Post-translational modifications of the insect sulfakinins - Sulfation, pyroglutamate-formation and O-methylation of glutamic acid

We identified and chemically characterized the two major forms of sulfakini ns from an extract of 800 corpora cardiaca/corpora allata complexes of the American cockroach, Periplaneta americana. Bioactivity during the purificat ion was monitored by measuring heart beat frequency in a preparation in sit u. By Edman degradation analysis and MS, these main forms were identified a s having the primary structures Pea-SK [EQFDDY(SO3H)GHMRFamide] and Lem-SK- 2 [pQSDDY(SO3H)GHMRFamide]. The sulfation was confirmed by UV, MS and pepti de synthesis. In addition,post-translationally modified sulfakinins of both major forms were isolated and identified. Firstly, nonsulfated forms of th ese peptides are present in considerable amounts in the corpora cardiaca/al lata. Secondly, the N-terminally blocked Pea-SK and the nonblocked Lem-SK-2 occur naturally in neurohaemal release sites. Thirdly, modified Pea-SK wit h O-methylated glutamic acid occurs which is not an artefact of peptide pur ification. The major forms of the sulfakinins were shown to be highly activ e on both the heart and hindgut with threshold concentrations of approximat e to 5 X 10(-10) M (heart) and 2 X 10(-9) M (hindgut).