Halvor N. Christensen

Organic ion transport during seven decades the amino acids

The amino acids are ions of various charge combinations, and one can argue that historically they were the first ions for which the ongoing problem of membrane transport was presented; also that among transported ions these may undergo a highly detailed molecular recognition. Furthermore, the distribution of charge on the amino acid molecule determines by what route or routes it is conducted across the biological membrane, with what directional and structural specificity, and therefore what regulation is imposed, and where. Cases where a presumably charged chemical group behaves as if it were somehow absent from the amino acid have been observed to fall into several categories: Straightforward cases where the pH has been low enough or high enough to remove the charge by protonation or deprotonation, even in free solution. Cases where that protonation or deprotonation is facilitated at the binding site, and perhaps by the total transport process. The cystine molecule can apparently thus be rendered either a tripolar anion or a tripolar cation for transport. Cases where an otherwise co-transported Na+ is omitted to redress charge, or where a Na+ serves as a surrogate for a missing charged group on the amino acid molecule. A case where the protonation occurs reversibly at the receptor site rather than on the amino acid molecule.

On the strategy of kinetic discrimination of amino acid transport systems

It was for renal epithelial transport that the logical conclusion was first reached that different agencies serve for the transport of cationic and zwitterionic amino acids [4]. Such partitions according to molecular charge have usually proved valid for other cells and tissues of the higher animal, given that attention is applied to identify the charge retained by the amino acid during transport, and also that allowance is made for the possibility that the transport system may have its charge changed by protonation or deprotonation (see review on the influence of molecular charge on the route of amino acid transport, ref. [7]). In the early sixties it came to be appreciated that a single agency would not, however, account for the transport of the zwitterionic amino acids, and thereafter the significant discrimination was made between the Na§ and Na+-independent agencies [17]. Each of these two transport systems showed other important distinguishing characteristics, for example, the extent to which inward transport was quickly offset by outward transport [17]. Furthermore, other limited tests suggested homogeneity of each of these two components in the Ehrlich cell. Already by the time we reported that result, it could be sensed that the Na§ component probably represented more than one system--note the change in title in going from our preliminary [161 to our final [17] report. An important feature of the latter report was the finding that parallel transport by two or more systems tended to be characteristic for any amino acid because of the heavy overlap in their amino acid selectivity. This discovery led ultimately to

Reactive Sites and Biological Transport

Publisher Summary Biological barriers are studded with a variety of reactive sites that favor the passage of suitable molecules; in some cases, mechanisms that seem inherently improbable deliver the molecules from these sites against electrochemical gradients, either normally or when a gradient of an analog is artifically produced. In their aggregate, these reactive sites give cytological barriers, their segregating action, and permit physiological and pharmacological control through the increase and decrease of such segregating effects. The coincident nature of such modifications of transport has suggested that the activity is inherent in a common matrix rather than a consequence of the shuttling of dissimilar carriers. Attempts should be intensified to identify these reactive sites on organelles, membranes, and intact cells by the chemical procedures now available for specific sites on macromolecules. Site subject to significant modification in conformation or charge distribution by hormone action should be investigated. Structures having the same binding characteristics as a given transport site should also be looked for in broken-cell preparations. This general search can take advantage of a critical property of the site on the intact cell and need not be deterred by immediate concern for the accessory equipment or mechanism of the transport.

Cysteine as a system-specific substrate for transport system ASC in rat hepatocytes

Abstract The rapid transport of L -cysteine into isolated rat hepatocytes escapes detectable inhibition by 2-(methylamino)-isobutyric acid at levels up to 50 mM. The system transporting cysteine instead is convincingly similar to the ASC system described for the Ehrlich cell in structural and steric specificity and in pH sensitivity. The Na+-dependent uptake of 2-aminoisobutyric acid is almost evenly divided between Systems A and ASC , showing better accommodation of its two α-methyl groups by ASC than in the Ehrlich cell. The hepatocyte ASC system tolerates Li+-for-Na+ substitution better than does System A , although the tolerance depends on amino acid structure. Adaptive regulation and insulin and glucagon stimulation were not seen under conditions producing these effects for System A .

Recognition Sites for Material Transport and Information Transfer

Publisher Summary This chapter reviews a list of some of the most conspicuous transport systems for the amino acids observed in cells of the higher animal. It tabulates the approximate range of the reactivity to transport of several of the well characterized systems, using mainly the results with the Ehrlich cell to compare in an approximate way how much each amino acid is transported by each of the several systems for neutral amino acids. Synthetic amino acids designed as model substrates for two transport systems mimic very closely the action of some typical substrates of these systems in causing hormone release. It seems to settle unequivocally the question whether amino acids need to be catabolized to have these effects. One metabolic consequence of the presence of the norbornane amino acid has been noted by Matschinsky et al.— a stimulation of lactate production. This stimulation is produced by a number of amino acids including both the levo- and the dextrorotatory isomers of the reactive form of the norbornane amino acid. Therefore, this metabolic effect does not parallel precisely the insulin-releasing potency of these amino acids. Another metabolic effect of the amino acids can, however be correlated with the insulin-releasing activity.

On the nature of the "non-saturable" migration of amino acids into Ehrlich cells and into rat jejunum.

Abstract The so-called non-saturable uptake of α-amino acids by the Ehrlich cell, even though it occurs at a characteristically slow rate for various neutral amino acids (whether they are in the d - or the l -form) is nevertheless structurally specific, since the uptake of β-alanine, taurine and betaine occurs only about one-third as rapidly as that of the α-amino acids. Furthermore the uptake shows a considerable sensitivity to pH, and a temperature sensitivity so high as to exclude simple diffusion as the rate-limiting step. The structural specificity is compatible with a reaction of the amino acid with a membrane site, either an abundant one or a relatively unreactive one, the reaction of the amino acid with which presumably need involve at most only its amino and carboxyl groups. Uptake of amino acids at high levels by rat-intestinal segments also showed high temperature sensitivities.

In vitro stimulation of insulin release by non-metabolizable, transport-specific amino acids

The insulin-releasing ability and uptake characteristics of non-metabolizable, transport-specific amino acids were studied in an in vitro system, using microdissected pancreatic islets with more than 90% β-cells. Among the four stereoisomers of 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH), only the b(−) form stimulated insulin release. This isomer is known as a specific substrate for transport system l in other cells. It was rapidly taken up by the islet cells and stimulated insulin release both in the presence and in the absence of glucose. 4-Amino-1-guanylpiperidine-4-carboxylic acid (GPA), a substrate for cationic transport systems, stimulated insulin release in the presence but not in the absence of glucose. In this respect GPA is similar to arginine. Like arginine, GPA also accumulated in the islet cells to yield distribution ratios well above unity. The results are consonant with the previous hypothesis that amino acids stimulate insulin release by binding to specific transport molecules.

Free Amino Acids and Peptides in Tissues

Publisher Summary This chapter presents an overview of free amino acids and peptides in tissues. Free amino acids are generally recognized to serve as the principal currency of protein metabolism in the multicellular organism, and their concentrations are low compared with the quantities present in the protein-bound form. The plasma level of each of the various amino acids is controlled by balances between the entry and exit from the plasma, as is the case for the blood sugar level. Under basal conditions each level is expected to tend to reach a characteristic value, as does the blood sugar. Although the interposition of a concentrative step and the multiplicity of the amino acids have discouraged the study of this behavior with the same care that the control of the blood sugar level has received, nevertheless a corresponding interplay of factors must be at work. Peptides were found to enter cells sluggishly, in comparison with free amino acids; in fact, peptides in partial protein hydrolyzates are to a large extent rejected by the human organism. Peptides are believed to have little importance in nutrition at the cellular level, and as free intermediates in protein synthesis.

Role of proton dissociation in the transport of acidic amino acids by the Ehrlich ascites tumor cells

Abstract The pH profile for the uptake of l -glutamic acid by the Ehrlich ascites tumor cell arises largely as a sum of the decline with falling pH of a slow, Na + -dependent uptake by System A, and an increasing uptake by Na + -independent System L. The latter maximizes at about pH 4.5, following approximately the titration curve of the distal carboxyl group. This shift in route of uptake was verified by (a) a declining Na + -dependent component. (b) an almost corresponding decline in the 2-(methylamino)-isobutyric acid-inhibitable component, (c) a rising component inhibited by 2-aminonorbornane-2-carboxylic acid. Other amino acids recognized as principally reactive with Systems A or L yielded corresponding inhibitory effects with some conspicuous exceptions: 2-Aminoisobutyric acid and even glycine become better substrates of System L as the pH is lowered; hence their inhibitory action on glutamic acid uptake is not lost. The above results were characterized by generally consistent relations among the half-saturation concentrations of the interacting amino acids with respect to: their own uptake, their inhibition of the uptake, one by another, and their trans stimulation of exodus, one by another. A small Na + -dependent component of uptake retained by l -glutamic acid but not by d -glutamic acid at pH 4.5 is inhibitable by methionine but by neither 2-(methylamino)-isobutyric acid nor the norbornane amino acid. We provisionally identified this component with System ASC, which transports l -glutamine throughout the pH range studied. No transport activity specific to the anionic amino acids was detected, and the unequivocally anionic cysteic acid showed neither significant mediated uptake nor inhibition of the uptake of glutamic acid or of the norbornane amino acid.

Role of System Gly in glycine transport in monolayer cultures of liver cells

Abstract The high-affinity component of glycine uptake by the hepatoma cell line HTC and by the ordinary rat hepatocyte corresponds to System Gly, the agency serving for glycine uptake by pigeon red blood cells and rabbit reticulocytes, and at most to only a minor extent to System ASC. This component was identified in HTC by its sensitivity to inhibition by sarcosine but scarcely by 2-(methylamino) isobutyric acid, by its insensitivity to lowering of the pH, and by the unique relation of its rate to the square of the Na+ concentration. The identity of the low-affinity component with System A was confirmed by opposite properties, and by its stimulation by insulin or amino acid starvation. Both components differed sharply from the System ASC uptake as measured with threonine.