B0,+, but not other known systems in blastocysts, became susceptible to activation as these conceptuses approached the time of implantation, so this activation could be involved in implantation. Although the data are consistent with the possibility that system B0,+ is also present in 2-cell and 8-cell conceptuses, the relatively slow L-alanine transport in conceptuses at these earlier stages of development precluded more detailed study of their ability to take up alanine. Similarly, the less conspicuous L-lysine-resistant component of L-alanine transport in blastocysts also may be present in conceptuses as early as the 2-cell stage. The L-lysine-resistant component of L-alanine transport could not be attributed to residual system B0,+ activity, however, because it was inhibited more strongly by trans-OH-L-proline than L-arginine, whereas the reverse was the case for system B0,+. Similarly, L-tryptophan and L-leucine each inhibited system B0,+ more strongly than L-serine or L-cysteine, whereas all four of these amino acids inhibited the L-lysine-resistant component equally well. Moreover, a Hofstee plot for L-alanine influx was consistent with the interpretation that at least two mediated components of Na(+)-dependent L-alanine transport are present in blastocysts. The less conspicuous component of L-alanine transport in blastocysts was relatively susceptible to inhibition by L-leucine and L-tryptophan, but it resisted inhibition by the 'model' system A substrate, MeAIB, and the system ASC inhibitors, L-penicillamine and cationic amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calmodulin-binding Locations on the Skeletal and Cardiac Ryanodine Receptors

Ryanodine receptor types 1 (RyR1) and 2 (RyR2) are calcium release channels that are highly enriched in skeletal and cardiac muscle, respectively, where they play an essential role in excitation-contraction coupling. Apocalmodulin (apo-CaM) weakly activates RyR1 but inhibits RyR2, whereas Ca(2+)-calmodulin inhibits both isoforms. Previous cryo-EM studies showed distinctly different binding locations on RyR1 for the two states of CaM. However, recent studies employing FRET appear to challenge these findings. Here, using cryo-EM, we have determined that a CaM mutant that is incapable of binding calcium binds to RyR1 at the apo site, regardless of the calcium concentration. We have also re-determined the location of RyR1-bound Ca(2+)-CaM using uniform experimental conditions. Our results show the existence of the two overlapping but distinct binding sites for CaM in RyR1 and imply that the binding location switch is due to Ca(2+) binding to CaM, as opposed to direct effects of Ca(2+) on RyR1. We also discuss explanations that could resolve the apparent conflict between the cryo-EM and FRET results. Interestingly, apo-CaM binds to RyR2 at a similar binding location to that of Ca(2+)-CaM on RyR1, in seeming agreement with the inhibitory effects of these two forms of CaM on their respective receptors.