Phenylalanine transport system

We found that LUCK irreversibly inhibited the neutral and general transport systems of Neurospora whereas LCK had little effect on these systems. Leucine is a substrate for both systems inactivated, whereas lysine is a substrate for only the general system. Both TCK and PCK inactivated the specific tyrosine/phenylalanine transport system of Bacillus as well the transport system (s) for neutral, aliphatic amino acids. 

We determined the apparent first-order rate constant for the inactivation of the tyrosine/phenylalanine transport system by several concentrations of PCK. By plotting these data in double-reciprocal form we were able to demonstrate the intermediacy of an enzyme-PCK complex in the inactivation reaction and determine K, values of 194 and 177 itM for PCK inhibition of tyrosine and phenylalanine transport, respectively. In addition, this analysis gives the actual first-order rate constants for the inactivation of tyrosine and phenylalanine transport by the enzyme-PCK complex of 0.016 and 0.012 M", respectively. Given the level of imprecision in the assays using whole-cell systems, these numbers are in agreement. The rate of loss of leucine transport activity is comparable to that for tyrosine and phenylalanine transport activity. 

Distortion of the plasma aminogram in individuals with an aminoaciduria also may lead to a relative failure of brain protein synthesis. Thus, in mice with a deficiency of phenylalanine hydroxylase, the blood concentration of phenylalanine is more than 20 times greater than the control value, leading to partial saturation of the transport system and a diminution in the brain level of neutral amino acids other than phenylalanine. Rates of protein synthesis were concomitantly reduced [8]. 

Fluoro amino acids have been incorporated into peptides, in order to ease the transport or reduce the systemic toxicity. Thus, trifluoroalanine, a powerful inhibitor of alanine racemase, is an essential enzyme for the biosynthesis of the cell wall of bacteria. It has a low antibiotic activity because of its very poor transport. In order to facilitate this transport, the amino acid has been incorporated into a peptide. This delivery allows a reduction of the doses, and thus the toxicity of the treatment is lowered.3-FIuorophenylaIanine (3-F-Phe) is a substrate of phenylalanine hydroxylase, which transforms it into 3-F-Tyr. 3-F-Tyr has a high toxicity for animals, due to its ultimate metabolization into fluorocitrate, a powerful inhibitor of the Krebs cycle (cf. Chapter 7). 3-F-Phe has a low toxicicity toward fungus cells, but when delivered as a tripeptide 3-F-Phe becomes an efficient inhibitor of the growth of Candida albicans. This tripeptide goes into the cell by means of the active transport system of peptides, where the peptidases set free the 3-F-Phe. ... 

There are instances in which toxicants have chemical or structural similarities to endogenous chemicals that rely on these special transport mechanisms for normal physiological uptake and can thus utilize the same system for membrane transport. Useful examples of drugs known to be transported by this mechanism include levodopa, which is used in treating Parkinson s disease, and fluorouracil, a cytotoxic drug. Levodopa is taken up by the carrier that normally transports phenylalanine, and fluorouracil is transported by the system that carries the natural pyrimidines, thymine, and uracil. Iron is absorbed by a specific carrier in the mucosal cells of the jejunum, and calcium by a vitamin D-dependent carrier system. Lead may be more quickly moved by a transport system that is normally involved in the uptake of calcium. 

Drags which have structures similar to that of endogenous nutrients may be taken up by a specialized transport system (carrier-mediated transport, receptor-mediated transcytosis) existing in the brain endothelium for nutrients. For example, drags having a molecular structure similar to a large neutral amino acid may cross the BBB via the neutral amino acid carrier such drags include melphalan (phenylalanine mustard), L-dopa, a-methyldopa, and p-chlorophenylalanine. 

As discussed above, certain nutrients are taken up into the brain by carrier-mediated systems. If a dmg possesses a molecular structure similar to that of a nutrient which is a substrate for carrier-mediated transport (Table 13.1), the pseudo-nutrient dmg may be transported across the BBB by the appropriate carrier-mediated system. For example, the dmg L-dopa crosses the BBB via the neutral amino acid carrier system. Other neutral amino acid dmgs that are transported through the BBB on this transport system are a-methyldopa, a-methylparatyrosine, and phenylalanine mustard. 

A. M. Lynch and J, D, McGivan, Evidence for a single common Na+-dependent transport system for alanine, glutamine, leucine and phenylalanine in brush-border membrane vesicles from bovine kidney, Biochim. Biophys. Acta, 899 176-184 (1987). 

Other data show that under disease state, transport systems may be less available to transport compounds into the brain. It was demonstrated that the transport of phenylalanine by LAT into the brain of patients with phenylketonuria was blocked (120). In addition, EEG analysis revealed that brain activity was acutely disturbed when phenylalanine was given orally without other LNAAs. Following administration with LNAAs, phenylalanine influx was completely blocked and no influence on EEG could be observed. 

Intracellular metabolism of amino acids requires their transport across the cell membrane. Transport of L-amino acids occurs against a concentration gradient and is an active process usually coupled to Na -dependent carrier systems as for transport of glucose across the intestinal mucosa (Chapter 12). At least five transport systems for amino acids (with overlapping specificities) have been identified in kidney and intestine. They transport neutral amino acids, acidic amino acids, basic amino acids, ornithine and cystine, and glycine and proline, respectively. Within a given carrier system, amino acids may compete for transport (e.g., phenylalanine with tryptophan). Na+-independent transport carriers for neutral and lipophilic amino acids have also been described, d-Amino acids are transported by simple diffusion favored by a concentration gradient. 

Cellular accumulation of D-fructose is inhibited by the presence of glycine, L-hydroxyproline, L-lysine, and L-phenylalanine. There is a well known relationship between transport systems for hexose and amino acid in intestine and kidney.22-25 D-Fruetose inhibits transport of L-alanine and glycine in rat intestine, but has no effect on transport... 

Conservation of amino acids filtered at the glomerulus is made possible by the existence of four main transport systems for specific amino acids that facilitate active reabsorption of these amino acids from the proximal tubule. A lack or deficiency of the transport system responsible for the absorption of valine, alanine, cystine, and tryptophan, and of the transport system for arginine, lysine, cystine, and ornithine, leads to excretion of these specific amino acids in urine, which is characterized as renal aminoaciduria to distinguish it from overflow aminoaciduria. In the latter situation, the production of amino acids far exceeds the proximal tubular reabsorption capacity, thus leading to overflow of amino acids into urine. This can occur due to defective metabolism of amino acids, as is the case when phenylalanine cannot be metabolized due to the deficiency of the enzyme phenylalanine hydroxylase, or to the inability to deaminate amino acids in liver disease. 

Figure 2. Mechanism for type II (facilitated) transport in a phenylalanine/chloride system.

The system examined in this study was that of a simple phenylalanine /chloride counter-transport system in which the interior phase was concentrated in chloride anion and the exterior phase contained concentrations of phenylalanine in the range of commercial fermentation titers. Since phenylalanine (like all a-amino acids) is predominantly a zwitterion at pH s between 3.5 and 9.0 (and is thus unable to be transported through the membrane), the pH of the exterior phase was kept above pH 10.0 to insure that the amino acid was present in its transportable anionic form. As mentioned above, the mechanism for this system is shown in Figure 2. 

Patients with classic PKU nsnally appear normal at birth. If the disease is not recognized and treated within the first month of life, the infant gradnally develops varying degrees of irreversible mental retardation (IQ scores freqnently nnder 50), delayed psychomotor matnration, tremors, seiznres, eczema, and hyperactivity. The nenrologic seqnelae may result in part from the competitive interaction of phenylalanine with brain amino acid transport systems and inhibition of neurotransmitter synthesis. These biochemical alterations lead to impaired myelin synthesis and delayed nenronal development, which result in the clinical picture in patients such as Piquet Yuria. Because of the simplicity of the test for PKU (elevated phenylalanine levels in the blood), all newborns in the United States are required to have a PKU test at birth. Early detection of the disease can lead to early treatment, and the nenrologic conseqnences of the disease can be bypassed. 

Large neutral amino acids (LNAA) are contraindicated as a sole source of protein in women with maternal PKU because LNAA do not sufficiently lower blood phenylalanine to within the desired treatment range of 120-360 pmol/L [16]. The proposed mechanism of action of LNAA is to block uptake of phenylalanine into the brain by supplementing other amino acids that share the LAT-1 transport system across the blood-brain barrier (Chap. 11). Some reduction in blood phenylalanine has been seen with LNAA use but not to the degree necessary to protect the fetus [17]. 

In the hyphal cells of P. cyclopium two pools exist for L-phenylalanine a low capacity, peripheral p6ol (the cytoplasm), and a central , expandable pool (the vacuoles) (Fig. 9). L-Phenylalanine is transported by several carrier systems through the plasma membrane and by an active transport system through the vacuole membrane. 

TCK and PCK appeared to give the same results, and we elected to continue our work with PCK since TCK was more difficult to prepare and purify. PCK is a competitive inhibitor of the transport of tyrosine and phenylalanine, and the rate of inactivation of the tyrosine/phenyla-lanine transport system can be effectively retarded by either natural ligand. On the other hand, the rate of inactivation of the transport system (s) for the neutral, aliphatic amino acids is unaffected by any of the substrates or by phenylalanine and tyrosine. Clearly, PCK is an affinity... 

Like the glucose carrier, the carriers for large neutral amino acids, the so-called L-system - now designated LAT - are present at both sides of the endothelial cell membranes and transport at least 10 essential amino acids. The L-transporter at the BBB has a much higher transport capacity than those in other tissues. Its marked preference for phenylalanine analogs explains why the anticancer drugs melphalan and d,l-NAM-7 are transported by the L-system, as is the L-Dopa used to treat Parkinson s disease [42]. 

A system exhibiting chiral recognition. The chiral macrotricyclic tet-raamide (250) (Lehn, Simon Moradpour, 1978) has been used for the complexation, extraction and transport of primary ammonium salts. The tetraamide was used rather than the corresponding tetraamine because of the lower basicity of the nitrogens in the former ligand. This avoids the possibility of proton transfer occurring from the primary ammonium substrates R-NH3+ used as guests. In a typical experiment, a solution of a primary ammonium salt, such as naphthylethyl ammonium or phenylalanine methylester hydrochloride in hydrochloric acid was... 

The other major class of transporter protein is the carrier protein. A prototypic example of a carrier protein is the large neutral amino acid transporter. An important function of the LNAA transporter is to transport molecules across the blood-brain barrier. As discussed previously, most compounds cross the BBB by passive diffusion. However, the brain requires certain compounds that are incapable of freely diffusing across the BBB phenylalanine and glucose are two major examples of such compounds. The LNAA serves to carry phenylalanine across the BBB and into the central nervous system. Carrier proteins, such as the LNAA transporter, can be exploited in drug design. For example, highly polar molecules will not diffuse across the BBB. However, if the pharmacophore of this polar molecule is covalently bonded to another molecule which is a substrate for the LNAA, then it is possible that the pharmacophore will be delivered across the BBB by hitching a ride on the transported molecule. 

As expected, system 13 did in fact bind and transport zwitterionic a-amino acids through a model membrane barrier with good selectivity under conditions where the porphyrin-derived control system (14), lacking the carboxylate anion chelation ability inherent in 13, would not. Specifically, it was found that at neutral pH compound 13 acts as a very efficient carrier for the through model membrane (H2O-CH2CI2-H2O) transport of phenylalanine and tryptophan. Further, in direct competition experiments, L-phenylalanine was found to be transported four times faster than L-tryptophan and 1000 times faster than L-tyrosine. As implied above, little or no transport was observed when a porphyrin control (14) was used. Nor was significant transport observed when a mixture of sapphyrin and lasalocid was used. 

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