Mechanistic studies on delta-aminolevulinic acid uptake and efflux in a mammary adenocarcinoma cell line.

Delta-aminolevulinic acid (ALA) is the precursor in the biosynthesis of porphyrins. The knowledge of both the regulation of ALA entrance and efflux from the cells and the control of porphyrin biosynthesis is essential to improve ALA-mediated photodynamic therapy. In this work, we studied the regulation of ALA uptake and efflux by endogenously accumulated ALA and/or porphyrins in murine mammary adenocarcinoma cells. Under our set of conditions, the haem synthesis inhibitor succinyl acetone completely prevented porphobilinogen and porphyrin synthesis from ALA, and led to an increase in the intracellular ALA pool. However, neither intracellular ALA nor porphyrin pools regulate ALA uptake or efflux during the first 15 min of the process. Based on temperature dependence data, ALA but not gamma-aminobutyric acid (GABA) efflux is mediated by a diffusion mechanism. Moreover, the addition of extracellular GABA not only did not influence the rate of ALA efflux but on the contrary it affected ALA uptake, showing the contribution of a saturable mechanism for the uptake, but not for the efflux of ALA from the cells.

Delta-Aminolevulinic acid transport in murine mammary adenocarcinoma cells is mediated by beta transporters.

Delta-aminolevulinic acid, the precursor of porphyrin biosynthesis has been used to induce the endogenous synthesis of the photosensitiser protoporphyrin IX for photodynamic therapy in the treatment of various tumours. The aim of this work was to characterise the delta-aminolevulinic acid transport system in the murine mammary adenocarcinoma cell line LM3 using (14)C-delta-aminolevulinic acid, to finally improve delta-aminolevulinic acid incorporation in mammalian cells. Our results showed that delta-aminolevulinic acid is incorporated into these cells by two different mechanisms, passive diffusion which is important at the beginning of the incubation, and active transport. Specificity assays suggested that the transporter involved in delta-aminolevulinic acid incorporation is a BETA transporter, probably GAT-2.

UGA4 gene expression in Saccharomyces cerevisiae depends on cell growth conditions.

In the yeast Saccharomyces cerevisiae, gamma-aminobutyric acid (GABA) transport is mediated by three permeases: the general amino acid permease GAP1, the proline permease PUT4 and the specific GABA permease UGA4. Expression of UGA4 gene is induced in the presence of GABA. We started a comparative study about UGA4 gene induction in cells grown on different culture media. Results presented here indicate that under certain growth conditions UGA4 permease is constitutive. Therefore, we demonstrate that in S. cerevisiae the UGA4 gene expression depends on cell growth conditions and that its synthesis not always depends on the presence of GABA.

Carbon and nitrogen sources regulate delta-aminolevulinic acid and gamma-aminobutyric acid transport in Saccharomyces cerevisiae.

Evidence has been obtained showing that transport of delta-aminolevulinic acid (ALA), a precursor of porphyrin biosynthesis in Saccharomyces cerevisiae, is mediated by the gamma-aminobutyric acid (GABA)-specific permease, UGA4. In yeast GABA is also incorporated by the general amino acid permease (GAP1) and the specific proline permease (PUT4). The aim of the present work was to carry out a comparative study on the regulation of ALA and GABA transport to confirm our proposal that both compounds share the UGA4 permease. ALA and GABA uptake were measured in cells grown on minimal media with different carbon and/or nitrogen sources. To study the effect of the carbon source on UGA4 permease, ALA and GABA incorporation were measured in D27 strain, lacking GAP1 permease, and grown in proline as the sole nitrogen source, so the activity of PUT4 permease was negligible. The effect of the nitrogen source on UGA4 permease was studied measuring ALA and GABA uptake rates in cells from media with ammonium, proline and urea as nitrogen sources. It was found that the regulation by the carbon source was similar on ALA and GABA transport; they depend equally on the energetic conditions of the cells. Moreover, regulation by the nitrogen source on ALA and GABA uptake was also similar, and identical to that described already for UGA4 permease. These results are further evidence that both compounds, ALA and GABA, share the GABA-specific permease, UGA4.

delta-Aminolevulinic acid uptake is mediated by the gamma-aminobutyric acid-specific permease UGA4.

There is evidence that delta-aminolevulinic acid (ALA), a precursor of porphyrin biosynthesis, and gamma-aminobutyric acid (GABA) would be incorporated into yeast cells by a common permease. The purpose of this work was to confirm this hypothesis and to identify the shared permease. The transport of GABA in Saccharomyces cerevisiae is mediated by three permeases: the general amino acid permease (GAP1), the specific proline permease (PUT4) and a fairly specific GABA permease (UGA4). To determine which of these permeases is also involved in ALA uptake, ALA and GABA incorporations were measured in strains lacking GAP1, UGA4 or GAP1 and UGA4 permeases. Results indicated that ALA is mainly incorporated by UGA4. This was also confirmed by regulatory studies, since ALA uptake was induced by GABA, and it is well known that UGA4 permease is induced by GABA. On the other hand, ALA did not induce the synthesis of this permease. Therefore, we demonstrate here that ALA, which cannot be used as a nitrogen source, is uptaken by S. cerevisiae cells mainly using a permease encoded by a gene subjected to a regulation typical of several nitrogen genes.

GABA uptake in a Saccharomyces cerevisiae strain.

The aim of this work was to characterize the 4-aminobutyric acid (GABA) transport in the Saccharomyces cerevisiae D27 strain, followed by the study of the relationship between 5-aminolevulinic acid (ALA) and GABA transport systems. It was found that the general amino acid permease (GAP) is not active in D27 strain, suggesting that GABA incorporation should be mediated by PUT4 and UGA4 permeases. However, after kinetic studies only one system was detected. It was also shown that GABA uptake is competitively inhibited by ALA. GABA incorporation is regulated by the carbon source but not by the nitrogen source. When cells were grown in the presence of GABA, its entrance was very low.

Evidence that 4-aminobutyric acid and 5-aminolevulinic acid share a common transport system into Saccharomyces cerevisiae.

It has been previously reported that 5-aminolevulinic acid (ALA) and 4-aminobutyric acid (GABA) share a common permease in Saccharomyces cerevisiae (Bermúdez Moretti et al., 1993). The aim of the present work was to determine the relationship between the transport of these compounds in isolated cells. Assessment of amino acid incorporation was performed in S. cerevisiae using 14C-ALA or 3H-GABA. Initial rates of ALA incorporation in cells grown in the presence of 5 mM ALA and 5 mM GABA, were three to four times lower than in cells grown without supplements. Kinetic studies indicate that GABA competitively inhibits ALA transport. During the growth phase GABA uptake was also inhibited by 74% and 60% in the presence of ALA and GABA, respectively. These findings indicate that in S. cerevisiae the structurally related compounds, ALA and GABA, may be incorporated into the cells by a common carrier protein. Should this occur in other lukaryotic cells it may explain the neurotoxic effect attributed to ALA in the pathogenesis of acute porphyrias.

Yeast porphobilinogen deaminase also forms enzyme-pyrrole intermediates.

The enzyme porphobilinogen deaminase (PBG deaminase, EC 4.3.1.8) catalyzes the condensation of four molecules of PBG to give the linear tetrapyrrol, hydroxymethylbilane. It has been shown that this enzyme forms stable mono-, di-, tri- and tetrapyrrole-enzyme covalent complexes. When the enzyme, partially purified in the absence or presence of phenylmethylsulfonyl fluoride (PMSF) and preincubated with PBG, was applied on DEAE-cellulose columns, three peaks with PBG deaminase activity were detected. Using Ehrlich's reagent, it was found that the active peaks corresponded to mono-, di- and tri-pyrrylmethane-enzyme complexes. Therefore, the mechanism of action of PBG deaminase from Saccharomyces cerevisiae also involves the sequential addition of four PBG units, leading to the formation of the enzyme-substrate intermediate complexes, as has already been described for the same enzyme from other sources.

Delta-aminolevulinic acid transport in Saccharomyces cerevisiae.

1. This work represents the first approach to characterize the transport system of haem pathway precursors, such as delta-aminolevulinic acid (ALA), in two strains of Saccharomyces cerevisiae, a wild type, D27, and a HEM R+ mutant. 2. ALA transport occurs unidirectionally by a sole active system with an apparent KM of 0.10 mM, at the optimum pH of 5.0. ALA uptake is influenced by both the carbon and nitrogen source; this suggests a rather complex regulation mechanism. 3. This transport is not mediated by the general amino acid permease (GAP). 4. ALA uptake is strongly inhibited by compounds harboring a methyl-amine terminus suggesting that this group is essential for ALA transport; however, the electric environment of the carboxylic group may be also important for the interaction between ALA and its transporter active site. 5. We have found differences in ALA transport which would indicate a different regulation mechanism for this system in both strain cells.

The role of ALA-S and ALA-D in regulating porphyrin biosynthesis in a normal and a HEM R+ mutant strain of Saccharomyces cerevisiae.

Catabolite repression and derepression on delta-aminolevulinate synthase (ALA-S) and delta-aminolevulinate dehydratase (ALA-D) in a normal yeast strain, D27, and its derived D27/C6 (HEM R+) were investigated. ALA-S and ALA-D activities and intracellular ALA (I-ALA) at different physiological states of the cells were measured. In YPD medium, under conditions of repression and when glucose was exhausted, both strains behaved identically as if the mutation was not expressed. In YPEt medium, however, both ALA-S and ALA-D activities were higher than in YPD, but the I-ALA content and the enzymic activity profiles shown by the two strains were quite different. It appears, therefore, that the mutation causes a deregulation of ALA-S, so that its activity is kept at a high level throughout the cell cycle. This would explain the increased levels of cytochromes present in the mutant. This mutation may affect some regulatory aspect of ALA formation and renders an ALA-S of high activity; moreover, this enzyme species seems to be more stable than in the normal strain.