Carboxypeptidase E (CPE), which cleaves C-terminal amino acid residues and is involved in neuropeptide processing, is itself subject to intracellular processing. Human CPE cDNA was isolated and sequence comparisons were made with those of a previously isolated brain cDNA (M1622) encoding rat CPE and of other human carboxypeptidases (M and N). Human (2.5 kb) and rat (2.1 kb) CPE cDNAs approximated to the size of their respective mRNAs; additional sequences were located in putative 5' and 3' untranslated regions of human CPE mRNA. There is 79% sequence similarity between human and rat CPE cDNAs, with greater similarity (89%) over the coding region and short sections of the non-coding sequence. The predicted 476-amino acid-residue sequences of human and rat preproCPEs are highly conserved (96% identity), with lower degree of similarity of the N-terminal signal peptide (76%). Human CPE showed 51% and 43% sequence similarity to human CPN and CPM respectively, with discrete regions of divergence dispersed between the highly conserved mechanistically implicated regions. Antiserum generated from a fusion protein, synthesized in Escherichia coli from constructs of the human cDNA, recognized an approx. 50 kDa membrane protein and a smaller soluble protein in rat and human brain preparations, corresponding to the two forms of native CPE. Human CPE mRNA transcripts directed the synthesis in reticulocyte lysate of a 54 kDa translation product, which in the presence of dog pancreas microsomal membranes was co-translationally processed with cleavage, insertion into membranes and glycosylation. Three processed forms were generated, the largest (56 kDa) and smallest (52 kDa) being equally glycosylated. The membrane association of the processed translation products and of native brain membrane CPE, detected immunologically, was resistant to moderate alkali but not pH 11.5 extraction. These results are consistent with secondary-structure predictions that CPE is a peripheral membrane protein. The dissimilar regions of human carboxypeptidases may provide information on sequences responsible for their different cellular disposition.

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The expression of the insulin-like growth factor-binding proteins (IGFBP) -3, -4, -5 and -6 was investigated in neonatal, in normal adult and in regenerating rat skeletal muscle. Semi-quantification was done by densitometric scannings of Northern blots. The expression of all investigated IGFBPs, with the exception of IGFBP-5, was higher in neonatal than in adult muscle. During postischaemic regeneration the expression of all IGFBPs increased, but with different time schedules. IGFBP-3 increased transiently during the early phase of regeneration, while IGFBP-4, -5 and -6 increased during the later phase of regeneration. In situ hybridisation on regenerating muscle showed that the expression of the various IGFBPs was cell specific; thus, IGFBP 3 was mainly expressed in macrophages, IGFBP-4 in connective tissue, IGFBP-5 in regenerating muscle cells, and IGFBP-6 in muscle cells, connective tissue and endothelium. Ligand blotting, using 125I-IGF-I as the ligand, showed a number of bands ranging between 24 and 44 kDa. Samples from neonatal and regenerating muscle contained much higher levels of all IGFBPs than those from normal adult muscle. An ordered and cell-specific expression of IGFBPs, allowing a strict regulation of IGF actions, is probably necessary to ensure an optimal regeneration process.