Isolation of the opioid peptide Leu-Val-Val-hemorphin-7 from bronchoalveolar lavage fluid of a patient with non-small cell lung cancer

The decapeptide Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe was isolated in high yield (1.5 nmol/ml) from bronchoalveolar lavage (BAL) fluid from a patient with an adenocarcinoma of the lung. This peptide, termed LVV-hemorphin-7 represents residues 32-41 of the beta-chain of hemoglobin and has been shown to be an endogenous ligand for opioid receptors. The N-terminal flanking peptide of LVV-hemorphin-7 [residues (1-31) of hemoglobin beta-chain] was also isolated in high yield. Neither peptide was detected in BAL fluid from the tumor-free lung of the same patient or from patients with non-neoplastic inflammatory lung disease. LVV-hemorphin-7 was not identified in BAL fluid from seven additional patients with non-small cell lung cancer, indicating that the formation of the peptide is unlikely to be of any diagnostic significance. However, the ability of LVV-hemorphin-7 to inhibit angiotensin-converting enzyme suggests that its formation may be of pathophysiological significance in the regulation of tumor blood flow in certain patients.     

Isolation of a hemoglobin-derived opioid peptide from cerebrospinal fluid of patients with cerebrovascular bleedings.

The hemorphins are peptides with opioid activity, which are enzymatically released from hemoglobin. A decapeptide identical to the sequence 32-41 of the beta-, delta-, gamma- or epsilon-chains of hemoglobin has been isolated from human ventricular cerebrospinal fluid (CSF). The peptide, designated LVV-hemorphin-7, was recovered in relatively high amounts (115-300 pmol per ml) from samples of patients with cerebrovascular bleedings, but was not detectable in control CSF. Its identity with the hemoglobin fragment was confirmed by mass spectrometry and gas-phase sequencing.     

LVV-hemorphin-7 lowers blood pressure in spontaneously hypertensive rats: radiotelemetry study

Cardiovascular effects of LVV-hemorphin-7, a member of the family of fragments from beta-chain of human or bovine hemoglobin, were studied in conscious spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats by radiotelemetry. Intraperitoneal injection of hemorphin in a dose of 100 microg/kg significantly decreased blood pressure in SHR, whereas negligible effect was seen in normotensive WKY rats. Blood pressure changes were accompanied by reduction of heart rate. In conclusion, a direct effect of LVV-hemorphin-7 on blood pressure was demonstrated in SHR. These biologically active peptides could be involved in blood pressure regulation especially in hypertensive rats, but the precise mechanism should be elucidated.     

Isolation and characterization of four antibacterial peptides from bovine hemoglobin

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields several intermediate peptide fractions after separation by reversed phase HPLC exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli, and Salmonella enteritidis. From these fractions, four new antibacterial peptides were isolated and analyzed by ESI-MS/MS. Three of these peptides correspond to fragments of the alpha-chain of bovine hemoglobin: alpha107-141, alpha137-141, and alpha133-141, and one peptide to the beta-chain: beta126-145. The minimum inhibitory concentrations (MIC) of these peptides towards the four strains and their hemolytic activity towards bovine erythrocytes were determined.     

Structure of hypothalamic coronaro-constrictory peptide factors

The following peptide structure in 3 of 5 coronaro-constrictory peptide factors isolated from bovine hypothalamus was determined by amino acid analysis and Edman degradation: 1) (P₁)-Val-Val-Tyr-Pro-Trp; 2) (P₂)-Val-Val-Tyr-Pro-Trp-Thr; 3) (P₃)-Leu-Val-Val-Tyr-Pro-Trp-Thr. A computer search for these amino acid sequences revealed that these peptides represent fragments 33-37; 33-38; 32-38 of the -chain of bovine hemoglobin. Solid phase peptide synthesis of 2 peptides (P₂ and P₃) was carried out. It was established that synthetic peptides had the properties of coronaro-constrictory peptides. The possibility of the formation of hypothalamic coronaro-constrictory peptides in vivo is discussed.     

Brain processing of hemorphin-7 peptides in various subcellular fractions from rats

Hemorphins are multifunctional peptides derived from hemoglobin or blood processing. They have been found at high levels within the central nervous system where they have a direct effect on neuronal cells via peptidergic receptors. As relatively few studies have examined their metabolic stability in the brain, such investigation was performed to locate the cellular distribution of enzymatic activity against these peptides. High-performance liquid chromatography (HPLC) combined with electrospray ionisation mass spectrometry (ESI-MS) allows identification of degradation products resulting from incubation of hemorphin-7 peptides (LVV-hemorphin-7, VV-hemorphin-7 and hemorphin-7) with subcellular fractions isolated from rat brain tissue. Metabolic activities were found against the three peptides in brain homogenate and subcellular fractions with the highest metabolic activity (<3% peptide remaining after 10 min) observed in the microsomal fraction which processed hemorphin-7 peptides mainly into N-terminal fragments (giving LVVH5) suggesting action of brain-membrane enzymes with C-terminal specificity. Incubation of the ACE inhibitor captopril (0.2 μM) with microsomal fraction, together with LVVH7, decreased the processing of LVVH7 to form LVVH5 by 85%.     

Partial nucleotide sequence of a novel bovine major histocompatibility complex class II β-chain gene, BoLA-DIB

A bovine genomic clone that hybridized to HLA-DQ beta cDNA was isolated and fragments containing the beta 1, beta 2 and transmembrane (TM) exons subcloned. The nucleotide sequences of the exons and flanking intron regions were determined. Comparisons of these exon nucleotide sequences and derived amino acid sequences to human class II beta-chain sequences showed that this gene is only 77% identical to HLA-DQ beta and about 75% identical to bovine DQ beta-like genes. The exon sequences were more divergent from other class II beta-chain genes. However, structural features such as conserved cysteines and regions of amino acids strongly suggest this to be a class II beta-chain gene. When exon-containing fragments were used as hybridization probes on Southern blots of bovine genomic DNA digested with Eco RI or Pvu II, each exon hybridized to a single band. Based on these results we have referred to this gene as a novel bovine class II beta-chain gene, BoLA-DIB.     

Peptide fragments derived from the beta-chain of hemoglobin (hemorphins) are centrally active in vivo

A novel tetrapeptide (hemorphin-4) and pentapeptide (hemorphin-5), derived from the beta-chain of hemoglobin, were synthesized by solid-phase methodology, purified and the amino acid sequences confirmed. The central (ICV) effects of hemorphin-4 and -5 were studied in two models of phasic and tonic nociception, the mouse warm water tail-flick assay and hindpaw formalin assay, respectively. Additionally, two physiological endpoints, central modulation of bladder motility and central effects on intestinal propulsion, were studied in rats and mice, respectively. In the tail-flick assay, both peptides (40-100 nmoles) produced a dose-related naloxone-reversible antinociceptive effect when tested 10 min after peptide administration, with the tetrapeptide being slightly more potent than the pentapeptide. No effect was noted for either peptide using the tonic nociception assay, except at a dose of 150 nmoles for hemorphin-5. Inhibition of gastrointestinal propulsion was also not affected by either peptide. However, both peptides (10-40 nmoles) inhibited micturition contractions in a dose-related and naloxone-reversible fashion, with the tetrapeptide being twice as potent as the pentapeptide. These findings provide evidence that hemorphin-4 and -5 exert naloxone-reversible opioid actions in vivo and, therefore, may be physiologically important blood-borne peptides.     

A Globin Fragment, LVV-Hemorphin-7, Induces [3H]Thymidine Incorporation in a Neuronal Cell Line via the AT4 Receptor

The AT4 receptor was characterized initially as a specific binding site for angiotensin IV, a C-terminal fragment of the vasoactive peptide angiotensin II. Recently, we found that LVV-hemorphin-7, a fragment of beta globin, is an abundant peptide in the brain and binds to the AT4 receptor with high affinity and specificity. In the neuroblastoma/glioma hybrid cell line, NG108-15, LVV-hemorphin-7 and angiotensin IV competed for 125I-angiotensin IV binding in a biphasic fashion with IC50 values of 1.2 x 10(-10) and 1.1 x 10(-9) M for the high-affinity site, respectively, and 6.7 x 10(-8) and 1.5 x 10(-8) M for the low-affinity site, respectively. Both peptides were internalized rapidly by the cells. However, LVV-hemorphin-7, but not angiotensin IV, elicited a 1.8-fold increase in DNA synthesis in a dose-dependent manner. Furthermore, co-incubation of the cells with an excess of angiotensin IV (10(-6) M) inhibited LVV-hemorphin-7-stimulated DNA synthesis. Therefore, whereas LVV-hemorphin-7 and angiotensin IV were capable of binding to the AT4 receptor, only LVV-hemorphin-7 elicited [3H]thymidine incorporation in NG108-15 cells. In contrast, angiotensin IV behaved as an antagonist. The current finding suggests that LVV-hemorphin-7 is a functional peptide in the central nervous system and in view of its abundance in neural tissue, compared with angiotensin IV, may be of significant physiological importance.     

Hemorphins derived from hemoglobin have an inhibitory action on angiotensin converting enzyme activity.

The hemorphins are opioid active peptides, which are enzymatically released from the beta-chain of hemoglobin. In this paper we report an inhibitory effect of these peptides on angiotensin converting enzyme (ACE) activity, known to be involved in blood pressure regulation. The hemorphins were found to be quite stable in tissue extracts containing ACE, and their importance as naturally occurring ACE inhibitors is discussed.     

Carnivora: the primary structure of the hemoglobin from the silver fox (Vulpes vulpes var., Canidae)

The primary structure determination of the hemoglobin alpha- and beta-chains from the silver fox (Vulpes vulpes var., Canidae) is described. The separation of the chains could be achieved directly from the hemoglobin by RP-HPLC as well as by column chromatography of the globin using carboxymethyl-cellulose. Following tryptic digestion of the chains, the peptides were isolated by RP-HPLC. Amino-acid sequences were determined by Edman degradation in liquid and gas phase sequencers. The peptides could be aligned by homology with human and other Carnivora hemoglobins. Compared to human hemoglobin the alpha- and beta-chains of the silver fox exhibit 24 and 13 amino-acid exchanges, respectively. They differ by one alpha- and two beta-chain replacements from the domestic dog and the coyote. The substitutions affecting contact positions are discussed.     

Identification of distinct binding site subunits of mu and delta opioid receptors

Iodinated human beta-endorphin was affinity-cross-linked to opioid receptors present in membrane preparations from bovine frontal cortex, bovine striatum, guinea pig whole brain, and rat thalamus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed covalently labeled peptides of 65, 53, 41, and 38 kilodaltons (kDa). The 65- and 38-kDa peptides were present in all four tissues. The 41-kDa peptide was seen only in bovine caudate and guinea pig whole brain while the 53-kDa peptide was absent in rat thalamus. All four labeled peptides were constituents of opioid receptors since their labeling was fully suppressed by the presence of excess opiates, such as bremazocine, during binding. The distribution and levels of the labeled species in the brain tissues examined and, in earlier work, in the neuroblastoma X glioma NG 108-15 cell line suggested that the 65-kDa peptide is a binding component of mu receptors while the 53-kDa peptide is a binding subunit of delta receptors. This result was strongly supported by the finding that the labeling of the 65-kDa peptide is selectively reduced by the presence of the highly mu-selective ligand Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol (DAMGE) during binding, while while the labeling of the 53-kDa peptide is selectively reduced or eliminated by the highly mu-selective ligand [D-Pen2, D-Pen5]enkephalin (DPDPE). The labeling of the 41- and 38-kDa bands was reduced by either DAMGE or DPDPE. The relationship of these lower molecular weight opioid-binding peptides to mu and delta receptors is not understood. Several possible explanations are presented.     

Hemoglobins. XXVII. The amino acid sequence of hemoglobin III from Myxine glutinosa L. A new hemecomplex: E7 glutamine, E11 isoleucine]

The sequence analysis of the main component, "HbIII", of the hemoglobins from the hagfish (Myxine glutinosa L.) is described. The hagfish belongs to the Cyclostomata, the most primitive class of the vertebrates. The hagfish hemoglobin displays a great heterogeneity, as described earlier. It consists of several monomeric hemoglobins. The globin of HbIII was isolated and used for the sequence analysis. The tryptic peptides as well as the cyanogen bromide and the BNPS-skatol fragments were separated. The sequences of the peptides were determined automatically by the help of a sequenator. Compared with other hitherto analyzed vertebral hemoglobins, also including other Cyclostomata, the primary structure of "HbIII" differs by more than 50%. The differences are so many that one can refer the Myxine hemoglobin neither as an alpha- nor as a beta-chain (of the tetrameric hemoglobins). The hagfish hemoglobin like other Cyclostomata has an additional segment of 9 residues at the amino terminus end compared with the mammalian hemoglobins. In the F-helix there is an insertion of 3 amino acid residues and in the interhelical gap, GH, there is a deletion of 9 residues. The substitutions of the residues forming the heme complex are of special interest. The distal histidine, E7, is substituted for glutamine. The proximal histidine, F8, is invariable. The valine E11 is substituted by isoleucine and the leucine FG3 by phenylalanine. These positions are involved in the contact with the heme group. This complex has never been described before.     

Conformational sensitivity of beta-93 cysteine SH to ligation of hemoglobin observed by FT-IR spectroscopy

The SH vibrational absorption of cysteine F9(beta-93) in concentrated aqueous solutions of native liganded hemoglobin (human HbA, horse, and bovine) has been observed by use of Fourier transform infrared spectroscopy. The pattern of beta-93 SH absorption intensity is ligand dependent. In bovine hemoglobin derivatives the SH absorption intensity pattern is (carbonmonoxy)hemoglobin (HbCO) greater than oxyhemoglobin (HbO2) = cyanomethemoglobin (HbCN) much greater than aquomethemoglobin (metHb) and deoxyhemoglobin (deoxyHb). In horse and human hemoglobin derivatives the pattern is HbCO greater than or equal to HbO2 greater than HbCN greater than metHb. The bovine metHb beta-93 SH shows a much lower absorptivity than that of horse or human metHb, and thus it has a different local tertiary equilibrium conformation than does horse or human hemoglobin. X-ray diffraction studies have shown the beta-93 SH in carbon monoxide or oxygen bound hemoglobin to be situated within a nonpolar pocket between the F, G, and H helices. The higher than usual SH absorption frequency (2592 cm-1) that we observe implies there is no hydrogen bonding for this thiol group while situated within this nonpolar pocket. A similar beta-93 SH absorption has been observed in the beta-chain tetramer (thalassemic hemoglobin H in vivo). The beta-112 SH stretching band, previously observed in the alpha 2 beta 2 tetramer, was observed for the first time in the beta-chain tetramer. A band at 2610 cm-1 that is not due to SH was resolved and found to be ligand dependent.     

LVV-hemorphin 7 and angiotensin IV in correlation with antinociception and anti-thermal hyperalgesia in rats

Hemorphins, a family of atypical endogenous opioid peptides, are produced by the cleavage of hemoglobin β-chain. Hemorphins were proved to bind to the μ-opioid receptors (agonist) and angiotensin IV receptors (insulin-regulated aminopeptidase; IRAP) (inhibitor). Among the hemorphins, LVV-hemorphin-7 (LVV-H7) was found to be abundant and with a longer half life in the CNS. Using intrathecal and intracerebroventricular injections, LVV-H7 and angiotensin IV were given to the rats, which were then subjected to the plantar test and the tail-flick test. Our results showed that LVV-H7 attenuated carrageenan-induced hyperalgesia at the spinal level, which could not be reversed by the co-administration of naloxone. At the supraspinal level, LVV-H7 also produced a significant anti-hyperalgesia effect but with a lower extent. Angiotensin IV showed a similar anti-hyperalgesia effect at the spinal level, but had no effect at the supraspinal level. In the tail-flick test and paw edema test, both peptides showed no effect. These results suggest that LVV-H7 mainly exert the anti-hyperalgesia effect at the spinal level, possibly through IRAP but not μ-opioid receptors. In addition, we observed the expression of IRAP in the CNS of animals with/without carrageenan-induced hyperalgesia. Our results showed a significant expression of IRAP in the spinal cord of rats. However, there was no significant quantitative change of IRAP after the development of hyperalgesia. The serum level of LVV-H7 was also found to be with no change caused by hyperalgesia. These results indicated that the endogenous LVV-H7 and IRAP may not regulate the severity of hyperalgesia through a quantitative change.     

Distribution Pattern of Metorphamide Compared with Other Opioid Peptides from Proenkephalin and Prodynorphin in the Bovine Brain

Metorphamide is a [Met]-enkephalin-containing opioid octapeptide with a C-terminal alpha-amide group. It is derived from proenkephalin and is, so far, the only endogenous opioid peptide with a particularly high affinity for mu opioid (morphine) receptors, a somewhat lesser affinity for kappa opioid receptors, and a relatively low affinity for delta opioid receptors. The concentrations of metorphamide in the bovine caudate nucleus, the hypothalamus, the spinal cord, and the neurointermediate pituitary were determined by radioimmunoassay and chromatography separation procedures. Metorphamide concentrations were compared with the concentrations of eight other opioid peptides from proenkephalin and prodynorphin in identical extracts. The other opioid peptides were [Met]-enkephalyl-Arg6-Phe7 and [Met]-enkephalyl-Arg6-Gly7-Leu8 from proenkephalin; alpha-neoendorphin, beta-neoendorphin, dynorphin A(1-8), dynorphin A(1-17), and dynorphin B from prodynorphin; and [Leu]-enkephalin, which can be derived from either precursor. All opioid peptides were present in all four bovine neural tissues investigated. Metorphamide concentrations were lower than the concentrations of the other proenkephalin-derived opioid peptides. They were, however, similar to the concentrations of the prodynorphin-derived opioid peptides in the same tissues. Marked differences in the relative ratios of the opioids derived from prodynorphin across brain regions were observed, a finding suggesting differential posttranslational processing. Differences in the ratios of the proenkephalin-derived opioids across brain regions were less pronounced. The results from this study together with previous findings on metorphamide's mu opioid receptor binding and bioactivities suggest that the amounts of metorphamide in the bovine brain are sufficient to make this peptide a candidate for a physiologically significant endogenous mu opioid receptor ligand.     

Interaction between bound cupric ion and spin-labeled cysteine beta-93 in human and horse hemoglobins

The location of the various copper binding sites for horse and human hemoglobin was probed using spin labels attached to the beta-93 cysteine residue. Dipole-dipole interactions between the spin label and bound copper produce a decrease in the amplitude of the spin label spectrum which was used to estimate the Cu(II) spin label distance. By comparing the results with horse and human hemoglobin at 298 and 77 K four different Cu(II) binding sites were identified. The low affinity horse hemoglobin site with the sulfhydryl blocked (site 1) was found to be located 10-13 A from the sulfhydryl spin label on the surface of the molecule. Only with a free sulfhydryl is the site (site 2) in the pocket between the F and H helices closer to the SH-group and the iron populated. It is site 2 which is responsible for the oxidation. In frozen solutions a Cu-nitroxide distance of about 17 A was determined with human hemoglobin. This distance is consistent with the previously postulated location of the "high affinity" human hemoglobin site near the amino terminus of the beta-chain. At 298 K a much shorter Cu-nitroxide distance of about 7 A was calculated for human hemoglobin. This shorter distance at higher temperature also correlated with a slightly smaller value of g11 and A11 for the Cu(II) ESR spectrum. It is postulated that in solution cross-linking between nitrogenous ligands in the region of the amino terminus of one beta-chain and the carboxyl terminus of the other beta-chain can explain this shorter distance. This cross-link could involve histidine beta-143, which is one of the ligands thought to be also involved in site 1. Binding to the "high-affinity" site in solution thus stabilizes the "low-affinity" site 2 relative to site 1 explaining the reported interaction between the "high-affinity" and "low-affinity" sites.     

Peptides from the N-terminal domain of chromogranin A (vasostatins) exert negative inotropic effects in the isolated frog heart

The negative inotropic effects of synthetic peptides derived from the N-terminus of chromogranin A (CgA) were studied in an avascular model of the vertebrate myocardium, the isolated working frog heart (Rana esculenta). The peptides were frog and bovine CgA(4-16) and CgA(47-66), and bovine CgA(1-40) with (CgA(1-40SS)) and without an intact disulfide bridge (CgA(1-40SH)). Under basal cardiac conditions, four of the peptides caused a concentration-dependent negative inotropism that was comparable to the negative inotropy reported for human recombinant vasostatin I (CgA(1-78)) and bovine CgA(7-57). By comparison of the structural characteristics of the bovine and frog sequences with their minimally effective concentrations ranging from 68 to 125 nM of peptide, the results were consistent with the natural structure (CgA(17-38SS)) being essential for the negative inotropism. In addition, the partial sequences of the frog and bovine vasostatin I were effective in counteracting the characteristic positive inotropism exerted by isoproterenol (1 nM) at minimally effective concentrations ranging from 45 to 272 nM. Taken together, these results extend the first evidence for a cardiosuppressive role of the N-terminal domain of chromogranin A known for its co-storage with catecholamines in the sympathoadrenal system of vertebrates.     

Hemorphins: substrates and/or inhibitors of dipeptidyl peptidase IV. Hemorphins N-terminus sequence influence on the interaction between hemorphins and DPPIV

Hemorphins are endogenous peptides belonging to the family of "atypical" opioid peptides released from sequentially hydrolyzed hemoglobin. In this paper, we report an inhibitory effect of these peptides on dipeptidyl peptidase IV (DPPIV) activity, known to be involved in regulatory functions such as the activation or inactivation of peptides. The structure activity research revealed that hemorphins N-terminus sequence influences nature of the interaction between hemorphins and DPPIV. Kinetic studies conducted with purified DPPIV demonstrated that hemorphin-7 (H7) constitutes a good substrate (K(cat)/K(m) of 137 mM(-1) s(-1)) for this enzyme but could also act as a selective competitive inhibitor by substrate binding site competition. These blood-derived peptides could represent endogenous regulators of this enzyme activity.     

Analysis of the beta-endorphin structure-related activity on human monocyte chemotaxis: importance of the N- and C-terminal

We evaluated the chemotactic activity of beta-endorphin and beta-endorphin-related peptides on human monocytes. We tested beta-endorphin(1-31) and fragments (1-16), (1-17), (1-27) in which the N-terminal of the opioid is preserved, N-acetyl-beta-endorphin(1-31) and fragments (6-31) and (28-31) in which the C-terminal is preserved, and fragment (2-17) that lacks both the N- and C-terminal. The fragments in which the N- and C-terminal were preserved [with the exception of fragment (28-31)] showed a chemotactic effect, while the lack of both terminals deprived the peptides of any activity. Moreover, only the N-terminal-mediated effects were naloxone reversible, while the C-terminal effects were not. These results indicate that while the intact N-terminal is necessary for opioid like effects, both N- and C-terminal can mediate effects on the immune system, thus offering evidence for a nonopioid receptor-mediated effect of opioid peptides on the immune system.