A neuropeptide with molt-inhibiting hormone activity from the sinus gland of the mexican crayfish Procambarus bouvieri (Ortmann)

Abstract

1. 1. By means of reverse-phase high-performance liquid chromatography (RP-HPLC) on a μBondapak-Phenyl column, a neuropeptide with molt-inhibiting hormone (MIH) activity was isolated from a crude extract of 2000 sinus glands from the crayfish Procambarus bouvieri (Ortmann) with a yield of 25 ng/SG. 2. 2. The activity was tested in a heterology in vitro assay as the depression of ecdysteroid biosynthesis by cultured Orconectes limosus' Y-organs. 3. 3. On SDS-PAGE, the peptide migrated as a single band of approximately 6000 Da, and on IEF, it migrated as a single band to a pI of 5.50. 4. 4. The N-terminus was shown to be blocked by means of the dansyl-Cl method, while the C-terminus was shown to be isoleucine by means of carboxypeptidase-Y digestion. 5. 5. The following amino acid average composition of MIH was obtained: (Asx) 7, (Val) 6, (Glx, Leu, Arg) 5, (Cys, Lys) 4, (Ala, Ile, Tyr, Phe) 3, (Ser) 2, (Thr, Gly) 1–2, (Pro) 1. MIH has 53–55 amino acid residues and its minimal molecular mass is calculated as 6243–6402 Da. It does not contain Trp, His nor Met. 6. 6. 37.7 μg (6.3 nmol) of the molt-inhibiting hormone (MIH) and 70.7 μg (11.8 nmol) of the major peak of the crustacean hyperglycemic hormone (CHH-B) were reduced, carboxymethylated and digested with trypsin. The tryptic peptides of each of the hormones were purified on an Ultrasphere-ODS column and the peptide maps compared with respect to retention times and the composition of each peak. 7. 7. 57.4% of the total amount of amino acid residues were accounted or in five peptides which coincided in elution time and composition. 8. 8. As we have found a clear-cut difference of only two residues between MIH and CHH-B (without taking into account the amides of aspartic and glutamic acids), the present results confirm that the two hormones belong to a family of neuropeptides comprising at least one molt-inhibiting hormone and two hyperglycemic hormones. 9. 9. Evidence is presented for the existence of microheterogeneity in the structure of each of these hormones.