Guanylyl cyclase-activating peptide II (GCAP-II), an endogenous ligand of particulate guanylyl cyclase C (GC-C), is processed from the precursor protein and circulates in human blood. GCAP-II consists of 24 amino acid residues and contains two disulfide bridges. The correct disulfide paring of GCAP-II is an absolute requirement for its biological activity. This study shows that the folding of the peptide from the reduced form yields a peptide with the native disulfide paring as a minor product and with non-native ones as major products, regardless of the presence or absence of reduced and oxidized glutathione. The results suggest that GCAP-II does not possess sufficient information to permit the adoption of the native conformation and to effectively form the correct disulfide pairing and, as a result, that GCAP-II is correctly folded by assistance of a factor(s) such as an intra- or intermolecular chaperone. We studied whether a peptide in the pro-leader sequence of the precursor protein (proGCAP-II) contains sufficient information to facilitate the folding of GCAP-II. For this purpose, we prepared proGCAP-II in Escherichia coli by a recombinant technique and examined the disulfide-coupled folding of proGCAP-II from the reduced form. proGCAP-II was quantitatively recovered with the correctly folded structure from the reduced form both in the presence and in the absence of reduced and oxidized glutathione. The protein contains only disulfide linkages at the same positions as the mature form of proGCAP-II, GCAP-II, and the biologically active isomer of GCAP-II in the molecule. These results provide evidence that the propeptide of proGCAP-II is a critical factor in the formation of the correct disulfide paring in the folding of the protein.

Carbon-13 spin-lattice relaxation times of the tripeptide methyl ester Boc-Gly-Pro-Pro-OMe and its polymer-bound analogues are reported. Line widths of the signals of -carbon atoms of Pro(2) and Pro(3) are also measured and an interpretation of the line broadening is presented. As polymeric macromolecules the following supports were employed: insoluble cross-linked polystyrene (PS), soluble poly(oxyethylene) (POE), and the graft copolymer poly(oxyethylene)-polystyrene-divinylbenzene (POE-PS). The molecular motions of the peptide moieties were determined by analysis of the relaxation time Tv The peptide methyl ester was synthesized classically and after saponification of the ester group it was coupled to the polymers according to known procedures. In the present studies for the measurements of the Tj values only the signals of the trans forms of the X-Pro sequence have been considered. The investigations revealed that the Tt values and hence the mobility of the C atoms of the peptide esters decrease in the sequence methyl ester > POE ester > POE-PS ester > PS ester.

An insoluble graft copolymer composed of the covalently bound polyoxyethylene to cross‐linked polystyrene‐divinylbenzene was employed as the solid support for peptide synthesis. The physical properties of this new polymeric support were similar to those of the cross‐linked polystyrene. Thus, identical manipulations such as shaking and filtration could be performed on this resin. The graft copolymer was used for the synthesis of the C‐terminal decapeptide of bovine insulin B‐chain in the usual solid‐phase manner. The protected peptide was cleaved from the polymeric support by photolysis and was purified by chromatography on silica gel and Sephadex LH‐20. Samples of the decapeptide were treated with liquid HF and the unprotected peptide was purified by ion‐exchange chromatography. The free peptide was shown to be homogeneous by HPLC, electrophoresis and t.l.c. The identity of this peptide was also confirmed by FAB‐MS and amino acid analysis. The synthesized peptides were shown to be free of racemization. The free peptide possesses a disordered conformation as revealed by the CD measurement.

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