Characteristics of taurine transport in rat liver lysosomes.

Taurine (2-aminoethanesulfonic acid) is a unique sulfur amino acid derivative that has putative nutritional, osmoregulatory, and neuroregulatory roles and is highly concentrated within a variety of cells. The permeability of Percoll density gradient purified rat liver lysosomes to taurine was examined. Intralysosomal amino acid analysis showed trace levels of taurine compared to most other amino acids. Taurine uptake was Na(+)-independent, with an overshoot between 5-10 minutes. Trichloroacetic acid extraction studies and detergent lysis confirmed that free taurine accumulated in the lysosomal space. Kinetic studies revealed heterogeneous uptake with values for Km1 = 31 +/- 1.82 and Km2 greater than 198 +/- 10.2 mM. The uptake had a pH optimal of 6.5 and was stimulated by the potassium specific ionophore valinomycin. The exodus rate was fairly rapid, with a t1/2 of 5 minutes at 37 degrees C. Analog inhibition studies indicated substrate specificity similar to the plasma membrane beta-alanine carrier system, with inhibition by beta-alanine, hypotaurine, and taurine. alpha-Alanine, 2-methylaminoisobutyric acid (MeAIB), and threonine were poor inhibitors. No effects were observed with sucrose and the photoaffinity derivative of taurine NAP-taurine [N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonate]. In summary, rat liver lysosomes possess a high Km system for taurine transport that is sensitive to changes in K+ gradient and perhaps valinomycin induced diffusional membrane potential. These features may enable lysosomes to adapt to changing intracellular concentrations of this osmotic regulatory substance.

Vitamin B12 transport by rat liver lysosomal membrane vesicles.

Vitamin B12 (hydroxycobalamin) is endocytosed by mammalian cells as a complex with transcobalamin II and then processed to free B12 in lysosomes. The mechanism by which free B12 becomes available for subsequent cellular metabolism has been uncertain. Lysosomal transport of cyanocobalamin (B12) was examined using membrane vesicles prepared from Percoll gradient purified lysosomes. B12 uptake by vesicles was dependent upon pH and was inhibited by the protonophore CCCP. Transport exhibited saturation kinetics with a Km of 3.5 microM and temperature dependence with a Q10 of 1.8. Uptake of B12 was dependent upon divalent cations and was inhibited by EDTA. Preparation of vesicles in the presence of 100 microM B12 resulted in stimulation of uptake consistent with a mechanism of countertransport. Excess cyanocobalamin, adenosylcobalamin, methylcobalamin, or cobinamide dicyanide inhibited uptake of B12. Trans-stimulation studies showed that only the first three compounds are actually transported species with cyanocobalamin as the preferred substrate. We conclude that lysosomes have a specific transport system for vitamin B12 that results in release of this enzyme cofactor to the cytoplasm.

Similarity of the E1 subunits of branched-chain-oxoacid dehydrogenase from Pseudomonas putida to the corresponding subunits of mammalian branched-chain-oxoacid and pyruvate dehydrogenases.

The genes encoding proteins responsible for activity of the E1 component of branched-chain-oxoacid dehydrogenase of Pseudomonas putida have been subcloned and the nucleotide sequence of this region determined. Open reading frames encoding E1 alpha (bkdA1, 1233 bp) and E1 beta (bkdA2, 1020 bp) were identified with the aid of the N-terminal sequence of the purified subunits. The Mr of E1 alpha was 45,158 and of E1 beta was 37,007, both calculated without N-terminal methionine. The deduced amino acid sequences of E1 alpha and E1 beta had no similarity to the published sequences of the E1 subunits of pyruvate and 2-oxoglutarate dehydrogenases of Escherichia coli. However, there was substantial similarity between the E1 alpha subunits of Pseudomonas and rat liver branched-chain-oxoacid dehydrogenases. In particular, the region of the E1 alpha subunit of the mammalian branched-chain-oxoacid dehydrogenase which is phosphorylated, was found to be highly conserved in the Pseudomonas E1 alpha subunit. There was also considerable similarity between the E1 beta subunits of Pseudomonas branched-chain-oxoacid dehydrogenase and human pyruvate dehydrogenase.

Comparative effects of adriamycin and DNA-non-binding analogues on DNA, RNA, and protein synthesis in vitro.

Drug-DNA binding is claimed to be the basis by which the antitumor antibiotic adriamycin (doxorubicin) inhibits DNA and RNA synthesis in vitro. However, in preliminary studies the DNA-non-binding adriamycin analogue N-trifluoroacetyladriamycin-14-valerate (AD 32) showed somewhat greater inhibition of DNA and RNA synthesis than adriamycin under identical conditions. The kinetics of macromolecule synthesis inhibition induced by adriamycin and AD 32, and the two principal DNA-non-binding metabolites of AD 32, N-trifluoroacetyladriamycin (AD 41) and N-trifluoroacetyladriamycinol (AD 92), have now been subjected to comparative study in cultured CEM (human leukemic lymphoblastic) cells. At equimolar concentrations (10 microM), or at concentrations related to their 50% growth-inhibitory values vs CEM cells, AD 32 was consistently found to be more inhibitory than adriamycin of DNA and RNA synthesis, as measured by the incorporation of tritiated thymidine and uridine, respectively, into acid-precipitable fractions relative to untreated controls. Marked inhibitory activity was apparent with 10 microM AD 32 even at the earliest sampling time (15 min); with adriamycin at the same concentration the maximal effect was not achieved until 3 h. AD 32 at 4.8 microM concentration continued to show strong inhibition of nucleic acid synthesis, whereas adriamycin at 1.0 microM was essentially inactive. Like AD 32, AD 41 and AD 92 showed greater inhibition than adriamycin of DNA and RNA synthesis at the early sampling times, although in all instances the effects of AD 32 were more profound. AD 32 at 10 microM concentration produced a moderate but significant inhibition of the incorporation of tritiated methionine into protein compared with adriamycin, which at this concentration was not active. Parallel HPLC analytical studies with similar drug-treated cultures indicated that, while small amounts of adriamycin were found in cells treated with 10 microM AD 32, the amount of adriamycin present at 15 min was only a small fraction (less than 5%) of the amount of adriamycin achieved at 3 h in cultures treated with 1.0 microM adriamycin, a concentration already shown to be only slightly inhibitory of nucleic acid synthesis under the culture conditions. The present study thus confirms the marked DNA and RNA synthesis-inhibitory effects of AD 32, and establishes that this inhibitory activity is not due to conversion of AD 32 into adriamycin. These findings accordingly call into question the validity of the drug-DNA binding mechanism as the explanation for the nucleic acid synthesis inhibitory effects seen with ADR.

Adriamycin analogues. Preparation and biological evaluation of some thio ester analogues of adriamycin and N-(trifluoroacetyl)adriamycin 14-valerate.

On the consideration that the highly active DNA-nonbinding adriamycin analogues N-(trifluoroacetyl)adriamycin 14-valerate and N-(trifluoroacetyl)adriamycin 14-O-hemiadipate undergo initial metabolic conversion to N-(trifluoroacetyl)adriamycin by the action of nonspecific serum and tissue esterases, a number of N-(trifluoroacetyl)adriamycin 14-thio esters have been prepared and studied for in vitro growth inhibition, vs. human-derived CCRF-CEM leukemic lymphocytes, and in vivo antitumor activity, vs. murine P388 leukemia, relative to the rate of thio ester deacylation induced by esterases present in mouse serum. Products were obtained by reaction of N-(trifluoroacetyl)-14-bromodaunorubicin with thioacetic, thiopropionic, thiobutyric, thiovaleric, and thiobenzoic acids in ethanol, in the presence of potassium carbonate. Because little is known about similar thio ester derivatives of adriamycin itself, the corresponding adriamycin 14-thio esters were also prepared and evaluated for antitumor activity; with these products, determination of their extent of interaction with calf thymus DNA was also performed. For the adriamycin thio ester products, significant in vivo anti-P388 activity was seen with the thioacetate, thiovalerate, and thiobenzoate derivatives, although no compound matched the curative effects of N-(trifluoroacetyl)adriamycin 14-valerate in this system. With respect to the N-(trifluoroacetyl)adriamycin 14-thio ester products, although the corresponding oxo ester analogues are all significantly biologically active, none of the thio ester derivatives showed activity in vitro or in vivo.

Adriamycin analogues. Preparation and biological evaluation of some N-(trifluoroacetyl)-14-O-[(N-acetylamino)acyl]adriamycin derivatives.

In connection with structure-activity studies related to the novel DNA-nonbinding adriamycin analogues N-(trifluoroacetyl)adriamycin 14-valerate (AD 32) and N-(trifluoroacetyl)adriamycin 14-O-hemiadipate (AD 143), we have now prepared a series of N-(trifluoroacetyl)adriamycin derivatives with N-acylamino acid esters at the 14-carbinol position. Target compounds were made by reaction of N-(trifluoroacetyl)-14-iododaunorubicin with the sodium salts of N-acylamino acids generally in dimethylformamide-ethylene glycol solvent. Products were evaluated for in vitro growth-inhibitory activity and, to a limited extent, in vivo antitumor activity in the murine P388 leukemia system. ID50 values for the target compounds vs. cultured CCRF-CEM cells were generally in the same range as those for the above-mentioned DNA nonbinding adriamycin analogues. Of the four compounds tested for in vivo activity, although none was as effective as N-(trifluoroacetyl)adriamycin 14-valerate, all showed significant activity in the P388 assay system, with three of the compounds, at the doses used, being essentially equiactive with an optimal dose of adriamycin. Studies on the rate of esterase-mediated deacylation of the products, in a defined system containing unfractionated mouse serum as the source of enzyme, showed no relationship between the in vitro and in vivo activities of these compounds and the relative ease at which the side-chain ester substituents were hydrolyzed.