Many synthetic compounds as well as almost all organisms including animals, plants and microorganisms have been screened for bioactive substances as starting materials for biomedicines. Although numerous substances have been isolated, there are probably many others, especially degradation products of proteins that have not been identified. Through a control of process parameters such as pH, time and enzyme-substrate ratio, it is possible to produce hydrolysates whose components may have retained various functional properties attached to the native molecules. Previous experiments performed using a large range of hydrolysates from various sources demonstrate the presence of calcitonin gene related peptide (CGRP) immunorelated molecules in various fish hydrolysates. The calcitonin gene related peptide, a 37 aminoacid neuropeptide, is derived from the same gene as calcitonin by a mechanism of alternative splicing. It is predominantly synthesised in neural tissue and is mainly involved in the control of heart metabolism but is also implicated in the regulation of gastric acid secretion. In addition, at high doses, CGRP induces the same effects as calcitonin, that is hypocalcemia and hypophosphatemia. In non-mammalian vertebrates, this peptide is mainly found in gills and intestine and is involved in the control of hydromineral metabolism by its specific action on gill membranes. The sequence similarity between calcitonin and CGRP suggests that both peptides may support identical biological effects mainly in the control of homeostasis and reproduction.

Among the various tested hydrolysates, the sardine (Sardina pilchardus) hydrolysates were characterised by the highest quantity of CGRP immunologically and biologically related molecules. In addition, we demonstrated that using increasing hydrolysis time and various alcalase concentrations, the CGRP like molecules were mainly found after 2 hours of hydrolysis using an alcalase concentration of 0.1%. Therefore, sardine hydrolysates were prepared using these conditions and purified by gel exclusion and HPLC chromatography. Finally, the purified CGRP like molecules were analysed for their CGRP like biological effect using the ability of CGRP to stimulate the adenylate cyclase activity in rat liver membranes.

Sardine hydrolysates were prepared from cooked head and guts using 0.1% alcalase 2.4L (Novo Nordisk Industri DK-2880 Bagsvaerd, Denmark) in phosphate buffer (0.1M, pH 8.1, 1/10, w/v). Hydrolysis was carried out during 2 hours at 40°C. The enzyme was inactivated by 15 min of boiling in a microwave oven. After centrifugation at 25000g, 20 min, the supernatant was filtrated through a polyethylene net, ultrafiltrated (cut off 10 KD) and freeze dried.

Sardine hydrolysates were prepurified by gel exclusion chromatography on a HW 40 toyopearl column (2.5 x 33.5 cm) using ammonium acetate 0.2 M, pH 5 as eluant. Immunoreactive fractions were analysed for CGRP immunoreactivity using an anti human CGRPI antibody. The positive fractions were further analysed by their ability to displace the labelled CGRP binding to rat liver membranes (radioreceptorassay).

Positive fractions were then subjected to HPLC on a C18 Prosphere column using a linear gradient of 10 to 60% acetonitrile in 0.1% TFA. The biological activity of these fractions was measured by radioreceptorassay and active fractions were repurified using the same column and a linear gradient of 30 to 60% acetonitrile in 0.1% TFA.

The fractions obtained from the final purification step were analysed for their ability to stimulate the adenylate cyclase activity in rat liver membranes. This enzyme activity was determined by measuring the synthesis of cAMP from non radioactive ATP. Cyclic AMP was quantified using the radioreceptor assay kit from Amersham. The adenylate cyclase activity was expressed as picomoles of cAMP synthesised by 1 mg membrane protein during 1 min of incubation.

Sardine hydrolysate (22 mg of protein suspended in 7 ml of eluant) was loaded on the HW40 Toyopearl column. 2 ml fractions were collected and a 0.5 ml aliquot was used to quantify the CGRP immunoreactivity. The CGRP immunoreactive profiles of this preparation demonstrated 5 main immunoreactive fractions. These fractions were further analysed using the CGRP radioreceptorassay. Only the second tested fraction was able to inhibit the labelled CGRP binding as the unlabeled hormone did. Fraction B was further purified by HPLC chromatography. 250 µg of proteins were loaded on the column, 1ml fractions were collected and a 0.4 ml aliquot used to determine the CGRP immunoreactivity. Immunoreactive profile of this separation gave three main immunoreactive peaks. Radioreceptor assays showed that the most efficient inhibiting effect resulted with the immunoreactive material present in the third peak.

53 µg proteins of this fraction were subjected to a second HPLC chromatography using the same column and a linear acetonitrile gradient from 30 to 60%. Two absorbance peaks were observed: only the first active fraction showed CGRP-like immunoreactivity. In the CGRP radioreceptorassay, this fraction displaced the labelled CGRP binding: 25% inhibition was observed with 370 ng of proteins. So, from 22 mg of sardine hydrolysate proteins, we obtained 14 µg of a molecule that present CGRP biological and immunological activity. The purification factor obtained was about 12.500. The different purification steps were followed using the CGRP radioreceptorassay. While 311 µg of proteins from crude extract were necessary to displace 50% of the initial CGRP binding to rat liver membranes (Table 1), only 0.9 µg of proteins from the final purification step produced the same inhibitory effect. The molecular weight of this molecule determined by mass spectrophotometry was 6000 daltons.

The biological activity of the fraction obtained from the second HPLC was analysed by referring to the capacity of CGRP to stimulate the adenylate cyclase activity in rat liver membranes. 25% inhibition was observed with 0.3 µg of proteins, that is a K_0.5 of 10⁶M^-1. In order to analyse the specificity of the observed inhibition, we compared the effect of the fraction purified after gel exclusion chromatography on the CGRP (4 ng/ml) and glucagon (200 ng/ml) stimulated adenylate cyclase activity in the same rat liver membrane preparation The inhibiting effect of the prepurified fraction was significant only on the CGRP stimulated adenylate cyclase activity. In the presence of 1.1 µg of proteins, the inhibiting effect observed was 14 and 35% of the glucagon and CGRP stimulated adenylate cyclase activity, respectively.

Many bioactive peptides have been isolated from enzymatic hydrolysates of proteins. Indeed, invertebrate and fish by-products might be the best source for such active biopeptides because these substances would make good starting materials for safer and less toxic medicines. In addition, these bioactive peptides may be used as adjuvants to stimulate food intake and to enhance growth and disease resistance of animals.

We reported here the purification from sardine hydrolysates of CGRP related molecules. In order to analyse the function of these molecules, we tested their ability to modulate the adenylate cyclase activity in rat liver membranes: a specific target tissue for CGRP. These experiments show that they have a specific inhibitory action on the CGRP stimulated adenylate cyclase activity These purified molecules are able to decrease the ¹²⁵I labelled CGRP binding to rat liver membranes and to inhibit the following biological effect. This action both at the receptor and the adenylate cyclase level is similar to that observed using a well known competitive inhibitor of CGRP action: the human CGRP 8-37.

So, these purified molecules may act as antagonists for peptides that bind to CGRP receptors in rat liver membranes. In addition, they are probably C terminal fragments as all CGRP antagonists, which were reported, belong to this class of molecules.

The development of new antagonists may be of particular importance in various aspects of the CGRP action mainly in the control of feeding where CGRP together with amylin exert a direct or indirect control of meal size and the control of meal initiation.