Nonopioid effects of beta-casomorphin-5 in guinea pig heart: alterations to the beta-adrenoceptor-G-protein complex and inhibition of myocardial responses to isoproterenol.

The influence of beta-casomorphin-5 on the beta-adrenoceptor complex in guinea pig heart membranes was studied by means of binding studies, G-protein investigations and isolated heart preparations. In nanomolar concentrations beta-CM-5 induced an increase in receptor affinity towards the agonist isoproterenol whereas the antagonist affinity was reduced. The isoproterenol-stimulated increase in cardiac contractility, moreover, is reduced by 10 nM beta-CM-5. Furthermore, beta-CM-5 was found to inhibit the isoproterenol-induced GDP/GTP exchange as well as the [35S]GTP[S] binding to guinea pig heart membranes, indicating an involvement of G-proteins. These findings suggest that low concentrations of beta-CM-5 modulate the functional properties of the myocardial beta-adrenoceptor-G-protein complex, presumably resulting in its desensitization. The observed effects of beta-CM-5 are not prevented by naloxone and, therefore, are nonopioid in nature.     

Effects of beta-casomorphin-5 on passive avoidance response in mice

The effects of intracerebroventricular (i.c.v.) injection of bovine beta-casomorphin-5 (beta-CM-5: Tyr-Pro-Phe-Pro-Gly), a micro-opioid agonist derived from milk beta-casein, on step-down type passive avoidance tasks were investigated in mice. Intracerebroventricular administration of a high dose (10 microg) of beta-CM-5 produced a significant decrease in step-down latency. beta-Funaltrexamine (5 microg, i.c.v.) almost completely reversed the beta-CM-5-induced shortening of step-down latency, although neither naltrindole (5 ng, i.c.v.) nor nor-binaltorphimine (5 microg, i.c.v.) had any significant influence on the effect of beta-CM-5. Meanwhile, a low dose (0.5 microg, i.c.v.) of beta-CM-5 inhibited scopolamine (1 mg/kg)-induced impairment of passive avoidance response. These results indicated that a high dose of beta-CM-5 induces amnesia, whereas a low dose ameliorates scopolamine-induced amnesia.     

Effects of systemically-administered beta-casomorphin-7 on nociception in rats]

The influence of food-derived heptapeptide beta-casomorphin-7 (beta-CM-7) on pain sensibility of white rats was studied by tail flick test. As shown for doses 10 and 20 mg/kg intraperitoneally, injected beta-CM-7 induced significant analgesia; lower peptide concentration (5 mg/kg) was ineffective. As a whole, there is a significant positive correlation between the intensity of analgesia and the quantity of administered exorphine. These changes of pain sensibility were observed for one hour after injection of heptapeptide; further measurements showed no significant difference of time reaction between control and experimental groups of rats. It was found out that animals with high native level of pain sensibility (4-8 sec) made the main contribution to manifestation of analgesia.     

Phenylalanine ammonia-lyase immobilized in microcapsules for the depletion of phenylalanine in plasma in phenylketonuric rat model

Microencapsulation of the enzyme phenylalanine ammonia-lyase was developed for in vivo depletion of systemic phenylalanine in phenylketonuric rats. Compared to normal rats, systemic phenylalanine blood levels in phenylketonuric rats was increased by 15-20-fold. Daily oral administration of 1 unit of phenylalanine ammonia-lyase-loaded artificial cells to phenylketonuric rats lowered the systemic phenylalanine level to 58% +/- 18% (mean + S.D.) in 7 days (P less than 0.010), while 5 units lowered the systemic phenylalanine level to 25% +/- 8%. 5 units of the immobilized enzyme lowered the systemic phenylalanine level to normal levels within 6 days. Phenylketonuric treated rats showed no signs of abnormal behavior and weight loss compared to phenylketonuric non-treated rats. The immobilized enzyme within artificial cells is therefore protected against low gastrointestinal pH and proteolytic enzymes.     

The nucleotide sequence of phenylalanine tRNA from the cytoplasm of Neurospora crassa.

The phenylalanine tRNA from the cytoplasm of Neurospora crassa has been purified and sequenced. The sequence is: pGCGGGUUUAm2GCUCA (N) GDDGGGAGAGCm22GpsiCAGACmUGmAAYApsim5CUGAAGm7GDm5CGUGUGTpsiCGm1AUCCACACAAACCGCACCAOH. Both in the nature of modified nucleotides which are present in this tRNA and in the overall sequence, this tRNA resembles more closely phenylalanine tRNAs of eukaryotic cytoplasm than those of prokaryotes. The sequence of this tRNA differs from those of the corresponding tRNAs of wheat germ and yeast by only 6 and 7 nucleotides respectively out of 76 nucleotides.U     

Characterization of a novel, non-peptidyl antagonist of the human glucagon receptor

We have identified a series of potent, orally bioavailable, non-peptidyl, triarylimidazole and triarylpyrrole glucagon receptor antagonists. 2-(4-Pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)p yrr ole (L-168,049), a prototypical member of this series, inhibits binding of labeled glucagon to the human glucagon receptor with an IC50 = 3. 7 +/- 3.4 nM (n = 7) but does not inhibit binding of labeled glucagon-like peptide to the highly homologous human glucagon-like peptide receptor at concentrations up to 10 microM. The binding affinity of L-168,049 for the human glucagon receptor is decreased 24-fold by the inclusion of divalent cations (5 mM). L-168,049 increases the apparent EC50 for glucagon stimulation of adenylyl cyclase in Chinese hamster ovary cells expressing the human glucagon receptor and decreases the maximal glucagon stimulation observed, with a Kb (concentration of antagonist that shifts the agonist dose-response 2-fold) of 25 nM. These data suggest that L-168,049 is a noncompetitive antagonist of glucagon action. Inclusion of L-168, 049 increases the rate of dissociation of labeled glucagon from the receptor 4-fold, confirming that the compound is a noncompetitive glucagon antagonist. In addition, we have identified two putative transmembrane domain residues, phenylalanine 184 in transmembrane domain 2 and tyrosine 239 in transmembrane domain 3, for which substitution by alanine reduces the affinity of L-168,049 46- and 4. 5-fold, respectively. These mutations do not alter the binding of labeled glucagon, suggesting that the binding sites for glucagon and L-168,049 are distinct.     

Demonstration of beta-casomorphin immunoreactive materials in in vitro digests of bovine milk and in small intestine contents after bovine milk ingestion in adult humans

Healthy young volunteers ingested one liter of cows' milk; then the contents of the small intestine were aspirated through an intestinal tube at various times and assayed for the presence of bovine beta-casomorphin immunoreactive materials. Considerable amounts of beta-casomorphin-7, but no beta-casomorphin-5 and only small amounts of beta-casomorphin-4 or -6 immunoreactive materials were found. Chromatographical characterization showed that most of the beta-casomorphin-7 immunoreactive material was not identical with beta-casomorphin-7, whereas the major part of the beta-casomorphin-4 or -6 immunoreactive materials might be identical with their corresponding beta-casomorphins. Analogous results were obtained for in vitro digestion of bovine milk which had been designed as a rough imitation of the gastrointestinal digestion process. A regulatory influence of beta-casomorphins as "food hormones" on intestinal functions is suggested.     

Neurite outgrowth-stimulating activities of beta-casomorphins in Neuro-2a mouse neuroblastoma cells

Endogenous opioid peptides and opiate drugs are known to affect the development of the nervous system. beta-Casomorphins (beta-CMs) belong to a family of exogenous opioid peptides derived from the milk protein beta-casein by proteolytic fragmentation. We investigated the effects of various fragments and analogues of beta-CM on neurite outgrowth in Neuro-2a mouse neuroblastoma cells. The fragments beta-CM-5 to -9 and beta-CM-5 amide stimulated neurite outgrowth. Fragments shorter than beta-CM-5 (beta-CM-3, -4, and beta-CM-4 amide) and longer than beta-CM-9 (beta-CM-13 and -21) had no effects. The activity of beta-CMs to promote neurite outgrowth does not correlate with their opioid activity in guinea-pig ileum. The effect of the most potent fragment, beta-CM-5, was prevented by the micro-opioid receptor-selective antagonist D-Phe-Cys(2)-Tyr(3)-D-Trp-Orn(5)-Thr(6)-Pen(7)-Thr(8)-NH(2) (CTOP), or by pretreatment with pertussis toxin. These results suggest that the stimulatory effects of beta-CMs on neurite outgrowth were mediated through G protein-coupled micro-opioid receptors.     

A bacterial phenylalanine aminotransferase lacking pyridoxal 5'-phosphate as cofactor.

A bacterium has been isolated from soil which metabolises phenylalanine initially through the action of a phenylalanine aminotransferase. This enzyme has been purified by conventional techniques and affinity chromatography and shown to be unusual among aminotransferases in not containing pyridoxal 5'-phosphate as cofactor.     

Spectroscopic analysis of [Trp3]-beta-casomorphin analogs. Comparative structure conformation-activity studies

A series of [3-tryptophan]-beta-casomorphin-5([Trp3]-beta-CM-5) analogs were investigated by circular dichroism (CD) and fluorescence spectroscopy to explore their structure-conformation properties in solution. In addition, the comparative opioid activities of these compounds were evaluated using the in vitro guinea pig ileum (GPI) and mouse vas deferens (MVD) assays. Specifically, the pentapeptide sequence of [Trp3]-beta-CM-5, H-Tyr-Pro-Trp-Pro-Gly-OH (I) was modified at Pro-2 and Pro-4 by D-Pro substitutions to provide two diastereometric analogs, [Trp3-D-Pro-4]-beta-CM-5 (II) and [D-Pro2,4,Trp3]-beta-CM-5 (III). In the GPI and MVD assays, beta-CM-5 effected IC50 values of 1.3 microM and 8.9 microM, respectively, which confirmed its known mu/delta-selectivity on these two peripheral opioid receptor subtypes. The potencies of compounds I, II, and III were 0.2, 2.0, and less than 0.005 relative to beta-CM-5 on the GPI assay. Compounds I and II exhibited pronounced mu/delta-selectivities (greater than 18.9- and 12.4-fold respectively), whereas compound III was essentially inactive in both the GPI and MVD assays. CD studies of beta-CM-5 and its [Trp3]-beta-CM-5 analogs showed striking differences in their near-UV and far-UV spectra in aqueous or organic solvents. In the far UV CD spectra, weak (20%) alpha-helicity (maximum at 193 nm and minima at 208 and 222 nm) for beta-CM-5 was obtained in trifluoroethanol (TFE); however, none of the [Trp3]-beta-CM-5 analogs showed such CD bands. Of potential relevance to gamma-turn or C7 secondary structure was the observation of a strong negative band at 245 nm for compounds II and III which was not solvent-dependent in H2O or TFE, whereas compound I showed this CD band exclusively in TFE. In the near-UV CD at 275 nm (Trp electronic transition), the relative order of intensities of this band were determined for the [Trp3]-beta-CM-5 compounds to be II greater than I greater than III, which was identical to their relative biological potencies in both the GPI and MVD assays. Fluorescence energy transfer (FET) experiments of compounds I-III provided the intramolecular distances (r) between their Tyr (donor) to Trp (acceptor) side-chains, by the F?rster method, and were as follows: [Trp3]-beta-CM-5, r = 10.6 A; [Trp3, D-Pro4]-beta-CM-5, r = 9.6 A; and [D-Pro2,4,Trp3]-beta-CM-5, r = 11.0 A.(ABSTRACT TRUNCATED AT 400 WORDS)     

A comparative study of Drosophila phenylalanine hydroxylase with a natural and a synthetic tetrahydropterin as cofactor.

1. Phenylalanine hydroxylase activity has been analyzed in Drosophila melanogaster using as cofactors the natural tetrahydropteridine 5,6,7,8-tetrahydrobiopterin (H4Bip) and the synthetic one 5,6-dimethyl-5,6,7,8-tetrahydropterin (H4Dmp). 2. The apparent Vmax and KM for substrate and cofactor showed that the enzyme has two times more affinity for the substrate when H4Bip is the cofactor in the reaction. Similarly to what was found with purified rat liver phenylalanine hydroxylase, H4Bip was the most effective cofactor, leading to 4-5 times more activity than that obtained with H4Dmp. 3. With the natural cofactor H4Bip, no activation of the enzyme with Phe was necessary (in contrast to mammalian phenylalanine hydroxylase), and this tetrahydropteridine inhibits phenylalanine hydroxylase activity when the enzyme is exposed to it before phenylalanine addition. With the synthetic H4Dmp, both types of preincubations led to an increase of phenylalanine hydroxylase activity. 4. The enzyme is highly unstable compared to mammalian phenylalanine hydroxylase, even at -20 degrees C. 5. Thorax and abdomen extracts caused significant inhibition of phenylalanine hydroxylase activity from third instar larvae or newborn adult head extracts, when assayed with the synthetic cofactor H4Dmp. This inhibition did not happen with H4Bip. The presence of the pteridine 7-xanthopterin in adult bodies was not the cause of this inhibition.     

Influence of human B-casomorphin-7 on specific binding of 3H-spiperone to the 5-HT2-receptors of rat brain frontal cortex

Using radio-receptor analysis, it has been demonstrated that human beta-casomorphin-7 (Tyr-Pro-Phe-Val-Glu-Pro-Ile) displaces 3H-spiperone from 5-HT2-receptors of rat brain frontal cortex. IC50 of human beta-casomorphin-7 was 8 microM. These data suggest that one of the mechanisms of neurotropic action of beta-casomorphin-7 is might be associated with its influence on the serotoninergic system.     

Synthesis of four diastereomeric enkephalins incorporating cyclopropyl phenylalanine

Four diastereomeric D-Ala2, Leu5-enkephalins have been synthesized and their CD spectra and rat brain binding affinities determined. Only the peptides containing Z-cyclopropyl phenylalanine residues showed strong binding affinity. No correlation between CD spectra and bioactivity could be made.     

Soymorphins, novel mu opioid peptides derived from soy beta-conglycinin beta-subunit, have anxiolytic activities

Based on the amino acid sequence YPFV found in the soy beta-conglycinin beta-subunit, which is common to an opioid peptide human beta-casomorphin-4, peptides YPFVV, YPFVVN, and YPFVVNA were synthesized according to their primary structure. On guinea pig ileum (GPI) assay, they showed opioid activity (IC50 = 6.0, 9.2 and 13 microM respectively) more potent than human beta-casomorphins, and were named soymorphins-5, -6, and -7, respectively. Their opioid activities on mouse vas deferens (MVD) assay were less potent than on GPI assay, suggesting that they are selective for the mu opioid receptor. Human beta-casomorphin-4 and soymorphin-5 were released from the soy 7S fraction (beta-conglycinin) by the action of gastrointestinal proteases. Soymorphins-5, -6, and -7 had anxiolytic activities after oral administration at doses of 10-30 mg/kg in the elevated plus-maze test in mice.     

Kinetic and crystallographic analysis of complexes formed between elastase and peptides from beta-casein.

Human beta-casomorphin-7 (NH2-Tyr-Pro-Phe-Val-Glu-Pro-Ile-CO2H) is a naturally occurring peptide inhibitor of elastase that has been shown to form an acyl-enzyme complex stable enough for X-ray crystallographic analysis at pH 5. To investigate the importance of the N-terminal residues of the beta-casomorphin-7 peptide for the inhibition of elastase, kinetic and crystallographic analyses were undertaken to identify the minimum number of residues required for effective formation of a stable complex between truncated beta-casomorphin-7 peptides and porcine pancreatic elastase (PPE). The results clearly demonstrate that significant inhibition of PPE can be effected by simple tri-, tetra-and pentapeptides terminating in a carboxylic acid. These results also suggest that in vivo regulation of protease activity could be mediated via short peptides as well as by proteins. Crystallographic analysis of the complex formed between N-acetyl-Val-Glu-Pro-Ile-CO2H and PPE at pH 5 (to 1.67 A resolution) revealed an active site water molecule in an analogous position to that observed in the PPE/beta-casomorphin-7 structure supportive of its assignment as the 'hydrolytic water' in the deacylation step of serine protease catalysis.     

Protein kinase inhibitor-(6-22)-amide peptide analogs with standard and nonstandard amino acid substitutions for phenylalanine 10. Inhibition of cAMP-dependent protein kinase

The minimal structure in the heat-stable inhibitor protein of cAMP-dependent protein kinase required for a low nanomolar potency of inhibition is the peptide Thr6-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-+ ++Ile22-NH2 (PKI-(6-22)-amide). While primary structural determinants for interaction with the protein kinase are distributed throughout the 17 residues of this peptide, we have previously shown that phenylalanine 10 in the NH2-terminal portion is a particularly important determinant for high affinity binding (Glass, D. B., Cheng, H.-C., Mende-Mueller, L., Reed, J., and Walsh, D. A. (1989) J. Biol. Chem. 264, 8802-8810). To investigate this requirement further, peptide analogs of PKI-(6-22)-amide in which various natural and nonstandard amino acids are substituted for phenylalanine 10 have been synthesized and tested for inhibitory potency against the catalytic subunit of the protein kinase. Consistent with the importance of the hydrophobicity of phenylalanine, an alanine 10 substitution analog exhibited a 270-fold decrease in inhibitory potency, whereas the leucine 10 analog lost only 33-fold in activity as compared to the parent peptide PKI-(6-22)-amide. Peptides containing the spatial conformation analogs D-phenylalanine, homophenylalanine, or phenylglycine were 60-120-fold less potent than the parent peptide. Peptides containing various para-substituted phenylalanines at position 10 were only 5-11-fold less potent. One exception to this was (4'-azidophenylalanine 10)PKI-(6-22)-amide, which was nearly equipotent with the parent inhibitor. The most potent analogs were those peptides containing highly aromatic residues at position 10. The 2'-thienylalanine 10, tryptophan (formyl) 10, tryptophan 10, and the 1'-naphthylalanine 10 analogs were 3-fold less potent, equipotent, slightly more potent, and 4-fold more potent than the parent peptide inhibitor, respectively. We conclude that phenylalanine 10 in PKI-(6-22)-amide, and presumably in the native protein inhibitor, interacts through specific hydrophobic and/or aromatic binding to a hydrophobic pocket or cleft near the active site of the protein kinase.     

Regulation of phenylalanine ammonia lyase in Rhodotorula glutinis.

In the red yeast Rhodotorula glutinis, phenylalanine ammonia lyase (PAL) was induced 10-fold during carbon starvation even in the absence of exogenous phenylalanine, although maximal induction occurred when phenylalanine was the nitrogen (40-fold) or carbon (100-fold) source. Apparent regulatory mutations that affected the expression of PAL were isolated by selecting mutants resistant to the analog p-fluoro-D,L-phenylalanine (PFP). One such mutant, designated FP1, could use phenylalanine as a nitrogen source but not as a carbon source. Similarly, FP1 failed to utilize intermediates of the phenylalanine degradative pathway, namely, benzoate, p-hydroxybenzoate, or 3,4-dihydroxybenzoate, as carbon sources. Although the PFP-resistant mutant contained a low level of PAL, no increase was found when it was grown with phenylalanine as the nitrogen source. A derivative of FP1, FP1a, was isolated that simultaneously regained an inducible PAL and the ability to use phenylalanine, benzoate, p-hydroxybenzoate, and 3,4-dihydroxybenzoate as carbon sources. In addition, when p-hydroxybenzoate was the carbon source, PAL was induced in the mutant FP1a but not in the PFP-sensitive parental strain. We propose that the mutation to PFP resistance occurred in a regulatory gene that controls the entire phenylalanine degradative pathway. Secondary mutations at this locus, as found in strain FP1a, not only restored expression of this pathway, but also altered the induction of PAL by metabolites of this pathway.     

Novel Dmt-Tic dipeptide analogues as selective delta-opioid receptor antagonists

A series of Dmt-Tic analogues with substitution on the Tic aromatic ring has been synthesized and evaluated for opioid receptor affinity and activation. Incorporation of large hydrophobic groups at position 7 of Tic did not greatly alter the delta opioid receptor binding affinities of the dipeptides whereas substitution at position 6 substantially diminished their affinity. These modified Dmt-Tic peptides showed binding affinities as low as 2.5 nM with up to 500-fold selectivity for the delta versus mu opioid receptor and proved to be delta receptor antagonists.     

Purification and biochemical characterization of recombinant rat liver phenylalanine hydroxylase produced in Escherichia coli.

Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms. To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to rat phenylalanine hydroxylase. Recombinant rat phenylalanine hydroxylase was placed into an expression vector in Escherichia coli. The enzyme has been purified to homogeneity and its physical and catalytic properties have been characterized. The molecular weight and the fluorescence emission spectrum of the recombinant enzyme were identical to those of the native enzyme. The recombinant enzyme could be activated by incubation with phenylalanine or lysolecithin or by phosphorylation, as is the rat liver enzyme. The extent of activation is the same as that for the native enzyme in each case except for phenylalanine, which activates the recombinant enzyme only 5- to 10-fold rather than the 15- to 30-fold activation observed with the native enzyme. The kinetic constants determined for the recombinant enzyme are also essentially the same as those reported for the native enzyme. We conclude that this enzyme is essentially identical to the native enzyme and should be very useful in the future study of this important hydroxylase.