Conformational behavior of [Trp4, Met5]-enkephalin: comparison of fluorescence parameters at different Ph.

The conformational behavior of the biologically active [Trp⁴,Met⁵]-enkephalin was elucidated by evaluation of intramolecular energy transfer between Tyr¹ and Trp⁴. Identical transfer efficiencies and tyrosine fluorescence quantum yields were observed in aqueous solution at pH 1.5 and 5.5 and the use of these parameters in Förster's equation resulted in the same average Tyr-Trp separation (9.3 Å) under these two conditions. The invariability of these sensitive parameters indicates the existence of very similar types of a folded conformation in the cationic and zwitterionic form of the analog at low concentrations.     

In vitro cyclic AMP-mediated lipolytic activity of endorphins, enkephalins and naloxone

The maximum lipolytic activity (L_max) of β-endorphin is two and one half times that of Leu⁵-enkephalin and twice that of Met⁵-enkephalin, D-Ala², Met⁵-enkephalinamide, α-endorphin and γ-endorphin in the rabbit adipocyte. D-Met², Pro⁵-enkephalin-amide, however, has an L_max 1.6 times greater than that of Met⁵-enkephalin. The potencies (A₅₀) of Met⁵-enkephalin and its analogs and that of Leu⁵-enkephalin lie between 1.4 and 3 μM. The A₅₀ values for α-endorphin, β-endorphin and γ-endorphin are significantly less (1.2 × 10^?1 μM). Naloxone acts as an agonist in this system (A₅₀ = 2.5 μM; L_max 1.4 × Met⁵-enkephalin). All of the peptides and naloxone stimulated adenylate cyclase activity.     

Tissue kallikrein processes small proenkephalin peptides

Tissue kallikrein may play a role in processing precursor polypeptide hormones. We investigated whether hydrolysis of natural enkephalin precursors, peptide F and bovine adrenal medulla docosapeptide (BAM-22P), by hog pancreatic kallikrein is consistent with this concept. Incubation of peptide F with this tissue kallikrein resulted in the release of Met⁵-enkephalin and Met⁵-Lys⁶-enkephalin. Met⁵-Lys⁶-enkephalin was the main peptide released, indicating that the major cleavage site was between two lysine residues. At 37°C and pH 8.5, the K_M values for formation of Met⁵-enkephalin and Met⁵-Lys⁶-enkephalin were 129 and 191 μM, respectively. Corresponding k_cat values were 0.001 and 0.03 s⁻¹ and k_cat/K_M ratios were 8 and 1.6·10² M⁻¹ · s⁻¹, respectively. Cleavage of peptide F at acidic pH (5.5) was negligible. When BAM-22P was used as a substrate, Met⁵-Arg⁶-enkephalin was released, thus indicating cleavage between two arginine residues. At pH 8.5, K_M was 64 μM, k_cat was 4.5 s⁻¹, and the k_cat/K_M ratio was 7 · 10⁴ M⁻¹ · s⁻¹. At 5.5, the pH of the secretory granules, K_M, k_cat and k_cat/K_M were 184 μM, 1.9 s⁻¹ and 10⁴ M⁻¹ · s⁻¹, respectively. It is unlikely that peptide F could be a substrate for kallikrein in vivo; however, tissue kallikrein could aid in processing proenkephalin precursors such as BAM-22P by cleaving Arg-Arg peptide bonds.     

Unsulfated C-terminal 7-peptide of cholecystokinin: a new ligand of the opiate receptor.

On the basis of structural and conformational similarities between the C-terminal 7-peptide of cholecystokinin (pancreozymin) (CCK-(27–33)) and the active enkephalin analog [Trp⁴,Met⁵]-enkephalin, the affinity of CCK-(27–33) for the opiate receptor was determined. With unsulfated CCK-(27–33) half-maximal inhibition of stereospecific binding of [³H]-naloxone in a rat brain membrane preparation was observed at a 200 times higher concentration than that required with [Met⁵]-enkephalin. Sulfated CCK-(27–33) did not bind at concentrations up to 4 × 10^?5M. In the bioassay based on inhibition of electrically evoked contractions of guinea pig ileum similar potency ratios were observed and the effect of CCK-(27–33) was shown to be naloxone-reversible. These findings are of interest in view of the recently demonstrated presence of CCK-fragments in the brain.     

The production and characterization of monoclonal antibodies against enkephalins

The hybridoma technology of Kohler and Milstein (1975) was utilized to produce monoclonal antibodies against the enkephalins. Two hybridomas, AD4 and DB4, produced monoclonal antibodies of the IgG type 1 class against Leu⁵-enkephalin that were highly specific for Leu⁵- and Met⁵-enkephalin. AD4 exhibited almost equal reactivity with either Leu⁵- or Met⁵-enkephalin, whereas DB4 exhibited only a 20% cross-reactivity with Met⁵-enkephalin. The IC₅₀ of these monoclonal antibodies were approximately two orders of magnitude greater than the IC₅₀ a polyclonal antiserum against enkephalins (A206; Miller et al 1978) used routinely in many immunochemical and immunocytochemical studies.The monoclonal antibodies, AD4 and DB4, exhibited specific sequence and size requirements for binding enkephalin-related peptides. The amino acid sequence Gly-Gly-Phe-Leu or Gly-Gly-Phe-Met was essential for recognition by AD4 and DB4. However, Tyr-Gly-Gly-Phe which lacks Leu or Met in the fifth position did not react with our monoclonal antibodies. Moreover, enkephalin-related peptides in which the enkephalin sequence was situated at the amino terminus and which contained six or more amino acids did not react significantly with AD4 or DB4. In particular, unlike the polyclonal antiserum A206, our monoclonal antibodies do not react with dynorphins 1–6 or 1–13. However, when the monoclonal antibody (AD4) was used to localize immunohistochemically the population of enkephalinergic amacrine cells in the chicken retina, it provided a staining pattern quite comparable to that observed in previous studies (Watt et al., 1983) using the polyclonal enkephalin antiserum A206. This finding therefore demonstrates that the immunoreactive products visualized in the enkephalin-immunoreactive amacrine cells of the chicken retina with the polyclonal antiserum correspond to authentic enkephalin or peptides very closely related to the enkephalins.     

Conformational energy studies on N-methylated analogs of thyrotropin releasing hormone, enkephalin, and luteinizing hormone-releasing hormone

Empirical conformational energy calculations have been carried out for N-methyl derivatives of alanine and phenylalanine dipeptide models and N-methyl-substituted active analogs of three biologically active peptides, namely thyrotropin-releasing hormone (TRH), enkephalin (ENK), and luteinizing hormone-releasing hormone (LHRH). The isoenergetic contour maps and the local dipeptide minima obtained, when the peptide bond (ω) preceding the N-methylated residue is in the trans configuration show that (1) N-methylation constricts the conformational freedom of both the ith and (i + 1)th residues; (2), the lowest energy position for both residues occurs around ? = ?135° ± 5° and ψ = 75° ± 5°, and (3) the α_L conformational state is the second lowest energy state for the (i + 1)th residue, whereas for the ith residue the C₅ (extended) conformation is second lowest in energy. When the peptide bond (ω_i) is in the cis configuration the ith residue is energetically forbidden in the range ? = 0° to 180° and ψ = ?180° to +180°. Conformations of low energy for ω_i = 0° are found to be similar to those obtained for the trans peptide bond. In all the model systems (irrespective of cis or trans), the α_R conformational state is energetically very high. Significant deviations from planarity are found for the peptide bond when the amide hydrogen is replaced by a methyl group. Two low-energy conformers are found for [(N-Me)His²]TRH. These conformers differ only in the ? and ψ values at the (N-Me)His² residue. Among the different low-energy conformers found for each of the ENK analogs [D -Ala²,(N-Me)Phe⁴, Met⁵]ENK amide and [D -Ala²,(N-Me)Met⁵]ENK amide, one low-energy conformer was found to be common for both analogs with respect to the side-chain orientations. The stability of the low-energy structures is discussed in the light of the activity of other analogs. Two low-energy conformers were found for [(N-Me)Leu⁷]LHRH. These conformations differ in the types of bend around the positions 6 and 7 of LHRH. One bend type is eliminated when the active analog [D -Ala⁶,(M-Me)Leu⁷]LHRH is considered.     

Radioimmunoassay of methionine5-enkephalin sulphoxide: Phylogenetic and anatomical distribution

A sensitive and specific radioimmunoassay (RIA) for the oxidised form of methionine⁵-enkephalin (Met⁵-Enk), Met⁵-Enk sulphoxide (Met⁵-Enk-S), has been developed. Antisera were raised in rabbits against Met⁵-Enk coupled to carrier proteins with glutaraldehyde or carbodiimide. Displacement of (¹²⁵I) Met⁵-Enk bound to antiserum by Met⁵-Enk was poor, but Met⁵-Enk-S displayed good displacement suggesting that the Met⁵-Enk immunogen was oxidised to Met⁵-Enk-S and that the antisera were formed against this compound. The sensitivity of the RIA for Met⁵-Enk-S was 0.02 pmole/tube using the most sensitive antiserum. The antisera showed negligible cross-reactivity with leucine⁵-enkephalin and with both native and oxidised endorphins. Cross-reactivity was between 15% and 28% with the fragment Met⁵-Enk (2–5) sulphoxide and between 9% and 25% with D-Ala²-Met⁵-Enk sulphoxide. The antisera showed<0.01% cross-reactivity with other Met⁵-Enk fragments and naturally occurring neuropeptides. Tissue extracts were oxidised with hydrogen peroxide prior to assay. Met⁵-Enk-S immunoreactivity (IMR) was detected in brain, pituitary gland, pancreas, and intestine extracts of the rat, chicken, toad and teleost, and in cerebral-suboesophageal ganglion extracts of the snail. All tissue extracts showed parallelism in serial dilution to synthetic mammalian Met⁵-Enk-S, suggesting possible immunological identity. The results indicate that spontaneous oxidation of Met⁵-Enk immunogen occurs such that antisera are produced against the sulphoxide analogue of Met⁵-Enk, and may account for the relative insensitivity of some published RIAs using Met⁵-Enk standard. Our findings demonstrate a wide phylogenetic and anatomical distribution of Met⁵-Enk IMR.     

Radioimmunoassay of methionine-enkephalin sulphoxide: Phylogenetic and anatomical distribution

A sensitive and specific radioimmunoassay (RIA) for the oxidised form of methionine⁵-enkephalin (Met⁵-Enk), Met⁵-Enk sulphoxide (Met⁵-Enk-S), has been developed. Antisera were raised in rabbits against Met⁵-Enk coupled to carrier proteins with glutaraldehyde or carbodiimide. Displacement of (¹²⁵I) Met⁵-Enk bound to antiserum by Met⁵-Enk was poor, but Met⁵-Enk-S displayed good displacement suggesting that the Met⁵-Enk immunogen was oxidised to Met⁵-Enk-S and that the antisera were formed against this compound. The sensitivity of the RIA for Met⁵-Enk-S was 0.02 pmole/tube using the most sensitive antiserum. The antisera showed negligible cross-reactivity with leucine⁵-enkephalin and with both native and oxidised endorphins. Cross-reactivity was between 15% and 28% with the fragment Met⁵-Enk (2–5) sulphoxide and between 9% and 25% with D-Ala²-Met⁵-Enk sulphoxide. The antisera showed<0.01% cross-reactivity with other Met⁵-Enk fragments and naturally occurring neuropeptides. Tissue extracts were oxidised with hydrogen peroxide prior to assay. Met⁵-Enk-S immunoreactivity (IMR) was detected in brain, pituitary gland, pancreas, and intestine extracts of the rat, chicken, toad and teleost, and in cerebral-suboesophageal ganglion extracts of the snail. All tissue extracts showed parallelism in serial dilution to synthetic mammalian Met⁵-Enk-S, suggesting possible immunological identity. The results indicate that spontaneous oxidation of Met⁵-Enk immunogen occurs such that antisera are produced against the sulphoxide analogue of Met⁵-Enk, and may account for the relative insensitivity of some published RIAs using Met⁵-Enk standard. Our findings demonstrate a wide phylogenetic and anatomical distribution of Met⁵-Enk IMR.     

Enkephalin analogues and naloxone modulate the release of growth hormone and prolactin - evidence for regulation by an endogenous opioid peptide in brain

Met⁵-enkephalin amide, D-Ala²-Met⁵-enkephalin amide, D-Ala²-Leu⁵-enkephalin amide, morphine sulfate and naloxone hydrochloride were examined for their effects on growth hormone and prolactin release $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. Subcutaneous injection of D-Ala²-Met⁵ enkephalin amide^a, D-Ala²-Leu⁵ enkephalin amide^b and morphine sulfate, but not Met⁵-enkephalin and amide^c, resulted in significant elevations in the serum growth hormone and prolactin of immature female rats. Naloxone blocked the hormone-stimulatory effect of the opioid receptor agonists and when administered alone significantly reduced serum growth hormone and prolactin concentrations. None of the drugs demonstrated a direct action on anterior pituitary tissue growth hormone or prolactin release $\text{in}$$\text{vitro}$.     

Nuclear magnetic resonance study of the impact of ribose 2′-O-methylation on the aqueous solution conformation of cytidylyl-(3′ → 5′)-cytidine

In order to obtain information about the conformational characteristics at the nearestneighbor level in the 2′-O-methylated region of t-RNA, as well as in the bizarre 5′-terminus of eucaryotic mRNA, a detailed nuclear magnetic resonance study of 2′-O-methyl-cytidylyl-(3′ → 5′)-cytidine (CmpC) was conducted. Proton spectra were recorded at 270 MHz in the Fourier mode in D₂O solutions, 0.01M, pD 7.3 in the temperature range 5–80°C. Complete accurate sets of nmr parameters were derived for each of the nucleotidyl units by a combination of homo-nuclear decouplings and simulation iteration methods. The data were translated into conformational parameters using procedures developed in earlier studies from these laboratories. It is shown that the ribofuranose ring exists at a ²E ? ³E equilibrium with clear preference [(75–80)%] for the ³E mode. The C(4′)-C(5′) and C(5′)-O(5′) bonds form a stable conformational network with outspoken preference for conformers in which Ψ₁, Ψ₂ ? 60° and ?₂ ? 180°. The orientation of the 3′-phosphate and 2′-O-methyl groups is such that ?₁′ ? 210° and ?″ ? 60°. The phosphodiester bonds are flexible and shift trends for base, H(1′), and H(5″) suggest the existence of a conformational blend of right-handed stack (g^?g^?), left-handed stack (g⁺g⁺), and unstacked arrays (tg^? and tg⁺). Elevation of temperature perturbs the ²E ? ³E equilibrium accompanied with modest depopulation of ψ₁, ψ₂ ? 60° and ?₂ ? 180° conformers. The major effect of elevation of temperature is in the increase of unstacked arrays at the expense of g^?g^? and g⁺g⁺ conformers. The shift trend of Cmp-H(3′) with temperature shows that torsional variation about O(3′)-P is facilitated by increase in temperature and the preferred rotamer about O(3′)-P in the unstacked form is t (ω₁′ = 180°). A detailed comparison of the aqueous solution conformations of CpC and CmpC reveals that 2′-O-methylation causes: (i) a reduction in the magnitude of _χ1; (ii) an increase in the population of ³E pucker at the 3′-nucleotidyl unit; and (iii) modest perturbations in the O(3′)-P and P-O(5′) bond conformations. Comparison of the aqueous solution conformations of AmpA and CmpC makes clear that the conformational properties of pyrimidine-pyrimidine and purine-purine dimers which carry a 2′-O-methylated 3′-nucleotidyl unit are significantly different.     

Experimental simulation of the environment of the δ opioid receptor. A 500 MHz study of enkephalins in CDCl3

Complexes of [Met⁵] and [Leu⁵]enkephalin amides with 18-crown-6-ether have been studied in CDCl₃ solution by means of 500 MHz NMR spectroscopy, in order to simulate two of the features of the opioid receptor: the apolar environment and the binding of the charged N atom. Contrary to all previus studies in polar solvents the NH resonances are spread in a huge range (ca. 4 ppm) as in the spectra of rigid cyclic peptides. The two observed intramolecular hydrogen bonds are consistent with the existence of a single, folded, conformation, i.e. a C₁₀β-turn in which the Phe⁴ NH is linked to the Tyr¹ CO group.     

Intramolecular Conformation of Puromycin in Solution As Studied by Proton Magnetic Resonance

Abstract

The intramolecular conformation of puromycin, a broad spectrum antiobiotic, in solution has been investigated by proton magnetic resonance (PMR) spectroscopy. A comparison of the proton chemical shift and proton-proton coupling constant data of puromycin with puromycin aminonucleoside suggests that puromycin in solution exists as an equilibrium blend of extended and folded conformers. These folded conformers are the result of flexibility around the C^α-C^β bond of the aminoacyl segment of puromycin. One of the folded conformers predicted by PMR is in excellent agreement with the x-ray data.     

Radical-induced purine lesion formation is dependent on DNA helical topology

Abstract

Herein we report the quantification of purine lesions arising from gamma-radiation sourced hydroxyl radicals (HO^?) on tertiary dsDNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation, along with single-stranded folded and non-folded sequences of guanine-rich DNA in selected G-quadruplex structures. We identify that DNA helical topology and folding plays major, and unexpected, roles in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA), along with tandem-type purine lesions 5′,8-cyclo-2′-deoxyguanosine (5′,8-cdG) and 5′,8-cyclo-2′-deoxyadenosine (5′,8-cdA). SC, OC, and L dsDNA conformers together with folded and non-folded G-quadruplexes d[TGGGGT]₄ (TG4T), d[AGGG(TTAGGG)₃] (Tel22), and the mutated tel24 d[TTGGG(TTAGGG)₃A] (mutTel24) were exposed to HO^? radicals and purine lesions were then quantified via stable isotope dilution LC-MS/MS analysis. Purine oxidation in dsDNA follows L?>?OC???SC indicating greater damage towards the extended B-DNA topology. Conversely, G-quadruplex sequences were significantly more resistant toward purine oxidation in their unfolded states as compared with G-tetrad folded topologies; this effect is confirmed upon comparative analysis of Tel22 (～50% solution folded) and mutTel24 (～90% solution folded). In an effort to identify the accessibly of hydroxyl radicals to quadruplex purine nucleobases, G-quadruplex solvent cavities were then modeled at 1.33?Å with evidence suggesting that folded G-tetrads may act as potential oxidant traps to protect against chromosomal DNA damage.     

Demonstration and Characterization of Opiate Inhibition of the Striatal Adenylate Cyclase

Abstract: The conditions in which Leu⁵-enkephalin inhibition of striatal adenylate cyclase was observed were defined. It was determined that enkephalin inhibition was dependent on GTP. The apparent K_m for GTP in opiate inhibition was determined to be 0.5 and 2 μM when 0.1 mM- and 0.5 mM-ATP were used as substrate. ITP, but not CTP or UTP, could substitute for GTP in the reaction. Though the addition of monovalent cations—Na⁺,K⁺, Li⁺, Cs⁺, and choline⁺—stimulated striatal adenylate cyclase activity, enkephalin inhibition of striatal adenylate cyclase did not require Na⁺ when theophylline was used as the phosphodiesterase inhibitor. Under optimal conditions, i.e., 20 μM-GTP and 100 mM-Na⁺, Leu⁵-enkephalin inhibited the striatal adenylate cyclase activity by 23–27%. When the enkephalin regulation of the cyclase activity was further characterized, it was observed that Leu⁵-enkephalin inhibited the rate of the enzymatic reaction. Kinetic analysis revealed that the opioid peptide decreases V_max values but not the K_m values for the substrates Mg²⁺ and Mg-ATP. Agents such as MnCl₂, NaF, and guanyl-5′-ylimido-diphosphate, which directly activated the adenylate cyclase, antagonized the opiate inhibition. Levorphanol and (–)naloxone were more potent than dextrorphan and (+)naloxone in inhibiting adenylate cyclase and in reversing the enkephalin inhibition, respectively. There were differences in the potencies of various opiate peptides in their inhibition of striatal adenylate cyclase activity, with Met⁵- > Leu⁵-enkephalin > β-endorphin. The opiate receptor through which the enkephalin inhibition was observed is most likely δ in nature, since in the presence of either Na⁺ or K⁺, the magnitude of the alkaloid inhibition was reduced, whereas the peptide inhibition was either potentiated or not affected.     

Recognition of altered E. coli formylmethionine transfer RNA by bacterial T factor

Treatment of $\text{E.}\text{coli}$ formylmethionine tRNA with sodium bisulfite produces six C → U base changes in the tRNA structure. Four of these modifications have no effect on the ability of tRNA_f^Met to be aminoacylated or formylated. Prior to bisulfite treatment, Met-tRNA_f^Met is not able to form a ternary complex with bacterial T factor and GTP, as measured by Sephadex G-50 gel filtration. After bisulfite treatment, a large portion of the modified tRNA is bound as T-GTP-Met-tRNA_f^Met. Formylation of bisulfite-modified Met-tRNA_f^Met completely eliminates T factor binding. Unmodified tRNA_f^Met is unique among the tRNAs sequenced to date in having a non-hydrogen-bonded base at the 5′ terminus. Bisulfite-catalyzed conversion of this unpaired C₁ to U₁ results in formation of a normal U₁-A₇₃ base pair at the end of the acceptor stem. It is likely that this structural alteration is responsible for the recognition of bisulfite-modified Met-tRNA_f^Met by T factor.     

Plant nuclear tRNAMet genes are ubiquitously interrupted by introns

We have isolated three independent clones for nuclear elongator tRNA^Met genes from an Arabidopsis DNA library using a tRNA^Met-specific probe generated by PCR. Each of the coding sequences for tRNA^Met in these clones is identical and is interrupted by an identical 11 bp long intervening sequence at the same position in the anticodon loop of the tRNA. Their sequences differ at two positions from the intron in a soybean counterpart. Southern analysis of Arabidopsis DNA demonstrates that a gene family coding for tRNA^Met is dispersed at at least eight loci in the genome. The unspliced precursor tRNA^Met intermediate was detected by RNA analysis using an oligonucleotide probe complementary to the putative intron sequence. In order to know whether introns commonly interrupt plant tRNA^Met genes, their coding sequences were PCR-amplified from the DNAs of eight phylogenetically separate plant species. All 53 sequences determined contain 10 to 13 bp long intervening sequences, always positioned one base downstream from the anticodon. They can all be potentially folded into the secondary structure characteristic for plant intron-containing precursor tRNAs. Surprisingly, GC residues are always present at the 5-distal end of each intron.     

Effect of formylation on the chromatographic behavior of methionyl transfer ribonucleic acid

The effect of formylation on the chromatographic behavior of Met-tRNA_f^Met on BD-cellulose has been investigated. Under conditions comparable to those routinely employed in analytical BD-cellulose chromatography, formylated Met-tRNA_f^Met was observed to elute at a significantly higher salt concentration than unformylated Met-tRNA_f^Met. Unformylated Met-tRNA_f^Met elutes well before Met-tRNA_m^Met, whereas fMet-tRNA_f^Met elutes slightly after Met-tRNA_m^Met; thus the net effect of formylation is an apparent inversion of the elution order of the isoaccepting methionyl tRNA species, tRNA_f^Met and tRNA_m^Met. Although aminoacylated tRNA_f^Met and tRNA_m^Met elute slightly later than their respective unacylated forms, aminoacylation alone does not produce the inverted elution order observed upon formylation of Met-tRNA_f^Met.     

Sequences of initiator and elongator methionine tRNAs in bean mitochondria

Summary Two bean mitochondria methionine transfer RNAs, purified by RPC-5 chromatography and two-dimensional gel electrophoresis, have been sequenced usingin vitro post-labeling techniques.One of these tRNAs^Met has been identified by formylation using anE. coli enzyme as the mitochondrial tRNA_F ^Met. It displays strong structural homologies with prokaryotic and chloroplast tRNA_F ^Met sequences (70.1–83.1%) and with putative initiator tRNA_m ^Met genes described for wheat, maize andOenothera mitochondrial genomes (88.3–89.6%).The other tRNA^Met, which is the mitochondrial elongator tRNA_F ^Met, shows a high degree of sequence homology (93.3–96%& with chloroplast tRNA_m ^Met, but a weak homology (40.7%) with a sequenced maize mitochondrial putative elongator tRNA_m ^Met gene.Bean mitochondrial tRNA_F ^Met and tRNA_m ^Met were hybridized to Southern blots of the mitochondrial genomes of wheat and maize, whose maps have been recently published (15, 22), in order to locate the position of their genes.