Association of newly synthesized islet prohormones with intracellular membranes.

Results from recent studies have indicated that pancreatic islet prohormone converting enzymes are membrane-associated in islet microsomes and secretory granules. This observation, along with the demonstration that proglucagon is topologically segregated to the periphery within alpha cell secretory granules in several species, led us to investigate the possibility that newly synthesized islet prohormones might be associated with intracellular membranes. Anglerfish islets were incubated with [3H]tryptophan and [14C]isoleucine for 3 h, then fractionated by differential and density gradient centrifugation. Microsome (M) and secretory granule (SG) fractions were halved, sedimented, and resuspended in the presence or absence of dissociative reagents. After membrane lysis by repeated freezing and thawing, the membranous and soluble components were separated by centrifugation. Extracts of supernatants and pellets were chromatographed by gel filtration; fractions were collected and counted. A high proportion (77-79%) of the newly synthesized proinsulin and insulin was associated with both M and SG membranes. Most of the newly synthesized proglucagons and prosomatostatins (12,000-mol-wt precursors) were also membrane-associated (86-88%) in M and SG. In contrast, glucagon- and somatostatin-related peptides exhibited much less membrane-association in SG (24-31%). Bacitracin, bovine serum albumin EDTA, RNAse, alpha-methylmannoside, N-acetylglucosamine, and dithiodipyridine had no effect on prohormone association with membranes. However, high salt (1 M KCl) significantly reduced membrane-association of prohormones. Binding of labeled prohormones to SG membranes from unlabeled tissue increased with incubation time and was inhibited by unlabeled prohormones. The pH optimum for prohormone binding to both M and SG membranes was 5.2. It is suggested that association of newly synthesized prohormones with intracellular membranes could be related to the facilitation of proteolytic processing of prohormones and/or transport from their site of synthesis to the secretory granules.

Comparison of prohormone-processing activities in islet microsomes and secretory granules: evidence for distinct converting enzymes for separate islet prosomatostatins.

In previous work we have examined the nature of converting enzymes for proinsulin, proglucagon, and prosomatostatin-I (PSS-I) in secretory granules isolated from anglerfish islets. The purpose of the present study was to extend the examination of precursor conversion to islet microsomes and to compare prohormone processing, including that of PSS-I and prosomatostatin-II (PSS-II), in islet secretory granules and microsomes. Microsomes (rough endoplasmic reticulum [RER] and Golgi complex) and secretory granules were prepared from anglerfish islets by differential and discontinuous density-gradient centrifugation. Microsomes were further fractionated into Golgi- and RER-enriched subfractions. Lysed secretory granule or microsome preparations were incubated in the presence of a mixture of radioactively labeled islet prohormones. Extracts of products generated were subjected to analysis by gel filtration and high-pressure liquid chromatography. Accuracy of product cleavage was monitored by comparing high-pressure liquid chromatography retention times from the radiolabeled in vitro conversion products with the retention times of labeled products from tissue extracts. All converting activity in microsomes was found to be similar to that in granules in that it had a pH optimum near pH 5 and was inhibited by p-chloromercuribenzoate. No significant differences in the converting activity of Golgi complex- and RER-enriched subfractions of microsomes was observed. The proinsulin, proglucagon, and PSS-II converting-enzymes, which were found in islet secretory granules, were also present and membrane-associated in islet microsomes. However, converting activity for PSS-I was displayed only in secretory granules. This suggests that two or more separate enzymes are involved in processing PSS-I and PSS-II, and that these enzymes have either differential distribution or differential activity in RER/Golgi complex and secretory granules. The demonstration of converting enzyme activity in islet microsomes supports the proposal that these enzymes may be synthesized at the RER and are internalized along with the prohormones.

Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level. I. Fractionation procedure and characterization of the subcellular fractions.

Anglerfish islets were homogenized in 0.25 M sucrose and separated into seven separate subcellular fractions by differential and discontinuous density gradient centrifugation. The objective was to isolate microsomes and secretory granules in a highly purified state. The fractions were characterized by electron microscopy and chemical analyses. Each fraction was assayed for its content of protein, RNA, DNA, immunoreactive insulin (IRI), and immunoreactive glucagon (IRG). Ultrastructural examination showed that two of the seven subcellular fractions contain primarily mitochondria, and that two others consist almost exclusively of secretory granules. A fifth fraction contains rough and smooth microsomal vesicles. The remaining two fractions are the cell supernate and the nuclei and cell debris. The content of DNA and RNA in all fractions is consistent with the observed ultrastructure. More than 82 percent of the total cellular IRI and 89(percent) of the total cellular IRG are found in the fractions of secretory granules. The combined fractions of secretory granules and microsomes consistently yield >93 percent of the total IRG. These results indicate that the fractionation procedure employed yields fractions of microsomes and secretory granules that contain nearly all the immunoassayable insulin and glucagons found in whole islet tissue. These fractions are thus considered suitable for study of proinsulin and proglucagon biosynthesis and their metabolic conversion at the subcellular level.

Studies on proinsulin and problucagon biosynthesis and conversion at the subcellular level. II. Distribution of radioactive peptide hormones and hormone precursors in subcellular fractions after pulse and pulse-chase incubation of islet tissue.

Anglerfish proinsulin and insulin were selectively labeled with [(14)C]isoleucine, while proglucagon, conversion intermediate(s), and glucagon were selectively labeled with[(3)H]tryptophan. After various periods of continuous or pulse-chase incubation, islet tissue was subjected to subcellular fractionation. Fraction extracts were analyzed by gel filtration for their content of precursor, conversion intermediate(s), and product peptides. Of the seven subcellular fractions prepared after each incubation, only the microsome and secretory granule fractions yielded significant amounts of labeled insulin-related and glucagon-related peptides. After short-pulse incubations, levels of both [(14)C]proinsulin and [(3)H]proglucagon (mol wt approximately 12,000) were highest in the microsome fraction. This fraction is therefore identified as the site of synthesis. With increasing duration of continuous incubation or during chase incubation in the absence of isotopes, proinsulin, proglucagon, and conversion intermediate(s) are transported to secretory granules. Conversion of proinsulin to insulin and proglucagon to a approximately 4,900 mol wt conversion intermediate and 3,500 mol wt glucagon occurs in the secretory granules. Converting activity also was observed in the microsome fraction. The recovery of most of the incorporated radioactivity in microsome and secretory granule fractions indicates that the newly synthesized islet peptides are relegated to a membrane-bound state soon after synthesis at the RER is completed. This finding supports the concept of intracisternal sequestration and intragranular maintenance of peptides synthesized for export from the cell of origin.

Cotranslational and posttranslational proteolytic processing of preprosomatostatin-I in intact islet tissue.

Preprosomatostatin-I (PPSS-I) is processed in anglerfish islets to release a 14-residue somatostatin (SS-14). However, very little is known regarding other processing events that affect PPSS-I. This is the first study to identify and quantify the levels of nonsomatostatin products generated as a result of processing of this somatostatin precursor in living islet tissue. The products of PPSS-I processing in anglerfish islet tissue were identified in radiolabeling studies using a number of criteria. These criteria included immunoreactivity, specific radiolabeling by selected amino acids, radiolabel sequencing, and chromatographic comparison to isolated, structurally characterized fragments of anglerfish PPSS-I using reverse-phase high performance liquid chromatography. Intact prosomatostatin-I (aPSS-I) was isolated from tissue incubated with [3H]tryptophan and [14C]leucine. Significant 14C radioactivity was observed in the products of 11 of the first 44 sequencer cycles in positions consistent with the generation of a 96-residue prosomatostatin. These results indicate that signal cleavage occurs after the cysteine located 25 residues from the initiator Met of PPSS-I, resulting in a signal peptide 25 amino acids in length. Nonsomatostatin-containing fragments of the precursor were also found in tissue incubated with a mixture of 3H-amino acids. Only a small quantity of the dodecapeptide representing residues 69-80 in the prohormone was found (10 nmol/g tissue). Two other fragments of aPSS-I, also observed to be present in low abundance, were found to correspond to residues 1-27 (16 nmol/g tissue) and to residues 1-67 (7 nmol/g tissue) of aPSS-I. No evidence for the presence of the fragment corresponding to residues 29-67 was found. However, large quantities of SS-14 were observed (287 nmol/g tissue), indicating that the major site of aPSS-I cleavage is at the basic dipeptide immediately preceding SS-14. Recovery of much lower levels of the nonsomatostatin fragments of aPSS-I suggests that prohormone processing at the secondary sites identified in this study occurs at a low rate relative to release of SS-14 from aPSS-I.

Characterization of proinsulin- and proglucagon-converting activities in isolated islet secretory granules.

The conversion of proglucagon and proinsulin by secretory granules isolated from both prelabeled and unlabeled anglerfish islets was investigated. Either granules isolated from tissue labeled with [3H]tryptophan and [14C]isoleucine or [35S]cysteine, or lysed granules from unlabeled tissue to which exogenously labeled prohormones had been added were incubated under various conditions. Acetic acid extracts of these granule preparations were analyzed for prohormone and hormone content by gel filtration. Both prelabeled and lysed, unlabeled secretory granules converted radiolabeled precursor peptides (Mr 8,000-15,000) to labeled insulin and glucagon. The accuracy of the cleavage process was established by demonstrating comigration of products obtained from in vitro cleavage with insulin and glucagon extracted from intact islets using electrophoresis and high-pressure liquid chromatography (HPLC). The pH optimum for granule-mediated conversion was found to be in the range of pH 4.5-5.5. Conversion of both proglucagon and proinsulin by secretory granules was significantly inhibited in the presence of antipain, leupeptin, p-chloromercuribenzoate (PCMB) or dithiodipyridine (DDP) but not chloroquine, diisopropyl fluorophosphate, EDTA, p-nitrophenyl guanidinobenzoate, soybean trypsin inhibitor, or N-p-tosyl-L-lysine chloromethyl ketone HCl. The inhibitory action of PCMB and DDP was reversed in the presence of dithiothreitol. Both membranous and soluble components of the secretory granules possessed significant converting activity. HPLC and electrophoretic analysis of cleavage products demonstrated that the converting activities of the membranous and soluble components were indistinguishable. The amount of inhibition of proinsulin and proglucagon conversion caused by 600 micrograms/ml porcine proinsulin was significantly lower than that caused by the same concentration of unlabeled anglerfish precursor peptides. These results indicate that the proinsulin and proglucagon converting enzyme(s) in the anglerfish pancreatic islet is a unique intracellular thiol proteinase(s) that may be granule membrane-associated and may require the presence of prohormone sequences in addition to the dibasic residues at cleavage sites for substrate recognition and/or binding.

Islet secretory granules contain cytochrome b561.

A cytochrome has been detected in secretory granules prepared from anglerfish islets of Langerhans. The heme moiety was determined to be of the b type, and the dithionite-reduced cytochrome exhibited an alpha-band maximum at 561 nm with an extinction coefficient of 13.8 mM-1 X cm-1. The protein was present at a concentration of 40 +/- 4 pmol/mg of secretory granule protein. The cytochrome was found to be an integral membrane protein and to be reduced by ascorbic acid but not by NADH, NADPH, reduced glutathione (GSH), or succinate. Because of the similarity to previously characterized secretory granule cytochrome b561's from neuroendocrine tissues, this cytochrome is also referred to as cytochrome b561. Although its function has not yet been elucidated, the apparent specificity for ascorbate suggests that it may be a component of the ascorbate-dependent peptidyl-glycine alpha-amidating monooxygenase system that functions in the amidation of islet hormones.

Evidence for redundancy in propeptide/prohormone convertase activities in processing proglucagon: an antisense study.

To further examine the physiological roles of the neuroendocrine prohormone convertases (PCs) in proglucagon processing, alpha TC1-6 cells were transiently transfected with PC1/3 and PC2 expression vectors containing either antisense or sense encoding cDNAs. PC1/3- and PC2-directed RIAs were used to determine that the PC1/3 antisense transfections lowered endogenous levels of PC1/3 by 40 +/- 7.9% but did not alter the levels of PC2. The PC2 antisense transfections decreased the endogenous levels of PC2 by 91 +/- 11.7% without affecting the levels of PC1/3. To quantitate the levels of proglucagon and proglucagon-derived products, transfected cells were metabolically labeled with [3H]tryptophan, and extracts were chromatographed by reversed-phase HPLC. Recovered peptides were then subjected to peptide mapping analyses, allowing precise quantification of 3H-radioactivity incorporated into proglucagon and its cleavage products. Product-precursor ratios were determined, and percent change in the proportion of products generated in antisense-transfected vs. sense-transfected cells was calculated. The decrease in PC1/3 after antisense treatment significantly reduced the amounts of glicentin produced and partially reduced the levels of all other proglucagon cleavage products. PC2 antisense treatment significantly reduced the levels of glicentin and 9K glucagon generated but had no significant effect on the remainder of the proglucagon-derived peptides. These results suggest the existence of redundant mechanisms that ensure the production of each of the intermediate and product peptides derived from proglucagon. PC1/3 is potentially an important enzyme in the processing of most proglucagon-derived peptides, whereas PC2-processing activity appears to predominate at only two of the four potential cleavage sites.

Evidence of sequential metabolic cleavage of proglucagon to glucagon in glucagon biosynthesis.

Following a 30 min preincubation in medium containing no isotopes, anglerfish islet tissue was incubated in the presence of [3H]tryptophan and [14C]isoleucine for 20 min. A portion of the tissue was removed for immediate extraction. The remainder was washed thoroughly with unlabeled medium and post-incubated in medium containing an excess of unlabeled tryptophan and isoleucine for varying periods of time. The distribution of radioactive proteins in alcoholic tissue extracts was analyzed by gel filtration and polyacrylamide gel electrophoresis. The distribution of immunoreactive glucagon was determined by radioimmunoassay. Following the 20 min pulse incubation, only proinsulin was labeled with [14C]isoleucine. Two glucagon immunoreactive molecules, one larger than proinsulin (mol wt near 11,400) and the other slightly smaller than proinsulin (mol wt near 9,000), were the primary proteins labeled with [3H]tryptophan following the 20 min. pulse. During chase incubations of increasing duration, 3H-radioactivity appeared in a glucagon immunoreactive molecule with the approximate molecular size of glucagon and increased with chase time while radioactivity in the 11,400 mol wt tryptophan-labeled molecule decreased. With increasing chase time, the 3H-radioactivity attributable to the 9,000 mol wt tryptophan-labeled molecule initially increased and subsequently decreased which is consistent with the pattern that would be expected for a conversion intermediate. The presence of glucagon immunoreactivity in [3H]tryptophan-labeled molecules having molecular weights near that of proinsulin was established by radioimmunoassay of alternate gel slices following electrophoresis of labeled proteins recovered from the proinsulin containing portions of gel filtration eluates. That [14C]isoleucine became incorporated into insulin and [3H]tryptophan became incorporated into glucagon was established by determination of the distribution of radioactivity in polyacrylamide gels following electrophoresis of labeled proteins recovered from the insulin and glucagon containing portions of gel filtration eluates. These results provide preliminary evidence for sequential metabolic cleavage of proglucagon in glucagon biosynthesis.

Characterization of an islet carboxypeptidase B involved in prohormone processing.

An islet carboxypeptidase B-like enzyme (CP B) has been identified and characterized in secretory granules of anglerfish islets. By employing several different column chromatography methods (gel filtration, ion exchange, and hydroxylapatite), it was determined that the islet secretory granules contained only one detectable CP B. This enzyme is present in both secretory granule- and microsome-enriched subcellular fractions and is membrane associated at pH 5.2. The specific activity of the islet CP B was approximately 4-fold higher in the secretory granule- and microsome-enriched subcellular fractions than in the lysosome-enriched fraction. It is a metallo-enzyme that is stimulated by Co++, and has a pH optimum in the range of 5.2-6.2. The isoelectric point of the islet CP B is at pH 4.9. The enzyme is a glycoprotein and has an approximate molecular size of Mr 30,000 by gel filtration. The substrate analogs guanidinoethylmercaptosuccinic acid, guanidinopropylsuccinic acid, and aminopropylmercaptosuccinic acid competitively inhibited the islet CP B with inhibition constant (Ki) values of 23, 21, and 230 nM, respectively. In experiments employing purified prohormone substrates it was demonstrated that the action of a CP B-like enzyme was required for the complete processing of anglerfish proinsulin and prosomatostatin-II. These results indicate that the anglerfish islet CP B is involved in prohormone processing and has properties which are very similar to those of enkephalin convertase.

The anglerfish somatostatin-28-generating propeptide converting enzyme is an aspartyl protease.

An enzyme that performs the conversion of anglerfish prosomatostatin-II (pro-SS-II) to anglerfish SS-28 has been identified using an improved two-dimensional electrophoresis procedure. The enzyme is a single chain 39 kDa polypeptide with its isoelectric point at pH 5.9. The converting enzyme has an acidic pH optimum, consistent with the lowered pH of the intracellular site of propeptide conversion. Secretory granule extracts were examined to determine the inhibitor sensitivity and pH optimum of the conversion of anglerfish pro-SS-II to anglerfish SS-28 in this organelle. Production of anglerfish SS-28 by secretory granules was maximal at pH 4.2 and was completely inhibited by the addition of pepstatin. Since pepstatin is a specific inhibitor of aspartyl proteases, these results indicate that the purified enzyme is a member of this enzyme family. This conclusion was supported by the data from partial amino acid sequence analysis. Because these results are consistent with the role of the purified enzyme in the in vivo production of anglerfish SS-28, the identified aspartyl protease has been termed the anglerfish SS-28-generating propeptide-converting enzyme.

Identification of a somatostatin-14-generating propeptide converting enzyme as a member of the kex2/furin/PC family.

A propeptide converting enzyme capable of producing somatostatin-14 has been identified and partially characterized from anglerfish pancreatic islets. Results from N-terminal protein sequence analysis indicate that this enzyme is a member of the kex2/furin/PC family. This observation provides strong corroborative evidence that the kex2/furin/PC protease family is involved in propeptide conversion. Comparison of the obtained protein sequence with the cDNA sequence of mammalian PC2 also suggests that the active enzyme is derived from a precursor by cleavage at a site containing four consecutive basic amino acids.

Isolation and characterization of somatostatin from anglerfish pancreatic islet.

Somatostatin was purified from anglerfish pancreatic islets using acetic acid extraction, gel filtration (Bio-Gel P-10), ion exchange chromatography (CM Bio-Gel A), and reversed phase high pressure liquid chromatography. The resulting peptide was characterized by RIA, bioassay, and determination of amino acid composition. Anglerfish islet somatostatin was found to possess an amino acid composition and immunological and biological activities equivalent to synthetic somatostatin. Sequence analyses revealed that the primary structure was H-Ala-Gly-cyclo-[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys]-OH. These results demonstrate that anglerfish islet somatostatin has the same primary structure as somatostatin from all other sources characterized to date.

Somatostatin biosynthesis occurs in pancreatic islets.

No information is at present available on the mode of SRIF biosynthesis. Since anglerfish pancreatic islet tissue is comprised of approximately 30% D cells, we have examined this tissue for SRIF synthesis . The following known differences in amino acid composition of islet peptides were used advantageously in this study: anglerfish proinsulin: Trp-0, Ile-2, Cys-6; anglerfish glucagon: Trp-1, Ile-0, Cys-0; mammalian SRIF: Trp-1, Ile-0, Cys-2. After incubating islet tissue with [3H]tryptophan and [14C]isoleucine or [35S]cystine for various time periods, proteins were extracted in 2 M acetic acid and desalted by Bio-Gel P-2 gel filtration. P-2 void volume proteins were then subjected to P-10 gel filtration and isolated by polyacrylamide gel electrophoresis (PAGE) at alkaline pH. The predominant amount of the immumoreactive SRIF in the extracts appeared in a peak eluting just before the salt volume on P-10 filtration and migrated slowly toward the cathode during PAGE. The behavior of synthetic SRIF was identical. The anglerfish SRIF immunoreactive peptide could be labeled with Trp and Cys but not Ile during incubations longer than 1 h. The Trp- and Cys-labeled peptide could be bound on columns to which the immunoglobulin fraction of antisera to SRIF had been complexed. Cycloheximide inhibited isotope incorporation into all islet proteins. These results indicate that islet SRIF is synthesized in situ. Moreover, the immunological activity, size, and charge characteristics of anglerfish islet SRIF appear to be similar to those of mammalian hypothalamic SRIF. When islets were subjected to short pulse incubations with labeled Trp and Cys, only peptides eluting in the 7,000-13,000 dalton portion of the filtration eluate became labeled. No appreciable isotope incorporation into SRIF was observed. However, when pulse incubations were followed by incubation in the presence of cycloheximide or excess unlabeled amino acids in isotope-free medium (chase), the incorporation of Trp and Cys into SRIF increased with the length of chase, suggesting the participation of a larger precursor in SRIF synthesis.

Biochemical and immunocytochemical characterization of neuropeptide Y in the immature rat ovary.

Neuropeptide Y (NPY)-like immunoreactivity has been found in nerves that innervate the rat ovary. In this study, we used immunohistochemical and biochemical methods to identify NPY in the prepubertal rat ovary. The normal distribution of NPY-containing nerve fibers and the route by which these nerves enter the ovary were analyzed with indirect immunofluorescence techniques. In ovaries with intact nerves, a profuse network of NPY-labeled fibers was observed surrounding blood vessels. Immunoreactive fibers were also seen in the interstitial tissue and coursing between follicles. Occasionally some fibers appeared to enter the follicles. Surgical transection of the superior ovarian nerve had no effect on NPY immunoreactivity; however, transection of the plexus nerve completely eliminated NPY-labeled nerve fibers in all ovarian compartments. The nature of this immunoreactivity was examined in extracts of pooled ovaries that were subjected to reverse phase HPLC and then analyzed by RIA. The major peak of NPY immunoreactivity in each extract eluted at the same time or slightly before synthetic porcine NPY. Two additional peaks of NPY-like immunoreactivity that eluted much earlier than porcine NPY were found in each extract. We conclude that the plexus nerve carries NPY afferents to the ovary and that the ovary contains NPY-like peptides, one of which has a retention time on reverse phase HPLC nearly identical to that of porcine NPY, whereas two others elute with earlier retention times. While the identity and composition of these substances remain to be determined, the presence of peptides that display NPY-like immunoreactivity in the ovary as well as the profuse network of NPY-containing fibers strongly imply a physiological involvement of NPY in the regulation of ovarian function.

Purified yeast aspartic protease 3 cleaves anglerfish pro-somatostatin I and II at di- and monobasic sites to generate somatostatin-14 and -28.

Anglerfish somatostatin-14 (SS-14) and somatostatin-28 (aSS-28) are derived from pro-somatostatin I (aPSS-I) and pro-somatostatin II (PSS-II), respectively. Purified yeast aspartic protease 3 (YAP3), was shown to cleave aPSS-I at the Arg81-Lys82 to yield SS-14 and Lys-1SS-14. In contrast, YAP3 cleaved aPSS-II only at the monobasic residue, Arg73 to yield aSS-28. Since the paired basic and monobasic sites are present in both precursors, the results indicate that the structure and conformation of these substrates dictate where cleavage occurs. Furthermore, the data show that YAP3 has specificity for both monobasic and paired basic residues.

Separate cell types that express two different forms of somatostatin in anglerfish islets can be immunohistochemically differentiated.

The somatostatin-related peptides somatostatin-14 (SS-14) and somatostatin-28 (aSS-28) are synthesized at the C-terminal end of two separate pre-pro-somatostatins in anglerfish pancreatic islets. The purpose of this study was to determine whether these peptides are expressed in the same or different cell types. Antisera R141 and R293, which recognize the central region of SS-14 and the C-terminal region of aSS-28 ([Tyr7,Gly10] SS-14), respectively, were used in an immunohistochemical examination of anglerfish islets. The R293 antiserum-labeled cells were distributed individually or in small clusters. These same cells, as well as a separate set of cells arranged in large clusters, were stained by the R141 antiserum. Pre-absorption of the R141 antiserum with [Tyr7,Gly10] SS-14 eliminated staining by R141 of only those cells also labeled by R293, whereas pre-absorption of R141 with SS-14 prevented all staining. Pre-absorption of R293 with [Tyr7,Gly10] SS-14 eliminated all staining, whereas pre-absorption with SS-14 had no effect on aSS-28-like immunoreactivity. These results suggest the existence of two separate cell types which express either SS-14 or aSS-28. The cells that contained the somatostatin-related peptides were found to be distinct from those cells that contained insulin, glucagon, or anglerfish peptide Y. However, the cells stained by the R293 antiserum were distributed in close association with glucagon-containing cells. The implications of the existence of separate cell types which express SS-14 or aSS-28 are discussed with regard to processing of the biosynthetic precursors to these peptides.

Primary structure and tissue distribution of anglerfish carboxypeptidase H.

Most peptide hormones are synthesized as part of larger precursor proteins which must be processed after translation to generate bioactive peptides. This usually involves cleavage of the precursor by an endopeptidase at sites marked by basic amino acids, followed by removal of N- or C-terminal basic residues by the action of an aminopeptidase or carboxypeptidase. These processing events have been observed in a variety of species, from yeast to mammals. As part of an effort to characterize prohormone processing enzymes in the anglerfish, Lophius americanus, we have cloned and sequenced a cDNA for the fish prohormone processing carboxypeptidase H (CPH). Polyadenylated RNA from anglerfish (AF) islet organs was used to construct a cDNA library in phage lambda gt11. The library was screened with a probe derived from the cDNA for rat CPH. A 2400 base pair AF cDNA clone was isolated. This cDNA encodes a polypeptide which is similar in size and composition to mammalian CPH. The sequence data indicate that the AF CPH precursor is a 454 amino acid polypeptide. The derived amino acid sequence of the putative fish CPH is 81% homologous to the rat and bovine CPH enzymes. Significantly, all of the amino acid residues thought to be important for metal ion and substrate binding, glycosylation, and catalytic activity of mammalian CPH are conserved in the fish enzyme. Northern hybridization using RNA from AF tissues indicates that a 2.5 kb fish CPH mRNA is expressed in brain, pituitary and islet organs, but not in other tissues which do not secrete peptide hormones.

Purification of prosomatostatin-converting enzymes.

The enzymes responsible for performing cleavage of propeptides at basic amino acids have proven difficult to characterize. Using the processing of anglerfish islet prosomatostatin (PSS) as a model system, we are pursuing the characterization of both a single basic amino acid-specific and a dibasic amino acid-specific converting enzyme. We describe here the model system and protein isolation methods that have allowed significant progress toward complete characterization of the somatostatin-generating propeptide converting enzymes (PCEs).

Specific glucagon-related peptides isolated from anglerfish islets are metabolic cleavage products of (pre)proglucagon-II.

Sequence analyses of cDNAs prepared from anglerfish islet mRNA have demonstrated the presence of mRNAs coding for two different preproglucagons, aPPG-I and aPPG-II. Each of these precursors was predicted to contain 29 residue and 34 residue glucagon-related peptides as potential cleavage products. Recently, several glucagon-related peptides found in extracts of anglerfish islets have been isolated and characterized. In order to determine whether any of these peptides could be identified as metabolic cleavage products in anglerfish islets, differentially radiolabeled Mr 2,500-8,000 peptides from islet extracts were subjected to reverse phase HPLC under varying conditions. The potential cleavage products aPPG-II[52-80] and aPPG-II[89-122] could be readily identified among the extract peptides. Both peptides became labeled appropriately (as predicted from their sequences) with 13 different amino acids and demonstrated glucagon-like immunoreactivity in a radioimmunoassay. Conversely, a third peptide (aPPG-II[89-119]) could be found among the labeled products in small amounts only. These results demonstrate that glucagon-II[52-80] and aGLP-II[89-112] are primary cleavage products of aPPG-II and suggest that aGLP-IIc[89-119] may be a peptide generated more slowly by post-translational modification of aGLP-II.