Radioligand binding properties of VV-hemorphin 7, an atypical opioid peptide.

Receptor binding properties of the hemoglobin-derived nonapeptide VV-hemorphin 7 (Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe-OH) were studied using both the unlabelled form and tritium-labelled derivative of the peptide. In binding studies using selective opioid radioligands, VV-hemorphin 7 exhibited a rank order of potency of mu > kappa >> delta. VV-hemorphin 7 was tritiated resulting in a compound with 1.03 TBq/mmol (27.8 Ci/mmol) specific radioactivity. The maximal number of binding sites was found to be 66.5 pmol/mg protein with an affinity of 82.1 nM in rat brain membranes. In competition studies, marked similarity was observed to the binding profile of the naturally occurring opioid heptapeptide Met-enkephalin-Arg-Phe (MERF) and its analogues to their naloxone-insensitive binding site. The common -Arg-Phe sequence at the carboxyl terminal end, which is similar to those of other endogenous peptides (-Arg-Phe-NH(2) in neuropeptide FF and FMRF-NH(2)) brings attention to the C-terminal end of the molecule and points to the possible existence of a common nonopioid binding site in mammals.

Proteolytic degradation of hemoglobin by endogenous lysosomal proteases gives rise to bioactive peptides: hemorphins.

Hemorphin generation by mice peritoneal macrophages has been recently reported, nevertheless no conclusive data exist to localize clearly the macrophage proteolytic activity implicated in their generation. Because lysosomes are believed to be the main site of degradation in the endocytic pathway, we have studied their potential implication in the generation of hemorphins from hemoglobin. When this protein is submitted to purified rat liver lysosomes, an early generation of hemorphin-7-related peptides, detected by a radioimmunoassay, was observed. These peptides seemed to be relatively stable during the first hours of hydrolysis.

Hemorphins inhibit angiotensin IV binding and interact with aminopeptidase N.

[125I]-Ang IV binding to rabbit collecting duct cell membranes was inhibited by hemorphins (H), a class of endogenous peptides obtained by hydrolysis of the beta chain of hemoglobin. The most potent competitors were those with a valine in their N-terminal part such as LVV-H7 and VV-H7 (IC50 = 1.3 nM) followed by VV-H8 and K6VV-H7 (5.1 nM). The same H, like Ang IV, interacted with aminopeptidase N (APN) as shown by their inhibitory effect (28-36%) on APN activity. HPLC analysis showed that only H with a N-terminal valine or leucine were hydrolyzed. Since H are detected in the body fluids, they are likely to act as endogenous competitors of Ang IV.

Cathepsin D is a good candidate for the specific release of a stable hemorphin from hemoglobin in vivo: VV-hemorphin-7.

Hemorphin peptides, issued from hemoglobin, are emerging as endogenous bioactive peptides derived from in vivo tissular degradation of hemoglobin. In order to find the enzymes which could be implicated in the in vivo release of these peptides, the major lysosomal enzyme cathepsin D was selected, and a study of its activity towards hemoglobin and hemorphins was performed. In this paper, it is shown that according to the primary specificity of cathepsin D towards hemoglobin, this enzyme could constitute a good candidate for the in vivo release of two hemorphins: LVV-hemorphin-7 and VV-hemorphin-7. Moreover, these products, especially VV-hemorphin-7, are resistant to an extended cleavage by the enzyme. Although LVV-hemorphin-7 exhibits a lower resistance, an extended incubation with cathepsin D led to the release of the stable peptide VV-hemorphin-7.