Elsevier

Peptides

Volume 18, Issue 2, 1997, Pages 293-300
Peptides

Hemorphin Peptides are Released from Hemoglobin by Cathepsin D. Radioimmunoassay Against the C-part of V-V-hemorphin-7: An Alternative Assay for the Cathepsin D Activity

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Abstract

Garreau, I., K. Cucumel, N. Dagouassat, Q. Zhao, A. Cupo and J. M. Piot. Hemorphin peptides are released from hemoglobin by Cathepsin D. radioimmunoassay against the C-part of V-V-hemorphin-7: An alternative assay for the Cathepsin D activity. Peptides 18(2) 293–300, 1997.—In order to investigate the putative physiological role of the in vivo release of hemorphins from hemoglobin in tissues, an immunological approach was developed. Specific and sensitive antiserum were raised against the C-part of the V-V-hemorphin-7. The antisera recognized to the same extent the two related hemorphins V-V-hemorphin-7 and L-V-V-hemorphin-7. The validity of our immunological approach was analyzed by studying the in vitro release of hemorphin from hemoglobin by cathepsin D and compared to the pepsin hydrolysis. These two enzymes led to the release of these same products suggesting that cathepsin D acted as an accurate pepsin-like enzyme. Moreover, considering the poor sensitivity of the available methods of detection for the in vitro Cathepsin D activity, our specific and sensitive V-V-hemorphin-7 radioimmunoassay seems to be a useful alternative assay for this enzymatic activity.

Introduction

Limited proteolysis of proteins appears to be a general mechanism for the generation of biologically active peptides. Morphinomimetic peptides have been detected and purified from enzymatic digests of various proteins 2, 3, 4, 14. Casomorphins were the first opioid-like peptides to be isolated from casein peptic digestion [2]. Subsequently, exorphins were purified from gluten enzymatical hydrolysates [13]. Cytochrophins derived from mitochondrial cytochrome b and hemorphins derived from hemoglobin were isolated from enzymatically treated bovine blood 3, 4. The biological activity and sequences of these peptides were found to be close to the casomorphins [19]. The Tyr-Pro-Trp-Thr and Tyr-Pro-Trp-Thr-Gln opioid-like peptides isolated by Brantl et al.[4] named hemorphin-4 and hemorphin-5 corresponded to the fragments 34–37 and 34–38 of bovine hemoglobin β chain, respectively and fragments 35–38 and 35–39 of human hemoglobin. A significant amount of evidence has been accumulated indicating that the generation of hemorphins might occur in vivo. For instance hemorphin peptides were isolated from human pituitary gland, cerebrospinal fluid 16, 17, 26, blood [18], bovine brain [21] and hypothalamic tissues [12]. In some cases, hemoglobin-derived peptides were found under physiological 16, 22 and pathological conditions (cerebrovascular bleedings, inflammation, Alzheimer disease) 17, 18, 27. The in vivo effects of hemorphins indicated that these fragments derived from hemoglobin exhibited an opioid activity [10]. These peptides injected intra-cerebroventricularly induced opioid effects such as antinociception and inhibition of gastrointestinal motility [10] which were mediated through the opioid receptors. Moreover, L-V-V-hemorphin-6 (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg) and L-V-V-hemorphin-4 exhibited an in vitro inhibitory activity towards the enkephalin-degradating enzymes (aminopeptidase, dipeptidyl-aminopeptidase and angiotensin-converting enzyme) [24]. The effects observed on angiotensin-converting enzyme suggested a putative implication of hemorphins in the in vivo blood pressure regulation 23, 29. In addition, it was demonstrated that L-V-V-hemorphin-7 (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe) may acted as a coronaro-constrictory peptide [1].

Previously we reported the generation of two hemorphins, V-V-hemorphin-7 (Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe) and L-V-V-hemorphin-7 (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe) from in vitro hemoglobin and globin peptic treatment suggesting that pepsin-like enzymes may be involved in the in vivo release of hemorphins 8, 25. Moreover, it was shown that V-V-hemorphin-7 was generated from globin by peritoneal macrophages [9]. The use of specific proteolytic inhibitors suggested that the pepsin-like acid protease, cathepsin D might be involved in this hemorphin release. In rat leukocytes, this lysosomal enzyme was found to be responsible for the release of xenopsin-related peptides from xenopsin precursor 5, 6. Therefore in order to study the in vivo putative physiological release of hemorphins from hemoglobin, we investigated the in vitro cathepsin D hydrolysis of hemoglobin.

However to investigate the in vivo physiological role of these peptides in discrete area of tissues, the natural concentration and the histological localization of these hemorphins ought to be determined. Therefore it was crucial to develop highly sensitive and specific analytical methods such as radioimmunoassays. The present work described the production of rabbit antibodies and development of a specific radioimmunoassay (RIA) against the C-terminal part of the V-V-hemorphin-7 and L-V-V-hemorphin-7 peptides. The detection and quantitation of the immunoreactive peptides were performed after HPLC purification of complex hemoglobin cathepsin D hydrolysates and compared to peptic hydrolysate.

Section snippets

Reagents

Hemorphin-7, V-V-hemorphin-7 and L-V-V-hemorphin-7 were synthetized by C. Guillon, Laboratoire de Technologie Enzymatique, (Compiègne, France). V-V-hemorphin-8 and [Lys6]V-V-hemorphin-7 were synthetized by Altergen (Strasbourg, France). Hemorphin-4 and -5, were purchased from France Biochem (Meudon, France). Pepsin (E.C. 3.4.23.1, 3670 units/mg), trypsin (E.C. 3.4.21.4, 250 units/mg), soybean trypsin inhibitor, 1 ethyl-3(3-dimethylaminopropyl) carbodiimide (ECD), cathepsin D (E. C. 3.4.23.5,

Characteristics of V-V-Hemorphin-7 Antibodies

The physicochemical characteristics of the V-V-hemorphin-7 antisera were investigated by performing a liquid phase radioimmunoassay with a 125I-labeled probe (Table 1). The titers (i.e., immunserum dilution able to bind to 50% of the radioactive probe) were 1/90,000 and 1/125,000, respectively and the IC50 (M) (i.e., the concentration able to inhibit 50% of the antigen-antibody binding) were 8.6 × 10−11 and 7.1 × 10−11 M, respectively. The minimum detectable values (corresponding to the amount

Discussion

Hemorphins are peptides with opioid activity, which are enzymatically released from hemoglobin. Several lines of evidence suggest that, under physiological and pathological conditions a processing may liberate L-V-V-hemorphin-7 peptide in vivo from neural fluids and tissues. However, in order to study a physiological release of this kind, the determination of the peptide contents requires a sensitive and specific method. Towards this goal, antibodies raised against the C-part of hemorphins were

Acknowledgements

We thank Dr. M. C. Parker for revising the english manuscript.

References (33)

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