A gene coding for a novel protein specific to the olfactory rosettes of Atlantic salmon.

We have isolated a 1.6 kb clone from a cDNA library made from the olfactory rosettes of the Atlantic salmon (Salmo salar). The clone contains a 1200 bp, open reading frame (named OSC) which codes for a protein with 400 amino-acid residues (Oscp). The mRNA corresponding to OSC is strongly expressed in the olfactory rosettes and weakly expressed in gills but is expressed in only these two tissues. This suggests that Oscp may have a specific and important role in olfaction. The sequence of Oscp suggests that it is not globular. Predictions show only a small fraction of alpha-helix. Oscp is hydrophilic but with the number of positively charged residues equal to the number of negatively charged residues. No closely similar protein can be found on the basis of homology searches or hydrophobicity comparisons. However, a 44 residue segment (G300 through K343) is significantly homologous to a segment of alpha-lactalbumin (G51 through K94). The similarities include the 19 residues of the "alpha- lactalbumin-lysozyme C signature," the ten residues of the Ca(2+) binding elbow and the four cysteine residues which provide two key disulfide links in alpha-lactalbumin and lysozyme C. Two more Cys residues are also very similarly placed. We conclude that the gene OSC codes for a unique protein which most likely contains a specific site for binding Ca(2+) and plays a unique role in the signal pathway of olfaction in salmon.

A calnaktin-like inhibitor of Na,K-ATPase in rat brain: regulation of alpha 1 and alpha 2 isozymes.

This study was designed to determine if a Ca(2+)-dependent, calnaktin-like inhibitor of Na,K-ATPase existed in rat brain and to compare the inhibition of different Na,K-ATPase isozymes in brain, heart and kidney. Based on the size and characteristics of human red blood cell calnaktin, a soluble protein fraction was obtained from rat brain and subjected to ultrafiltration and gel filtration to restrict the proteins to an appropriate molecular range of 6-50 kDa (6/50 fraction) for a crude calnaktin preparation. The 6/50 fraction was reconstituted with semipurified rat brain Na,K-ATPase and resulted in Ca(2+)-dependent inhibition of Na,K-ATPase activity. A 6/50 fraction was also prepared from rat heart ventricles, and, in its presence, Ca(2+)-dependent inhibition of cardiac Na,K-ATPase activity was observed. With brain preparations, the threshold for inhibition was approximately 100 nM free Ca2+, and inhibition was half maximal at 3-10 microM free Ca2+. Different isozymes of Na,K-ATPase were examined using differential sensitivity to ouabain and differential tissue distribution in brain, heart and kidney. The alpha 1 activity was inhibited in all three tissues. The alpha 2 activity of heart and the alpha 2 and/or alpha 3 activity of brain were also inhibited by the brain 6/50 fraction. In synaptosomal preparations from rat forebrain, resting intracellular (intrasynaptosomal) free Ca2+ was close to the threshold for calnaktin-like inhibition. The results are consistent with the presence of a calnaktin-like inhibitor of Na,K-ATPase in rat brain and indicate that calnaktin could be a widespread regulator of the alpha 1 isozyme. In addition, this study provides the first evidence that calnaktin also inhibits the alpha 2 activity of heart and the alpha 2 and/or alpha 3 isozymes of brain.

Chemoreceptive control of feeding processes in hydra.

Cnidarians are the simplest metazoans to exhibit satiety after feeding. When hydra are fed to repletion, they close their mouths and cease to capture prey. As feeding stops, contractions of the tentacles and body column increase. Our earlier experiments showed that a gel chromatographic fraction of prey substances inhibits prey capture. We now present evidence that the same fraction reduces the duration of mouth opening induced by reduced glutathione (GSH) and inhibits the binding of GSH to its putative receptor. The fraction also induces column contractions which are similar to those normally seen in sated animals. Prey substances, of unfractionated homogenate, also induce post-feeding tentacle contractions similar to those seen in sated animals. Gut distention does not appear to induce behavior associated with satiety. Therefore, these experiments suggest that chemoreception of prey substances induce satiety in hydra.

A subtype of the metabotropic glutamate receptor family in the olfactory system of Atlantic salmon.

A plasma membrane rich fraction was prepared from olfactory rosettes of Atlantic salmon and used to study binding of L-glutamic acid and activation of phospholipase C (PLC). Glutamate binding was saturable, high affinity, and inhibited by aspartic acid and taurocholate but not by alanine and lysine. Binding of glutamate was potently inhibited by various ligands for rat brain metabotropic glutamate receptors (mGluR) and also by kainate and N-methyl-D-aspartate. Glutamate stimulated phosphatidylinositol 4,5-bisphosphate breakdown consistent with G protein-dependent activation of PLC. Northern blot analyses demonstrated the presence of olfactory rosette RNA that hybridizes with cDNA probes for mGluR1 and mGluR4 under low stringency conditions. The results indicate the salmon olfactory system includes a subtype of the metabotropic glutamate receptor family.

Affinity purification of Hydra glutathione binding proteins.

The association of glutathione (GSH) with putative external chemoreceptors elicits feeding behavior in Hydra. In the present study, solubilized membrane proteins were chromatographed on an affinity column of immobilized GSH in order to isolate GSH-binding proteins that may represent the Hydra GSH chemoreceptor. The most abundant of the affinity-purified proteins was a triplet of peptides ranging in molecular weight from 24.5-26 kDa. Antiserum generated against the 24.5-26 kDa triplet peptides inhibited GSH-stimulated feeding behavior by 47%, implicating a role for one or more of these peptides in Hydra chemoreception.

Signal transduction for taurocholic acid in the olfactory system of Atlantic salmon.

Conjugated bile acids such as taurocholic acid (TChA) are potent olfactory stimuli for Atlantic salmon (Salmo salar). A plasma membrane rich fraction was derived from salmon olfactory rosettes and used to investigate TChA signal transduction and receptor binding. In the presence of GTP gamma S, TChA caused dose-dependent stimulation of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown, half maximal at less than 10(-7) M TChA. Stimulation of PIP2 breakdown by TChA required GTP gamma S, was blocked by GDP beta S, and was mimicked by A1F4-, consistent with a G protein requirement. A1F4- and Ca2+ stimulated breakdown of PIP2, but not phosphatidylcholine, arguing against a non-specific lipase activation. Stimulation of PIP2 breakdown by TChA was maximal at low Ca2+ concentration, < or = 10 nM. Conventional binding analysis with 3H-TChA was inconclusive due to a high degree of non-specific binding and to lack of tissue specificity expected for an olfactory receptor. Analysis of odorant amino acid binding indicated possible interaction of TChA with a putative acidic amino acid receptor but no interaction of TChA with a putative neutral amino acid receptor. We conclude that olfactory discrimination between amino acids and bile acids occurs in part at the receptor level while both classes of odors appear to use the same signal transduction mechanism, G protein mediated activation of phosphoinositide specific phospholipase C (PLC).

Stimulation of synaptosomal Na+,K(+)-ATPase by ethanol: possible involvement of an isozyme-specific inhibitor of Na+,K(+)-ATPase.

In synaptosomal preparations from rat cerebral cortex, ouabain-sensitive Rb+ uptake was stimulated by ethanol (20-80 mM). Based on differential sensitivity to ouabain, 80% of this Na+,K(+)-ATPase activity represented activity of the alpha 1 isozyme while 20% was due to the alpha 2 and/or alpha 3 isozymes (alpha 2/ alpha 3). Stimulation of Na+,K(+)-ATPase was selective for the activity of alpha 2/alpha 3 which was increased by 167% in the presence of 80 mM ethanol. In this concentration range, ethanol had no effect on alpha 1 activity. Exposure of synaptosomal preparations to EGTA increased basal (no ethanol) alpha 2/alpha 3 activity with no effect on alpha 1 activity. Further, ethanol no longer stimulated alpha 2/alpha 3 activity after EGTA treatment. An EGTA extract was concentrated and desalted to yield a fraction that selectively inhibited alpha 2/alpha 3 activity when reconstituted with EGTA-treated synaptosomal preparations. This inhibition was trypsin-sensitive, suggesting protein involvement, and was prevented by 80 mM ethanol. In the presence of the inhibitory protein fraction, ethanol stimulated Na+, K(+)-ATPase activity in EGTA-treated membranes with a dose-response like that observed with the crude (no EGTA) synaptosomes. We propose that the alpha 2/alpha 3 activity of Na+,K(+)-ATPase is subject to inhibitory regulation and that ethanol stimulates this activity by releasing it from inhibition, an effect that may mimic in vivo deregulation of the enzyme by ethanol.

High-affinity Ca2+,Mg(2+)-ATPase in plasma membrane-rich preparations from olfactory epithelium of Atlantic salmon.

High-affinity Ca2+,Mg(2+)-ATPase was identified in a plasma membrane-rich fraction of olfactory epithelium from Atlantic salmon (Salmo salar). The enzyme required both Ca2+ and Mg2+ for activation. The apparent Km for Ca2+ was 9.5 nM and Vmax was 0.85 mumol Pi/mg of protein per min. Stimulation by Ca2+ was optimal at 5-100 microM MgCl2. Bovine brain calmodulin had no effect on Ca2+,Mg(2+)-ATPase, even after multiple washes of the membrane preparation with EDTA or EGTA. Endogenous calmodulin was somewhat resistant to removal and could be detected with immunoblotting after multiple washes of the membrane preparation with EDTA or EGTA. This endogenous calmodulin may regulate Ca2+,Mg(2+)-ATPase activity because the activity was inhibited by calmidazolium. Vanadate inhibited Ca2+,Mg(2)-ATPase activity and thapsigargin, a specific inhibitor for Ca2+,Mg(2+)-ATPase of endoplasmic reticulum, had no effect on the enzyme activity. High affinity Ca2+,Mg(2+)-ATPase exists in both ciliary and nonciliary membranes with a similar Km for Ca2+. Ca2+,Mg(2+)-ATPase activity is greater in cilia preparations than in membranes from the deciliated olfactory epithelium. As a putative plasma membrane Ca2+ pump, this high-affinity Ca2+,Mg(2+)-ATPase may play an important role in the regulation of intracellular Ca2+ in olfactory epithelia. In particular, the ciliary membrane may play a prominent role in the removal of Ca2+ from ciliated olfactory receptor cells after odorant stimulation.

Stimulation of Ca(2+)-regulated olfactory phospholipase C by amino acids.

L-Amino acids are potent olfactory stimuli for Atlantic salmon. A plasma membrane fraction, previously shown to be rich in amino acid binding sites, was prepared from olfactory rosettes of Atlantic salmon (Salmo salar) and utilized to investigate the role of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis in olfactory signal transduction. A cocktail of L-amino acids (Ser, Glu, Lys, and Gly) stimulated PIP2 hydrolysis by phospholipase C (PLC) in a dose-dependent manner with half-maximal stimulation when all amino acids were present at approximately 1 microM. Stimulation of PIP2 hydrolysis by amino acids required GTP gamma S, which alone had no effect on PLC activity. Unlike GTP gamma S, AlF4- and Ca2+ stimulated PIP2 breakdown. Preincubation with 1 mM GDP beta S eliminated the effect of amino acids and AlF4- on PIP2 hydrolysis, suggesting the involvement of G protein regulation. The lack of stimulation by GTP gamma S alone suggested that there was negligible exchange of GTP gamma S for GDP in the absence of odorant. There were no significant effects of amino acids on either adenylate cyclase or guanylate cyclase activities in the membrane preparation under these conditions. The effect of the amino acid cocktail was maximal at 1-10 nM free Ca2+. At or above 100 nM free Ca2+, no effect of amino acids on PIP2 hydrolysis was found. However, between 100 nM and 100 microM, Ca2+ directly stimulated PLC activity in a dose-dependent manner. This stimulation by Ca2+ appeared to be G protein independent because it did not require GTP gamma S and was not inhibited by GDP beta S.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial characterization and detergent solubilization of the putative glutathione chemoreceptor from hydra.

Feeding behavior in hydra is initiated by the association of glutathione (GSH) with a putative external chemoreceptor. In the present study, the binding of [35S]GSH to hydra membranes has been characterized. Nondisplaceable [35S]GSH binding which compromised previous analyses [Grosvenor, W., Bellis, S., Kass-Simon, G., & Rhoads, D. (1992) Biochim. Biophys. Acta (in press)] was eliminated by treating membranes with an inhibitor of GSH metabolism, borate in combination with L-serine. The specific binding which was not inhibited by borate/serine demonstrated many of the characteristics expected of a ligand/receptor interaction. The binding was rapid, reversible, and saturable. A Scatchard analysis of saturation isotherms indicated a dissociation constant (KD) of 3.4 microM, a value which is in good agreement with concentrations of glutathione which are known to induce feeding behavior. Hydra membranes were detergent-solubilized with 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 100 mM KCl, and 10% glycerol. The soluble fraction contained 40% of the original saturable, reversible GSH binding activity. The KD for GSH binding to the solubilized preparation was estimated as 2.7 microM, a valuable which is not appreciably different from the KD for binding to intact membranes. The fidelity of GSH binding in the solubilized preparation suggests that this preparation will be useful in further characterization of the putative glutathione chemoreceptor.

Effects of ethanol on Na(+)-dependent amino acid uptake: dependence on rat age and Na+, K(+)-ATPase activity.

Acute effects of ethanol on Na(+)-dependent transport of gamma-aminobutyric acid (GABA) and glutamic acid (GLU) were investigated in crude synaptosomal preparations from rat cerebral cortex. In experiments with 30-40-day-old (peripubertal) rats, the overall dose responses of the GABA and GLU transport systems to ethanol were biphasic. Stimulation was observed at ethanol concentrations (40-160 mM) relevant to intoxication. Inhibition was observed at higher concentrations of ethanol. The stimulatory phase of the dose response was not observed in 60-100-day-old (adult) rats. In preparations from peripubertal rats, other alcohols also had biphasic dose response curves with stimulation at low alcohol concentrations. The relative efficacy of the different alcohols appeared to correlate with the relative membrane-buffer partition coefficient. In synaptosomal membrane vesicles, where artificial ion concentration gradients rather than Na+,K(+)-ATPase activity provide the driving force for uptake, ethanol did not stimulate GABA uptake. In direct measures of Na+,K(+)-ATPase activity, both Rb+ uptake and ATP hydrolysis were enhanced by 80 mM ethanol. We conclude that stimulation of Na(+)-dependent uptake of amino acids by ethanol was secondary to enhanced Na+,K(+)-ATPase activity and may be associated with a specific developmental stage in the rat.

Chemoreception in hydra: specific binding of glutathione to a membrane fraction.

Specific binding of reduced [35S]glutathione (GSH) was measured using a crude membrane fraction of Hydra vulgaris (attenuata). The specific binding shows both rapid displaceable and nondisplaceable components. Rapid displaceable binding accounted for 20% of the total specific binding. Data from saturation binding experiments indicates half maximal total specific binding occurs at 2 microM GSH which is similar to reported EC50 values from behavioral experiments. Calcium is required for displaceable binding of GSH to the putative receptor. Oxidized glutathione (GSSG), an antagonist of the GSH-activated feeding response, and S-methylglutathione (GSM), an agonist of the feeding response, inhibit the binding of radiolabeled GSH to the putative receptor. Glutamate, a putative competitive antagonist of the GSH-activated feeding response in hydra, does not inhibit the specific binding of radiolabeled GSH to the receptor and must therefore block the feeding response by a mechanism other than competitive inhibition.

Chemoreception in Hydra vulgaris (attenuata): initial characterization of two distinct binding sites for L-glutamic acid.

To elucidate the relationship between L-glutamic acid and the putative chemoreceptor for glutathione, binding of L-[3H]glutamate to a crude membrane fraction from Hydra vulgaris (attenuata) has been characterized. The binding of L-[3H]glutamate was rapid, reversible and saturable. A Scatchard analysis of the specific binding revealed values of 10 microM for the dissociation constant (Kd) and 170 pmol/mg for the maximal capacity of binding sites (Bmax). A maximum of 65% of the specific L-[3H]glutamate binding was inhibited by the chemostimulatory peptide, glutathione. This glutathione-sensitive glutamate binding presumably represents the association of glutamate with a putative chemoreceptor which modulates feeding behavior in hydra. The remaining 35% of the specific L-[3H]glutamate binding may be due to a second class of glutamate binding sites which is insensitive to glutathione. The identification of glutathione-insensitive glutamate binding is the first indication of a putative glutamate receptor, which may mediate an action independent of the glutathione-induced feeding response. The glutathione-insensitive and glutathione-sensitive sites must have similar affinities for glutamate since these sites were indistinguishable by Scatchard analysis. A preliminary characterization of the glutathione-insensitive site, performed in the presence of saturating levels of glutathione, revealed inhibition of glutathione-insensitive glutamate binding by kainate and quisqualate, but not by N-methyl-D-aspartate. A glutathione-insensitive L-[3H]glutamate binding suggests that kainate and alpha-aminoadipate may be selective ligands for the glutathione-insensitive and glutathione-sensitive glutamate binding sites, respectively.

L-alanine binding sites and Na+, K(+)-ATPase in cilia and other membrane fractions from olfactory rosettes of Atlantic salmon.

1. Membrane fractions were obtained from homogenates of olfactory rosettes from Atlantic salmon (Salmo salar) or from isolated olfactory cilia and homogenates of deciliated olfactory rosettes. 2. Specific binding of L-[3H]alanine was saturable, high-affinity, and effectively inhibited by L-threonine, L-serine and L-alanine but not by L-lysine or L-glutamic acid. Comparable results were obtained with L-[3H]serine except for the presence of a second, lower affinity, binding site for L-alanine but not L-serine. 3. Specific binding of L-[3H]alanine was inhibited by low concentrations of mercury ion, acidic pH, and high concentrations of cadmium, copper or zinc ions. Aluminum had no effect. 4. Specific binding sites for L-alanine were present in membranes from isolated cilia at a level 2-fold that of membranes prepared from the deciliated rosette. 5. Ouabain sensitive Na+, K(+)-ATPase activity was also determined in cilia preparations. This enzyme was present in cilia at a level approximately 3-fold that of membranes prepared from the deciliated rosette. 6. The results are consistent with the presence of an olfactory alanine receptor in S. salar with binding characteristics similar to those of a variety of other fish species and with a localization on olfactory cilia as well as non-ciliated receptor cell membranes.

Release of immunoreactive insulin from rat brain synaptosomes under depolarizing conditions.

Synaptosome preparations were utilized to characterize the release and compartmentalization of immunoreactive insulin (IRI) in the adult rat brain. Depolarization of synaptosomes by elevation of the external potassium ion concentration elicited release of IRI from the synaptosomes into the incubation medium. This release was reduced or eliminated under three conditions known to prevent depolarization-induced Ca2+ flux: elevating the external MgCl2, adding CoCl2, and eliminating external Ca2+ with EGTA. Depolarization of synaptosomes by veratridine also elicited release of synaptosomal IRI. This release was inhibited by tetrodotoxin. The amount of IRI released under depolarizing conditions represented 3-7% of that contained in the synaptosomes. High levels of IRI release also were observed upon removal of external Na+ to allow depolarization-independent influx of external Ca2+ into the synaptosomal compartment. The Ca2+ dependency of synaptosomal IRI release suggests IRI is stored in the adult rat brain in synaptic vesicles within nerve endings from which it can be mobilized by exocytosis in association with neural activity.

Specific binding of insulin to membranes from dendrodendritic synaptosomes of rat olfactory bulb.

The external plexiform layer of the olfactory bulb is among the brain regions where insulin receptors are most abundant. In vitro binding of porcine 125I-insulin to membranes of dendrodendritic synaptosomes isolated from adult rat olfactory bulbs was studied to test the hypothesis that dendrodendritic synapses are major insulin-receptive sites in the external plexiform layer of olfactory bulbs. Of the specific insulin binding sites present in a total particulate fraction from the olfactory bulbs, approximately half were recovered in the dendrodendritic synaptosome fraction. The only other subcellular fraction to which substantial insulin binding was observed was the conventional (axodendritic/axosomatic) synaptosome fraction. Analysis of equilibrium binding of insulin to dendrodendritic synaptosomal membranes, at total insulin concentrations of 0.5-1,000 nM, revealed binding site heterogeneity consistent with a two-site model for insulin binding to a high-affinity (KD = 6 nM), low-capacity (Bmax = 110 fmol/mg of protein) site and a low-affinity (KD = 190 nM), high-capacity (Bmax = 570 fmol/mg of protein) site. The results indicate that the intense labeling of the external plexiform layer of the olfactory bulb in autoradiographic studies of insulin binding can be attributed to insulin receptors on dendrodendritic synaptic membranes in this region.

The inositol lipids of Paramecium tetraurelia and preliminary characterizations of phosphoinositide kinase activity in the ciliary membrane.

Inositol glycerolipids make up less than 10% of total phospholipids of Paramecium tetraurelia cells. Unlike inositol lipids found in mammalian and other cell types, these lipids from Paramecium lack arachidonic acid. It was demonstrated that kinase and possibly phosphatase enzymes that interconvert phosphatidylinositol (PI), phosphatidylinositol phosphate (PI-P) and phosphatidylinositol-bis-phosphate (PI-P2) exist in ciliary membranes of this ciliate. When exogenous soybean PI and [gamma-32P]ATP were provided as substrates, isolated cilia preparations exhibited PI and PI-P kinase activities as demonstrated by the incorporation of radiolabel into PI-P and PI-P2. Kinase activity was activated by millimolar [Mg2+] and inhibited by millimolar [Ca2+]. Significant inhibition of kinase activity in the presence of unlabeled excess ATP suggested that ATP is the preferred phosphate donor for this reaction. Of 4 suborganellar fractions of isolated cilia, the membrane fraction had the greatest kinase activity indicating that the enzyme(s) is membrane-associated.

Metabolism of saturated fatty acids by Paramecium tetraurelia.

Paramecium requires oleate for growth. The phospholipids of the ciliate contain high concentrations of palmitate and 18- and 20-carbon unsaturated fatty acids. We previously showed that radiolabeled oleate is desaturated and elongated to provide these 18- and 20-carbon unsaturated acids. We now report on saturated fatty acid (SFA) metabolism in Paramecium. Radiolabeled palmitate and stearate were incorporated directly into cellular phospholipids with little or no desaturation and/or elongation. Radiolabeled acetate, malonate, pyruvate, citrate, or glucose added to cultures were not incorporated into cellular phospholipid fatty acids indicating that these exogenously supplied putative precursors were not utilized for fatty acid synthesis by Paramecium. Radiolabel from octanoate or hexanoate appeared in fatty acyl groups of phospholipids, possibly by partial beta-oxidation and reincorporation of the label. Under oleate-free conditions in which cultures do not grow, radiolabel from these shorter chain SFA were beta-oxidized and preferentially used for the formation of arachidonate, the major end-product of fatty acid synthesis in Paramecium. Cerulenin inhibited culture growth apparently by inhibiting de novo fatty acid synthesis. Cerulenin-treated cells did not incorporate radioactivity from [1-14C]octanoate into esterified palmitate. However, total saponifiable phospholipid fatty acids, including SFA, per cell increased under these conditions.

The glyceryl ethers of Paramecium phospholipids and phosphonolipids.

Two glyceryl ethers, 1-O-hexadecyl glycerol and 1-O-cis-octadec-11-enyl glycerol, chimyl and paramecyl alcohol, respectively, were quantified in total phospholipids and five glycerophospholipid classes from cells and cilia of the ciliated protozoon, Paramecium tetraurelia. The ether content of 2-aminoethyl phosphonoglycerolipid was 85-90 mole %. Concentrations of ethers were greatest in the ethanolamine phosphonolipids greater than phosphatidylcholines greater than phosphatidylserines greater than phosphatidylethanolamines greater than phosphatidylinositols. The glyceryl ether concentrations in total cellular phospholipids increased with culture age in P. tetraurelia and P. multimicronucleatum cells. The glyceryl ether concentrations in the phospholipids of P. tetraurelia cilia remained constant from mid log to stationary phase of culture growth. Paramecium tetraurelia phospholipid glyceryl ether concentrations were made greater by supplementation of cultures with chimyl alcohol.

Interaction of enkephalins and des-tyrosyl-enkephalins with synaptosomal plasma membrane vesicles: enkephalin binding and inhibition of proline transport.

Leucine- and methionine-enkephalins inhibit the Na+-dependent transport of proline into plasma membrane vesicles derived from synaptosomes. Glycine transport is weakly inhibited by enkephalins whereas there is no inhibition of transport of glutamic acid, aspartic acid, or gamma-aminobutyric acid. The inhibition of proline uptake is observed with des-tyrosyl-enkephalins but not with morphine, dynorphin(1-13), or beta-endorphins. Furthermore, enkephalin-induced inhibition of proline transport is not antagonized by naloxone. [Leu]enkephalinamide and modified [Leu]enkephalins with greater selectivity for the delta-subclass of enkephalin binding sites are less effective than [Leu]enkephalin in the inhibition of proline transport. Specific binding of [3H]Leu-enkephalin to the plasma membrane vesicles is demonstrated, and des-Tyr-[Leu]enkephalin competes with Leu-enkephalin for [Leu]enkephalin binding sites. The similarity in the concentrations of des-Tyr-[Leu]enkephalin required to compete for specific [Leu]enkephalin binding and to inhibit proline transport suggests that a specific subclass of enkephalin binding sites, distinguished by their recognition of both the enkephalins and their des-tyrosyl derivatives, may be associated with the synaptic proline transport system.