MeAIB Transport Research Articles

Etoposide inhibition of methylaminoisobutyric acid transport and its recovery in K562 cells

Sodium-dependent amino acid transport of methylaminoisobutyric acid (MeAIB) in a human myelogenous leukemic cell line K562 was inhibited by 30 nM etoposide (VP-16). Cell growth in suspension was almost completely suppressed by 30 nM VP-16 after 72h. Colony formation by K562 cells markedly decreased after 24h in a culture medium containing 30 nM VP-16, whereas MeAIB transport was inhibited after 72 h. K562 cell proliferation was restored in drug-free medium after recovery from 72-h culture with 30 nM VP-16. Colony formation and MeAIB transport were restored to pretreatment levels after 1–2 weeks. Thus, two conclusions may be drawn. (1) MeAIB transport may be correlated with cellular growth and (2) the effects of VP-16 on MeAIB transport were partially reversible.

Chloride-dependence of amino acid transport in rabbit ileum

Chloride-dependence of influx across the brush-border membrane of distal rabbit ileum was examined for β-alanine, 2-methylaminoisobutyric acid (MeAIB), leucine, lysine, proline and d-glucose. Influx of leucine at 2 mM and of d-glucose at 0.5 mM was chloride-independent indicating that substitution of isethionate for chloride has no unspecific effect on sodium gradient driven transport processes. In contrast influx of β-alanine and MeAIB was totally dependent on the presence of chloride ions. In the absence of chloride, proline transport was reduced to 20% of its control level. This remaining transport can be accounted for by the function of the carrier of α-amino-monocarboxylic acids. Transport of leucine at 0.1 mM was reduced by absence of chloride. This is in accordance with the observation of leucine transport by the β-alanine carrier. The kinetics of chloride and sodium activation of transport of MeAIB were examined at 1 mM MeAIB. Chloride activation was characterized by a Hill coefficient of 1 and a K 1 2 of 23.5 mM, and sodium activation by a Hill coefficient of 2 and a K 1 2 of 51 mM. Thus cotransport of chloride with an imino acid would be compatible with the known rheogenic nature of this transport. This study adds the imino acid carrier and the β-alanine carrier to the group of chloride-dependent, epithelial amino acid transport systems.

Protein kinase C activators selectively inhibit insulin-stimulated system A transport activity in skeletal muscle at a post-receptor level

We have investigated the role of phorbol esters on different biological effects induced by insulin in muscle, such as activation of system A transport activity, glucose utilization and insulin receptor function. System A transport activity was measured by monitoring the uptake of the system A-specific analogue alpha-(methyl)aminoisobutyric acid (MeAIB), by intact rat extensor digitorum longus muscle. The addition of 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.5 microM) for 60 or 180 min did not modify basal MeAIB uptake by muscle, suggesting that insulin signalling required to stimulate MeAIB transport does not involve protein kinase C activation. However, TPA added 30 min before insulin (100 nM) markedly inhibited insulin-stimulated MeAIB uptake. The addition of polymyxin B (0.1 mM) or H-7 (1 mM), protein kinase C inhibitors, alone or in combination with TPA leads to impairment of insulin-stimulated MeAIB uptake. This paradoxical pattern is incompatible with a unique action of Polymyxin B or H-7 on protein kinase C activity. Therefore these agents are not suitable tools with which to investigate whether a certain insulin effect is mediated by protein kinase C. TPA did not cause a generalized inhibition of insulin action. Thus both TPA and insulin increased 3-O-methylglucose uptake by muscle, and their effects were not additive. Furthermore, TPA did not modify insulin-stimulated lactate production by muscle. In keeping with this selective modification of insulin action, treatment of muscles with TPA did not modify insulin receptor binding or kinase activities. In conclusion, phorbol esters do not mimic insulin action on system A transport activity; however, they markedly inhibit insulin-stimulated amino acid transport, with no modification of insulin receptor function in rat skeletal muscle. It is suggested that protein kinase C activation causes a selective post-receptor modification on the biochemical pathway by which insulin activates system A amino acid transport in muscle.

NEUTRAL AMINO ACID TRANSPORT IN SHEEP EXTERNAL INTERCOSTAL MUSCLE

The transport of two amino acid anologs, aminoisobutyric acid (AIB) and N-methylaminoisobutyric acid (MeAIB), was studied in external intercostal muscle bundles taken from 12 wether lambs to examine the significance of these transport systems in sheep muscle tissue. Competition for transport between AIB and other amino acids was evaluated to determine proportions of AIB transported on individual carriers. Rates of AIB transport into EIC muscle in relation to previous liveweight gains of lambs and animal weights were also examined. Aminoisobutyric acid was concentrated to approximately fourfold extracellular AIB concentrations via a sodium gradient-dependent process within 2 h of incubation. However, MeAIB was not concentrated by this tissue. Kinetic analyses of MeAIB transport indicated that its entry was entirely nonsaturable. Since MeAIB is a model substrate for transport system A (Na gradient-dependent, hormone and substrate concentration regulated) and since AIB may be transported by both systems A and ASC (Na gradient-dependent, hormone and substrate concentration insensitive), the active transport of AIB is attributed primarily to a system ASC-like agency. Kinetic analyses of the Na gradient-dependent transport of AIB revealed Jmax and Kt values of 2.48 ± 0.35 μmoles g tissue−1 30 min−1 and 11.4 ± 4.2 mM, respectively. Competition studies between AIB and other analogs provided further support for the existence of an ASC-like transport agency and the possible absence of or a repressed system A. A significant relationship between previous liveweight gain and amino acid transport was not detected; however, AIB transport in muscle taken from more mature animals was significantly lower than in muscle taken from younger animals. Key words: Sheep, muscle, amino acid transport, system A, system ASC

Characteristics of L-alanine uptake in freshly isolated hepatocytes of elasmobranch Raja erinacea.

The specific transport mechanisms that mediate the hepatic uptake of L-[3H]alanine and of an unnatural homologue, alpha-[14C]methylaminoisobutyric acid (MeAIB), were analyzed in hepatocyte suspensions from Raja erinacea. Aminooxyacetate, an inhibitor of aminotransferase activity was used to prevent alanine metabolism. After 3 h of incubation with either 0.5 mM alanine or MeAIB, hepatic concentrations of these amino acids were significantly higher in the presence than absence of Na+ (8 vs. 1 and 1 vs. 0.1 mM, respectively). Kinetic studies indicated that both alanine and MeAIB transport occurred via sodium-dependent saturable mechanisms. [14C]MeAIB uptake was completely inhibited by excess L-alanine. Uptake of [3H]alanine was inhibited by a 40-fold excess of serine and cysteine (53-54%), by MeAIB and methylalanine (26-31%), and by leucine (14%), whereas D-alanine, beta-alanine, taurine, and glutamate had no effect. Insulin and glucagon were unable to stimulate [3H]alanine uptake. Glucose release from hepatocytes was unaffected by 10 mM alanine or 2 mM aminooxyacetate, indicating that alanine is not a major gluconeogenic precursor in this marine elasmobranch. These results suggest that uptake of L-alanine by skate hepatocytes occurs predominantly via a sodium-dependent system, with properties similar to those exhibited by the ASC neutral amino acid transport system previously characterized in Ehrlich ascites tumor cells and rat hepatocytes.

Evidence for an alanine, serine, and cysteine system of transport in isolated brain capillaries.

Brain capillaries isolated from 2-month-old male and female Sprague-Dawley rats were used to study the transport of neutral amino acids. The uptake of alanine, leucine, and alpha-methylaminoisobutyric acid (MeAIB) was a linear function of time for the first minute of incubation. Based on these observations, an incubation time of 1 min was used to measure transport activities. The intracellular water volume of the isolated capillaries was 2.2 microliters/mg protein. This value was significantly lower (1.8 microliter/mg protein) when measured in the absence of sodium. L-Alanine, L-serine, and L-cysteine were taken up from the abluminal surface of brain capillaries by a sodium- and energy-dependent, carrier-mediated system. This uptake, for the most part, was not inhibited by MeAIB. System ASC (alanine-serine-cysteine) appeared to be of primary importance for the transport of these amino acids in isolated brain capillaries. The apparent Km and Vmax values for L-alanine uptake by ASC transport, based on the Hofstee plot presentation, were 1.3 mM and 0.975 nmol/microliter water content/min, respectively. The results also indicate that the transport of MeAIB and 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) was limited to the sodium-dependent system A (alanine) and the sodium-independent system L (leucine), respectively.

Regulation of amino acid transport in isolated rat hepatocytes during development.

The effect of amino acid depletion or supplementation and the effect of glucagon and insulin on the amino acid transport mediated by system A were investigated by determining the uptake of either 2-amino [1-14C]isobutyric acid (AIB) or N-methyl 2-amino [1-14C]isobutyric acid (MeAIB) in rat hepatocytes, freshly isolated at different stages of pre- and postnatal development. The data obtained show that the Na+-dependent uptake was higher at the earliest developmental stages, and steadily decreased until the adult level. The hormones increased AlB and MeAIB uptake enhancing the Vmax, while the Km was unchanged. This effect was evident in cells from adult and 18-20-day-old fetuses, while no response was present before the 18th day of fetal life and in the perinatal period. Actinomycin D or cycloheximide abolished this hormone-dependent increase. A decrease in AlB and MeAIB transport after incubation in an amino acid-rich medium was demonstrated at all ages tested, but was particularly evident in the prenatal life. The increase in the activity of the system following amino acid starvation was shown to be mostly dependent from de novo protein synthesis in the fetal life; on the contrary in the adult the increase appeared to be more linked to the release from transinhibition of the transport.

Sodium-amino acid cotransport by type II alveolar epithelial cells.

Type II alveolar epithelial cell monolayers have been shown to actively transport sodium (Na+). Coupling to amino acid uptake could be an important mechanism for Na+ entry into these cells. This study demonstrates the presence of such a coupled cotransport mechanism in the plasma membrane of isolated type II cells by use of the nonmetabolizable amino acid analogue alpha-methylaminoisobutyric acid (MeAIB). Transport of MeAIB in 137 mM Na+ is saturable, with the uptake constant (Vmax) equaling 13.9 pmol X mg prot-1 X s-1 and the Michaelis-Menten constant (Km) equaling 0.13 mM. In the presence of Na+, MeAIB is accumulated against a concentration gradient. MeAIB uptake in the absence of Na+ is linear with MeAIB concentration, as expected for simple diffusion. The Hill coefficient for Na+-MeAIB cotransport is 1.11, suggesting a 1:1 stoichiometry. Proline inhibits Na+-MeAIB cotransport, with Ki equaling 0.5 mM. These findings suggest that Na+-amino acid cotransport may be an important pathway for Na+ (and/or amino acid) uptake into type II alveolar epithelial cells.

Identification of amino acid transport systems stimulated by FSH in rat testes.

Testes of 12-day-old rats preincubated (90 min) and incubated (60 min) with FSH in Krebs-Ringer bicarbonate buffer (KRB) increased their uptake of [14C]methylaminoisobutyric acid and [14C]aminoisobutyric acid. The hormone was ineffective in sodium-free KRB. Transport of [14C]aminoisobutyric acid in the presence of methylaminoisobutyric acid (MeAIB; 30 mmol/l) was not stimulated by FSH, and uptake of [14C]cycloleucine was not increased by FSH in any of the experimental conditions used. It was concluded that in immature rat testes FSH affects only the sodium-dependent system which permits the transport of MeAIB (transport system A) and has no effect on the sodium-independent system which prefers leucine (system L) or the sodium-dependent system which does not transport MeAIB (system ASC).

Neutral amino acid transport in embryonal carcinoma cells.

Neutral amino acid transport was characterized in the pluripotent embryonal carcinoma (EC) cell line, OC15. Ten of the thirteen amino acids tested are transported by all three of the major neutral amino acid transport systems--A, L, and ASC--although one system may make a barely measurable contribution in some cases. The characterization of N-methyl-aminoisobutyric acid (meAIB) transport points to this model amino acid as a definitive substrate for System A transport by OC15 cells. Thus, high concentrations of meAIB can be used selectively to block System A transport, and the transport characteristics of meAIB represent system A transport. Kinetic analysis of System A, with a Km = 0.79mM and Vmax = 14.4 nmol/mg protein/5 min, suggests a single-component transport system, which is sensitive to pH changes. While proline transport in most mammalian cells is largely accomplished through System A, it is about equally divided between Systems A and ASC in OC15 cells, and System A does not contribute at all to proline transport by F9 cells, an EC cell line with limited developmental potential. Kinetic analysis of System L transport, represented by Na+-independent leucine transport, reveals a high-affinity, single-component system. This transport system is relatively insensitive to pH changes and has a Km = 0.0031 mM and Vmax = 0.213 nmol/mg protein/min. The putative System L substrate, 2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH), inhibits Systems A and ASC as well as System L in OC15 cells. Therefore, BCH cannot be used as a definitive substrate for System L in OC15 cells. Phenylalanine is primarily transported by Na+-dependent Systems A and ASC (83% Na+-dependent; 73% System ASC) in OC15 cells, while it is transported primarily by the Na+-independent System L in most other cell types, including early cleavage stage mouse embryos and F9 cells. We have also found this unusually strong Na+-dependency of phenylalanine transport in mouse uterine blastocysts (82% Na+-dependent). There is no evidence for System N transport by OC15 cells, since histidine is transported primarily by a Na+-independent, BCH-inhibitable mechanism.

Transport of imino acids and non-alpha-amino acids across the brush-border membrane of the rabbit ileum.

The transport of beta-alanine and MeAIB and their effects as inhibitors of the transport of alanine, leucine and lysine across the brush-border membrane of the intact epithelium from the rabbit's distal ileum has been examined. Two separate transport systems have been characterized: 1) A sodium-dependent, beta-alanine-accepting system, which is a high-affinity transport system for alpha-amino-monocarboxylic acids (neutral a.a.) and for cationic a.a., accepts non-alpha-amino acids as well as non-alpha-imino acids, is moderately stereospecific, and for which the affinity of a neutral a.a. is greatly reduced by N-methylation. 2) A sodium-dependent transport system for imino acids, which is inaccessible to cationic amino acids and non-alpha-amino acids but accepts cyclic, non-alpha-imino acids, is moderately stereospecific, and for which neutral a.a. have much lower affinities than their N-methylated derivatives. On the basis of the observations of this and the preceding paper five transport systems for amino acids are ascribed to the rabbit ileum. Some discrepancies between the present results and those obtained with brush-border membrane microvesicles from the rabbit small intestine are discussed.

Amino acid transport in a kidney epithelial cell line (MDCK): Characteristics of Na +/amino acid symport in membrane vesicles and basolateral localization in cell monolayers

Na +-stimulated amino acid transport was investigated in MDCK kidney epithelial cell monolayers and in isolated membrane vesicles. When transport polarity was assessed in confluent polarized epithelial cell monolayers cultured on Nucleopore filters and mounted between two lucite chambers, Na +-stimulated transport of 2-(methylamino)isobutyric acid (MeAIB), a substrate specific for the A system, was predominantly localized on the basolateral membrane. Na +-stimulated amino acid transport activity was maximal in subconfluent cultures, and was substantially reduced after confluence. A membrane vesicle preparation was isolated from confluent MDCK cell cultures which was enriched in Na +-stimulated MeAIB transport activity and Na +,K +,ATPase activity, a basolateral marker, but was not enriched in apical marker enzyme activities or significantly contaminated by mitochondria. Na +-coupled amino acid transport activity assayed in vesicles exhibited a marked dependence on external pH, with an optimum at pH 7.4. The pattern of competitive interactions among neutral amino acids was characteristic of A system transport. Na +-coupled MeAIB and AIB transport in vesicles was electrogenic, stimulated by creation of an interior-negative membrane potential. The Na + dependence of amino acid transport in vesicles suggested a Na + symport mechanism with a 1:1 stoichiometry between Na + and amino acid.

Neutral amino acid transport by plasma membrane vesicles of the rabbit choroid plexus

Uptakes of neutral l-amino acids into rabbit choroid plexus apical membrane vesicles were studied using l-[ 14C]amino acids. Apical membrane vesicles were prepared using Ca 2+ precipitation and uptakes of 14C-labeled substrates were measured by a rapid mixing and filtration procedure. Na-dependent, concentrative uptake was observed for proline, histidine and methylaminoisobutyric acid (MeAIB). Phenylalanine and d-glucose uptakes showed no significant Na dependence. Proline uptake was a saturable function of the proline concentration with J max 3.5 nmol/(mg min) and K t 0.3 mM. Competition experiments in the presence of Na indicated that proline and MeAIB are mutually competetive. Proline uptake was also inhibited 25% by phenylalanine, but MeAIB uptake was relatively unaffected. Neither proline nor MeAIB transport was significantly inhibited by glycine. We conclude that proline uptake across rabbit choroid plexus apical membrane vesicles is via an Na-dependent pathway which is shared by MeAIB and, to a minor extent, by phenylalanine, but from which glycine is excluded. Histidine uptake was inhibited by glycine, GABA, phenylalanine, proline and MeAIB. This suggests that histidine may utilize a different pathway in addition to that shared with proline and MeAIB. These transporters should play an active role in the regulation of amino acids in the CSF.

Neutral amino acid transport in isolated rat pancreatic islets.

The neutral amino acid transport systems of freshly isolated rat pancreatic islets have been studied by first examining the transport of L-alanine and the nonmetabolizable analogue 2-(methylamino)isobutyric acid (MeAIB). By comparing the uptake of MeAIB and L-alanine for their pH dependency profile, choline and Li+ substitution for Na+, tolerance to N-methylation, and competition with other amino acids, the existence in pancreatic islets of both A and ASC amino acid transport systems was established. The systems responsible for the inward transport of five natural amino acids was studied using competition analysis and Na+ dependency of uptake. These studies defined three neutral amino acid transport systems: A and ASC (Na+-dependent) and L (Na+-independent). L-Proline entered rat islet cells mainly by system A; L-leucine by the Na+-independent system L. The uptake of L-alanine, L-serine, and L-glutamine was shared by systems ASC and L, the participation of system A being negligible for these three amino acids. An especially broad substrate specificity for systems L and ASC is therefore suggested for the rat pancreatic islet cells. The regulation of amino acid transport was also investigated in two conditions differing as to glucose concentration and/or availability, i.e. islets from fasted rats and islets maintained in tissue culture at high or low glucose concentrations. Neither alanine nor MeAIB transport was altered by fasting of the islet-donor rats. On the other hand, pancreatic islets maintained for 2 days in tissue culture at high (16.7 mM) glucose transported MeAIB at twice the rate of islets maintained at low (2.8 mM) glucose. Amino acid starvation of pancreatic islets during 11 h of tissue culture resulted in a 2-fold increase in MeAIB transport.

Transport of alpha-aminoisobutyric acid across brain capillary and cellular membranes.

The transport of alpha-aminoisobutyric acid (AIB), N-methyl-AIB (MeAIB), and diethylenetriaminepentaacetic acid (DTPA) from blood to brain was measured over different experimental periods in eight regions of the rat brain. Unidirectional transfer rate constants were determined from multiple-time/graphical and single-time analysis of the experimental data; values of 0.0018, 0.00057, and 0.000021 ml g-1 min-1, respectively, were obtained for the thalamus by graphical analysis. The initial distribution volume of AIB and MeAIB in brain tissue was several-fold greater than that of DTPA and the tissue plasma volume, and this difference was not accounted for by red blood cell uptake. This discrepancy could be due to rapid transport of AIB and MeAIB into brain endothelial cells in addition to the relatively rapid uptake by choroidal, meningeal, and ependymal associated tissues that was demonstrated by autoradiography. Thus, it may be misleading and erroneous to consider the blood-brain barrier (BBB) to be a simple, single-membrane structure when analyzing the blood-brain transfer data of solutes such as amino acids. The data from the ventriculocisternal perfusion experiments and previously published AIB uptake data in mouse brain slices were used to estimate the transfer rate constants across brain cell membranes. These studies indicated that the transport of AIB into brain cells was approximately 110 to 265 times greater than that across normal brain capillaries per unit mass of brain tissue, and that the BBB limits blood-to-brain cell transport of this amino acid. These observations (low rate of transport across normal brain capillaries and rapid concentrative uptake by brain cells) indicate that AIB is a good marker for measuring moderate to large increases in BBB permeability by experiments that require unidirectional flux of the tracer.

Antigen-specific stimulation of amino acid transport in bovine lymphocytes.

Treatment of bovine lymphocytes isolated from animals which were either infected with Mycobacterium bovis or sensitized to a purified protein derivative (PPD-B) from this organism induced an increase in the transport of alpha-aminoisobutyric acid (AIB) and alpha-methylaminoisobutyric acid (MeAIB). PPD-B did not stimulate these transport activities in lymphocytes from nonsensitized animals. The transport stimulation was first measurable after about 7 hours of treatment, reached about a two-fold enhancement after 20 hours, and continued to increase to 30- to 40-fold after 6 days. The stimulation of AIB transport was inhibited by both ouabain and cycloheximide. Experiments to determine transport system specificities in nonstimulated lymphocytes showed that MeAIB transport was primarily by the Na+-dependent, A-system, and leucine transport was mostly by Na+-independent system(s). In contrast, AIB transport was about 25% by the A-system, 25% by at least one Na+-dependent, non-A-system, and 50% by one or more Na+-independent system(s). Analysis of the three components of AIB transport after treatment with PPD-B showed that: 1) transport by both the A-system and the Na+-independent system(s) was stimulated; 2) A-system transport was stimulated to a larger extent than Na+-independent transport; and 3) Na+-dependent, non-A-system transport was not stimulated significantly.

Changes of 2-(methylamino) isobutyric acid transport and intracellular pH upon preincubation of rat hepatocyte primary cultures.

When rat hepatocyte monolayers were preincubated for 4 h in Hanks' salt solutions at pH 7.0, 7.4 and 8.0, and the Na+-dependent uptake of 2-(methylamino) isobutyric acid (MeAIB) was measured at the same pH values, a stimulation of transport in the order pH 7.0 less than pH 7.4 less than pH 8.0 was observed. Estimations of the intracellular pH from the distribution of DMO revealed a decrease in the internal pH during the preincubation period. The MeAIB transport velocities appear to parallel with the proton gradients across the cell membrane rather than with external (or internal) pH. Analyses of the lactate/pyruvate concentrations in the media indicated that the fall in the intracellular pH is presumably due to an enhanced glycolysis. Suppressive concentrations of system A-reactive amino acids did not prevent the decrease in the internal pH nor did they alter the metabolic data.

Anti-inflammatory drugs alter amino acid transport in HTC cells.

Following the addition of indomethacin to exponentially growing rat hepatoma cell (HTC) cultures, cells accumulated in the G1 phase. Over the course of several hours, specific changes in membrane transport accompanied this inhibition. Indomethacin stimulated the uptake of 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH), inhibited the Na+-dependent active transport of alpha-aminoisobutyric acid (AIB), and methylaminoisobutyric acid (MeAIB), but had no effect on the uptake of thymidine, uridine, or 2-deoxyglucose. The stimulation of BCH transport was immediate and reversible. The uptake of BCH was Na+-independent and only slightly depressed by metabolic inhibitors (10 to 20%). Inhibition of AIB and MeAIB uptake, which was of gradual onset, and was observed with several anti-inflammatory drugs and was dependent on the concentration of drugs. Upon removal of drug, AIB and MeAIB transport gradually increased and reached a maximum prior to resumption of DNA synthesis and cell division. Thee effects involved changes in Vmax only. Neither the apparent affinity (Km) for amino acids nor rate of exodus (k, 0.7h-1 for BCH, 1.2h-1 for AIB) were altered. The uptake of MeAIB (and AIB) occurred by a low and a high affinity (Km = 0.27 mM) component. Indomethacin inhibited specifically the high affinity component which was shown to be Na+- and energy-dependent. Although this component was inhibited by treatment with agents that lowered ATP levels or blocked protein synthesis, the anti-inflammatory drugs appeared not to act through these mechanisms.

Evidence supporting a two-receptor model for insulin binding by cultured embryonic heart cells.

Curvilinear Scatchard plots for insulin binding by cultured embryonic heart cells at 15 C were interpreted assuming a two-receptor model (Santora II, A. C., F.B. Wheeler, R.L. DeHaan, and L.J. Elsas II, Endocrinology 104:1059, 1979). The present studies test this hypothesis further. Both 125I-labeled hormone binding and 2-methylamino-[1-14C]isobutyric acid ([14C]meAIB) transport were assayed at 24 C in identical buffers. Equilibrium chicken insulin-binding experiments yielded curvilinear Scatchard functions, consistent with binding to both high affinity, low capacity and low affinity, high capacity receptor sites at 24 C. Insulin-stimulated transport of the model amino acid [14C]meAIB (a specific alanine-preferring system substrate) paralleled the expected occupancy of the low affinity receptors. In competitive binding assays, the abilities of multiplication-stimulating activity (MSA), proinsulin, and insulin to compete for primarily high affinity insulin binding were compared with their abilities to stimulate transport. The relative potencies for binding competition with 34 pM chicken [125I]insulin were insulin greater than proinsulin greater than or equal to MSA (1:0,05:0.03). In contrast, the relative potencies for stimulating [14C]meAIB transport were MSA greater than insulin greater than proinsulin (3:1:0.28). Maximal stimulation of transport by insulin was not additive to that by MSA or proinsulin. The relative potency profile for binding competition with [125I]MSA was the same as that for stimulation of transport: MSA greater than insulin greater than proinsulin (2-3:1:0.2-0.3). Proinsulin, added in excess of insulin at a constant molar ratio, reduced low affinity insulin binding to a greater extent than it reduced high affinity insulin binding. Moreover, the extent of binding to each receptor was predicted from, and thus directly supported, our biological potency data, in which proinsulin was nearly one third as potent as insulin at the low affinity receptor yet possessed only 5% of insulin's potency at the high affinity receptor. The different relative affinity profiles suggest that these two receptors had binding site specificities for different portions of the insulin molecule. Each experiment with proinsulin and insulin in a constant molar ratio was carried out in parallel with an experiment in which insulin was the only unlabeled hormone. Analysis of the results from weighted nonlinear least squares regression fits of data to Michaelis-Menten equations showed that a two-receptor model was necessary to fit the data obtained with insulin as the only unlabeled hormone. Low affinity site binding parameters were also needed when data within each parallel experiment were pooled. The following kinetic experiments indicated the absence of enhanced dissociation of bound [125I]insulin by unlabeled insulin...