Biochemistry of Amino Acids - A. Maister 1961

General Biochemistry and Physiology of Amino Acid Metabolism
Amino Acid Transport into the Cell

Intracellular Amino Acid METABOLISM is naturally preceded by their Transport Across the Cell membrane. The mechanisms underlying this process remain to be fully elucidated, although A number of participating factors have been investigated. Various experimental models are employed to study amino acid uptake, including intestinal preparations, tissue slices, erythrocytes, free tumor Cells, and bacterial Suspensions. One experimental approach involves incubating isolated cells in an amino acid-containing medium and determining the initial and final concentrations of Amino Acids in both the solution and the cells. Another method consists of measuring The amino acid appearing in the solution bathing the serserosal surface of an intestinal loop into which an amino acid solution has been introduced.

Available data indicate that Amino acids can enter cells both via simple diffusion and through an active process that concentrates them intracellularly. The existence of Active Transport is supported by experiments showing that the intracellular amino acid concentration significantly exceeds that of the extracellular fluid, and that L-isomers enter cells much faster than their corresponding D-isomers. The transport of a specific amino acid into different cell types may occur via distinct mechanisms; conversely, within a single cell type, the uptake mechanism for different amino acids may also vary. This phenomenon of amino acid concentration plays a vital role in intestinal absorption, renal reabsorption, and The transfer of amino acids from maternal Blood to fetal blood [1].

Compelling evidence has been obtained confirming the capacity of mammalian cells to concentrate amino acids. The concentration of amino acids in Tissues is significantly higher than in Body Fluids [2]. When introduced into the digestive tract or bloodstream, Amino acids are rapidly cleared from the blood and accumulate in tissues. Van Slyke and Meyer [3] long ago concluded that the high concentration of amino acids in tissues cannot be explained by simple diffusion alone; they suggested potential transport mechanisms involving amino acid adsorption or The formation of loose molecular compounds between Amino Acids and Proteins. In 1912, Van Slyke and Meyer wrote that "Structure/133.html">Discussion of this subject is currently premature." Although the 45 years since that time have seen numerous experimental studies on Amino Acid Transport, a definitive Conclusion regarding The Mechanism of this process has still not been reached.

It appears that the majority of dietary proteins undergo Hydrolysis in the digestive tract into their constituent amino acids; consequently, amino acids are absorbed from the intestine predominantly, if not exclusively, in a free state. There is no evidence entirely ruling out the possibility that certain proteins may be absorbed intact.

Data indicating the absorption of egg albumin via the thoracic lymphatic duct have been obtained using immunological Methods [4]. Fisher [5] and several other authors suggest that amino acids may be absorbed as Peptides; it is possible that peptide absorption in small amounts does indeed occur (p. 483). Although the concentration of peptides in Blood Plasma is extremely low [2], this does not exclude the participation of peptides or acylamino acids in the absorption process.

A number of studies have focused on the intestinal Absorption of Amino acids. Experiments with intact animals have demonstrated that the blood amino nitrogen content rises rapidly following the ingestion of individual amino acids (e.g., glutamic acid, leucine) [6, 7]. While some studies [8–10] yielded data consistent with simple diffusion, it is evident that an active absorption mechanism also exists. For instance, the absorption of Alanine, Glycine, and valine was found to be disproportionate to their concentration in the intestinal lumen [11]. Furthermore, when DL-amino acid solutions were introduced into an isolated loop of the rat Small Intestine, the L-isomers were absorbed at a significantly greater rate than the corresponding D-isomers [12]. Other experiments using small intestinal preparations similarly demonstrated more rapid absorption of L-amino acids compared to their D-isomers [13–20]. For example, after introducing racemic alanine

into the small intestinal loop of a cat, the venous blood draining the loop is richer in L-alanine than D-alanine [14, 15]; identical results were obtained with racemic phenylalanine and leucine [13]. Research of this type has also shown that L-glutamic and L-aspartic acids are not absorbed from the intestine against a concentration gradient [17].

In in vitro experiments where isolated segments of the small intestine were immersed in a suspension medium [21], L-Histidine and L-phenylalanine—unlike their D-isomers—were found to be transported across the intestinal wall against a concentration gradient. L-Glutamic acid is transported at the same rate as the Water in which it is dissolved [19]. This uphill Transport of Amino acids is inhibited by cyanide and 2,4-dinitrophenol; in the presence of these inhibitors, the absorption rate of L-isomers was approximately equal to that of D-isomers [20]. In experiments using isolated guinea pig small intestine segments, the L-isomers of histidine and alanine were transferred from the intestinal lumen to the serosal bathing solution much faster than the corresponding D-isomers; L-amino acid transport was inhibited under anaerobic conditions and in the presence of 2,4-dinitrophenol.

Phlorhizin has no effect on the active absorption of L-amino acids [18]. It had been noted even earlier that in rats treated with phlorhizin, the absorption of glycine and alanine occurred faster than under normal conditions, whereas Glucose Absorption was impaired [22]. Furthermore, 4-deoxypyridoxine was found to inhibit DL-alanine absorption from guinea pig intestinal preparations [18] and, like 2,4-dinitrophenol, to inhibit glucose and fructose uptake [23]. Taken together, these data indicate that the absorption mechanism depends on intact Respiration and may be linked to phosphorylation processes. The fact that phlorhizin inhibits sugar absorption without suppressing amino acid uptake suggests that amino acids and sugars are absorbed via different mechanisms. The inhibitory effect of 4-deoxypyridoxine points to the potential involvement of vitamin B₆ in amino acid absorption; this hypothesis is also supported by findings from other studies (p. 169). Experiments on amino acid absorption by intestinal preparations showed that uptake is not delayed in the presence of glucose, except at very high concentrations, which further confirms the existence of separate absorption mechanisms for amino acids and sugars. It is noteworthy that glutamine is absorbed unhydrolyzed and significantly faster than glutamic acid. Asparagine is also absorbed faster than aspartic acid, although it undergoes hydrolysis during the absorption process [18]. These findings align with other research indicating that glutamine penetrates various tissues, especially Brain tissue, much faster than glutamic acid (p. 174).

Amino acid uptake has also been observed in other experimental systems. For instance, guinea pig Cerebral Cortex slices were found to accumulate L-glutamic acid against a concentration gradient [24]. Although L-glutamine was taken up by the cerebral cortex much faster, the final tissue concentrations of the amino acid in experiments with Glutamic Acid and Glutamine were approximately equal. In experiments with glutamic acid, the increase in its tissue concentration ceased when the concentration difference between the tissue and the surrounding medium reached about 0.02 M. Human and duck erythrocytes, as well as rabbit reticulocytes, are capable of concentrating amino acids. The activity of rabbit reticulocytes is inhibited by 2,4-dinitrophenol and cyanide; these agents have virtually no effect on amino acid concentration by human and duck erythrocytes [25, 26].

Isolated rat Diaphragm accumulates glycine; this process is sensitive to cyanide and 2,4-dinitrophenol [27, 28]. Active glycine uptake by tissues has also been observed in in vivo experiments on guinea pigs fed glycine [29–31]. The amount of glycine taken up by tissues decreased when animals were administered Other Amino Acids simultaneously; these data point to competitive interactions among amino acids during transport. There is good reason to believe that many amino acids are concentrated in tissues via the same mechanism.

Christensen et al. [32–35] utilized a highly successful experimental model to study amino acid transport. These researchers discovered that free cells of mouse Ehrlich carcinoma take up amino acids more actively than cells from most other mammalian tissues. Under appropriate conditions, glycine concentration gradients between the tumor cells and the suspension medium exceeded 60 mmol per liter of water. Active amino acid concentration was inhibited under anaerobiosis, as well as by cyanide, arsenate, 2,4-dinitrophenol, malonate, aureomycin, and chloromycetin. Glycine uptake was accompanied by the efflux of potassium ions from the cells and the influx of water and sodium ions [32].

When interpreting data related to amino acid transport, the intracellular state of the amino acids is of paramount importance. Clearly, the uptake of a given amino acid by a cell may depend on its extracellular concentration, the activity of the transport system, and the metabolic transformations the amino acid undergoes. While free amino acids can be extracted from cells using various methods, the possibility that they exist in a bound form within intact cells cannot be ruled out. Such bonds might be relatively labile and capable of dissociating even under mild extraction conditions. Meanwhile, data from Christensen [32–34] and Heinz [35] indicate that easily extractable cellular amino acids exist in a free state. Maintaining glycine at the high concentrations observed in ascites tumor cells would require equally high concentrations of a binding agent; no evidence for such an agent has been found to date. Observations showing that water enters cells along with amino acids also support the presence of free amino acids intracellularly. In experiments with free Cancer cells, a direct correlation was observed between the glycine concentration gradient and the increase in cellular water content (an osmotic effect). Heinz [35], studying The kinetics of glycine influx and efflux during transport in ascites tumor cells, found that the relationship between The rate of glycine influx and its environmental concentration can be described by the Michaelis–Menten Equation. The rate of glycine influx does not decrease and may even increase upon prior saturation of the cells with glycine. The author concludes that the rate-limiting factor in glycine uptake is its binding to a Cell wall component. His results are consistent with the concept that intracellular glycine exists in a free state and indicate that glycine efflux occurs primarily via diffusion.

The Nature of the primary amino acid binding event indicated by kinetic data remains unknown. Findings that pyridoxal stimulates amino acid accumulation by mouse carcinoma cells suggest the potential involvement of vitamin B₆ in this process [36]. Ascites cells obtained from B₆-deficient mice exhibit a markedly lower amino acid transport activity than tumor cells from control mice. Although these observations suggest that vitamin B₆ participates in amino acid transport, Pyridoxal phosphate was found to be less active than free pyridoxal. Furthermore, 4-nitrosalicylaldehyde stimulated amino acid uptake to the same extent as pyridoxal [37].

Christensen and co-workers [34, 38–42, 696–698] investigated the accumulation of a wide range of amino acids by mouse carcinoma cells. They found that both L- and D-isomers are concentrated in these cells, with L-isomers taken up more actively. As a rule, lengthening the side chain hinders amino acid transport; amino acids bearing electron-withdrawing substituents (e.g., Ornithine, Methionine, Oxyproline) are concentrated more actively. The presence of a methyl group at the α-position enhances accumulation, whereas a second carboxyl group in the molecule generally reduces it. Diamino acids, such as ornithine, Lysine, α,γ-diaminobutyric acid, and α,β-diaminopropionic acid, are concentrated in cells more readily than the corresponding monoamino acids. These results are consistent with the view that transport reactions proceed much more easily when the amino group is uncharged—i.e., in a form that readily reacts to yield acyl derivatives or Schiff bases. Christensen hypothesizes that Schiff base formation may serve as an intermediate step in the amino acid transport mechanism. The participation of α-methylamino acids in such reactions suggests that an α-hydrogen atom is not essential for transport; indeed, the absence of an α-hydrogen atom may enhance the Stability of the intermediate Schiff base. The rapid uptake of diamino acids also Supports the idea that they form stable Schiff base-type intermediate complexes with pyridoxal [34]. It was likewise found that the absence of a free carboxyl group or acylation of the amino group reduces or completely abolishes the cellular accumulation of a given amino acid.

Amino acid uptake by bacterial cells has been investigated across a wide range of species. The mechanism of this process in Bacteria differs substantially from that in mammalian cells, and sharp differences are also observed among various microorganisms. Studying Bacterial Amino Acid uptake is complicated by simultaneous intracellular metabolic transformations, including amino acid degradation and Protein Synthesis pathways. Research conducted by Roberts et al. [43] on Escherichia coli demonstrates that The Cell membrane of this microorganism is readily permeable to many compounds. According to these authors, the sole mechanism for Amino Acid and nutrient uptake in E. coli is simple diffusion, with environmental components freely diffusing into the cellular "water space." Consequently, identical concentrations of substances should be established inside the "water space" and in the surrounding medium. Experiments measuring the "water space" volume by equilibrating cells with solutions of various isotope-labeled compounds (including amino acids) followed by analysis of centrifuged cells and supernatant yielded approximately the same values. These experiments imply that the concentrations of diffusible substances within E. coli cells and in the external environment are roughly equal, except when these substances are bound intracellularly or utilized in metabolic processes.

At the same time, conflicting data indicate that E. coli and certain other microorganisms are capable of accumulating amino acids against a concentration gradient. For instance, Gale and Rodwell [44] discovered that Streptococcus faecalis cells suspended in lysine solutions accumulate lysine; under these conditions, the intracellular lysine concentration reaches levels significantly higher than in the surrounding fluid. These results appeared to indicate active lysine uptake by the cells. However, it was found that under certain experimental conditions, lysine can freely enter and exit Str. faecalis cells [45, 46]. The authors concluded that the observed phenomena can be explained by established physical laws governing the distribution of substances across a semipermeable membrane.

It is possible that E. coli employs a fixation process for accumulated amino acids prior to their incorporation into proteins [47]. When E. coli was incubated in a medium containing ¹⁴C-valine, the intracellular valine concentration reached levels 1000 times higher than in the medium. The absorbed valine could be competitively displaced by leucine or isoleucine. This uptake process was specific for L-valine and proceeded much faster than the incorporation of this amino acid into proteins. Although the Nature of the fixation process remains unclear, it has been suggested that E. coli cells possess specific amino acid receptors.

Gale and co-workers [45, 46, 48, 49] also studied the accumulation of glutamic acid by Streptococcus faecalis and Staphylococcus aureus cells. As in the experiments on lysine uptake, data were obtained indicating that glutamic acid can move easily between the cellular "water space" and the external environment. However, in the presence of glucose, cellular accumulation of glutamic acid does occur. Clearly, a number of experimental difficulties must be overcome to clarify this issue; some of these have been discussed in detail by Gale [45] and Christensen [34].