Preparation of tritiated oostatic peptides for study of radioactivity incorporation in flesh fly Neobellieria bullata

Abstract

A series of insect oostatic peptides containing 3,4-dehydroproline in the C-terminal part or inside of the peptide chain was synthesized and tritiated by addition of (3)H2 to double bond of 3,4-dehydroproline residue. (3)H-label was introduced also into tyrosine residue of oostatic tetra- and pentapeptides by isotopic exchange of benzyl beta-hydrogens. In this way, three types of tritiated peptides were prepared, different in the radiolabeled amino acid position: [(3)H] Tyr-Asp-Pro-Ala-OH, H-Tyr-Asp-[(3)H] Pro-Ala-OH, [(3)H] Tyr-Asp-Pro-Ala-Pro-OH, H-Tyr-Asp-[(3)H] Pro-Ala-Pro-OH, H-Tyr-Asp-Pro-Ala-[(3)H] Pro-OH, H-Tyr-Asp-Pro-Ala-Pro(5)-[(3)H] Pro-OH and H-Asp-[(3)H] Pro-OH. These peptides made possible a highly sensitive comparative study on radioactivity incorporation into head and ovaries of the flesh fly Neobellieria bullata, which revealed this process to proceed differently. The reasons of the found differences are discussed.

Study of Oostatic Peptide Uptake and Metabolism in Developing Ovaries of the Flesh Fly,Neobellieria bullata

“…The total radioactivity in the ovaries (Figure 2) was determined, and the radioactive components in extracts of ovaries were analyzed. Radiolabeled metabolites of [ 3 H-Pro 3 ]5P found in ovaries were identified using synthetic standards of non-labeled sequences (Hlaváček et al 2007) as illustrated in Figure 3.…”

“…Tritiated oostatic pentapeptide (5P) H-Tyr-Asp-[ 3 H]Pro-Ala-Pro-OH, [ 3 HPrO 3 ] 5P, 1.44TBq/mmol, with radiochemical purity > 98%, as described earlier Hlaváček et al (2007) was used in the study. Different stages of yolk deposition were classified according to the scale developed by King (1970).…”

“…Standard unlabeled peptides (Hlaváček et al 2007) as well as non-active proline were detected by UV, and their retention times were compared to that of the peaks in the radiochromatogram. The retention times of radioactive fractions were corrected to the time delay between the UV and the radiometric detector (0.55 min).…”

“…In studies of the effects of 5P on vitellogenic stages (Hlaváček et al 1997, 1998; Bennettová et al 2002; Slaninová et al 2004) differences were found between morphological changes of both egg chambers: in the first one there were no visible effects, while in the second, proliferation of the follicular epithelium into the inner space of egg chamber, followed by resorption was observed (Figure 1). Therefore, its 3 H-labeled forms were used for further studies on radioactivity accumulation and degradation of the oostatic peptides in the flesh fly N. bullata and other insects (Tykva et al 1999, 2007; Slaninová et al 2004; Hlaváček et al 2007). On the other hand, the native TMOF of N. bullata , hexapeptide H-Asn-Pro-Thr-Asn-Leu-His-OH (Bylemans et al 1994; de Loof et al 1995), has no structural similarity to the above oostatic peptides and lacks any oostatic effects, nor does its isosteric analogue H-Asn-Proψ[CH 2 O]-D-Thr-Asn-Leu-His-OH, (Bennettová, unpublished results; Hlaváček et al 2004).…”

Oostatic peptides containing d-amino acids: synthesis, oostatic activity, degradation, accumulation in ovaries and NMR study

“…We subsequently used the 3 H-labeled form (3a) (see Table 1) of the most active pentapeptide 1a in studies on its degradation and radioactivity accumulation in organs (ovaries, hemolymph) of the N. bullata (Tykva et al 1999;Slaninová et al 2004Slaninová et al , 2007Hlaváček et al 2007). We found that the radioactivity incorporates into the organs in minutes and the pentapeptide is quickly degraded to tyrosine and the H-Asp-Pro-Ala-Pro-OH tetrapeptide in the ovaries and to proline and the H-Tyr-Asp-Pro-Ala-OH tetrapeptide in the hemolymph.…”