A simple spectrophotometric method for the determination of beta-blockers in dosage forms.

A simple, extraction-free spectrophotometric method is proposed for the analysis of some beta-blockers, namely atenolol, timolol and nadolol. The method is based on the interaction of the drugs in chloroform with 0.1% chloroformic solutions of acidic sulphophthalein dyes to form stable, yellow-coloured, ion-pair complexes peaking at 415 nm. The dyes used were bromophenol blue (BPB), bromothymol blue (BTB) and bromocresol purple (BCP). Under the optimum conditions, the three drugs could be assayed in the concentration range 1-10 microg ml(-1) with correlation coefficient (n = 5) more than 0.999 in all cases. The stoichiometry of the reaction was found to be 1:1 in all cases and the conditional stability constant (K(F)) of the complexes have been calculated. The free energy changes (DeltaG) were determined for all complexes formed. The interference likely to be introduced from co-formulated drugs was studied and their tolerance limits were determined. The proposed method was then applied to dosage-forms the percentage recoveries ranges from 99.12-100.95, and the results obtained were compared favorably with those given with the official methods.

Identification of the human mitochondrial S-adenosylmethionine transporter: bacterial expression, reconstitution, functional characterization and tissue distribution.

The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites and cofactors through this membrane, and connect cytoplasmic functions with others in the matrix. SAM (S-adenosylmethionine) has to be transported into the mitochondria where it is converted into S-adenosylhomocysteine in methylation reactions of DNA, RNA and proteins. The transport of SAM has been investigated in rat liver mitochondria, but no protein has ever been associated with this activity. By using information derived from the phylogenetically distant yeast mitochondrial carrier for SAM and from related human expressed sequence tags, a human cDNA sequence was completed. This sequence was overexpressed in bacteria, and its product was purified, reconstituted into phospholipid vesicles and identified from its transport properties as the human mitochondrial SAM carrier (SAMC). Unlike the yeast orthologue, SAMC catalysed virtually only countertransport, exhibited a higher transport affinity for SAM and was strongly inhibited by tannic acid and Bromocresol Purple. SAMC was found to be expressed in all human tissues examined and was localized to the mitochondria. The physiological role of SAMC is probably to exchange cytosolic SAM for mitochondrial S-adenosylhomocysteine. This is the first report describing the identification and characterization of the human SAMC and its gene.

Kinetics of glutamate efflux in rat liver mitochondria.

The transport of glutamate was studied in isolated rat liver mitochondria preloaded with glutamate in the presence of respiratory inhibitors. Glutamate efflux was initiated by dilution of the loaded mitochondria into a glutamate-free medium, and the rate of transport was measured by following the disappearance of glutamate from the mitochondrial matrix following rapid centrifugation through silicone oil. Glutamate efflux was inhibited extensively by bromcresol purple and partially by N-ethylmaleimide, compounds which are both known to inhibit mitochondrial glutamate uptake. The efflux process was stereospecific for L-glutamate and exhibited an activation energy of 19.2 kcal/mol. The rate of glutamate efflux was not affected by changes in the mitochondrial membrane potential. However, a good correlation was observed between the rate of glutamate efflux and the matrix pH, the efflux rate being stimulated by a decrease in matrix pH in the range from 8.0 to 7.2. In contrast, acidification of the incubation medium in the pH range 7.4 to 6.5 inhibited the rate of glutamate efflux. A kinetic analysis was made of the efflux reaction by a computer curve-fitting procedure which fits the experimental data to an integrated rate equation (Williamson, J.R., and Viale, R.O. (1979) Methods Enzymol. 56, 252-278). The results indicated that a fall in the matrix pH primarily caused a decrease in the K'm for matrix glutamate, with little change in V'max. In contrast, a low external pH had an effect on the V'max but not on the K'm for intramitochondrial glutamate. The results are in agreement with a symmetrical sequential model of glutamate transport where the glutamate anion binds to the protonated carrier.

Phenolsulfophthalein dyes as probes in the study of lysozyme activity towards cell wall substrates.

Difference spectra have shown that the dissociation constant associated with the dominant species, formed by the binding of bromophenol blue or bromocresol purple to lysozyme, is not sensitive to pH in the range 6-9.5. This was confirmed from temperature-jump studies. However, the inhibition of lysozyme catalysed cell lysis by these dyes is dependent on pH and ionic strength. In the reaction scheme, which takes note of both these observations, we have to consider the formation of an enzyme-dye substrate complex (EDS) which has the same kcat as does the enzyme-substrate complex (ES). The formation of EDS from ES and free dye (D) is controlled by an ionisable group. Analysis of the data using an equation similar to that of Maurel and Douzou gives a pK of approx. 5.6 for this group and this pK is close to that of histidine-15. The inhibition mainly comes from the difference in the formation constants of EDS and ES. The initial binding site of the substrate (S) in ES is not in the cleft region A-F. The cell lysis takes place after S binds in the cleft, in a subsequent step. The rate constants of this step are included in kcat. (kcat is obtained by analysing activity using the simple Michaelis-Menten kinetics). Inhibition by chitotriose also supports this conclusion. The dye binding site is also suggested to be close to histidine-15. Experimental results support the contention that the electrostatic potential due to the negatively charged cell wall substrate could alter the effective pK of ionisable groups on the enzyme in ES.

Possible mechanisms of the efflux of glutamate from kidney mitochondria generated by the activity of mitochondrial glutaminase.

The transport of glutamate across the inner membrane of kidney mitochondria and the influx of glutamine into the mitochondria was studied using an oxygen electrode, the swelling technique and by continous recording of the activity of the mitochondrial glutaminase by an NH4+-sensitive electrode. It is well known that the enzyme is activated by inorganic phosphate and strongly inhibited by glutamate. 1. Avenaciolide, Bromocresal purple and Bromothymol blue inhibited the respiration of the mitochondria almost completely in the presence of glutamate as substrate but not in the presence of glutamine. Production of aspartate during the oxidation of glutamine was not significantly inhibited by avenaciolide but it was markedly suppressed by Bomocresol purple and Bromothymol blue. 2. Swelling of kidney mitochondria in an isosmotic solution of glutamine and ammonium phosphate was not inhibted by avenaciolide or Bromocresol purple indicating that these substances do not inhibit the penetration of the mitochondrial membrane by glutamine or phosphate. 3. The activity of the mitochondrial glutaminase was strongly inhibited by avenaciolide or Bromocresol purple in the presence of inhibitos of respiration or an uncoupler but not in ther absence. Experimental data suggest that this was caused by the inhibition of glutamate efflux. The addition of a detergent removed this inhibition. On the basis of these observations it was concluded that two mechanisms exist which enable glutamate to leave the inner space of kidney mitochondria: (a) an electrogenic efflux coupled to the respiration-driven proton translocation and the presence of a membrane potential (positive outside) and (b) an electroneutral glutamate-hydroxyl antiporter which is inhibted by avenaciolide and which operates in both directions. Our observations do not support the existence of the electrogenic glutamine-glutamate antiporter or glutamate-aspartate exchange in the mitochondria studied.