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Chapter 6 Biosynthesis of Insulin and Glucagon
METHODS IN CELL BIOLOGY, VOLUME
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Chapter 6 Biosynthesis of Insulin und Glucagon HOWARD S. TAGER, DONALD F. STEINER, CHRISTOPH PATZELT
AND
Department of Biorhemistry, University of Chicago, Chicago, Illinois
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Insulin Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Glucagon Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . References
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Introduction
The discovery of proinsulin some 13 years ago (Steiner et al., 1967; Steiner, 1977) initiated an important area of investigation concerning the biosynthesis of peptide hormones via the selective proteolytic conversion of higher-molecularweight precursors. These posttranslational modifications, which result in the cleavage of the hormone from a longer amino acid sequence, occur during the biosynthesis of a great number of bioactive peptides and often yield complex mixtures of hormone-related forms (for reviews, see Tager and Steiner, 1974; Tager e6 a / ., 1975; and Chapter 5 by J . F. Habener et al. and Chapter 8 by E. Herbert et al. in this volume for detailed examples). Although the sites for proteolytic cleavage in many hormone precursors are remarkably similar, the pathway for conversion of each precursor appears to follow a unique course that leads to the production of the correct hormonal product. In many cases, the nonhormonal sequence is secreted along with the hormone product, and measurements of its formation and release have provided important clues regarding endocrine cell function as well as precursor processing and hormone biosynthesis. More recent investigations have demonstrated that proteolytic modification plays an additional role in the biosynthesis of most peptides and proteins destined for secretion (Milstein et al., 1972; Devillers-Thiery et al., 1975; Blobel and 73 Copynghl 0 1981 by Academic Preaa. In' All nghb of reproducuon in any form reqcrved ISBN 0-12 564123-0
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Dobberstein, 1975). An NH,-terminal signal peptide or leader sequence on the prohormone structure participates in the segregation of the precursor from the cytosolic compartment to the lumen of the rough endoplasmic reticulum prior to both its passage to the Golgi and its conversion to final products (Kemper er al., 1976; Chan et al., 1976). Further studies of hormone biosynthesis at the level of nucleic acid structure and function have suggested that the initial mRNA transcripts of many genes-including those for insulin (Lomedico et al., 1979; Duguid and Steiner, 1978; Bell et al., 1980)-are larger molecules, which undergo posttranscriptional processing to their mature forms. Thus, the biosynthesis of many peptide hormones appears to require several stages of macromolecular processing, which involve the participation of both proteases and nucleases. Investigations of peptide hormone biosynthesis in the broadest sense thus rely heavily on the techniques of cell biology, protein chemistry, and nucleic acid chemistry. This contribution will summarize aspects of our understanding of the biosynthesis of the islet cell hormones insulin and glucagon.
11. Insulin Biosynthesis Until 1967, it was widely believed that insulin was formed by the combination of separately synthesized A and B chains. The discovery of proinsulin (Steiner et al., 1967) disproved this hypothesis, however, and it is now known that a connecting peptide attaches the COOH terminus of the B chain to the NH, terminus of the A chain in the single-chain precursor (Steiner et al., 1969; Steiner, 1977). As shown diagrammatically at the top of Fig. 1, the folded proinsulin molecule has correctly formed disulfide bonds both within the A-chain region and between the A-chain and B-chain regions. The C peptide connects the two insulin chains by pairs of dibasic amino acid residues (Lys-Arg and Arg-Arg at the A and B chains, respectively) and forms a bridge between the two portions of the insulin molecule (Steiner et al., 1969; Chance, 1971). Although insulin itself displays a high degree of structural conservation during animal evolution, the C-peptide region shows a great deal of interspecies variability in terms of both its length and its primary structure. The region contains between 21 (dog) and 31 (human, rat, and horse) amino acid residues (Oyer et al., 1971; Tager and Steiner, 1972). Studies on these and other C peptides have shown a high degree of mutation acceptance throughout their sequences (Peterson et al., 1972; Steiner, 1977). Notwithstanding the usual appearance of acidic residues at their NH2termini (forming the partial proinsulin sequence X-Arg-Arg-Glu or X-Arg-Arg-Asp) and the appearance of glutamine residues at their COOH termini (giving the partial sequence Gln-Lys-Arg-Gly), conserved structures are difficult to identify. Since the crystal structure of insulin suggests that the NH2 terminus of the A
6.
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\-AI
& + a000
insulin FIG.1. Diagrammatic scheme for the conversion of proinsulin to insulin. The figure shows the branched pathway by which proinsulin is converted to insulin plus C peptide by trypsin-like (T) and carboxypeptidase B-like (C) enzymes. The dibasic amino acid residues at the conversion sites are illustrated by circles: 0, Arg; Lys.
*,
chain and the COOH terminus of the B chain could be bridged by a connector only 8 h; long (Blundell et al., 1972), in one respect the C peptide seems longer than necessary. Studies showing the efficient oxidation of cysteine residues of both reduced proinsulin (Steiner and Clark, 1968) and model insulin analogs in which LysBZ9and GlyA1are bridged by small chemical linkers (Brandenberg and Wollmer, 1973; Busse et al., 1974) suggest that the C peptide maintains an important role in reducing the folding event to an unimolecular process. As
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previously suggested, however, the “extra” length of the C peptide may permit the nascent peptide to span the large ribosomal subunit and microsomal membrane during the biosynthesis and segregation of the insulin precursor (Patzelt et al., 1978a). The conversion of proinsulin to insulin by limited proteolysis, as illustrated in Fig. 1, has been examined both by pulse-chase, biosynthetic labeling experiments (Steiner et al., 1969; Tager and Steiner, 1979) and by isolation and characterization of presumed biosynthetic intermediates (Steiner et al., 1969; Chance, 1971). Once proinsulin has entered the cisterna of the rough endoplasmic reticulum and correct disulfide bond formation has occurred, the precursor is transferred in an energy-requiring step to the Golgi apparatus and from there to the secretion granule fraction of the /3 cell (Steiner, 1977). Although conversion begins during the formation of these granules, it proceeds, usually to about 95% completion, during secretion granule maturation. Since the order of the proinsulin molecule is given by NH2-B chain-Arg-Arg-C peptide-Lys-Arg-A chainCOOH, and since both pairs of dibasic amino acids must be removed during the formation of insulin and C peptide, two enzyme activities are required for the overall process. The model predicts a trypsin-like enzyme, which would cleave proinsulin either at the connecting region between the B chain and C peptide (Fig. 1, compound 2) or between the C peptide and the A chain (compound 3), and a carboxypeptidase B-like enzyme to remove COOH-terminal basic residues, producing compounds 5 and 7, respectively. Since intermediates related in structure to those illustrated in Fig. 1 have been isolated, it appears that both courses for conversion occur in normal tissue. The further conversion of intermediates 5 and 7 then proceeds by the actions of trypsin-like and carboxypeptidase B-like enzymes on the contralateral sides of the respective molecules, producing insulin, C peptide, and four basic amino acid residues. The C peptide is retained within the secretion granule and is cosecreted with insulin upon appropriate stimulation of the p cell (Rubenstein et al., 1977). Although a mixture of trypsin and carboxypeptidase B is effective in converting proinsulin to insulin plus C peptide in the test tube (Kemmler et al., 1971), the nature of the enzymes actually involved in cellular conversion is not known. Lysed secretion granules disclose the presence of a carboxypeptidase B-like activity (Kemmler ef al., 1973; Zuhlke et al., 1976), but fail to demonstrate clearly a trypsin-like activity. Although cathepsin B has been proposed as the natural endopeptidase, recent studies on proparathyroid hormone have shown that the purified enzyme does not have the appropriate cleavage specificity (MacGregor et al., 1979). Similarly, experiments suggesting glandular kallikrein as the converting enzyme (Ole-Moi et al., 1979) have not yet explored in detail the chemistry of the proposed conversion. The complexity of the conversion process illustrated in Fig. 1 is further heightened, at least for the rat, pig, and human (Tager et al., 1973; Chance, 1971; DeHaen et al., 1978), by the occur-
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rence of a chymotrypsin-like cleavage in the C-peptide region of proinsulin prior to conversion by trypsin- and carboxypeptidase B-like enzymes. In many cases, more than 15 intermediates are either known or can be inferred to participate in the overall process by which proinsulin is converted to insulin. Studies on the translation of insulin mRNA extracted from rat islets in cell-free systems have shown that the product is not proinsulin, but is a larger peptide, which bears a 24-residue, NH,-terminal extension on the prohormone sequence (Chan et al., 1976). This molecule, called preproinsulin, has also been identified during the cell-free translation of insulin mRNA from cattle, anglerfish, sea raven, hagfish, and humans (Lomedico et al., 1977; Shields and Blobel, 1977; Tager et al., 1980a). The NH,-terminal extensions (called both leader sequences and signal peptides) contain a preponderance of hydrophobic amino acids and undoubtedly play a crucial role in the vectorial translocation of peptides destined for secretion to the cisterna of the rough endoplasmic reticulum (see Milstein et al., 1972; Blobel and Dobberstein, 1975; Steiner et al., 1980). After the removal of the leader sequence by as yet unidentified proteolytic enzymes or “signal peptidases, proinsulin is transferred to subsequent cellular compartments and eventually to secretion granules for further processing. Two models for the translocation event have been developed: one early model proposes the passage of the leader sequence into the microsomal lumen (Blobel and Dobberstein, 1975), whereas the other strongly suggests a looping of the sequence through the microsomal membrane so that its NH, terminus remains in the cytosolic compartment (Steiner et al., 1980). In both cases, cleavage of the leader sequence from the secretory protein is generally believed to be a cotranslational rather than a posttranslational event. Thus, proinsulin and proinsulin intermediates represent the vast majority of the early biosynthetic products of the pancreatic p cell. Recent studies using rat islets incubated for very short periods with radioactive amino acids, however, have demonstrated the cellular synthesis of preproinsulin (Patzelt et al., 1978b). Figure 2 illustrates the separation of radioactively labeled islet proteins by SDS slab gel electrophoresis. Although preproinsulin appears as a minor component, this 11,500-dalton peptide (identified by both peptide mapping and sequence determination) is clearly separable from 9000-dalton proinsulin. As expected, the biosynthesis of preproinsulin in isolated islets is highly sensitive to the concentration of extracellular glucose (Fig. 2) and is converted to proinsulin with a half-time of only about 1 minute (Patzelt et al., 1978b). Preproinsulin has also been detected during biosynthetic experiments using catfish islets, where the processing of the presecretory form appears to occur over a more extended period (Albert and Permutt, 1979). Although little is known about the insulin gene at the level of transcription, much structural information has recently accumulated on both the gene and its corresponding mRNA . Insulin mRNA isolated from X-ray-induced @cell tumors of the rat is known to contain a 5’ 7-methylguanosine pyrophosphate or “cap” ”
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FIG.2 . Synthesis of proinsulin and preproinsulin by rat islets. Islets were incubated with r5S]methionine at 25 mM or 2.5 mM glucose for 60 minutes, and the newly synthesized proteins were separated by SDS-polyacrylamide gel electrophoresis. The figure shows a fluorogram of the dried slab gel. Molecular weights of the major proteins are shown in kilodaltons. Preproinsulin, identified by sequence analysis and tryptic mapping, is labeled ppi, and proinsulin is labeled pi.
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structure and a 3’ tail of polyadenylic acid (Duguid et al., 1976). The sequences of most of the coding regions of the mRNAs for the two nonallelic insulin genes of the rat (as determined by analysis of the corresponding complementary DNAs) are also known (Ulbrich et al., 1977; Chan et al., 1979). Determination of the structures of the rat insulin genes (Lomedico et al., 1979) and of the human insulin gene (Bell et al., 1980) have provided important new information: all three of these genes contain an intervening sequence in the 5’ untranslated region, whereas the rat insulin I1 gene and the human insulin gene contain an additional intervening sequence within the C-peptide region. Interestingly, these second intervening sequences occur at the same position in the two species, but the sequence for the human (786 base pairs) is longer than that for the rat (499 base pairs). These sequences predict the existence of larger mRNA precursors, which would mature by excision and ligation to their smaller, translationally effective cytoplasmic forms; such larger forms of insulin mRNA have been detected.in X-ray-induced p-cell tumors of the rat (Duguid and Steiner, 1978). The process of information transfer during the biosynthesis of insulin is presented schematically in Fig. 3 . As outlined, transcription of the gene followed by U
I :
I
I
US B C 1) 11
1 I
)
1 1
, I
1 - 1
C A U ‘
I
I
U S B
C
S B
C
4-1
1-1
B
1-1
C
B
A U
A
A
A
INSULIN GENE
INSULIN mRNA
PREPROINSULIN
PROINSULIN
INSULIN
FIG. 3. Information transfer from the insulin gene to insulin. The uppermost entry illustrates the structure of the human insulin gene as determined by Bell and co-workers (1980). Noncoding regions are shown as lines, and coding regions as heavier bars. Transcription of the gene and maturation of the mRNA precursor leads to excision of the intervening sequences (I) and formation of insulin mRNA. Translation of the mRNA leads to formation of preproinsulin and cotranslational processing to proinsulin. Finally, posttranslational processing leads to the formation of insulin. Letter codes identify the different portions of the gene and its products: U, untranslated region; I , intervening sequence; S , signal peptide region; B, B-chain region; C, C-peptide region; A, A-chain region.
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maturation of the mRNA precursor results in the loss of the intervening sequences. Translation of the mature mRNA on the rough endoplasmic reticulum, accompanied by cotranslational segregation, results in loss of the NH,-terminal leader sequence. Finally, posttranslational conversion of proinsulin to insulin results in the loss of the C peptide. Thus, several stages of macromolecular processing-involving nucleases as well as proteases-are required for the eventual expression of the hormone product. Studies of abnormal, insulin-related proteins have disclosed two probable mutations in the human insulin gene. The first apparently occurs at a DNA sequence coding for one of the dibasic amino acid conversion sites of proinsulin and results in the secretion of a split proinsulin intermediate (Gabbay et al., 1979; Robbins et al., 1981);the second occurs within the B-chain region of the gene and results in the secretion of an abnormal insulin with a leucine for phenylalanine substitution at position B24 (Tager et al., 1979, 1980b). Bell and co-workers (1 980) have also found sequence variability within the 3' untranslated region of the human insulin gene. Although our understanding of gene function is still at an early stage, it is probable that additional mutations in the insulin gene (or in the genes for the processing enzymes) will be found, and determination of their consequences will provide important tools for the further study of insulin biosynthesis.
111. Glucagon Biosynthesis Although studies on pancreatic glucagon biosynthesis were initiated shortly after the discovery of insulin, a clear understanding of the pathway has emerged only recently. A number of investigators have examined the heterogeneity of glucagon-related peptides in islet tissues of both mammals (Rigopoulou et a l . , 1970; Heilerstrom et al., 1972; O'Connor and Lazarus, 1976) and fish (Noe and Bauer, 1971; Traketellis et a l . , 1975), but the sizes and immunological properties of these forms vary considerably (see Tager and Steiner, 1974, for a review). An early indication of proglucagon structure arose from an examination of crystalline glucagon for higher-molecular-weight, hormone-related peptides (Tager and Steiner, 1973). As illustrated in Fig. 4, one such peptide contains the primary structure of glucagon with an eight-residue, COOH-terminal extension. Since the COOH-terminal sequence is connected to the hormone structure by a pair of dibasic amino acid residues (Lys-Arg), both trypsin- and carboxypeptidase B-like enzymes would be necessary to convert this proglucagon fragment to glucagon. The use of trypsin and carboxypeptidase B digestions to probe the structures of glucagon-related peptides is shown schematically in the lower part of Fig. 4. Development of an antiserum specifically directed against the COOHterminal tryptic fragment of glucagon (Tager and Markese, 1979) permits the
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proglucagon fragment
4
NH2 I
glucagon
IArg-Arg
b
1-Lys -A rg -As n-A sn - Lys- A sn - I Ie - A Ia - COOH
I
I
trypsin [I-Lys-Arg
I
carboxypeptidase B
-
0
-
COOH terminal tryptic peptide FIG.4. Structure of a proglucagon fragment. The upper portion of the figure illustrates the structure of a fragment of proglucagon containing the entire sequence of glucagon at its NH, terminus (open bar; arginine residues shown explicitly) and an eight-residue extension at its COOH terminus. The lower portion of the figure shows how sequential digestion of the fragment with trypsin and carboxypeptidase B releases the COOH-terminal tryptic peptide of glucagon. (See text for details.)
radioimmunometric detection of the unlabeled fragment and the immunoprecipitation of the biosynthetically labeled fragment after enzyme digestion of larger forms. These procedures thus allow an immunochemical mapping of glucagonrelated peptides at femtomole to picomole concentrations. Studies of larger glucagon-related peptides using both centrally directed and COOH-terminally directed antisera have revealed a high degree of heterogeneity in tissue forms (Tager and Markese, 1979). Application of both direct immunoassays and immunoassays after trypsin or trypsin plus carboxypeptidase B digestion (see Fig. 4) to gel-filtered pancreatic extracts showed that (1) the tissue contains 12,000- and 8000-dalton peptides that react with centrally directed antiglucagon sera, but not with the antiserum directed against the COOHterminal tryptic fragment of the hormone; (2) a mixture of trypsin plus carboxypeptidase B (but neither enzyme alone) releases the immunoreactive COOH-terminal tryptic fragment of glucagon from both higher-molecular-weight peptides; and (3) the tissue contains, in addition, a 9000-dalton peptide that reacts with both antisera equally well. These studies thus suggested that the 8000- and 12,000-molecular-weight peptides contain the COOH-terminal sequence of glucagon, but that this sequence is extended from its COOH terminus
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by a trypsin-sensitive site. On the other hand, the 9000-molecular-weight peptide appears to lack the COOH-terminal extension. Our conclusions on the structures of the higher-molecular-weight forms of pancreatic glucagon were confirmed in two ways. First polyacrylamide gel electrophoresis showed that digestion of the 9000-dalton peptide with trypsin resulted in a fragment having the electrophoretic mobility of the native COOH-terminal fragment of glucagon, whereas digestion with trypsin plus carboxypeptidase B was necessary to obtain the fragment from the two COOH-terminally extended peptides. Second, we found that the immunoreactive determinant of the 9000dalton peptide was degraded by carboxypeptidase A alone, but that, as predicted from the sequence shown in Fig. 4, a mixture of carboxypeptidases A and B was necessary to degrade the determinant in the 8000- and 12,000-dalton forms. A careful determination of the rates of degradation of the 8000- and 12,000-dalton peptides by the carboxypeptidases further suggested that they bear COOHterminal extensions of both similar length and similar amino acid composition (Tager and Markese, 1979). Thus, the different molecular weights of these two peptides likely result from alterations in their NH,-terminal regions. In all, we have identified five glucagon-containing peptides of pancreas ranging in molecular weight from 12,000 to 3500. Many of these forms have NH,-terminal and/or COOH-terminal extensions on the hormone sequence. Their structures suggest both their probable roles as intermediates in the biosynthesis of glucagon and a likely course by which they might be converted to the hormone in the biosynthetic scheme (Tager et al., 1980b). Studies of radiolabeled amino acid incorporation into islet proteins have often yielded confusing results with regard to glucagon biosynthesis. Investigations using either avian or mammalian islets have resulted in the assignment of peptides ranging in molecular weight from 9000 to >50,000 as the glucagon precursor, and in many cases conversion of the putative precursor to glucagon has been difficult to prove (for reviews, see Tager and Steiner, 1974; Tager, 1980). Nevertheless, in a number of well-designed studies, Noe and his co-workers have identified a 12,000-dalton peptide as anglerfish proglucagon (Noe and Bauer, 1971, 1975) and have proposed a biosynthetic conversion that might progress through both a 9000-dalton intermediate and a short, COOH-terminally extended peptide such as the one illustrated in Fig. 4 (Noe, 1977). Although little information is available on the structures of these peptides, the kinetics of their processing is consistent with their proposed roles, and their molecular weights are similar to those of the major glucagon-related peptides identified in mammalian pancreas. We recently examined the biosynthesis of glucagon in rat pancreatic islets using SDS-polyacrylamide gel electrophoresis to analyze the biosynthetically labeled products. Our methods, which included ( I ) very short periods of pulselabeling followed by longer periods of chase, (2) disintegration of islet tissue
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directly in SDS-containing electrophoresis buffer, (3) fluorography of SDS slab gels for the localization of labeled products, and (4)analysis of peptides by both immunological and chemical means, were designed to achieve the most sensitive results. Figure 5 illustrates the results from an experiment in which rat islets were incubated with [35S]methioninefor 2 minutes (the pulse) and then with unlabeled methionine for the periods indicated (the chase). Although proinsulin remains a major product during these incubations, our attention was directed toward higher-molecular-weight peptides, which had the kinetics of appearance and
FIG. 5 . Pulse-chase study of protein biosynthesis in rat pancreatic islets. Isolated islets were pulsed with [35S]methionine for 2 minutes and were chased with the unlabeled amino acids for the indicated periods. The figure shows a fluorogram of an SDS slab gel used to separate the radioactively labeled products. (See text for further details.)
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disappearance consistent with what would be expected for a peptide hormone precursor; that is, these peptides should be synthesized rapidly and should be processed to smaller forms with a half-life of about 1 hour. Two such peptides were identified. The first has a molecular weight of about 12,500 and was later shown to be prosomatostatin (Patzelt et al., 1980); the second (indicated by the left-hand arrow in Fig. 5) has a molecular weight of about 18,000 and has been identified as the glucagon precursor (Patzelt et a l ., 1979; Tager et al., 1980b). Experiments that identified the 18,000-dalton peptide as proglucagon can be summarized as follows. (1) Of the approximately ten rapidly synthesized islet cell peptides and proteins examined (irrespective of the kinetics of their subsequent processing during the period of chase) only the 18,000-dalton peptide was specifically immunoprecipitated by a variety of antiglucagon sera. (2) Digestion of this peptide with trypsin plus carboxypeptidase B, but not by either enzyme alone, resulted in an immunoprecipitable, methionine-labeled fragment, which had the electrophoretic mobility of the COOH-terminal tryptic fragment of glucagon. (3) Two-dimensional mapping of the phenylalanine-labeled, 18,000-dalton peptide after trypsin digestion showed that it contained the NH,-terminal and central tryptic peptides of pancreatic glucagon. These results, as well as others, suggested that the hormone sequence within the precursor contained both NH2terminal and COOH-terminal extensions and that these extensions were abutted by trypsin-sensitive sites. The proposed structure is thus consistent with the structures of the smaller, glucagon-containing peptides identified in pancreas extracts. Interestingly, the 18,000-molecular-weight precursor appears to undergo a posttranslational modification to a slightly larger form during short periods of chase: both peptides of the resulting 18,000-dalton doublet (see Fig. 5) contain the structure of the hormone (Patzelt et al., 1979; Tager et al., 1980b). Although the nature of this modification remains unknown, failure of either of these precursor forms to be labeled by radioactive sugars or to bind to any of a variety of lectins suggests that the modification is not related to glycosylation. Further studies on the processing of the 18,000-dalton glucagon precursor have suggested the existence of an approximately 13,000-dalton intermediate of conversion (identified by the uppermost, right-hand arrow in Fig. 5) and a 10,000-dalton product (middle right-hand arrow) as well as glucagon itself (lowermost, right-hand arrow). The 10,000-dalton peptide, like glucagon, appears only later in the pulse-chase experiment. Although the 10,000-dalton peptide does not contain the structure of glucagon, peptide mapping studies have shown that it is indeed present in the 18,000-dalton precursor (Patzelt et al., 1979). It thus appears that this peptide represents the NH,-terminal portion of proglucagon and that it remains as a stable product after the conversion of the precursor to the hormone. A tentative scheme for the conversion of proglucagon to glucagon is presented in Fig. 6. The figure illustrates at the top the initial posttranslational modification of proglucagon resulting in an apparent increase in its molecular
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proglucagon l6K
g luca gon
FIG. 6 . Diagrammatic scheme for the conversion of proglucagon to glucagon. The solid bars indicate the sequence of glucagon within the larger forms, and the wavy lines indicate the nonhormonal extensions on that sequence. Approximate molecular weights for the intermediates (in kilodaltons) are shown above the pictorial representations.
weight. The lower portion of the figure illustrates a branched pathway for the conversion based on our biosynthetic studies and on our identification of related peptides in extracts of pancreatic tissue. Whether the 10,000-dalton peptide is cleaved from the precursor or from a subsequent intermediate of conversion is not yet known. Although this question and the firm identification of biosynthetic intermediates in the conversion of proglucagon to glucagon are important matters for further study, the nonterminal position of glucagon within the precursor, the complexity of the conversion process, and the requirements for both trypsin-like and carboxypeptidase B-like enzymes in the mechanism for conversion are now recognized. Tissue heterogeneity of glucagon-related peptides is by no means unique to the pancreas. Glucagon-like immunoreactive peptides are also known to occur throughout the gastrointestinal tract, and it is only recently that their structural relationships to pancreatic glucagon have been clarified. The immunoreactivity of these forms with only selected antiglucagon sera appears to result from the fact
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that they, like some peptides of the pancreas, bear COOH-terminal extensions on the glucagon sequence (Tager and Markese, 1979). In terms of their immuiloreactivity with centrally directed antisera, their immunoreactivity with COOH-terminally directed antisera only after digestion with trypsin and carboxypeptidase B , their molecular weights, and their electrophoretic mobilities, the major glucagon-containing peptides of the intestine are indistinguishable from their 8000- and 12,000-molecular-weightpancreatic counterparts (Tager and Markese, 1979). In addition, a 12,000-dalton glucagon-like peptide of porcine intestine bears a COOH-terminal amino acid sequence identical (except for an apparent inversion) to that of the proglucagon fragment shown in Fig. 4 (Sundby et al., 1976; Jacobsen et al., 1977). Thus, although the biosynthetic intermediates illustrated in Fig. 6 represent only minor components of the glucagon-like peptides of pancreas, they represent the vast majority of the glucagon-like peptides of intestine. These structural studies suggest that the biosynthetic precursors for pancreatic glucagon and for intestinal glucagoncontaining peptides are identical, but that proteolytic modifications resulting in the loss of the COOH-terminal extension are lacking in intestinal tissue (Tager and Markese, 1979; Tager et al., 1980b). Whether or not such tissue-specific processing plays a regulatory role remains to be seen. Nevertheless, the specificity and complexity of macromolecular processing during the biogenesis of glucagon, as during the biogenesis of insulin, suggest the participation of both highly evolved and generally applied biosynthetic mechanisms.
ACKNOWLEDGMENTS Research performed in the authors’ laboratories was supported by Grants AM 18347, AM 13914, and AM 20295 from the National Institutes of Health. H.S.T. is the recipient of Research Career Development Award AM 00145.
REFERENCES Albert, S . G., and Permutt, A. (1979). J . Biol. Chem. 254, 3483-3491. Bell, G. I . , Pictet, R. L., Rutter, W.J., Cordell, B., Tischer, E., and Goodman, H. M. (1980). Nature (London) 284, 26-32. Blobel, G., and Dobberstein, B. (1975). J . Cell Biol. 67, 852-862. Blundell, T., Dodson, G., Hodgkin, D., and Mercola, D. (1972). Adv. Prorein Chem. 26,279-402. Brandenberg, D., and Wollmer, A. (1973). Hopper-Seyler’s Z. Physiol. Chem.354, 613-623. Busse, W . D., Hansen, S. R., and Carpenter, F. H. (1974). J . Am. Chem. Soc. %, 5947-5950. Chan, S. J . , Keim, P., and Steiner, D. F. (1976). Proc. Narl. Acad. Sci. U . S . A . 7 3 , 1964-1968. Chan, S . J . , Noyes, B. E., Agarwal, K. L., and Steiner, D. F. (1979). Proc. Natl. Acad. Sci. U . S . A . 76, 5036-5040. Chance, R. (1971). In “Diabetes” (R. R. Rodriguez and J . Vallance-Owen, eds.), pp. 292-305. Excerpta‘Medica, Amsterdam.
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DeHaen, C . , Little, S. A., May, J. M., and Williams, R. H. (1978). J. Clin. Invest. 62, 727-732. Devillers-Thieny, A . , Kindt, T., Scheele, G . , and Blobel, G . (1975). Proc. Narl. Acad. Sci. U.S.A. 72, 5016-5020. Duguid, J . R., and Steiner, D. F. (1978). Proc. Narl. Acad. Sci. U.S.A. 75, 3249-3253. Duguid, J. R., Steiner, D. F., and Chick, W. L. (1976). Proc. Narl. Acad. Sci. U.S.A. 73, 3539-3543. Gabbay, K . H . , Bergenstal, R. H . , Wolf, J., Mako, M. E . , and Rubenstein, A. H. (1979). Proc. Narl. Acad. Sci. U . S . A . 76, 2881-2885. Hellerstrom, C., Howell, S. L., Edwards, J. C . , Andersson, A., and Ostenson, C.-G. (1974). Biochem. J . 140, 13-23. Jacobsen, H . , Demandt, A., Moody, A. J . , and Sundby, F. (1977). Biochim. Biophys. Acra 493, 452-459. Kemmler, W., Peterson, J. D., and Steiner, D. F. (1971). J. Biol. Chem. 246, 6786-6791. Kemmler, W., Steiner, D. F., and Borg, J . (1973). J. Biol. Chem. 248. 4544-4551. Kemper, B., Habener, J . , Emst, M. D., Potts, J . , and Rich, A. (1976). Biochemistry 15, 15-19. Lomedico, P., Rosenthal, N., Efstratiadis, A , , Gilbert, W., Kolodner, R., and Tizard, R. (1979). Cell 18, 545-558. Lomedico, P. T., Chan, S . J . , Steiner, D. F., and Saunders, G . F. (1977). J. Biol. Chem. 252, 797 1-7978. MacGregor, R. R., Hamilton, J . W.,Kent, G. N., Shofstall, R. E., and Cohn, D. V. (1979). J. Biol. Chem. 254, 4428-4433. Milstein, C . , Brownlee, G . G . , Harrison, T. M., and Mathews, M. B. (1972). Nature (London), New Biol. 239, 117-120. Noe, B. D. (1977). I n “Glucagon: Its Role in Physiology and Clinical Medicine” (P. P. Foa, J. S. Bajaj, and W. L. Foa, eds.), pp. 31-41. Springer-Verlag, Berlin and New York. Noe, B. D., and Bauer, G. E. (1971). Endocrinology (Baltimore) 89, 642-651. Noe, B. D.. and Bauer, G. E. (1975). Endocrinology (Balrimore) 97, 868-877. O’Connor, K. J . , and Lazarus, N. R. (1976). Biochem. J. 156, 265-277. Ole-Moi, Y. 0.. Pinkus, G. S., Spragg, J . , and Austen, K . F. (1979). N . Engl. J . Med. 300, 1289- 1294. Oyer, P. E., Cho, S., Peterson, J. D., and Steiner, D. F. (1971). J. Biol. Chem. 246, 1375-1386. Patzelt, C., Chan, S. J . , Duguid, J . , Horton, G., Keim, P., Heinrikson, R. L., and Steiner, D. F. (1978a). I n “Regulatory Proteolytic Enzymes and Their Inhibitors” (S. Magnusson, ed.), pp. 69-78. Pergamon, Oxford. Patzelt, C., Labrecque, A. D., Duguid, J . R., Carroll, R. J . , Keim, P. S., Heinrikson, R. L., and Steiner, D. F. (1978b). Proc. Narl. Acad. Sci. U . S . A . 75, 1260-1264. Patzelt, C . , Tager, H. S., Carroll, R. J . , and Steiner, D. F. (1979). Nature (London) 282,260-266. Patzelt, C . , Tager, H. S . , Carroll, R. J., and Steiner, D. F. (1980). Proc. Narl. Acad. Sci. U.S.A. 77, 2410-2414. Peterson, J . D., Nerlich, S., Oyer, P. E., and Steiner, D. F. (1972). J . Biol. Chem. 247,4866-4871. Rigopoulou, D., Valverde, 1. Marco, J . , Faloona, G . , and Unger, R. H. (1970). J. B i d . Chem. 245, 496-501. Robbins, D. C., Blix, P. M., Rubenstein, A. H . , Kanazawa, Y., Kosaka, K., and Tager, H. S. (1981). Narure (London) 291, 679-681. Rubenstein, A. H . , Steiner, D. F., Horwitz, D. L., Mako, M. E., Block, M. B., Starr, J . I . , Kuzuya, H . , and Melani, F. (1977). Recent f r o g . Horm. Res. 33, 435-468. Shields, D., and Blobel, G. (1977). Proc. Narl. Acad. Sci. U.S.A. 74, 2059-2063 Steiner, D. F. (1977). Diaberes 26, 322-340. Steiner, D. F . , and Clark, J. L. (1968). Proc. Narl. Acad. Sci. U.S.A. 60, 622-629. Steiner, D. F., Cunningham, D. D., Spigelman, S., and Aten, B. (1967). Science 157, 697-700.
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Steiner, D . F., Clark, J . L., Nolan, C . , Rubenstein, A. H., Margoliash, E., Aten, B., and Oyer, P. E. (1969). Recent Prog. Horm. Res. 25, 207-282. Steiner, D. F., Quinn, P. S., Patzelt, C., Chan, S. J.. Marsh, J., and Tager, H. S. (1980). In “Cell Biology: A Comprehensive Treatise” (L. Goldstein and D. M. Prescott, eds.), Vol. 4 , pp. 175-201. Academic Press, New York. Sundby, F., Jacobson, H . , and Moody, A. J . (1976). Horm. Metab. Res. 8, 366-371. Tager, H . S. (1980). In “Glucagon: Physiology, Pathophysiology and Morphology of the Pancreatic A-Cells” (R. H. Unger and L. Orci, eds.), pp. 39-54. American Elsevier, New York. Tager, H. S., and Markese, J . (1979). J . B i d . Chem. 254, 2229-2233. Tager, H. S . , and Steiner, D. F. (1972). J . B i d . Chem. 247, 7936-7940. Tager, H. S . , and Steiner, D. F. (1973). Proc. Natl. Acad. Sci. U.S.A. 70, 2321-2325. Tager, H. S . , and Steiner, D . F. (1974). Annu. Rev. Biochem. 43, 509-538. Tager, H . S . , Emdin, S. O., Clark, J . L., and Steiner, D. F. (1973). J . B i d . Chem. 248,3476-3482. Tager, H . S . , Rubenstein, A. H., and Steiner, D. F. (1975). In “Methods in Enzymology” (B. W. O’Malley and J . G. Hardman, eds.), Vol. 37, Part B, pp. 326-345. Academic Press, New York. Tager, H. S., Given, B., Baldwin, D., Mako, M., Rubenstein, A,, Olefsky, J., Kolterman, O., Kobayashi, M., and Poucher, R. (1979). Nature (London) 281, 122-125. Tager, H. S . , Patzelt, C . , Assoian, R. K., Chan, S. J., Duguid, J . R., and Steiner, D. F. (1980a). Ann. N . Y . Acad. Sci. 343, 133-147. Tager. H. S . , Thomas, N., Assoian, R., Rubenstein, A , , Olefsky, J., and Kaiser, E. T. (1980b). Proc. Natl. Acad. Sci. U.S.A. 77, 3181-3185. Trakatellis, A. C., Tada, K., Yamaji, K . , and Gondiki-kouidov, P. (1975). Biochemistry 14, 1508- I51 2. Ulbrich, A., Shine, J., Chirguin, J., Pictet, R., Tischer, E., Rutter, W. J., and Goodman, H. M. (1977). Science 1%, 1313-1319. Zuhlke, H., Steiner, D. F., Lernmark, di., and Lipsey, C. (1976). Ciba Found. Symp. [N.S.] 41, 183- 195.
23
Chapter 6 Biosynthesis of Insulin und Glucagon HOWARD S. TAGER, DONALD F. STEINER, CHRISTOPH PATZELT
AND
Department of Biorhemistry, University of Chicago, Chicago, Illinois
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Insulin Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Glucagon Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . References
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
73 74 80 86
Introduction
The discovery of proinsulin some 13 years ago (Steiner et al., 1967; Steiner, 1977) initiated an important area of investigation concerning the biosynthesis of peptide hormones via the selective proteolytic conversion of higher-molecularweight precursors. These posttranslational modifications, which result in the cleavage of the hormone from a longer amino acid sequence, occur during the biosynthesis of a great number of bioactive peptides and often yield complex mixtures of hormone-related forms (for reviews, see Tager and Steiner, 1974; Tager e6 a / ., 1975; and Chapter 5 by J . F. Habener et al. and Chapter 8 by E. Herbert et al. in this volume for detailed examples). Although the sites for proteolytic cleavage in many hormone precursors are remarkably similar, the pathway for conversion of each precursor appears to follow a unique course that leads to the production of the correct hormonal product. In many cases, the nonhormonal sequence is secreted along with the hormone product, and measurements of its formation and release have provided important clues regarding endocrine cell function as well as precursor processing and hormone biosynthesis. More recent investigations have demonstrated that proteolytic modification plays an additional role in the biosynthesis of most peptides and proteins destined for secretion (Milstein et al., 1972; Devillers-Thiery et al., 1975; Blobel and 73 Copynghl 0 1981 by Academic Preaa. In' All nghb of reproducuon in any form reqcrved ISBN 0-12 564123-0
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Dobberstein, 1975). An NH,-terminal signal peptide or leader sequence on the prohormone structure participates in the segregation of the precursor from the cytosolic compartment to the lumen of the rough endoplasmic reticulum prior to both its passage to the Golgi and its conversion to final products (Kemper er al., 1976; Chan et al., 1976). Further studies of hormone biosynthesis at the level of nucleic acid structure and function have suggested that the initial mRNA transcripts of many genes-including those for insulin (Lomedico et al., 1979; Duguid and Steiner, 1978; Bell et al., 1980)-are larger molecules, which undergo posttranscriptional processing to their mature forms. Thus, the biosynthesis of many peptide hormones appears to require several stages of macromolecular processing, which involve the participation of both proteases and nucleases. Investigations of peptide hormone biosynthesis in the broadest sense thus rely heavily on the techniques of cell biology, protein chemistry, and nucleic acid chemistry. This contribution will summarize aspects of our understanding of the biosynthesis of the islet cell hormones insulin and glucagon.
11. Insulin Biosynthesis Until 1967, it was widely believed that insulin was formed by the combination of separately synthesized A and B chains. The discovery of proinsulin (Steiner et al., 1967) disproved this hypothesis, however, and it is now known that a connecting peptide attaches the COOH terminus of the B chain to the NH, terminus of the A chain in the single-chain precursor (Steiner et al., 1969; Steiner, 1977). As shown diagrammatically at the top of Fig. 1, the folded proinsulin molecule has correctly formed disulfide bonds both within the A-chain region and between the A-chain and B-chain regions. The C peptide connects the two insulin chains by pairs of dibasic amino acid residues (Lys-Arg and Arg-Arg at the A and B chains, respectively) and forms a bridge between the two portions of the insulin molecule (Steiner et al., 1969; Chance, 1971). Although insulin itself displays a high degree of structural conservation during animal evolution, the C-peptide region shows a great deal of interspecies variability in terms of both its length and its primary structure. The region contains between 21 (dog) and 31 (human, rat, and horse) amino acid residues (Oyer et al., 1971; Tager and Steiner, 1972). Studies on these and other C peptides have shown a high degree of mutation acceptance throughout their sequences (Peterson et al., 1972; Steiner, 1977). Notwithstanding the usual appearance of acidic residues at their NH2termini (forming the partial proinsulin sequence X-Arg-Arg-Glu or X-Arg-Arg-Asp) and the appearance of glutamine residues at their COOH termini (giving the partial sequence Gln-Lys-Arg-Gly), conserved structures are difficult to identify. Since the crystal structure of insulin suggests that the NH2 terminus of the A
6.
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75
\-AI
& + a000
insulin FIG.1. Diagrammatic scheme for the conversion of proinsulin to insulin. The figure shows the branched pathway by which proinsulin is converted to insulin plus C peptide by trypsin-like (T) and carboxypeptidase B-like (C) enzymes. The dibasic amino acid residues at the conversion sites are illustrated by circles: 0, Arg; Lys.
*,
chain and the COOH terminus of the B chain could be bridged by a connector only 8 h; long (Blundell et al., 1972), in one respect the C peptide seems longer than necessary. Studies showing the efficient oxidation of cysteine residues of both reduced proinsulin (Steiner and Clark, 1968) and model insulin analogs in which LysBZ9and GlyA1are bridged by small chemical linkers (Brandenberg and Wollmer, 1973; Busse et al., 1974) suggest that the C peptide maintains an important role in reducing the folding event to an unimolecular process. As
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previously suggested, however, the “extra” length of the C peptide may permit the nascent peptide to span the large ribosomal subunit and microsomal membrane during the biosynthesis and segregation of the insulin precursor (Patzelt et al., 1978a). The conversion of proinsulin to insulin by limited proteolysis, as illustrated in Fig. 1, has been examined both by pulse-chase, biosynthetic labeling experiments (Steiner et al., 1969; Tager and Steiner, 1979) and by isolation and characterization of presumed biosynthetic intermediates (Steiner et al., 1969; Chance, 1971). Once proinsulin has entered the cisterna of the rough endoplasmic reticulum and correct disulfide bond formation has occurred, the precursor is transferred in an energy-requiring step to the Golgi apparatus and from there to the secretion granule fraction of the /3 cell (Steiner, 1977). Although conversion begins during the formation of these granules, it proceeds, usually to about 95% completion, during secretion granule maturation. Since the order of the proinsulin molecule is given by NH2-B chain-Arg-Arg-C peptide-Lys-Arg-A chainCOOH, and since both pairs of dibasic amino acids must be removed during the formation of insulin and C peptide, two enzyme activities are required for the overall process. The model predicts a trypsin-like enzyme, which would cleave proinsulin either at the connecting region between the B chain and C peptide (Fig. 1, compound 2) or between the C peptide and the A chain (compound 3), and a carboxypeptidase B-like enzyme to remove COOH-terminal basic residues, producing compounds 5 and 7, respectively. Since intermediates related in structure to those illustrated in Fig. 1 have been isolated, it appears that both courses for conversion occur in normal tissue. The further conversion of intermediates 5 and 7 then proceeds by the actions of trypsin-like and carboxypeptidase B-like enzymes on the contralateral sides of the respective molecules, producing insulin, C peptide, and four basic amino acid residues. The C peptide is retained within the secretion granule and is cosecreted with insulin upon appropriate stimulation of the p cell (Rubenstein et al., 1977). Although a mixture of trypsin and carboxypeptidase B is effective in converting proinsulin to insulin plus C peptide in the test tube (Kemmler et al., 1971), the nature of the enzymes actually involved in cellular conversion is not known. Lysed secretion granules disclose the presence of a carboxypeptidase B-like activity (Kemmler ef al., 1973; Zuhlke et al., 1976), but fail to demonstrate clearly a trypsin-like activity. Although cathepsin B has been proposed as the natural endopeptidase, recent studies on proparathyroid hormone have shown that the purified enzyme does not have the appropriate cleavage specificity (MacGregor et al., 1979). Similarly, experiments suggesting glandular kallikrein as the converting enzyme (Ole-Moi et al., 1979) have not yet explored in detail the chemistry of the proposed conversion. The complexity of the conversion process illustrated in Fig. 1 is further heightened, at least for the rat, pig, and human (Tager et al., 1973; Chance, 1971; DeHaen et al., 1978), by the occur-
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rence of a chymotrypsin-like cleavage in the C-peptide region of proinsulin prior to conversion by trypsin- and carboxypeptidase B-like enzymes. In many cases, more than 15 intermediates are either known or can be inferred to participate in the overall process by which proinsulin is converted to insulin. Studies on the translation of insulin mRNA extracted from rat islets in cell-free systems have shown that the product is not proinsulin, but is a larger peptide, which bears a 24-residue, NH,-terminal extension on the prohormone sequence (Chan et al., 1976). This molecule, called preproinsulin, has also been identified during the cell-free translation of insulin mRNA from cattle, anglerfish, sea raven, hagfish, and humans (Lomedico et al., 1977; Shields and Blobel, 1977; Tager et al., 1980a). The NH,-terminal extensions (called both leader sequences and signal peptides) contain a preponderance of hydrophobic amino acids and undoubtedly play a crucial role in the vectorial translocation of peptides destined for secretion to the cisterna of the rough endoplasmic reticulum (see Milstein et al., 1972; Blobel and Dobberstein, 1975; Steiner et al., 1980). After the removal of the leader sequence by as yet unidentified proteolytic enzymes or “signal peptidases, proinsulin is transferred to subsequent cellular compartments and eventually to secretion granules for further processing. Two models for the translocation event have been developed: one early model proposes the passage of the leader sequence into the microsomal lumen (Blobel and Dobberstein, 1975), whereas the other strongly suggests a looping of the sequence through the microsomal membrane so that its NH, terminus remains in the cytosolic compartment (Steiner et al., 1980). In both cases, cleavage of the leader sequence from the secretory protein is generally believed to be a cotranslational rather than a posttranslational event. Thus, proinsulin and proinsulin intermediates represent the vast majority of the early biosynthetic products of the pancreatic p cell. Recent studies using rat islets incubated for very short periods with radioactive amino acids, however, have demonstrated the cellular synthesis of preproinsulin (Patzelt et al., 1978b). Figure 2 illustrates the separation of radioactively labeled islet proteins by SDS slab gel electrophoresis. Although preproinsulin appears as a minor component, this 11,500-dalton peptide (identified by both peptide mapping and sequence determination) is clearly separable from 9000-dalton proinsulin. As expected, the biosynthesis of preproinsulin in isolated islets is highly sensitive to the concentration of extracellular glucose (Fig. 2) and is converted to proinsulin with a half-time of only about 1 minute (Patzelt et al., 1978b). Preproinsulin has also been detected during biosynthetic experiments using catfish islets, where the processing of the presecretory form appears to occur over a more extended period (Albert and Permutt, 1979). Although little is known about the insulin gene at the level of transcription, much structural information has recently accumulated on both the gene and its corresponding mRNA . Insulin mRNA isolated from X-ray-induced @cell tumors of the rat is known to contain a 5’ 7-methylguanosine pyrophosphate or “cap” ”
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FIG.2 . Synthesis of proinsulin and preproinsulin by rat islets. Islets were incubated with r5S]methionine at 25 mM or 2.5 mM glucose for 60 minutes, and the newly synthesized proteins were separated by SDS-polyacrylamide gel electrophoresis. The figure shows a fluorogram of the dried slab gel. Molecular weights of the major proteins are shown in kilodaltons. Preproinsulin, identified by sequence analysis and tryptic mapping, is labeled ppi, and proinsulin is labeled pi.
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structure and a 3’ tail of polyadenylic acid (Duguid et al., 1976). The sequences of most of the coding regions of the mRNAs for the two nonallelic insulin genes of the rat (as determined by analysis of the corresponding complementary DNAs) are also known (Ulbrich et al., 1977; Chan et al., 1979). Determination of the structures of the rat insulin genes (Lomedico et al., 1979) and of the human insulin gene (Bell et al., 1980) have provided important new information: all three of these genes contain an intervening sequence in the 5’ untranslated region, whereas the rat insulin I1 gene and the human insulin gene contain an additional intervening sequence within the C-peptide region. Interestingly, these second intervening sequences occur at the same position in the two species, but the sequence for the human (786 base pairs) is longer than that for the rat (499 base pairs). These sequences predict the existence of larger mRNA precursors, which would mature by excision and ligation to their smaller, translationally effective cytoplasmic forms; such larger forms of insulin mRNA have been detected.in X-ray-induced p-cell tumors of the rat (Duguid and Steiner, 1978). The process of information transfer during the biosynthesis of insulin is presented schematically in Fig. 3 . As outlined, transcription of the gene followed by U
I :
I
I
US B C 1) 11
1 I
)
1 1
, I
1 - 1
C A U ‘
I
I
U S B
C
S B
C
4-1
1-1
B
1-1
C
B
A U
A
A
A
INSULIN GENE
INSULIN mRNA
PREPROINSULIN
PROINSULIN
INSULIN
FIG. 3. Information transfer from the insulin gene to insulin. The uppermost entry illustrates the structure of the human insulin gene as determined by Bell and co-workers (1980). Noncoding regions are shown as lines, and coding regions as heavier bars. Transcription of the gene and maturation of the mRNA precursor leads to excision of the intervening sequences (I) and formation of insulin mRNA. Translation of the mRNA leads to formation of preproinsulin and cotranslational processing to proinsulin. Finally, posttranslational processing leads to the formation of insulin. Letter codes identify the different portions of the gene and its products: U, untranslated region; I , intervening sequence; S , signal peptide region; B, B-chain region; C, C-peptide region; A, A-chain region.
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maturation of the mRNA precursor results in the loss of the intervening sequences. Translation of the mature mRNA on the rough endoplasmic reticulum, accompanied by cotranslational segregation, results in loss of the NH,-terminal leader sequence. Finally, posttranslational conversion of proinsulin to insulin results in the loss of the C peptide. Thus, several stages of macromolecular processing-involving nucleases as well as proteases-are required for the eventual expression of the hormone product. Studies of abnormal, insulin-related proteins have disclosed two probable mutations in the human insulin gene. The first apparently occurs at a DNA sequence coding for one of the dibasic amino acid conversion sites of proinsulin and results in the secretion of a split proinsulin intermediate (Gabbay et al., 1979; Robbins et al., 1981);the second occurs within the B-chain region of the gene and results in the secretion of an abnormal insulin with a leucine for phenylalanine substitution at position B24 (Tager et al., 1979, 1980b). Bell and co-workers (1 980) have also found sequence variability within the 3' untranslated region of the human insulin gene. Although our understanding of gene function is still at an early stage, it is probable that additional mutations in the insulin gene (or in the genes for the processing enzymes) will be found, and determination of their consequences will provide important tools for the further study of insulin biosynthesis.
111. Glucagon Biosynthesis Although studies on pancreatic glucagon biosynthesis were initiated shortly after the discovery of insulin, a clear understanding of the pathway has emerged only recently. A number of investigators have examined the heterogeneity of glucagon-related peptides in islet tissues of both mammals (Rigopoulou et a l . , 1970; Heilerstrom et al., 1972; O'Connor and Lazarus, 1976) and fish (Noe and Bauer, 1971; Traketellis et a l . , 1975), but the sizes and immunological properties of these forms vary considerably (see Tager and Steiner, 1974, for a review). An early indication of proglucagon structure arose from an examination of crystalline glucagon for higher-molecular-weight, hormone-related peptides (Tager and Steiner, 1973). As illustrated in Fig. 4, one such peptide contains the primary structure of glucagon with an eight-residue, COOH-terminal extension. Since the COOH-terminal sequence is connected to the hormone structure by a pair of dibasic amino acid residues (Lys-Arg), both trypsin- and carboxypeptidase B-like enzymes would be necessary to convert this proglucagon fragment to glucagon. The use of trypsin and carboxypeptidase B digestions to probe the structures of glucagon-related peptides is shown schematically in the lower part of Fig. 4. Development of an antiserum specifically directed against the COOHterminal tryptic fragment of glucagon (Tager and Markese, 1979) permits the
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proglucagon fragment
4
NH2 I
glucagon
IArg-Arg
b
1-Lys -A rg -As n-A sn - Lys- A sn - I Ie - A Ia - COOH
I
I
trypsin [I-Lys-Arg
I
carboxypeptidase B
-
0
-
COOH terminal tryptic peptide FIG.4. Structure of a proglucagon fragment. The upper portion of the figure illustrates the structure of a fragment of proglucagon containing the entire sequence of glucagon at its NH, terminus (open bar; arginine residues shown explicitly) and an eight-residue extension at its COOH terminus. The lower portion of the figure shows how sequential digestion of the fragment with trypsin and carboxypeptidase B releases the COOH-terminal tryptic peptide of glucagon. (See text for details.)
radioimmunometric detection of the unlabeled fragment and the immunoprecipitation of the biosynthetically labeled fragment after enzyme digestion of larger forms. These procedures thus allow an immunochemical mapping of glucagonrelated peptides at femtomole to picomole concentrations. Studies of larger glucagon-related peptides using both centrally directed and COOH-terminally directed antisera have revealed a high degree of heterogeneity in tissue forms (Tager and Markese, 1979). Application of both direct immunoassays and immunoassays after trypsin or trypsin plus carboxypeptidase B digestion (see Fig. 4) to gel-filtered pancreatic extracts showed that (1) the tissue contains 12,000- and 8000-dalton peptides that react with centrally directed antiglucagon sera, but not with the antiserum directed against the COOHterminal tryptic fragment of the hormone; (2) a mixture of trypsin plus carboxypeptidase B (but neither enzyme alone) releases the immunoreactive COOH-terminal tryptic fragment of glucagon from both higher-molecular-weight peptides; and (3) the tissue contains, in addition, a 9000-dalton peptide that reacts with both antisera equally well. These studies thus suggested that the 8000- and 12,000-molecular-weight peptides contain the COOH-terminal sequence of glucagon, but that this sequence is extended from its COOH terminus
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by a trypsin-sensitive site. On the other hand, the 9000-molecular-weight peptide appears to lack the COOH-terminal extension. Our conclusions on the structures of the higher-molecular-weight forms of pancreatic glucagon were confirmed in two ways. First polyacrylamide gel electrophoresis showed that digestion of the 9000-dalton peptide with trypsin resulted in a fragment having the electrophoretic mobility of the native COOH-terminal fragment of glucagon, whereas digestion with trypsin plus carboxypeptidase B was necessary to obtain the fragment from the two COOH-terminally extended peptides. Second, we found that the immunoreactive determinant of the 9000dalton peptide was degraded by carboxypeptidase A alone, but that, as predicted from the sequence shown in Fig. 4, a mixture of carboxypeptidases A and B was necessary to degrade the determinant in the 8000- and 12,000-dalton forms. A careful determination of the rates of degradation of the 8000- and 12,000-dalton peptides by the carboxypeptidases further suggested that they bear COOHterminal extensions of both similar length and similar amino acid composition (Tager and Markese, 1979). Thus, the different molecular weights of these two peptides likely result from alterations in their NH,-terminal regions. In all, we have identified five glucagon-containing peptides of pancreas ranging in molecular weight from 12,000 to 3500. Many of these forms have NH,-terminal and/or COOH-terminal extensions on the hormone sequence. Their structures suggest both their probable roles as intermediates in the biosynthesis of glucagon and a likely course by which they might be converted to the hormone in the biosynthetic scheme (Tager et al., 1980b). Studies of radiolabeled amino acid incorporation into islet proteins have often yielded confusing results with regard to glucagon biosynthesis. Investigations using either avian or mammalian islets have resulted in the assignment of peptides ranging in molecular weight from 9000 to >50,000 as the glucagon precursor, and in many cases conversion of the putative precursor to glucagon has been difficult to prove (for reviews, see Tager and Steiner, 1974; Tager, 1980). Nevertheless, in a number of well-designed studies, Noe and his co-workers have identified a 12,000-dalton peptide as anglerfish proglucagon (Noe and Bauer, 1971, 1975) and have proposed a biosynthetic conversion that might progress through both a 9000-dalton intermediate and a short, COOH-terminally extended peptide such as the one illustrated in Fig. 4 (Noe, 1977). Although little information is available on the structures of these peptides, the kinetics of their processing is consistent with their proposed roles, and their molecular weights are similar to those of the major glucagon-related peptides identified in mammalian pancreas. We recently examined the biosynthesis of glucagon in rat pancreatic islets using SDS-polyacrylamide gel electrophoresis to analyze the biosynthetically labeled products. Our methods, which included ( I ) very short periods of pulselabeling followed by longer periods of chase, (2) disintegration of islet tissue
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directly in SDS-containing electrophoresis buffer, (3) fluorography of SDS slab gels for the localization of labeled products, and (4)analysis of peptides by both immunological and chemical means, were designed to achieve the most sensitive results. Figure 5 illustrates the results from an experiment in which rat islets were incubated with [35S]methioninefor 2 minutes (the pulse) and then with unlabeled methionine for the periods indicated (the chase). Although proinsulin remains a major product during these incubations, our attention was directed toward higher-molecular-weight peptides, which had the kinetics of appearance and
FIG. 5 . Pulse-chase study of protein biosynthesis in rat pancreatic islets. Isolated islets were pulsed with [35S]methionine for 2 minutes and were chased with the unlabeled amino acids for the indicated periods. The figure shows a fluorogram of an SDS slab gel used to separate the radioactively labeled products. (See text for further details.)
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disappearance consistent with what would be expected for a peptide hormone precursor; that is, these peptides should be synthesized rapidly and should be processed to smaller forms with a half-life of about 1 hour. Two such peptides were identified. The first has a molecular weight of about 12,500 and was later shown to be prosomatostatin (Patzelt et al., 1980); the second (indicated by the left-hand arrow in Fig. 5) has a molecular weight of about 18,000 and has been identified as the glucagon precursor (Patzelt et a l ., 1979; Tager et al., 1980b). Experiments that identified the 18,000-dalton peptide as proglucagon can be summarized as follows. (1) Of the approximately ten rapidly synthesized islet cell peptides and proteins examined (irrespective of the kinetics of their subsequent processing during the period of chase) only the 18,000-dalton peptide was specifically immunoprecipitated by a variety of antiglucagon sera. (2) Digestion of this peptide with trypsin plus carboxypeptidase B, but not by either enzyme alone, resulted in an immunoprecipitable, methionine-labeled fragment, which had the electrophoretic mobility of the COOH-terminal tryptic fragment of glucagon. (3) Two-dimensional mapping of the phenylalanine-labeled, 18,000-dalton peptide after trypsin digestion showed that it contained the NH,-terminal and central tryptic peptides of pancreatic glucagon. These results, as well as others, suggested that the hormone sequence within the precursor contained both NH2terminal and COOH-terminal extensions and that these extensions were abutted by trypsin-sensitive sites. The proposed structure is thus consistent with the structures of the smaller, glucagon-containing peptides identified in pancreas extracts. Interestingly, the 18,000-molecular-weight precursor appears to undergo a posttranslational modification to a slightly larger form during short periods of chase: both peptides of the resulting 18,000-dalton doublet (see Fig. 5) contain the structure of the hormone (Patzelt et al., 1979; Tager et al., 1980b). Although the nature of this modification remains unknown, failure of either of these precursor forms to be labeled by radioactive sugars or to bind to any of a variety of lectins suggests that the modification is not related to glycosylation. Further studies on the processing of the 18,000-dalton glucagon precursor have suggested the existence of an approximately 13,000-dalton intermediate of conversion (identified by the uppermost, right-hand arrow in Fig. 5) and a 10,000-dalton product (middle right-hand arrow) as well as glucagon itself (lowermost, right-hand arrow). The 10,000-dalton peptide, like glucagon, appears only later in the pulse-chase experiment. Although the 10,000-dalton peptide does not contain the structure of glucagon, peptide mapping studies have shown that it is indeed present in the 18,000-dalton precursor (Patzelt et al., 1979). It thus appears that this peptide represents the NH,-terminal portion of proglucagon and that it remains as a stable product after the conversion of the precursor to the hormone. A tentative scheme for the conversion of proglucagon to glucagon is presented in Fig. 6. The figure illustrates at the top the initial posttranslational modification of proglucagon resulting in an apparent increase in its molecular
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proglucagon l6K
g luca gon
FIG. 6 . Diagrammatic scheme for the conversion of proglucagon to glucagon. The solid bars indicate the sequence of glucagon within the larger forms, and the wavy lines indicate the nonhormonal extensions on that sequence. Approximate molecular weights for the intermediates (in kilodaltons) are shown above the pictorial representations.
weight. The lower portion of the figure illustrates a branched pathway for the conversion based on our biosynthetic studies and on our identification of related peptides in extracts of pancreatic tissue. Whether the 10,000-dalton peptide is cleaved from the precursor or from a subsequent intermediate of conversion is not yet known. Although this question and the firm identification of biosynthetic intermediates in the conversion of proglucagon to glucagon are important matters for further study, the nonterminal position of glucagon within the precursor, the complexity of the conversion process, and the requirements for both trypsin-like and carboxypeptidase B-like enzymes in the mechanism for conversion are now recognized. Tissue heterogeneity of glucagon-related peptides is by no means unique to the pancreas. Glucagon-like immunoreactive peptides are also known to occur throughout the gastrointestinal tract, and it is only recently that their structural relationships to pancreatic glucagon have been clarified. The immunoreactivity of these forms with only selected antiglucagon sera appears to result from the fact
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that they, like some peptides of the pancreas, bear COOH-terminal extensions on the glucagon sequence (Tager and Markese, 1979). In terms of their immuiloreactivity with centrally directed antisera, their immunoreactivity with COOH-terminally directed antisera only after digestion with trypsin and carboxypeptidase B , their molecular weights, and their electrophoretic mobilities, the major glucagon-containing peptides of the intestine are indistinguishable from their 8000- and 12,000-molecular-weightpancreatic counterparts (Tager and Markese, 1979). In addition, a 12,000-dalton glucagon-like peptide of porcine intestine bears a COOH-terminal amino acid sequence identical (except for an apparent inversion) to that of the proglucagon fragment shown in Fig. 4 (Sundby et al., 1976; Jacobsen et al., 1977). Thus, although the biosynthetic intermediates illustrated in Fig. 6 represent only minor components of the glucagon-like peptides of pancreas, they represent the vast majority of the glucagon-like peptides of intestine. These structural studies suggest that the biosynthetic precursors for pancreatic glucagon and for intestinal glucagoncontaining peptides are identical, but that proteolytic modifications resulting in the loss of the COOH-terminal extension are lacking in intestinal tissue (Tager and Markese, 1979; Tager et al., 1980b). Whether or not such tissue-specific processing plays a regulatory role remains to be seen. Nevertheless, the specificity and complexity of macromolecular processing during the biogenesis of glucagon, as during the biogenesis of insulin, suggest the participation of both highly evolved and generally applied biosynthetic mechanisms.
ACKNOWLEDGMENTS Research performed in the authors’ laboratories was supported by Grants AM 18347, AM 13914, and AM 20295 from the National Institutes of Health. H.S.T. is the recipient of Research Career Development Award AM 00145.
REFERENCES Albert, S . G., and Permutt, A. (1979). J . Biol. Chem. 254, 3483-3491. Bell, G. I . , Pictet, R. L., Rutter, W.J., Cordell, B., Tischer, E., and Goodman, H. M. (1980). Nature (London) 284, 26-32. Blobel, G., and Dobberstein, B. (1975). J . Cell Biol. 67, 852-862. Blundell, T., Dodson, G., Hodgkin, D., and Mercola, D. (1972). Adv. Prorein Chem. 26,279-402. Brandenberg, D., and Wollmer, A. (1973). Hopper-Seyler’s Z. Physiol. Chem.354, 613-623. Busse, W . D., Hansen, S. R., and Carpenter, F. H. (1974). J . Am. Chem. Soc. %, 5947-5950. Chan, S. J . , Keim, P., and Steiner, D. F. (1976). Proc. Narl. Acad. Sci. U . S . A . 7 3 , 1964-1968. Chan, S . J . , Noyes, B. E., Agarwal, K. L., and Steiner, D. F. (1979). Proc. Natl. Acad. Sci. U . S . A . 76, 5036-5040. Chance, R. (1971). In “Diabetes” (R. R. Rodriguez and J . Vallance-Owen, eds.), pp. 292-305. Excerpta‘Medica, Amsterdam.
6.
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87
DeHaen, C . , Little, S. A., May, J. M., and Williams, R. H. (1978). J. Clin. Invest. 62, 727-732. Devillers-Thieny, A . , Kindt, T., Scheele, G . , and Blobel, G . (1975). Proc. Narl. Acad. Sci. U.S.A. 72, 5016-5020. Duguid, J . R., and Steiner, D. F. (1978). Proc. Narl. Acad. Sci. U.S.A. 75, 3249-3253. Duguid, J. R., Steiner, D. F., and Chick, W. L. (1976). Proc. Narl. Acad. Sci. U.S.A. 73, 3539-3543. Gabbay, K . H . , Bergenstal, R. H . , Wolf, J., Mako, M. E . , and Rubenstein, A. H. (1979). Proc. Narl. Acad. Sci. U . S . A . 76, 2881-2885. Hellerstrom, C., Howell, S. L., Edwards, J. C . , Andersson, A., and Ostenson, C.-G. (1974). Biochem. J . 140, 13-23. Jacobsen, H . , Demandt, A., Moody, A. J . , and Sundby, F. (1977). Biochim. Biophys. Acra 493, 452-459. Kemmler, W., Peterson, J. D., and Steiner, D. F. (1971). J. Biol. Chem. 246, 6786-6791. Kemmler, W., Steiner, D. F., and Borg, J . (1973). J. Biol. Chem. 248. 4544-4551. Kemper, B., Habener, J . , Emst, M. D., Potts, J . , and Rich, A. (1976). Biochemistry 15, 15-19. Lomedico, P., Rosenthal, N., Efstratiadis, A , , Gilbert, W., Kolodner, R., and Tizard, R. (1979). Cell 18, 545-558. Lomedico, P. T., Chan, S . J . , Steiner, D. F., and Saunders, G . F. (1977). J. Biol. Chem. 252, 797 1-7978. MacGregor, R. R., Hamilton, J . W.,Kent, G. N., Shofstall, R. E., and Cohn, D. V. (1979). J. Biol. Chem. 254, 4428-4433. Milstein, C . , Brownlee, G . G . , Harrison, T. M., and Mathews, M. B. (1972). Nature (London), New Biol. 239, 117-120. Noe, B. D. (1977). I n “Glucagon: Its Role in Physiology and Clinical Medicine” (P. P. Foa, J. S. Bajaj, and W. L. Foa, eds.), pp. 31-41. Springer-Verlag, Berlin and New York. Noe, B. D., and Bauer, G. E. (1971). Endocrinology (Baltimore) 89, 642-651. Noe, B. D.. and Bauer, G. E. (1975). Endocrinology (Balrimore) 97, 868-877. O’Connor, K. J . , and Lazarus, N. R. (1976). Biochem. J. 156, 265-277. Ole-Moi, Y. 0.. Pinkus, G. S., Spragg, J . , and Austen, K . F. (1979). N . Engl. J . Med. 300, 1289- 1294. Oyer, P. E., Cho, S., Peterson, J. D., and Steiner, D. F. (1971). J. Biol. Chem. 246, 1375-1386. Patzelt, C., Chan, S. J . , Duguid, J . , Horton, G., Keim, P., Heinrikson, R. L., and Steiner, D. F. (1978a). I n “Regulatory Proteolytic Enzymes and Their Inhibitors” (S. Magnusson, ed.), pp. 69-78. Pergamon, Oxford. Patzelt, C., Labrecque, A. D., Duguid, J . R., Carroll, R. J . , Keim, P. S., Heinrikson, R. L., and Steiner, D. F. (1978b). Proc. Narl. Acad. Sci. U . S . A . 75, 1260-1264. Patzelt, C . , Tager, H. S., Carroll, R. J . , and Steiner, D. F. (1979). Nature (London) 282,260-266. Patzelt, C . , Tager, H. S . , Carroll, R. J., and Steiner, D. F. (1980). Proc. Narl. Acad. Sci. U.S.A. 77, 2410-2414. Peterson, J . D., Nerlich, S., Oyer, P. E., and Steiner, D. F. (1972). J . Biol. Chem. 247,4866-4871. Rigopoulou, D., Valverde, 1. Marco, J . , Faloona, G . , and Unger, R. H. (1970). J. B i d . Chem. 245, 496-501. Robbins, D. C., Blix, P. M., Rubenstein, A. H . , Kanazawa, Y., Kosaka, K., and Tager, H. S. (1981). Narure (London) 291, 679-681. Rubenstein, A. H . , Steiner, D. F., Horwitz, D. L., Mako, M. E., Block, M. B., Starr, J . I . , Kuzuya, H . , and Melani, F. (1977). Recent f r o g . Horm. Res. 33, 435-468. Shields, D., and Blobel, G. (1977). Proc. Narl. Acad. Sci. U.S.A. 74, 2059-2063 Steiner, D. F. (1977). Diaberes 26, 322-340. Steiner, D. F . , and Clark, J. L. (1968). Proc. Narl. Acad. Sci. U.S.A. 60, 622-629. Steiner, D. F., Cunningham, D. D., Spigelman, S., and Aten, B. (1967). Science 157, 697-700.
88
HOWARD S . TAGER
et a!.
Steiner, D . F., Clark, J . L., Nolan, C . , Rubenstein, A. H., Margoliash, E., Aten, B., and Oyer, P. E. (1969). Recent Prog. Horm. Res. 25, 207-282. Steiner, D. F., Quinn, P. S., Patzelt, C., Chan, S. J.. Marsh, J., and Tager, H. S. (1980). In “Cell Biology: A Comprehensive Treatise” (L. Goldstein and D. M. Prescott, eds.), Vol. 4 , pp. 175-201. Academic Press, New York. Sundby, F., Jacobson, H . , and Moody, A. J . (1976). Horm. Metab. Res. 8, 366-371. Tager, H . S. (1980). In “Glucagon: Physiology, Pathophysiology and Morphology of the Pancreatic A-Cells” (R. H. Unger and L. Orci, eds.), pp. 39-54. American Elsevier, New York. Tager, H. S., and Markese, J . (1979). J . B i d . Chem. 254, 2229-2233. Tager, H. S . , and Steiner, D. F. (1972). J . B i d . Chem. 247, 7936-7940. Tager, H. S . , and Steiner, D. F. (1973). Proc. Natl. Acad. Sci. U.S.A. 70, 2321-2325. Tager, H. S . , and Steiner, D . F. (1974). Annu. Rev. Biochem. 43, 509-538. Tager, H . S . , Emdin, S. O., Clark, J . L., and Steiner, D. F. (1973). J . B i d . Chem. 248,3476-3482. Tager, H . S . , Rubenstein, A. H., and Steiner, D. F. (1975). In “Methods in Enzymology” (B. W. O’Malley and J . G. Hardman, eds.), Vol. 37, Part B, pp. 326-345. Academic Press, New York. Tager, H. S., Given, B., Baldwin, D., Mako, M., Rubenstein, A,, Olefsky, J., Kolterman, O., Kobayashi, M., and Poucher, R. (1979). Nature (London) 281, 122-125. Tager, H. S . , Patzelt, C . , Assoian, R. K., Chan, S. J., Duguid, J . R., and Steiner, D. F. (1980a). Ann. N . Y . Acad. Sci. 343, 133-147. Tager. H. S . , Thomas, N., Assoian, R., Rubenstein, A , , Olefsky, J., and Kaiser, E. T. (1980b). Proc. Natl. Acad. Sci. U.S.A. 77, 3181-3185. Trakatellis, A. C., Tada, K., Yamaji, K . , and Gondiki-kouidov, P. (1975). Biochemistry 14, 1508- I51 2. Ulbrich, A., Shine, J., Chirguin, J., Pictet, R., Tischer, E., Rutter, W. J., and Goodman, H. M. (1977). Science 1%, 1313-1319. Zuhlke, H., Steiner, D. F., Lernmark, di., and Lipsey, C. (1976). Ciba Found. Symp. [N.S.] 41, 183- 195.