Iminoglycine transport system in synaptosomes and its interaction with enkephalins

Evidence is presented which suggests that proline, pipecolic acid, and glycine are accumulated by a common transport system in rat brain cortical synaptosomes and synaptosomal plasma membrane vesicles. This system is Na+ dependent and appears to be similar to the iminoglycine transport system present in renal tubules and in renal brush border membranes. The opioid pentapeptides Leu- and Met-enkephalin specifically inhibit the uptake of these three imino/amino acids, presumably by interaction with a nonopioid receptor, since the inhibition is not affected by the opiate antagonist naloxone and occurs with des-tyrosyl enkephalins as well as with the intact pentapeptides.     

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.     

Effects of intravenously administered Leu- or Met-enkephalin on arterial blood pressure

The purpose of these studies was to determine if two endogenous opioids, leucine (Leu) and methionine (Met) -enkephalin, alter blood pressure and, if so, by what mechanisms. Studies from our laboratory show that intravenous administration of Leu-enkephalin in doses of 0.032–320 μg/kg induced a biphasic response in pentobarbital-anesthetized cats. A transient rise in mean arterial pressure was followed by a more prolonged decline. Administration of Met-enkephalin caused only a decline in mean arterial pressure. Neither agent significantly altered heart rate, venous pressure or the EKG. Having determined that both enkephalins altered blood pressure and observed that the responses were qualitatively different, selected pharmacological antagonists were employed to see if the alterations in blood pressure could be blocked. Naloxone blocked the hypertensive responses and antagonized the hypotensive effects seen with the administration of Leu-enkephalin. Naloxone also shifted the dose-effect curve of Met-enkephalin to the right. Diphenhydramine attenuated both the hypertensive and hypotensive responses of Leu-enkephalin. However, diphenhydramine pretreatment did not alter the decline in blood pressure seen with the higher doses of Met-enkephalin. Propranolol exerted some antagonistic activity in association with the rise in blood pressure seen with Leu-enkephalin, but propranolol did not alter the drop in pressure observed with the administration of either enkephalin. These results show that intravenous administration of the enkephalins can alter blood pressure and these effects are not alike for each enkephalin. Additionally, the enkephalins are not blocked in the same fashion by antagonists, giving support to the hypothesis that the two enkephalins interact with different receptors.     

The significance of mu- and delta-receptors in rat pancreatic islets for the opioid-mediated insulin release

The binding and the insulinotropic effects of enkephalin analogs and of morphine were investigated in rat pancreatic islets. Binding of [3H]Met-enkephalin was saturable, specific and reversible; the rank order for inhibition competition of [3H]Met-enkephalin binding by various compounds was Met-enkephalin = D-Ala2-MePhe4, Met(0)ol enkephalin) greater than Leu-enkephalin greater than morphine with half-maximal inhibitory constants (IC50) of approx. 0.3, 0.3, 100 and greater than 100 nM, respectively. Both the natural enkephalins exerted their insulinotropic effect only at stimulatory glucose concentrations. They had a dual action; whereas insulin secretion was increased at low enkephalin concentration, this effect was reversed at higher concentrations. However, the various enkephalins exerted this effect at different concentrations; only the EC50 values (half-maximal effective concentrations) of their insulinotropic effect were in the same range as the IC50 values of inhibition of [3H]met-enkephalin binding. Cysteamine pretreatment of rats (depletion of somatostatin containing D-cells and decrease in somatostatin secretion) did not change the Met-enkephalin effect on insulin secretion. In contrast to Met-enkephalin, binding of [3H]morphine to islets was not saturable, and morphine had no effect on insulin secretion unless at unphysiologically high concentrations. The data, therefore, indicate that: mu-receptors (affinity for morphine) do not play a role in rat pancreatic islets; delta-receptors (binding site for Met-enkephalin when mu-receptors are not present) mediate the insulinotropic effect of low Met-enkephalin concentrations; and the insulinotropic action of Met-enkephalin is not mediated indirectly via the paracrine effect of an inhibition of somatostatin secretion.     

Enkephalins are transported by a novel eukaryotic peptide uptake system

We have identified an oligopeptide transporter in the yeast Saccharomyces cerevisiae which mediates the uptake of tetra- and pentapeptides, including the endogenous opioids leucine enkephalin (Tyr-Gly-Gly-Phe-Leu) and methionine enkephalin (Tyr-Gly-Gly-Phe-Met). The transporter is encoded by the gene OPT1. Yeast expressing OPT1 can utilize enkephalins to satisfy amino acid auxotrophic requirements for growth. The transport of radiolabeled leucine enkephalin exhibits saturable kinetics, with a K(m) of 310 microM. Transport activity is optimum at acidic pH and sensitive to reagents which uncouple oxidative phosphorylation, suggesting an energy dependence on the proton gradient. Growth, transport, and chromatographic data indicate that leucine enkephalin is not hydrolyzed in the extracellular medium and as such is translocated intact across the cell membrane. The system is specific for tetra- and pentapeptides and can be inhibited by the opioid receptor antagonists naloxone and naltrexone. To date, this is the first example of a eukaryotic transport system which can use enkephalins as a substrate, opening the possibility that a homologue exists in higher eukaryotes.     

Initial Processing of Human Proenkephalin in Bovine Chromaffin Cells

Abstract: The opioid peptide precursor preproenkephalin (PPE) contains seven enkephalin sequences and is synthesized by epinephrine-producing adrenal chromaffin cells and various peripheral and central neurons. After removal of its signal peptide. PPE undergoes processing at dibasic amino acid sites to yield its final opioid products—Met-enkephalin, Leu-enkephalin, and various larger, enkephalin-containing peptides. Processing of PPE was examined in bovine chromaffin cells using a plasmid containing the human PPE (hPPE) cDNA under the control of the cytomegalovirus immediate early enhancer/promoter. Following transfection of this hPPE-containing plasmid into bovine chromaffin cells, several proenkephalin-immunoreactive bands were observed on western blots with monoclonal antibodies that recognize human, but not bovine, proenkephalin sequences. The pattern of hPPE-derived peptides observed was similar to that of bovine PPE processing products. A series of recombinant plasmids containing mutations in the hPPE sequence at putative processing sites was then constructed. Conversion of Lys-Lys and Lys-Arg sequences to Lys-Gln and of Arg-Arg to Arg-Gln altered initial hPPE processing at only three of the putative processing sites. When hPPE cDNA containing mutations at all of these initially processed sites was expressed, one or more alternative processing sites were revealed. These data suggest the importance of structural features in addition to the dibasic sequences that limit the processing of proenkephalin.     

Dermorphins, Opioid Peptides from Amphibian Skin, Act on Opioid Receptors of Mouse Neuroblastoma x Rat Glioma Hybrid Cells

Dermorphin and its Hyp6 analogue are opiate-like heptapeptides originally discovered in frog skin and characterized by the presence of a D-Ala2 residue in their sequence. They were assayed for their capacity to compete with [3H]Leu-enkephalin for binding to opioid receptors in membranes of neuroblastoma x glioma hybrid cells. In the presence of 7 nM-[3H]Leu-enkephalin, the concentrations at which they caused 50% inhibition of [3H]enkephalin binding (IC50 values) are 0.1 micro M and 0.3 micro M, respectively. In contrast, the synthetic L-Ala2-dermorphin shows very low affinity for the opioid receptors. In addition, like other opioid peptides, dermorphin and hyp6-dermorphin inhibit the elevation by prostaglandin E1 (PGE1) of the level of adenosine 3':5'-cyclic monophosphate (cyclic AMP) (IC50 values 0.2 micro M and 0.4 micro M, respectively). The inhibition is prevented by the opiate antagonist naloxone, L-Ala2-dermorphin is at least three orders of magnitude less potent in inhibiting the PGE1-evoked increase in the level of cyclic AMP. The results show that peptides with an amino acid sequence quite different from that of the enkephalins can bind to opioid receptors of the hybrid cells.     

Biphasic competition between opiates and enkephalins: does it indicate the existence of a common high affinity ("mu-1") binding site?

Displacement from brain membranes of labeled opiates by low concentrations of enkephalins and of labeled enkephalins by low concentrations of opiates has been previously explained by the existence of a common high affinity site termed mu-1. An alternative interpretation of the same results is that the trough seen in the low concentration zone of the displacement curves represents cross binding of mu and delta opioid ligands to delta and mu receptors, respectively. In three sets of experiments with brain membranes, the size of the trough is shown to be dependent on the labeled ligand used: The ratio between the size of troughs seen with [3H]D-Ala, D-Leu enkephalin and with [3H]morphine varies with experimental conditions (storage of membranes at 4 degrees C for 72 h), with ratio of mu:delta receptors (e.g. in thalamus and cortex which are enriched in mu and delta sites, respectively) and with pretreatment of membranes with naloxonazine. These results can not be explained by a common high affinity site, but rather by binding of [3H]D-Ala, D-Leu enkephalin to mu and of [3H]morphine to delta opioid receptors.     

Cloning and expression in Escherichia coli of the synthetic proenkephalin analogue gene

Enkephalins are pentapeptides with opioid activity that have been found in brain and other neural tissues. They are released by proteolytic processing of the proenkephalin, which contains several enkephalin sequences each flanked by pairs of basic amino acid (aa) residues. We have constructed an artificial variant of the proenkephalin gene by concatenation of synthetic oligodeoxynucleotides (oligo) coding for Met-enkephalin preceded by two arginines. One of the resulting structures, containing eleven enkephalin sequences separated by pairs of arginine codons, was cloned in the expression vector pRE31. The biological activity of enkephalin was detected after the digestion of the isolated plasmid-coded protein with trypsin and carboxypeptidase B. The product of the synthetic gene may thus serve as a defined simplified substrate for the study of the not yet fully understood enzymatic mechanisms of proenkephalin processing.     

A unique proenkephalin-converting enzyme purified from bovine adrenal chromaffin granules

A unique proenkephalin converting enzyme specifically generating enkephalin was partially purified from lysates of adrenal chromaffin granules. The enzyme, whose molecular weight is estimated as ca. 220,000, is thiol-dependent protease, with optimal pH at around 5.5. The enzyme converts proenkephalin to enkephalins by cleaving specifically at the sites of consecutive basic amino acid residues. The enzyme also converts BAM-12P, an adrenal “big” Met-enkephalin, to Met-enkephalin in a similar manner. During the enzyme reaction, formation of [Arg⁶]-Met-enkephalin was not observed. Additionally, [Arg⁶]-enkephalins were not converted to enkephalins by the enzyme. Consequently, the enzyme was proved to be a unique converting enzyme distinct from either trypsin-like or carboxypeptidase B-like proteases.     

N-cyclo-[Leu5]enkephalin: a rational approach for the synthesis of conformationally restricted cyclic pentapeptides

The enkephalins are neuropeptides belonging to the class of endogenous opioids. The conformationally restricted analog, N-cyclo-[Leu5]enkephalin, was recently synthesized. Since the synthesis of cyclic pentapeptides which lack proline and contain amino acids with bulky side chains is problematic, the synthesis, purification, and analytical characterization of N-cyclo-[Leu5]enkephalin is described in detail. This conformationally restricted cyclic pentapeptide was prepared from H-Gly-Phe-Leu-[O-(2,6-dichlorobenzyl)-Tyr]-Gly-NHNH2, which was synthesized by the solid-phase method of peptide synthesis. Cyclization was accomplished through an azide intermediate at high dilution, using high-pressure liquid chromatography to monitor the reaction. The desired cyclic monomer was isolated and purified by semipreparative HPLC. The structure of the purified cyclic product was confirmed by multiple chemical techniques including amino acid analysis, lack of an amino terminus (as assessed by reaction with ninhydrin and Edman reagent), and mass spectroscopy.     

Characterization of opioid receptors on isolated canine gallbladder smooth muscle cells

Smooth muscle cells were isolated from the fundus of the canine gallbladder and examined for the presence of opioid receptors. The cells contracted in a concentration-dependent manner in response to three opioid peptides (Met-enkephalin, dynorphin1-13 and Leu-enkephalin), which are known derivatives of opioid precursors present in myenteric neurons of the gut. The order of potency was Met-enkephalin greater than dynorphin1-13 greater than Leu-enkephalin. The contractile response to opioid agonists was selectively inhibited by opioid antagonists (naloxone and Mr2266) but not by muscarinic, CCK/gastrin or tachykinin antagonists. Equivalent responses to the three opioid peptides exhibited differential sensitivity to preferential antagonists of mu (naloxone) and kappa (Mr2266) opioid receptors consistent with the presence of the three main types of opioid receptors (mu, delta and kappa) on canine gallbladder muscle cells.     

The actions of naloxazone on the binding and analgesic properties of morphiceptin (NH2Tyr-Pro-Phe-Pro-CONH2), a selective mu-receptor ligand

Active in both binding and biological assays, morphiceptin (NH₂ Tyr-Pro-Phe-Pro-CONH₂), a potent opioid peptide derivative of β-casamorphine, binds specifically and selectively to mu or morphine-type receptors with little affinity for delta sites. Displacement studies of a variety of ³H-labeled opiates and enkephalins show biphasic curves. Naloxazone, which blocks irreversibly and selectively high affinity opiate and enkephalin binding, abolishes morphiceptin's inhibition of binding at low concentrations, suggesting that the high affinity binding of enkephalins and opiates represents a mu or morphine-type receptor. Unlike the reversible antagonist naloxone, naloxazone treatment $\text{in}$$\text{vivo}$ inhibits for over 24 hours the analgesic activity of morphiceptin like it inhibits morphine, β-endorphin and enkephalin analgesia. Together, these studies imply that opiates and enkephalins bind with highest affinity to a mu receptor which mediates their analgesic activity. The ³H-D-ala²-D-leu⁵-enkephalin binding remaining after naloxazone treatment, representing a lower affinity site (K_D 4 nM), is quite insensitive to morphiceptin inhibition and has the characteristics of a delta receptor. However, the ³H-dihydromorphine binding present after naloxazone treatment, which also represents a lower affinity site (K_D 6 nM), is far more sensitive to both morphine and morphiceptin and may represent a second morphine-like, or mu, receptor.     

A comparison of the ability of opioid peptides and opiates to affect active avoidance conditioning in rats

Enkephalins reduce acquisition of an active avoidance response when administered intraperitoneally shortly before training. The present study examined whether microgram or delta opiate receptors are involved in this enkephalin effect. This was done by comparing the efficacy of micro- and delta-receptor agonists; by attempting to block the enkephalin effect with micro- and delta-receptor antagonists; and by comparing the characteristics of the effects of Met-enkephalin and Leu-enkephalin. In addition, the efficacy of kappa-agonists in reducing acquisition was assessed. It was found that micro-agonists are inactive in this assay; several delta- and kappa-agonists are active. However, not all of the data are consistent with the adequacy of this receptor classification. The micro-receptor antagonist naloxone did not readily block the effect of Met- or Leu-enkephalin but neither did the micro/delta-antagonist, diprenorphine. An additional complexity is the emergence of differences in behavioral activity of Met- snd Leu-enkephalin.     

Oxidative and nitrative modifications of enkephalins by human neutrophils: effect of nitroenkephalin on leukocyte functional responses

Neutrophils play a major role in acute inflammation by generating reactive oxygen/nitrogen species. Opioid peptides, including enkephalins, are present at inflammation sites. Neutrophils contribute to protect against inflammatory pain by releasing opioid peptides. In this investigation, the ability of human polymorphonuclear cells to induce oxidative and nitrative modifications of Leu-enkephalin has been investigated in vitro. Activated human neutrophils mediate the oxidation of Leu-enkephalin resulting in the production of dienkephalin. In the presence of nitrite at concentrations observed during inflammatory and infectious process (10-50 μM), nitroenkephalin, a nitrated derivative of Leu-enkephalin, is additionally formed. The yield of nitroenkephalin increases with nitrite concentration and is significantly inhibited by the addition of catalase or 4-aminobenzoic acid hydrazide (ABAH), a specific inhibitor of peroxidases. These results suggest that neutrophils induce nitration of Leu-enkephalin by a mechanism that is dependent on myeloperoxidase activity and hydrogen peroxide. Oxidative/nitrative modifications of Leu-enkephalin have been also evidenced when cells were treated with the NO-donor molecule, DEANO. The nitrated enkephalin has been examined for its effect on leukocyte functional responses. The data reveal that nitroenkephalin at micromolar concentrations inhibits superoxide anion generation and degranulation of azurophilic granules of human polymorphonuclear cells. Moreover, nitroenkephalin inhibits spontaneous apoptosis of neutrophils, as evaluated by measuring caspase-3 activity. Collectively, our data indicate that the nitrated enkephalin attenuates neutrophil activation and promotes the short-term survival of these cells, suggesting a possible role of the nitrocompound in the efficiency and resolution of inflammatory processes.     

Organization of proenkephalin in amphibians: cloning of a proenkephalin cDNA from the brain of the anuran amphibian, Spea multiplicatus

Cloning of a proenkephalin cDNA from the pelobatid anuran amphibian, Spea multiplicatus, provides additional evidence that Leu-enkephalin, although present in the brain of anuran amphibians, is not encoded by the proenkephalin gene. The S. multiplicatus proenkephalin cDNA is 1375 nucleotides in length, and the open reading frame contains the sequences of seven opioid sequences. There are five copies of the Met-enkephalin sequence, as well as an octapeptide opioid sequence (YGGFMRNY) and a heptapeptide opioid sequence (YGGFMRF). In the proenkephalin sequence of S. multiplicatus the penultimate opioid is a Met-enkephalin sequence rather than the Leu-enkephalin present in mammalian sequences. The same order of opioid sequences also is observed for the proenkephalin sequence of the pipid anuran amphibian, Xenopus laevis. Hence, from a phylogenetic standpoint the organization of tetrapod proenkephalin has been remarkably conserved. What remains to be resolved is whether the Leu-enkephalin sequence found in mammalian proenkephalin is an ancestral trait or a derived trait for the tetrapods. Unlike the proenkephalin precursor of X. laevis, all of the opioid sequences in the S. multiplicatus proenkephalin cDNA are flanked by paired-basic amino acid proteolytic cleavage sites. In this regard the proenkephalin sequence for S. multiplicatus is more similar to mammalian proenkephalins than the proenkephalin sequence of X. laevis. However, a comparison of the proenkephalin sequences in human, X. laevis, and S. multiplicatus revealed several conserved features in the evolution of the tetrapod proenkephalin gene. By contrast, a comparison of tetrapod proenkephalin sequences with the partial sequence of a sturgeon proenkephalin cDNA indicates that the position occupied by the penultimate opioid sequence in vertebrate proenkephalins may be a highly variable locus in this gene.     

Opioid receptor affinity and selectivity effects of second residue and carboxy terminal residue variation in a cyclic disulfide-containing opioid tetrapeptide.

The previously described cyclic, delta opioid receptor-selective tetrapeptide H-Tyr-D-Cys-Phe-D-Pen-OH, where Pen, penicillamine, is beta-beta-dimethylcysteine, was modified at residues 2 and 4 by varying combinations of D- and L-Cys and D- and L-Pen, and effects on mu and delta opioid receptor binding affinities and on potency in the mouse vas deferens (MVD) smooth muscle assay were evaluated. A comparison was drawn between consequences of alterations in this series of analogs and those of analogous modifications in the related cyclic pentapeptide series which includes the highly delta receptor-selective [D-Pen2,D-Pen5]enkephalin, DPDPE. Unlike effects observed in the cyclic pentapeptide series, the mu receptor binding affinities of the cyclic tetrapeptides are not dramatically influenced by substitution of Pen for Cys at residue 2. Conversely, while binding of the pentapeptides is only slightly affected by alteration of the chirality of the carboxy-terminal residue, modification of stereochemistry at the carboxy terminus in the tetrapeptides critically alters binding behavior at both mu and delta sites. In contrast with the pentapeptide series, the tetrapeptides appear to be highly dependent upon primary sequence for binding and activity, as only the lead compound binds with high affinity to the delta site. Results suggest that the less flexible cyclic tetrapeptides, lacking the Gly3 residue, display more stringent structural requirements for binding and activity than do the corresponding cyclic pentapeptides.     

Modulation of Opioid Receptor Binding by Cis and Trans Fatty Acids

In synaptosomal brain membranes, the addition of oleic acid (cis), elaidic acid (trans), and the cis and trans isomers of vaccenic acid, at a concentration of 0.87 mumol of lipid/mg of protein, strongly reduced the Bmax and, to a lesser degree, the binding affinity of the mu-selective opioid [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ([3H]DAMGO). At comparable membrane content, the cis isomers of the fatty acids were more potent than their trans counterparts in inhibiting ligand binding and in decreasing membrane microviscosity, both at the membrane surface and in the core. However, trans-vacenic acid affected opioid receptor binding in spite of just marginally altering membrane microviscosity. If the receptors were uncoupled from guanine nucleotide regulatory protein, an altered inhibition profile was obtained: the impairment of KD by the fatty acids was enhanced and that of Bmax reduced. Receptor interaction of the delta-opioid [3H](D-Pen2,D-Pen5)enkephalin was modulated by lipids to a greater extent than that of [3H]DAMGO: saturable binding was abolished by both oleic and elaidic acids. The binding of [3H]naltrexone was less susceptible to inhibition by the fatty acids, particularly in the presence of sodium. In the absence of this cation, however, cis-vaccenic acid abolished the low-affinity binding component of [3H]naltrexone. These findings support the membrane model of opioid receptor sequestration depicting different ionic environments for the mu- and delta-binding sites. The results of this work show distinct modulation of different types and molecular states of opioid receptor by fatty acids through mechanisms involving membrane fluidity and specific interactions with membrane constituents.     

Interaction of D-Ala2-[Tyr-3,5-3H]enkephalin(5-D-Leu) with opiate receptors of rat brain

Some kinetic features of D-Ala2-[Tyr-3.5-3H]enkephalin (5-D-Leu) binding to opiate receptors of rat brain were studied. It was shown that the Leu-enkephalin D analog interacts with the high and low affinity binding sites of opiate receptors, the equilibrium constants being equal to 0.71 and 8.4 nM, respectively. The rate constant for the label association with the high affinity binding sites in 2 . 10(8) M-1 min-1; those for the label dissociation from the opiate receptor binding sites with high and low affinities are 7.2 . 10(-3) and 0.16 min-1, respectively. Hence, the half-life time of these complexes is 95.7 and 4.3 min, respectively. Na+, K+ and Li+ markedly decrease the specific finding of the label, while Mg2+, Mn2+ and Ca2+ at the concentrations studied markedly increase its specific binding. It is concluded that the Leu-enkephalin D-analog under study acts as a morphine agonist and reveals a much higher affinity for rat brain opiate receptors than does Leu- or Met-enkephalin. This makes it a useful tool for study of the enkephalin reception under normal and pathological conditions.     

Effects of Free Fatty Acids on Synaptosomal Amino Acid Uptake Systems

Abstract: The Na⁺-dependent synaptosomal uptakes of proline, aspartic acid, glutamic acid, and γ-aminobutyric acid were strongly inhibited by monounsaturated fatty acids. With oleic acid, half-maximal inhibition was observed at about 15 μM. The Na⁺-independent uptakes of leucine, phenylalanine, histidine, and valine were less sensitive to inhibition by the unsaturated fatty acids. In contrast, the uptakes of all of these amino acids were unaffected by saturated fatty acids. The inhibition of proline uptake (and that of the other Na⁺-dependent amino acids) by oleic acid was overcome by the addition of serum albumin and the data presented further indicate that the previously reported stimulation of proline uptake by albumin could be related to its fatty acid binding properties.