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A Chemically Synthesized Radiolabeled Signal Peptide: Design, Preparation, and Biological Evaluation of an lodinated Analog of Preproparathyroid Hormone

Parathyroid Hormone Research Laboratory, Department of Biological Research and Molecular Biology Merck, Sharp and Dohme Research Laboratories West Point, Pennsylvania 19486
Department of Medicine, Harvard Medical School Boston, Massachusetts 02114

Address requests for reprints to: Dr. Michael Caulfield, Merck, Sharp and Dohme Research Laboratories, W26-207, West Point, Pennsylvania 19486.

Abstract

The chemically synthesized signal peptide (nativesequence signal peptide) of preproparathyroid hormone exhibits signal sequence-like activity by inhibiting the translocation/processing of precursor proteins to their mature forms in an in vitro translation system. In order to prepare a biologically functional radiolabeled form of this peptide, we undertook structure-function studies of the native-sequence signal peptide. Since conventional iodination of peptides is performed under oxidizing conditions, chemical design efforts were focused on the oxidationlabile residues, methionine and cysteine, present in the native sequence. Substitution of the three methionines with norleucine and the single cysteine with alanine yielded a surfur-free analog, [Nle-(-25), Nle-(–21),Nle-(–18),Ala-(–14),D-Tyr-(+1)]pre-proPTH- (–29–+1)amide, which is resistant to oxidation and active in the inhibition of processing assay. An interaction between the signal region and one of the components of the intracellular secretory apparatus, signal recognition particle (SRP), was demonstrated: iodinated sulfur-free analog was cross-linked (using the homo-bifunctional reagent disuccinimidyl suberate) to the 54 kilodalton (kDa) subunit of SRP. The 68 kDa and 72 kDa subunits of SRP were also labeled, but to a lesser extent, by the iodinated peptide.

FOOTNOTES

This investigation was supported in part by NIADDK Grant AM-31356 and a grant from Merck, Sharp and Dohme Research Laboratories.

Received for publication December 15, 1987. Accepted for publication February 10, 1988.