Enzyme immunoassay of pancreatic glucagon at the picogram level using beta-D-galactosidase as a label.

An enzyme immunoassay of pancreatic glucagon was established by using E. coli beta-D-galactosidease [EC 3.2.1.23] as a marker. In order to increase the sensitivity of the immunoassay, different peptides obtained from glucagon fragments were used to produce the enzyme conjugate and the immunogen. Antiserum N6E raised against C-terminal fragment peptide (15-29) could be diluted to more than 1 : 100,000 in the assay and was highly specific for pancreatic glucagon. The antiserum reacted well with the C-terminal fragment peptide (21-29) as well as another fragment peptide (15-29) and pancreatic glucagon. The enzyme immunoassay using antiserum N6E and fragment peptide (21-29)-enzyme conjugate could detect as little as 1 to 2 pg of glucagon. The mean recovery of glucagon added to serum specimens was 104% and the coefficients of variation were 3.7-14.5% (within assay) and 9.0-18.5% (between assay).     

Production of anti-glucagon sera with a C-terminal fragment of pancreatic glucagon.

The C-terminal region-sepcific anti-glucagon sera were raised in rabbits using as immunogen, and conjugate of BSA and a C-terminal fragment of pancreatic glucagon. The hapten was prepared by trypsin digestion of the glucagon, which was proved to be a 1:3 mixture of glucagon (18--29) and (19--29). Six rabbits were immunized by subcutaneous injection of an emulsion of the conjugate with complete Freund's adjuvant and five of the rabbits produced antibodies to the glucagon (GC-1, GC-2, GC-3, GC-5 and GC-6). For comparison, rabbit antisera were also produced against glucagon polymer (GA-10) and syrupy glucagon fibrils (PGA-2). All these antisera as well as the pancreatic glucagon-specific antiserum 30 K were characterized with dog gut-extract (gut-GLI) and glucagon-related peptide fragments in the radioimmunoassay systems. The assay systems utilized 125 I-monosubstituted pancreatic glucagon as tracer and human mono-component glucagon as standard. All sera of the GC-series crossreacted with the dog gut-extract very weakly and antisera GC-5 and GC-6 exhibited the lowest crossreactivities with the extract, which were shown to be as low as that of 30k. Characterization of the antiserum GC-5 with purified glucagon related fragments indicated that the major antigenic determinant located exactly in the C-terminal region of glucagon. The present results clearly showed high efficiency of the use of the glucagon C-terminal fragment as hepatenic immunogen in obtaining the C-terminal region-specific, i.e., pancreatic glucagon-specific antisera.     

Pharmacological and biochemical characterization of cholecystokinin/gastrin receptors in developing rat pancreas. Age-related expression of distinct receptor glycoforms.

Cholecystokinin/gastrin receptors in the pancreas of newborn (3-day-old) rats are of type A, as in control mature rats, revealed by pharmacological analysis of specific 125I-Bolton-Hunter-reagent-labelled [Thr34,Ahx37]cholecystokinin(31-39) (Ahx, aminohexanoic acid) binding. Also, by 1 day post-partum, pancreatic cholecystokinin receptors were shown to be coupled to guanine-nucleotide-binding regulatory (G) proteins. Scatchard analysis of 125I-Bolton-Hunter-reagent-labelled [Thr34,Ahx37]cholecystokinin(31-39) binding to pancreatic membranes from rats at different times after birth showed a slight increase in the binding capacity of cholecystokinin receptors between days 3 and 14 and a sixfold increase in 21-day-old rats, with no change in receptor affinity during development. SDS/PAGE analysis of pancreatic membranes affinity labelled with the photoactivable ligand 125I-[2-(p-azidosalicylamido)-1,3'-dithiopropionate]-labelled [Thr34,Ahx37]cholecystokinin-(31-39) identified cholecystokinin receptors of 100-135 kDa in 3-day-old rats, 96-130 kDa in 7-day-old rats, 90-125 kDa in 10-day-old rats and 85-100 kDa in 14-day-old and 21-day-old rats, as found in control adult rats. Endo-beta-N-acetylglucosaminidase F treatment yielded a core protein of 42 kDa in all developmental stages. These findings are consistent with an age-related postnatal expression of distinct glycoforms of pancreatic cholecystokinin receptors. Furthermore, it was observed that the period 2-3 weeks after birth, characterized by stabilization of the mass of the cholecystokinin receptor, precedes the dramatic increase in the receptor number.     

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.     

Isolation and chemical characterization of glicentin C-terminal hexapeptide in porcine pancreas

Using a radioimmunoassay specific for porcine glicentin C-terminal hexapeptide, we isolated a peptide from porcine pancreas and characterized it as the C-terminal 64-69 sequence of glicentin: H-Asn-Lys-Asn-Asn-Ile-Ala-OH. The purification steps included gel filtration, ion-exchange chromatography and HPLC. In each step, the recovery of the desired peptide, radioimmunologically estimated from the respective elution profile, was 71.4-91.7%. The final yield of the hexapeptide was 22 micrograms (4.3%) from 800 g pancreas. The pancreatic content of this peptide was estimated to be approximately equimolar to that of pancreatic glucagon. No hexapeptide-like component was detected in porcine intestinal extracts. The data confirmed that the processing of pancreatic proglucagon liberates the C-terminal hexapeptide of the intramolecular glicentin sequence in a tissue-specific manner during the production of glucagon.     

Presence of glucagon-(1-21)-Like immunoreactive substance in the dog small intestinal mucosa

By using an antiserum (K291) specifically directed to the C-terminal of glucagon-(1-21)-peptide, we demonstrated the presence of glucagon-(1-21)-like immunoreactivity (G21-IR) in the dog intestine. G21-IR was found to be widely distributed throughout the small intestine and colon in parallel with the distribution of glucagon-like immunoreactivity (GLI), measured by N-terminal glucagon antiserum (OAL196). The subsequent analyses by gel filtration and three HPLC columns (reverse phase, ion exchange and further reverse phase columns) showed that G21-IR consisted of three main peaks, and the smallest molecular form of G21-IR is identical to glucagon-(1-21)-peptide.     

Glucagon-(19-29), a Ca2+ pump inhibitory peptide, is processed from glucagon in the rat liver plasma membrane by a thiol endopeptidase

Glucagon-(19-29) is 1000-fold more potent that glucagon as an inhibitor of the liver plasma membrane calcium pump, which suggests that this peptide fragment is naturally occurring. Since glucagon-(19-29) is undetectable in plasma, the processing of glucagon into its (19-29) fragment may occur upon interaction of glucagon with its target tissues. The use of a specific radioimmunoassay for glucagon-(19-29) in association with the separation and identification of peptides by high performance liquid chromatography revealed that, upon incubation at 37 degrees C with hepatic plasma membranes, glucagon is processed into its (19-29) C-terminal fragment. The identity of the fragment was confirmed by amino acid sequencing. The processing activity was inhibited by reagents of the thiol group and by 1,10-phenanthroline, suggesting that a thiol endopeptidase containing a catalytically active metal is involved in this processing. Following its production, glucagon-(19-29) was degraded with a half-life of less than 10 s. This degradation was inhibited by bacitracin and by the aminopeptidase inhibitors bestatin and amastatin. When glucagon was incubated with liver plasma membranes in the absence of inhibitors, the accumulation of glucagon-(19-29) reached a maximum at 2 min (1% of initial glucagon), followed by a slow decline. In the presence of bacitracin and bestatin, the amounts of glucagon-(19-29) obtained from glucagon increased continuously, 1 and 2% of glucagon being transformed after 10 and 30 min, respectively. The production of glucagon-(19-29) did not appear to be associated with the binding of glucagon to its receptors, since (i) guanosine 5'-(3-O-thio)triphosphate, a compound which decreases the glucagon-receptor interaction, could not decrease the conversion of glucagon into glucagon-(19-29); (ii) a glucagon analogue which displays a strongly decreased affinity for the hepatic glucagon receptors was processed similarly to glucagon. The conversion also occurs upon incubation with intact hepatoma cells in monolayer culture. These observations suggest that, under physiological conditions, glucagon is processed in liver by cleavage of the Arg17-Arg18 basic doublet, leading to the production of a fragment which is known to display an original biological specificity, namely the modulation of the hepatocyte plasma membrane calcium pump.     

Isolation and characterization of a tumor-derived human protein related to chromogranin A and its in vitro conversion to human pancreastatin-48

A protein with pancreastatin-like immunoreactivity has been isolated and purified from liver metastasis of a patient with insulinoma. NH2-terminal residue analysis, in conjunction with the use of antibodies that are specific for the C-terminal amide peptide of porcine pancreastatin, identified this protein as a 186-amino-acid protein corresponding to human chromogranin A-116-301 (the fragment corresponding to the positions from 116 to 301 of human chromogranin A). Digestion of this protein with trypsin yielded a 48-amino-acid peptide with the retention of full pancreastatin activity. Serum from patient with insulinoma contains a peptide specie(s) that comigrates with the 48-amino-acid pancreastatin, suggesting that this peptide might be a physiologically important circulation form of pancreastatin in humans. A sensitive radioimmunoassay was established using antibody developed against a synthetic 29-amino-acid peptide amide of pancreastatin. Immunocytochemical staining revealed that a major population of human pancreatic islet cells were immunoreactive to the antiserum but with varying intensity of staining. Pancreastatin-like immunoreactivity was not observed in exocrine acinar cells.     

On the effect of xenin and xenin fragments on exocrine pancreas secretion in vivo.

A stimulatory effect on exocrine pancreas secretion could be demonstrated with high concentrations of the 25-amino-acid peptide xenin in non-anesthetized dogs. This peptide has been isolated from gastric mucosa and it is part of a structural coat protein. It has close structural similarities to neurotensin. The longer C-terminal fragments xenin-(13--25) and xenin-(18--25) are essential for the stimulation of exocrine pancreas secretion in vivo. The smaller peptide fragments xenin-(21--25) and xenin-(22--25) failed to stimulate the pancreas as well as the N-terminal peptide fragment xenin-(1--23). The stimulatory effects of xenin may be mediated via neural neurotensin pathways, because neurotensin receptor blockade abolished the stimulatory effect on pancreatic secretion. Cholinergic pathways are not involved, because atropine had no inhibiting effect.     

Immunocytochemical localization of insulin- and glucagonlike peptides in rat salivary glands

An avidin-biotin immunocytochemical technique was used to localize cells containing an insulin- or glucagon-like peptide in the major salivary glands of Sprague-Dawley rats. Cells with insulin-like staining were observed in the intercalated ducts of both the parotid and submandibular glands, but none were found in the sublingual gland. A discrete population of cells with intense glucagon-like immunostaining was associated with the acini of all three major salivary glands. This immunostaining only followed use of a glucagon antiserum with N-terminal specificity and not after incubation of tissues with an anti-glucagon serum having C-terminal specificity. These results suggest that rat salivary glands may contain peptides potentially capable of influencing substrate metabolism. In addition, the present findings indicate that the glucagon-like peptide found in salivary glands has a greater immunocytochemical similarity to glicentin (gut-type glucagon) and/or glucagon precursors than to the 3500 molecular weight pancreatic glucagon.     

Importance of N- and C-terminal regions of gastrin-Gly for preferential binding to high and low affinity gastrin-Gly receptors

G17-Gly has been shown to stimulate the growth of DLD-1 human colon cancer cells in a biphasic manner via high and low affinity receptors. In the current study, the existence of heterogeneous receptor populations for G17-Gly on the HT-29 human colon cancer cell line was investigated. The effect of either N- or C-terminal peptide truncation on receptor binding and cell growth stimulation was also explored. [Leu15]G17-Gly bound to both high (nM) and low (microM) affinity sites on HT-29 cells. The peptide stimulated cell growth in a dose-dependent and biphasic manner with maximal stimulation at 10(-9) M peptide concentration, suggesting that, as in the case of DLD-1 cells, it is the high affinity receptor which is responsible for the growth-promoting effects. In contrast, G17(1-12) stimulated the growth of HT-29 cells in a sigmoidal fashion with an EC50 of 4.6x10(-9) M. Sequential N-terminal truncation of [Leu15]G17-Gly results in decreased binding to the high affinity G17-Gly receptor on DLD-1 cells. [Leu15]G17(11-17)Gly bound to the low affinity G17-Gly receptor with an affinity similar to that of the full sequence peptide but was unable to displace the radioligand from high affinity sites. G17(1-6)-NH2 was unable to displace [3H]G17-Gly from either site. These results suggest that the important residues for binding to the low affinity receptor are in the C-terminal region of the peptide while those required for interaction with the high affinity receptor lie further towards the N-terminus.     

Importance of the C-terminal alpha-helical structure for glucagon's biological activity

The synthetic glucagon analogues [Glu21]glucagon, 2, and [Lys17,18,Glu21]glucagon, 3, were designed using Chou-Fasman calculations for the purpose of enhancing the probability for the formation of a C-terminal amphipathic alpha-helical conformation. Circular dichroism indicates increased alpha-helical content for these analogues in solution relative to glucagon. Analogues 2 and 3 also exhibit a 3-fold and 5-fold increase in receptor binding potency, respectively. The adenylate cyclase stimulating potencies of 2 and 3 relative to glucagon are 2.1 and 7 times greater, respectively. Attempts were made at further alpha-helical enhancement by further substitutions in the 10-13 region of glucagon, as represented by the glucagon analogues [Phe13,Lys17,18 Glu21]glucagon, 4, and [Phe10,13,Lys17,18,Glu21]glucagon, 5. These latter substitutions resulted in lowered receptor binding and adenylate cyclase potencies for 4 and 5 relative to 3 despite increased alpha-helical content in solution as observed by circular dichroism spectroscopy.     

Preproglucagon gene expression in pancreas and intestine diversifies at the level of post-translational processing

Glucagon is a pancreatic hormone of 29 amino acids that regulates carbohydrate metabolism and glicentin is an intestinal peptide of 69 amino acids that contains the sequence of glucagon flanked by peptide extensions at the amino and carboxy termini. The glucagon gene encodes a precursor containing glucagon and two additional, structurally related, glucagon-like peptides separated by an intervening peptide. These peptides are encoded in separate exons. To determine whether the pancreatic and intestinal forms of glucagon arise by alternative RNA and/or protein processing, we used antisera to synthetic glucagon-like peptides and exon-specific, complementary oligonucleotides for analyses of proteins and mRNAs in pancreatic and intestinal extracts. Preproglucagon mRNAs are identical, but different and highly specific peptides are liberated in the two tissues. Immunocytochemistry shows colocalization of glucagon and the two glucagon-like peptides in identical cells. We conclude that diversification of preproglucagon gene expression occurs at the level of cell-specific post-translational processing.     

The novel VIP-like hypothalamic polypeptide PACAP interacts with high affinity receptors in the human neuroblastoma cell line NB-OK

We investigated the ability of two forms of Pituitary Adenylate Cyclase Activating Polypeptide [PACAP-38, the 38 amino acid peptide isolated from ovine hypothalamus, and PACAP-27, a shorter N-terminal (1-27) amidated version] to interact with specific receptors in membranes from the human neuroblastoma cell line NB-OK. [125I]PACAP-27 bound rapidly and specifically to one class of high affinity sites (Kd 0.5 nM). VIP inhibited [125I]PACAP-27 binding 300- to 1000-fold less potently than PACAP-27 and PACAP-38. One microM PHI prevented tracer binding only partially and secretin, glucagon and GRF(1-29)NH2 were ineffective in this respect. PACAP-27 and PACAP-38 stimulated adenylate cyclase activity dose dependently and with similar efficacy (Kact 0.2-0.3 nM), this activation being compatible with the occupancy of specific high affinity PACAP receptor. VIP was markedly less potent and less efficient on this enzyme than PACAP. Chemical cross-linking of [125I]PACAP-27 followed by SDS-PAGE and autoradiography revealed specific cross-linking with a 68 kDa protein.     

Immune recognition of affinity-labelled cholecystokinin receptor

The present study was undertaken to characterize the immune recognition of pancreatic cholecystokinin receptor by an anti-cholecystokinin antibody. Cholecystokinin receptor from pancreatic plasma membranes was photoaffinity labelled using the specific, cleavable probe 125I-labelled 2-(p-azidosalicylamido)-1,3-dithiopropionate-[Thr28,Ahx31 ]CCK(25-33) [CCK(25-33) is the C-terminal nonapeptide of the 33-amino-acid form of cholecystokinin]. Labelled receptor was then solubilized and subsequently prepurified on immobilized wheat-germ agglutinin. The C-terminal-directed anti-cholecystokinin serum (8E) specifically immunoprecipitated a fraction of affinity-labelled cholecystokinin receptor which was identified at Mr 85,000 - 100,000 on SDS/PAGE. The binding affinity of antiserum 8E for covalently labelled cholecystokinin receptor was lower (Kd 0.11 +/- 0.02 nM) than for cholecystokinin (Kd 3.65 +/- 0.55 pM). The compound L364-718, an A-subtype cholecystokinin-receptor antagonist did not interfere with the immune recognition of cholecystokinin. However, the recognition of affinity-labelled cholecystokinin receptor was enhanced as a result of an increasing availability of cholecystokinin molecules. Indeed, the amount of immunoprecipitated receptor was doubled in the presence of 10 microM L364-718. This study offers the possibility of using an anti-cholecystokinin antibody for cholecystokinin-receptor purification and demonstrates that prepurified affinity-labelled cholecystokinin receptor retains A-subtype specificity.     

Fusogenic Alzheimer's peptide fragment Abeta (29-42) in interaction with lipid bilayers: secondary structure, dynamics, and specific interaction with phosphatidyl ethanolamine polar heads as revealed by solid-state NMR

The interaction of the native Alzheimer's peptide C-terminal fragment Abeta (29-42), and two mutants (G33A and G37A) with neutral lipid bilayers made of POPC and POPE in a 9:1 molar ratio was investigated by solid-state NMR. This fragment and the lipid composition were selected because they represent the minimum requirement for the fusogenic activity of the Alzheimer's peptide. The chemical shifts of alanine methyl isotropic carbon were determined by MAS NMR, and they clearly demonstrated that the major form of the peptide equilibrated in membrane is not in a helical conformation. (2)H NMR, performed with acyl chain deuterated POPC, demonstrated that there is no perturbation of the acyl chain's dynamics and of the lipid phase transition temperature. (2)H NMR, performed with alanine methyl-deuterated peptide demonstrated that the peptide itself has a limited mobility below and above the lipid phase transition temperature (molecular order parameter equal to 0.94). MAS (31)P NMR revealed a specific interaction with POPE polar head as seen by the enhancement of POPE phosphorus nuclei T(2) relaxation. All these results are in favor of a beta-sheet oligomeric association of the peptide at the bilayer interface, preferentially recruiting phosphatidyl ethanolamine polar heads.     

Localization of glucagon-like peptide (GLP) immunoreactants in human gut and pancreas using light and electron microscopic immunocytochemistry

The distribution of peptide immunoreactivities predicted from the sequence of the human preproglucagon gene in enteroglucagon (EG; glicentin-like immunoreactant-containing) cells of the human gut and A cells of the pancreas has been determined by light and electron microscopic immunocytochemistry. At light microscopy the application of peroxidase-antiperoxidase and immunogold-silver staining methods has revealed that glucagon-like peptide (GLP-1 and GLP-2) immunoreactivities coexist with a glicentin-related immunodeterminant in human colorectal EG cells and pancreatic A cells. Using single and double colloidal gold probe electron immunocytochemistry, we have been able to show the coexistence of glicentin, GLP-1, and GLP-2 immunoreactivities within single EG cell secretory granules. No morphologic segregation of the proglucagon immunoreactants was observed in EG cells of the colonic mucosa. In pancreatic A cells we have localized GLP-1, GLP-2, and glucagon-[16-29] immunoreactivities solely to the electron-dense core of the secretory granules, whereas glicentin-related immunoreactivity was restricted to the electron-lucent halo. The results obtained in the present study have shown that the peptide immunoreactivities predicted from cDNA sequencing of the human preproglucagon gene are indeed expressed in colorectal EG and pancreatic A cells. The topographical segregation of immunoreactivities in the A cell secretory granule shows that antigenic determinants derived from the C-terminal portion of proglucagon are stored with glucagon in the core of the secretory granule.     

Light and electron microscopical immunocytochemical localization of pancreastatin-like immunoreactivity in porcine tissues

Pancreastatin is a 49 amino acid comprising peptide isolated from porcine pancreas that is derived by proteolytic processing from chromogranin A. Using an antibody against the synthetic C-terminal fragment pancreastatin (33-49), we examined the light and electron microscopical immunocytochemical localization of this peptide in porcine tissues. Pancreastatin-like immunoreactivity (PLI) was found in pancreatic somatostatin-, insulin- and glucagon cells in varying intensities; pancreatic polypeptide cells were always negative. At the electron microscopical (EM) level the immunoreactivity was confined to the electron dense core of the secretory granules in the case of somatostatin and insulin cells or to the less electron dense "halo" of the glucagon granules. In the antrum PLI positive cells represented gastrin (G), somatostatin (D) and enterochromaffin (EC) cells, in the duodenum in addition to EC- and G-cells a small number of PLI positive cells showed a positive immunoreaction for glucagon-like peptide (GLP) I and secretin in serial sections. Both norepinephrine and epinephrine containing cells of the adrenal medulla exhibited a strong reaction for PLI. In the pituitary several cell populations stained with varying intensities, including gonadotrophs and thyrotrophys. PLI is present in a distinct and characteristic subpopulation of neuroendocrine cells in various organs. The subcellular localization may indicate a function in the granular concentration, packaging and storage of peptides and amines in the brain-gut endocrine system.     

Hormonal regulation of Gi2 alpha-subunit phosphorylation in intact hepatocytes.

Hepatocytes contain the Gi2 and Gi3 forms of the 'Gi-family' of guanine-nucleotide-binding proteins (G-proteins), but not Gi1. The anti-peptide antisera AS7 and I3B were shown to immunoprecipitate Gi2 and Gi3 selectively, and the antiserum CS1 immunoprecipitated the stimulatory G-protein Gs. Treatment of intact, 32P-labelled hepatocytes with one of glucagon, TH-glucagon ([1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), Arg-vasopressin, angiotensin-II, the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) and 8-bromo-cyclic AMP elicited a time- and dose-dependent increase in the labelling of the alpha-subunit of immunoprecipitated Gi2 which paralleled the loss of ability of low concentrations of the non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity ('Gi'-function). The immunoprecipitation of phosphorylated Gi-2 alpha-subunit by the antiserum AS7 was blocked in a dose-dependent fashion by the inclusion of the C-terminal decapeptide of transducin, but not that of Gz (a 'Gi-like' G-protein which lacks the C-terminal cysteine group which is ADP-ribosylated by pertussis toxin in other members of the Gi family), in the immunoprecipitation assay. No labelling of the alpha-subunits of either Gi3 or Gs was observed. alpha-Gi2 was labelled in the basal state and this did not change over 15 min in the absence of ligand addition. In contrast to the monophasic dose-effect curves seen with vasopressin, angiotensin and TPA, the dose-effect curve for the glucagon-mediated increase in the labelling of alpha-Gi2 was markedly biphasic where the loss of Gi function paralleled the high-affinity component of the labelling of alpha-Gi2 caused by glucagon. TPA, TH-glucagon, angiotensin-II and vasopressin achieved similar maximal increases in the labelling of alpha-Gi2, which was approximately half that found after treatment of hepatocytes with either high glucagon concentrations (1 microM) or 8-bromocyclic AMP. Analysis of the phosphoamino acid content of immunoprecipitated alpha-Gi2 showed the presence of phosphoserine only. Incubation of hepatocyte membranes with [gamma-32P]ATP and purified protein kinase C, but not protein kinase A, led to the incorporation of label into immunoprecipitated alpha-Gi2. This labelling was abolished if membranes were obtained from cells which had received prior treatment with ligands shown to cause the phosphorylation of alpha-Gi2 in intact cells. We suggest that there are two possible sites for the phosphorylation of alpha-Gi2; one for C-kinase and the other for an unidentified kinase whose action is triggered by A-kinase activation.     

Conformational and biological properties of glucagon fragments containing residues 1-17 and 19-29

The 29 amino acid polypeptide hormone glucagon was cleaved into two large fragments by the enzyme clostripain. The conformational properties of these two fragments were monitored by circular dichroism at pH 2 and 12 in both the presence and absence of sodium dodecyl sulfate. Both glucagon (1-17) and glucagon (19-29) have reduced abilities to fold in aqueous solution. However, both fragments can take on structure of higher apparent helical content in acidic solution in the presence of sodium dodecyl sulfate but only the glucagon (19-29) retains this conformation at high pH. Neither of the two fragments react with dimyristoylphosphatidylcholine as the intact peptide does. Only the carboxyl terminal fragment was capable of reacting with an antibody specific for glucagon. The glucagon (1-17) has markedly reduced affinity for binding to the glucagon receptor as well as markedly reduced ability to stimulate adenylate cyclase activity which is not affected by the presence of glucagon (19-29). It is proposed that the intact sequence provides specific groups required for activity as well as the potential for forming a stable amphipathic helix, both of which are necessary for full biological activity at low peptide concentrations.