A neurosecretoru hyperglycemic hormone from the sinus gland of the mexican crayfish Procambarus bouvieri (Ortmann)—I. Purification and biochemical characterization of the most abundant form of the hormone

Abstract

1. 1. The hyperglycemic activity of a crude extract (CE) from the sinus gland (SG) of Procambarus bouvieri (Ortmann) was recovered as a single asymmetric peak by means of reverse phase high performance liquid chromatography (RP-HPLC) on a Nova-Pak C 18 column. This peak was resolved into three symmetric peaks on rechromatography on a μBondapak-Phenyl column. Peaks were designated A, B, and C in the order of elution. Peak A had no hyperglycemic activity, while Peak B was as active as Peak C. 2. 2. Protein determination showed the following results (in ng/SG): CE—907.5, Peak A—14.5, Peak B—30.0 and Peak C—16.5. The protein content of Peaks A + B + C was 61 ng/SG, which is 6.7% of the CE protein. 3. 3. The bioassay was conducted on destalked Procambarus bouvieri. One unit of hyperglycemic activity was found to correspond to 9.4 ng of CE, 860 pg of B and 750 pg of C. 4. 4. Peak B, the most abundant form, was further characterized. On SDS-PAGE it migrated as a single band of approximately 6000 M.W. 5. 5. On I.E.F., peptide B migrated as a single band to a p I of 4.79. 6. 6. By means of the DNS-Cl method, it was shown that peptide B is N-terminally blocked. 7. 7. The carboxyl end of peptide B was found to be isoleucine by carboxypeptidase Y digestion and dansylation. 8. 8. The following amino acid average composition of peptide B was obtained from the acid hydrolysate of 2 nmoles for 20, 48 and 72 hr: (ASX) 7, (VAL) 6, (GLX) 5, (LEU, TYR, CYS, ARG) 4, (ALA, ILE, LYS) 3, (THR, SER, PRO, PHE) 2, (GLY) 1. 9. 9. Peptide B has 52 amino acid residues and its minimal M.W. was calculated as 6042 Da. It does not contain TRP, HIS or MET. 10. 10. While this work was in process, a simplified procedure utilizing only one RP-HPLC step on a μBondapak-Phenyl column was introduced. The same results as above were obtained.