Abstract
An antiserum to a synthetic peptide corresponding to residues 95-117 of bovine proenkephalin recognizes all the major intermediates of this prohormone in bovine adrenal medulla (Birch, N. P. and Christie, D. L. (1986) J. Biol. Chem. 261, 12213-12221). This antiserum enabled an investigation of the stability and molecular properties of intermediates in the processing of proenkephalin in bovine adrenal medullary chromaffin granules. Intact and hypotonic lysates of chromaffin granules were incubated at 37 degrees C and the stability of intermediates assessed by gel filtration followed by radioimmunoassay and gel electrophoresis in combination with immunoblotting. Processing was slow in intact granules compared with incubations of hypotonic lysates which resulted in the selective cleavage of an Mr 27,000 intermediate and increases in the amounts of immunoreactivity of lower molecular weight. Protease inhibitors increased the stability of the 27-kilodalton intermediate, the most effective being p-chloromercuribenzoate. Preliminary evidence was obtained for the regulation of the processing of this intermediate by soluble factors present in chromaffin granules. It appears that membrane-associated intermediates of proenkephalin are relatively stable, although analysis of soluble immunoreactivity released during the incubation of chromaffin granule membranes showed a decrease in the 27-kilodalton intermediate and increased amounts of lower molecular weight intermediates. Analysis of hypotonic lysates by two-dimensional gel-electrophoresis showed that proenkephalin intermediates exhibit significant microheterogeneity. It will be important to compare the products of proenkephalin generated by purified proteases with a putative role in the processing of this prohormone with the properties of endogenous intermediates as revealed in this study.
Highlights
An antiserum to a synthetic peptide corresponding atively large amounts of EC’ polypeptides which are interto residues95-1 1 7of bovine proenkephalin recognizes mediates in the biosynthesis of enkephalins [4]
Intact and hypotonic lysatesof chromaffin granules were incubated at 3 7 “C and the stability of intermediates assessed by gel filtration followed by radioimmunoassay and gel electrophoresis in combinationwithimmunoblottingP. rocessing was
Met-enkephalin-Arg6-Phe7T. he sequence of each enkephalin peptide isbordered by paired basic amino acid residues (-LysArg, -Lys-Lys, and -Arg-Arg-) which are believed to act as recognition sequences for processingenzymes
Summary
Most biologically active peptides are synthesized as partof larger precursor molecules (I), and the conversion of these prohormones toactive peptides requires packaginginto secretory granules [2] and limitedproteolysis by highly specific proteases [3]. Chromaffin granules were prepared as described in Ref. 14. Pellets of chromaffin granules (12 pellets were obtained from g of adrenal medullary tissue after the final ultracentrifugation step) were lysed by the addition of 5 mM Tris succinate, pH 5.9, followed by freezing and thawing (4 “C). The abbreviations used are: EC, enkephalin-containing; SDS, sodium dodecyl sulfate;PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethanesulfonyl fluoride. Intact chromaffin granules were resuspended in 1.5 ml of 0.1 M sodium phosphate, 0.3 M sucrose, pH 6.5 [13], and incubated 3a7t "C. Samples were heated a t 95 "C for 20 min, centrifuged (20,000 X g, 30 min, 4 "C), and subjected to gel filtration. Aliquots were removed at various times andsubjected to heating in1ht acetic acid, 2 mM PMSF and gel filtration as described for intact granules. SDS-polyacrylamide gel was the same as that used for the analysis of proenkephalinintermediates.Proteinbands were visualized by staining withCoomassie Blue and proenkephalin intermediateswere detected by immunoblotting
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