Journal
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY
Volume 35, Issue 8, Pages 1105-1110Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/S1872-2040(07)60072-3
Keywords
Aplysia; endopeptidease; mass spectrometry; attraction; insulin
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Endopeptidease of Aplysia egg (AEE) was separated and purified by column chromatography using separation gel both DEAE-52 cellulose. The purity of AEE and its molecular weight of the subunit approximately 39. 0 kDa) were determined by Sephadex G-1 50 and SDS-polyacrylamid gel electrophoresis PAGE) methods. The results from MALDI-TOF mass spectrometry indicated that AEE(39) consisted of single subunit type ( M and its ratio of mass to charge ( m/z) was 12738. 17, 18108. 79 and 38221. 42, called [M3+], [M2+], and [M+] respectively. Using probe of insulin ( INS), a combined technology with both metal chelator EDTA and MALDI-TOF mass spectrometry was employed to measure molecular weight of AEE39 and to identify an endopeptidease with metal zinc, showing molecular weight with 38221. 42Da, called AEE,,. It was found that AEE 39 has the capacity for lysating INS, giving enzymolysis produces with m/z 1449. 51, 2085. 84, 4080. 41, 4165. 42. The software designed with ourselves was used to analyze the produces sequences and to identify the cleavage of INS. As a conclusion, It was found that the best sensitive cleavage for enzymolysis was Leu ( leucine) -X ( X: residues of amino acid), next one is Glu ( glutamic acid) -X. In addition, Phe(phenylalanine)-X, Asn(asparagine)-X and Ser(serine)-X in INS can be lysated once in a while. Compared to molecular structure both INS and attraction, it indicated that one of main functions of AEE39 was responsible for lysating the attraction in egg, which played an important role in informational intercourse, recalling recognizing, and mating. In addition, AEE(39) had another novel function for enzymolysis Leu-Leu in acidic peptide, which was a multifunctional ednopeptidease.
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