Glucagon Biosynthesis in Anglerfish Islets: Identification of A 4,900 Dalton Metabolic Conversion Intermediate

Noe, Bryan D.

doi:10.1007/978-1-4612-6366-1_3

Cited by 4 publications

(13 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Concomitant with the increased radiolabeling of the smaller peptides, there is a diminution of both 3H and`"C radioactivity in the precursor peptides . The elution patterns generated by granule-mediated conversion of both endogenously and exogenously labeled prohormones were indistinguishable from those obtained in extracts of intact tissue subjected to continuous or pulse-chase incubation (10,(13)(14)(15) .…”

Section: Conversion Of Exogenously Labeled Prohormonesmentioning

confidence: 72%

“…Proposed converting enzymes include "trypsinlike" enzymes (5,33,36), cathepsin B (37, 38), pancreatic kallikrein kininase (39), and plasminogen activator-plasmin (40) . The role of enzymes with similar specificity in the conversion of anglerfish islet prohormones has been demonstrated by investigations in which trypsin treatment of anglerfish proglucagon (15,41) and proinsulin (15,42) yielded peptides having some of the characteristics of glucagon and insulin, respectively . However, on the basis of the proteinase inhibitor data presented in this paper and in a previous paper (10), it is concluded that none of the converting enzymes that have been implicated previously is involved in the conversion of anglerfish prohormones by secretory granules .…”

Section: Discussionmentioning

confidence: 99%

“…This accounts for the difference in the pattern shown in Fig . 5 A as compared with those obtained when extraction is in acid ethanol and gel filtration is performed using 1 M acetic acid (14,15) . The enhanced mobility is in accordance with the results of Bromer et al (24), who examined the electrophoretic mobility of deamidated glucagons at high pH using 200Io acrylamide gels .…”

Section: Assessment Of the Accuracy Of Cleavagementioning

confidence: 99%

“…Percent accumulation of glucagon was obtained by calculation of the ratioof 'H radioactivity in the M, 2,500-6,000 portion of the gel filtration eluates to total post-void volume 'H radioactivity. It has been demonstrated previously that 81% of the [3H]tryptophan-induced disintegrations per minute (dpm) eluting in M. 2,500-6,000 portion of the fractionation range can be attributed directly to labeled glucagon, monodesamidoglucagon and a M, 4,900 glucagon immunoreactive peptide presumed to be a conversion intermediate between proglucagon and glucagon (7,15). Total post-void volume 'H dpm were used rather than the 'H dpm that elute at Mr 11,000-13,500, because it is now known that prosomatostatin (M, 13,328), which also contains tryptophan, coelutes with proglucagon under the conditions employed (10,16).…”

Section: Determination Of Percent Conversionmentioning

confidence: 99%

“…Tryptophan is incorporated into anglerfish proglucagon, glucagon, prosomatostatin, and somatostatin but not into proinsulin (10,(13)(14)(15) . Percent accumulation of glucagon was obtained by calculation of the ratioof 'H radioactivity in the M, 2,500-6,000 portion of the gel filtration eluates to total post-void volume 'H radioactivity.…”

Section: Determination Of Percent Conversionmentioning

confidence: 99%

See 4 more Smart Citations

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

Fletcher¹,

Quigley²,

Bauer³

et al. 1981

The Journal of Cell Biology

116

View full text Add to dashboard Cite

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 [14 C]isoleucine or ["S]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 (M r 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 HCI. 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 p,g/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.Although the subcellular conversion of proinsulin to insulin has been the subject of numerous investigations (1-9), the mechanisms ofproglucagon biosynthesis and conversion at the subcellular level have been the focus of only one study. Using anglerfish pancreatic islet, Noe et al. (7) demonstrated that both proglucagon and proinsulin are synthesized in the microsomes and then transported to the secretory granules. The appearance of radiolabeled glucagon and insulin in the micro-

show abstract

Section: Conversion Of Exogenously Labeled Prohormonesmentioning

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Section: Assessment Of the Accuracy Of Cleavagementioning

confidence: 99%

Section: Determination Of Percent Conversionmentioning

confidence: 99%

Section: Determination Of Percent Conversionmentioning

confidence: 99%

See 3 more Smart Citations

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

Fletcher¹,

Quigley²,

Bauer³

et al. 1981

The Journal of Cell Biology

116

View full text Add to dashboard Cite

show abstract

Studies on proinsulin and proglucagon 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

Noe¹,

Baste²,

Bauer³

1977

The Journal of Cell Biology

View full text Add to dashboard Cite

Anglerfish proinsulin and insulin were selectively labeled with [14C]isoleucine, while proglucagon, conversion intermediate(s), and glucagon were selectively labeled with [aH]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 [14C]proinsulin and [3H]proglucagon (mol wt -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 -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 membranebound 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.Results from recent studies designed to ascertain the mode of biosynthesis of glucagon indicate that a biosynthetic precursor is involved (8,(27)(28)(29)(30)(31)(32)(33)47). Evidence from other studies performed over the last 10 yr indicates that many, if not all, peptide hormones are synthesized as peptides larger than the biologically active circulating hormone and are cleaved enzymatically before secre-

show abstract

Somatostatin Biosynthesis Occurs in Pancreatic Islets*

et al. 1978

View full text Add to dashboard Cite

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.

show abstract

Glucagon Biosynthesis in Anglerfish Islets: Identification of A 4,900 Dalton Metabolic Conversion Intermediate

Cited by 4 publications

References 39 publications

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

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

Studies on proinsulin and proglucagon 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

Somatostatin Biosynthesis Occurs in Pancreatic Islets*

Contact Info

Product

Resources

About