Pancreatic Membranes Research Articles

Pharmacological and biochemical characterization of cholecystokinin/gastrin receptors in developing rat pancreas

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.

Chemical modification of cholecystokinin‐A receptors in rat pancreatic membranes

Chemical modification of amino acids was used to probe the molecular structure of the cholecystokinin-A (CCK-A) receptor on rat pancreatic membranes. Radioligand binding studies with [3H]N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3- yl)1H- 2-carboxamide [(+/-)-[3H]L-364,718], a tritiated highly potent CCK-A receptor antagonist, enabled the evaluation of the effects caused by the modifying reagents. The apparent fragility of the receptor protein necessitated the development of a modification procedure without wash and centrifugation steps. Treatment of a concentrated membrane preparation with the group-specific agents N-ethylmaleimide, phenylglyoxal and diethylpyrocarbonate, subsequent dilution and incubation at lower temperatures (20 degrees C instead of the more generally used 37 degrees C) proved successful. All modifiers affected the binding characteristics for both agonists and antagonists considerably. CCK-A receptor coupling to guanosine-nucleotide-binding proteins was substantially diminished upon modification with N-ethylmaleimide and diethylpyrocarbonate, as could be concluded from the effects on the (+/-)-[3H]L-364,718 displacement by the cholecystokinin C-terminal octapeptide (CCK-8). The ligand-binding site was affected by all three reagents, as could be inferred from the specific protection obtained with the CCK-A receptor antagonist, lorglumide. It therefore appears that sulfhydryl, arginyl, and histidyl residues form an essential part of the ligand-binding domain on the CCK-A receptor and that sulfhydryl and histidyl residues are also involved in the signal-transduction pathway.

Bombesin, neuromedin B and neuromedin C interact with a common rat pancreatic phosphoinositide-coupled receptor, but are differentially regulated by guanine nucleotides

Bombesin (BB), neuromedin C (NMC) and neuromedin B (NMB) stimulated amylase secretion to similar maximum levels, with EC50 values (concentrations causing 50% of maximum effect) of 0.2, 0.3 and 2 nM respectively. Treatment of pancreatic acini with BB or NMB (10 nM) for 30 min resulted in cross-desensitization of secretory responses to subsequent BB and NMB, but not to acetylcholine, which suggests that NMB and BB activate the same receptor. BB, NMC and NMB stimulated production of similar maximum amounts of inositol mono-, bis- and tris-phosphates, with EC50 values of 3, 5 and 141 nM respectively. The bombesin receptor antagonist [Leu13-psi(CH2NH)Leu14]BB inhibited stimulation of amylase secretion and inositol phosphate formation by BB, NMC and NMB. Binding of 125I-labelled gastrin-releasing peptide (GRP; 200 pM) to rat pancreatic membranes at 22 degrees C was inhibited with relative potencies and IC50 (concn. causing 50% of maximal inhibition; nM) as follows: NMC (0.4) = BB (0.5) greater than NMB (1.8 = GRP (2.6). IC50 values for BB, NMC and NMB inhibition of 125I-GRP binding to intact acini were 5-, 19- and 68-fold higher than their respective values in membranes. The guanine nucleotide analogue guanosine 5'-[beta gamma-imido]triphosphate (Gpp[NH]p) produced rightward shifts of NMC and NMB competition curves by 3.5- and 16-fold respectively, but had little effect on the BB and GRP curves. Elevation of the temperature to 37 degrees C or inclusion of NaCl (40 mM) produced quantitatively similar effects to those of Gpp[NH]p. In the presence of both NaCl and Gpp[NH]p the affinities of peptides for membrane receptors were similar to those for intact cells. Modulation of NMB competition curves by Gpp[NH]p was not attenuated by prior treatment of acini with activated pertussis toxin. These results suggest that BB, NMB and NMC stimulate pancreatic secretion by interaction with a common phosphoinositide-linked receptor. Differences in guanine nucleotide regulation suggest that secretagogue-induced receptor-protein interactions may not be identical for NMB and BB.

Characterization of cDNA encoding the mouse hepatic triglyceride lipase and expression by in vitro translation

A cDNA coding for the mouse hepatic triglyceride lipase has been isolated from a mouse liver cDNA library. The nucleotide sequence of the cDNA shows an open reading frame encoding a polypeptide of 510 amino acids that is 91.5% and 86% homologous to rat and human hepatic lipase, respectively. The most drastic protein sequence divergence is found at the carboxyterminal end which was speculated to harbour one heparin-binding site. By in vitro translation of cRNA in the presence of pancreatic membranes the hepatic lipase was shown to be glycosylated and to have an electrophoretic mobility of 53 kDa.

Elevated in vitro translation of a 25-kDa protein in renal cell carcinoma

As a first step in understanding the changes in protein synthesis that occur in renal cell carcinoma, we have prepared poly(A)+ RNA from surgically removed tumors and from their normal tissue counterpart. These RNAs were then translated in vitro in the rabbit reticulocyte lysate system and the synthesized labeled polypeptides were separated by one- and two-dimensional gel electrophoresis. A major 25-kDa primary translation product was observed with all renal cell carcinomas. The synthesis of this protein was barely detectable with the RNA from normal tissue adjacent to the tumor. To determine if this protein could be further processed (removal of signal peptide and (or) core glycosylation), canine pancreatic microsomal membranes were added to the system. This addition resulted in the formation of a vertical row of three additional spots, with the same isoelectric point as the primary translation product and with molecular masses ranging from 27 to 31 kDa. The 31-kDa protein was retained on Concanavalin A. After digestion with endoglycosidase H, it was no longer visible on sodium dodecyl sulfate gels and a new 27-kDa band was generated suggesting that the mature protein was indeed a glycoprotein. Future experiments will be aimed at identifying this protein and examining its potential value as a marker of renal cell carcinoma.

Prostaglandin E2 inhibits secretagogue-induced enzyme secretion from rat pancreatic acini

Prostaglandins of the E type may have a potential role in pancreatic physiology and pathophysiology. Because prostaglandins of the E type inhibit HCl secretion in parietal cells via a specific receptor by inhibition of adenylylcyclase, we studied whether a similar mechanism exists in the exocrine pancreas. Isolated rat pancreatic acini were incubated with various concentrations of secretagogues, such as cholecystokinin-octapeptide (CCK-8), bombesin, carbachol, and vasoactive intestinal peptide (VIP), in the absence or presence of prostaglandin E2 (PGE2), and amylase secretion was measured. For receptor binding studies, acini and pancreatic membranes were incubated with [3H]PGE2 and either unlabeled PGE2 or other types of prostaglandins. PGE2 (10(-13) to 10(-5) M) did not inhibit basal amylase secretion. However, CCK-8-stimulated secretion was significantly inhibited. Stimulation of secretion by bombesin, carbachol, VIP, and secretin was also inhibited by PGE2, but not as pronounced as CCK-8-stimulated secretion. The formation of inositol 1,4,5-trisphosphate induced by CCK-8 was markedly inhibited by simultaneous incubation with PGE2. Furthermore, PGE2 slightly but significantly reduced the CCK-8-induced efflux of 45Ca2+ from prelabeled acini. Intact acini and a membrane fraction bound [3H]PGE2 and this function could be equally competed by either unlabeled PGE2 or PGE1 in contrast to less-related prostaglandins such as PGF2 alpha, PGD2, and prostacyclin. We conclude that prostaglandins of the E type inhibit pancreatic enzyme secretion stimulated by various secretagogues. This function is mediated via specific receptors for PGE. With regard to CCK-8-stimulated secretion this function may be mediated by an inhibition of formation of inositol 1,4,5-trisphosphate.

Isolation of a gastrin releasing peptide receptor from normal rat pancreas.

The presence of a putative GRP receptor on rat pancreatic particulate membranes was demonstrated by covalent cross-linking to 125I-gastrin releasing peptide (GRP), which revealed a radioactive band with Mr = 80-90 kDa on reduced SDS-PAGE. Fresh rat pancreatic membranes contained a GRP receptor which was solubilized with Triton X-100 as assessed by its failure to sediment at 100,000 x g for one hour and its ability to pass through a 0.22 mu filter. When 125I-GRP binding was studied using Sephadex G50 gel filtration chromatography to separate bound from unbound ligand, substantial amounts of 125I-GRP binding were observed in rat crude solubilized pancreatic membranes, but essentially no specific binding was observed until the crude solubilized membranes were fractionated by ammonium sulfate precipitation. Specific 125I-GRP binding was 500, 700 and 1400 fmol/mg protein, respectively, in the 0-25%, 25-50% and 50-80% saturated ammonium sulfate fractions (125I-GRP concentration = 1 nM). Specific binding was temperature dependent, saturable and of high affinity, (KD = 2.3 nM). A unique 70 kDa band was visualized by silver staining of the SDS-PAGE of eluates of GRP(14-27) affinity gel compared with eluates of control affinity gels incubated with the 25-50% (NH4)2SO4 fraction. The lower Mr than that observed with covalent cross-linking may represent the binding subunit of a larger receptor protein. This ligand-affinity isolated protein is thus a good candidate for the GRP receptor, or the binding subunit of it, from normal rat pancreas.

Characterization of binding sites for VIP-related peptides and activation of adenylate cyclase in developing pancreas

HPLC-purified 125I-labeled vasoactive intestinal peptide (VIP) bound in a specific, saturable, and reversible manner to pancreatic plasma membranes isolated from newborn calves, from milk-fed calves at 28 and 119 days, and from weaned calves at 119 days. A series of VIP analogues, including pituitary adenylate cyclase-activating polypeptide (PACAP), displaced 125I-VIP binding and activated adenylate cyclase in the same order of relative potency: PACAP-38 greater than helodermin greater than VIP, PACAP-27 greater than PHM (human peptide with NH2-terminal histidine and COOH-terminal methionine amide). At maximally effective concentrations, these five peptides produced the same two- to threefold increase of adenylate cyclase activity in pancreatic membranes from newborn and 28-day-old calves, and fourfold in ruminant or preruminant animals at 119 days. The activation constant for PACAP-38 ranged from 0.1 to 0.34 nM throughout the postnatal development. Helospectin I and II were three times less potent than VIP in inhibiting 125I-VIP binding. At concentrations up to 0.1 microM, secretin, rat and human growth hormone-releasing factors, glucagon, oxyntomodulin, the truncated form of glucagon-like peptide-1 lacking the 6 NH2-terminal amino acid sequence (TGLP-1), GLP-2, gastric inhibitory peptide, gastrin, CCK, and insulin had no effect on binding. Scatchard plots from 28- and 119-day-old calves were compatible with the presence of two classes of 125I-VIP binding sites: one with a high affinity for VIP and a low binding capacity (Kd = 0.11-0.4 nM, Bmax = 66-174 fmol/mg protein) and the other with a low affinity and high binding capacity. At birth, only one class of binding sites was observed (Kd = 0.4 nM, Bmax = 858 fmol/mg protein). The covalently cross-linked PACAP-preferring 125I-VIP binding site is a glycoprotein of 55 kDa with higher sensitivity to PACAP vs. helodermin and VIP. Our results suggest that calf pancreatic functions might be regulated at an early stage of postnatal development by PACAP receptors linked to cAMP generation.

Characterization of cholecystokinin receptors on guinea pig gastric chief cell membranes

The binding of cholecystokinin (CCK) to its receptors on guinea pig gastric chief cell membranes were characterized by the use of 125I-CCK-octapeptide (CCK8). At 30 °C optimal binding was obtained at acidic pH in the presence of Mg 2+, while Na + reduced the binding. In contrast to reports on pancreatic and brain CCK receptors, scatchard analysis of CCK binding to chief cell membranes revealed two classes of binding sites. Whereas, in the presence of a non-hydrolyzable GTP analog, GTPγS, only a low affinity site of CCK binding was observed. Chief cell receptors recognized CCK analogs, with an order of potency of: CCK8> gastrin-I> CCK4. Although all CCK receptor antagonists tested (dibutyryl cyclic GMP, L-364718 and CR1409) inhibited labeled CCK binding to chief cell membranes, the relative potencies of these antagonists in terms of inhibiting labeled CCK binding were different from those observed in either pancreatic membranes or brain membranes. The results indicate, therefore, that on gastric chief cell membranes there exist specific CCK receptors, which are coupled to G protein. Furthermore, chief cell CCK receptors may be distinct from pancreatic or brain type CCK receptors.

Translocation of the yeast Dolichol-phosphate-mannose synthase into microsomal membranes

Dolichol-phosphate-mannose synthase catalyzes the formation of Dolichol-phosphate-mannose from Dolichol-phosphate and GDP-mannose. Analysis of the primary amino acid sequence of the yeast enzyme predicts a luminal orientation of the enzyme in the endoplasmic reticulum. We analysed the translocation of the Dolichol-phosphate-mannose synthase into dog pancreatic microsomal membranes: resistance to proteolytic attack provides evidence of its luminal orientation and asks for a reevaluation of the topology of the reaction.

Functional and molecular characterization of CCK receptors in the rat pancreatic acinar cell line AR 4-2J

Competitive inhibition binding studies on membranes from the rat pancreatic AR 4-2J cell line revealed the predominance (80%) of low selectivity CCK receptors ( K D of 1 nM and 4 nM for, respectively, CCK-8 and gastrin-17 I (G-17 I)) over selective receptors (20% with a K D of 1 nM and 1 μM for, respectively, CCK-8 and G-17 I). Amylase secretion was stimulated by low concentrations of CCK-8, G-17 I and CCK-4. G-17 I-induced amylase secretion was unaffected by 100 nM of the selective peripheral CCK-A receptor antagonist L-364,718, suggesting that amylase hypersecretion followed non-selective CCK receptor activation, a function normally assumed by selective CCK-A receptors in rat pancreatic acini. Direct ultraviolet irradiation of AR 4-2J cell membranes preloaded with 125I-BH-CCK-33 or 125I(Leu)G(2–17) I resulted in covalent cross-linking with, respectively, a 90 kDa protein and a 106 kDa protein, both distinct from the 81 kDa CCK binding species revealed in normal rat pancreatic membranes. Gpp[NH]p increased the dissociation rate of CCK-8 and G-17 I from AR 4-2J cell membranes, indicating a coupling of receptors with guanyl nucleotide regulatory protein(s) G. [ 32P]ADP-ribosylation of AR 4-2J cell membranes allowed to detect the presence of two G sα (the 50 kDa form predominating over the 45 kDa form) and one G iα (41 kDa). However, G i and G s may not be involved in gastrin stimulation of amylase secretion, as Bordetella pertussis toxin and cholera toxin pretreatment of cells did not suppress G-17 I-dependent amylase secretion.

Relationship between Ca2+-transport and ATP hydrolytic activities in guinea-pig pancreatic acinar plasma membranes

Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca(2+)-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca(2+)-transporter showed high affinity for Ca2+ (KCa2+ = 0.04 +/- 0.01 microM), an apparent requirement for Mg2+ and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca2+ or Mg2+ but showed a low affinity for these cations. At low concentrations, Mg2+ appeared to inhibit the Ca(2+)-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg2+ concentration (0.5-1 mM), a high affinity Ca(2+)-dependent ATPase activity was observed (KCa2+ = 0.08 +/- 0.02 microM). The hydrolytic activity showed little specificity towards ATP. Neither the Ca(2+)-transport nor high affinity Ca(2+)-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca2+ (or Mg2+)-ATPase activity, the presence of both a high affinity Ca(2+)-pump and high affinity Ca(2+)-dependent ATPase. However, the high affinity Ca(2+)-ATPase activity does not appear to be the biochemical expression of the Ca(2+)-pump.

The cell-wall glycoproteins of Chlamydomonas reinhardtii: analysis of the in vitro translation products

An antibody raised against a salt-soluble cell-wall glycoprotein with an apparent molecular weight of 150 kDa cross-reacted with several polypeptide components synthesized by Chlamydomonas reinhardtii wild-type cells and also by the wall-deficient mutant CW-15. These polypeptides were enriched in LiCl extracts from intact wild-type cells but missing in LiCl extracts from the wall-deficient mutant. The same set of wall-related polypeptides was detected in the culture medium of the mutant CW-15 but not in the culture medium of wild-type cells. These findings indicate that the wall-deficient mutant CW-15 is able to synthesize the same set of wall-related polypeptides as the wild-type. Immunochemical analyses of in vitro translation products of poly(A)-containing RNA showed that at least four polypeptides with apparent molecular weights of 94 kDa, 56 kDa, 56 kDa and 31 kDa were specifically precipitated by antibodies raised against individual cell-wall glycoproteins with apparent molecular weights of 150 kDa and 35 kDa, respectively. The same wall-related polypeptides were detected in the in vitro translation products of poly(A)-containing RNA from wild-type and CW-15 cells. The presence of leader peptide which could be cleaved off by dog pancreatic membranes could be demonstrated only for the 94-kDa component. The pattern of wall-related polypeptides detected in the in vitro translation products of poly(A)-containing RNA corresponded to the pattern of chemically deglycosylated salt-soluble cell-wall glycoproteins. These findings indicate that the cell-wall polypeptides of C. reinhardtii are encoded by different mRNA species and that the polypeptide backbones of these cell-wall glycoproteins are structurally related.

Functional and Morphological Changes of the Exocrine Pancreas in Ciclosporin-Treated Rats

We have examined whether or not ciclosporin A (CsA) affects structure and function of the exocrine pancreas. Male Wistar rats were treated once a day for 7 days with intraperitoneal injections of vehicle or 5, 15, 30, or 50 mg/kg of CsA. Following the CsA treatment, incorporations of [3H]thymidine into DNA and [3H]orotic acid into RNA were found to be significantly reduced in a dose-dependent manner up to 30 and 15 mg/kg, respectively, at which doses the incorporations reached a plateau. The incorporation of [3H]-L-leucine into protein also decreased by 36% at the dose of 5 and by 53% at the dose of 50 mg/kg of CsA. The activities of amylase and lipase in the pancreas also decreased in the CsA-treated rats. Furthermore, a significant reduction in the specific binding of [3H]N-methylscopolamine to muscarinic receptor was observed following the administration of CsA. The Scatchard analysis for the [3H]N-methylscopolamine binding to the pancreatic membrane revealed a significant decrease in Bmax, but not in Kd values in the CsA-treated animals. In addition, it was found histologically that administration of CsA induced cellular atrophy, cytoplasmic vacuolization, and cellular necrosis in the exocrine pancreas. These results strongly suggest that prolonged treatment with CsA may induce the suppression of pancreatic exocrine functions by decreasing the contents of amylase and lipase as well as the number of muscarinic receptors, possibly by the inhibition of the syntheses of protein and DNA in the exocrine pancreas.

Secretion of killer toxin encoded on the linear DNA plasmid pGKL1 from Saccharomyces cerevisiae.

By the kar1-mediated cytoduction, linear double-stranded DNA plasmids pGKL1 and pGKL2, encoding killer toxin complex, have been successfully transferred to the recipient strains with about 30% frequency. The killer toxin was found to be secreted through the normal yeast secretory pathway by introducing pGKL plasmids into the several Saccharomyces cerevisiae sec mutants and examining the secretion of killer toxin. S. cerevisiae cells, harboring newly isolated deletion plasmid pGKL1D, expressed only the 28K protein among three killer subunits, and secreted the 28K subunit at a level of zero to 20% efficiency of the cells containing intact pGKL1 plasmid. These data indicated that subunit interaction (cosecretion) of killer proteins is required for the efficient secretion of 28K subunit. The 28K precursor protein was found to translocate across the canine pancreatic endoplasmic reticulum membrane under the direction of its own signal peptide in vitro without any other subunits. From kex2 mutant cells harboring pGKL1 plasmid, the 97K subunit, and its precursor 128K protein were not secreted, however, the 28K subunit was secreted in the same amount as that secreted from KEX2 cells. These lines of evidence suggest that the final assembly of killer toxin complex after KEX2 site of Golgi apparatus is not essential for the secretion of 28K subunit, and therefore, that putative interaction between 128K protein and 28K subunit for the transport between endoplasmic reticulum and Golgi apparatus may be required for the efficient secretion of 28K subunit.

Rat PHI, PHI-GLY and PHV(1–42) stimulate adenylate cyclase in six rat tissue and cell membranes

PHI and the two C-terminally extended forms PHI-GLY and PHV(1–42) coexist in rat tissues. We compared the relative potency and efficacy of these three PHI forms and of VIP to stimulate adenylate cyclase activity and, when feasible, to occupy VIP receptors in six rat tissue and cell membranes. With the exception of lung membranes, all three PHI forms were markedly less potent than VIP but all were systematically as efficacious. PHI-GLY and PHV(1–42) were never more potent than PHI itself and their relative potencies revealed four spectra, depending on the membrane preparation tested: 1) PHI = PHI-GLY = PHV(1–42) in hepatic, pulmonary and pancreatic membranes; 2) PHI > PHV(1–42) = PHI-GLY in membranes from circulating lymphocytes; 3) PHI = PHV(1–42) > PHI-GLY in membranes from the thymocyte cell line 51E; and 4) PHI > PHI-GLY = PHV(1–42) in anterior pituitary membranes. These results indicate that the two naturally observed C-terminal extensions of rat PHI variously affected peptide potency on 6 rat membrane preparations.

Preparation and Characterization of a Fully Active Biotinylated Probe of Cholecystokinin

In this study we describe the synthesis and purification of biotinylated cholecystokinin-8 (Bio-CCK-8) and characterize its use as a probe for the pancreatic cholecystokinin receptor. CCK-8 (0.1 umoles) was reacted with either radiolabeled d-[8,9(-3)H]biotin succinimide ester (0.5 umoles) or N-hydroxysuccinimidyl-biotin in dimethylformamide and triethylamine, and purified by anion exchange chromatography. Concentrations of Bio-CCK-8 and CCK-8 needed for half-maximal inhibition of [125]I-CCK-8 binding to pancreatic membranes were the same (1.0 and 1.3 nM). Bio-CCK-8 retained full biological activity as determined by stimulation of pancreatic protein secretion from rats, and the biotin group bound to CCK-8 retained its high sensitivity for avidin.

Cholecystokinin activates Gi1-, Gi2-, Gi3- and several Gs-proteins in rat pancreatic acinar cells

On separation of rat pancreatic plasma membrane proteins by two-dimensional gel electrophoresis, 15 GTP-binding protein (G-protein) alpha-subunits could be detected immunochemically using an alpha common antibody. These consisted of five 48 kDa proteins (pI 5.70, 5.80, 5.90, 6.10 and 6.25) and five 45 kDa proteins (pI 5.90, 6.05, 6.25, 6.30 and 6.70), presumably corresponding to low- and high-molecular mass forms of the Gs-protein, as well as three 40/41 kDa proteins (pI 5.50, 5.70 and 6.00) and two 39 kDa proteins (pI 5.50 and 6.00). All of these proteins except for the more acidic 39 kDa protein were ADP-ribosylated by cholera toxin (CT). In addition, the three 40/41 kDa proteins and the more alkaline 39 kDa protein were also ADP-ribosylated by pertussis toxin (PT). CT- and PT-induced ADP-ribosylation changed the pI values of G-protein alpha-subunits by 0.2 pI units to more acidic values. Preincubation of isolated pancreatic membranes with cholecystokinin octapeptide (CCK-OP), which stimulates phospholipase C in acinar cells, decreased CT-induced as well as PT-induced ADP-ribosylation of the three 40/41 kDa proteins, whereas CT-induced ADP-ribosylation of one 45 kDa (pI 5.80) and all 48 kDa proteins was enhanced in the presence of CCK. Carbachol, another stimulant of phospholipase C, had no effect. The three 40/41 kDa proteins and one 48 kDa protein could be labelled with the GTP analogue [alpha-32P]GTP-gamma-azidoanilide. CCK, but not carbachol, stimulated incorporation of the GTP analogue into all of these four proteins. Using different anti-peptide antisera specific for alpha-subunits of G-proteins we identified the three 40/41 kDa Gi-proteins as Gi1 (pI 6.00), Gi2 (pI 5.50) and Gi3 (pI 5.70). The Gi3-protein was found to be the major Gi-protein of pancreatic plasma membranes. One of the 39 kDa proteins (pI 6.0) was identified as Go. These results indicate that CCK receptors functionally interact with six Gs-proteins and with Gi1, Gi2 and Gi3-proteins. Since evidence suggests that a 40/41 kDa CT substrate is involved in the stimulation of phospholipase C in pancreatic acinar cells, it is likely that one, two or all three 40/41 kDa Gi-proteins are involved in the coupling of CCK receptors with phospholipase C.

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.

Thyrotropin receptor processing and interaction with thyrotropin

In vitro transcription/translation, using rat thyrotropin receptor cDNA, results in the formation of nonglycosylated proteins able to bind thyrotropin, one of which approximates the 87 Kd size predicted for the receptor. In the presence of canine pancreatic microsomal membranes, putative glycosylation sites are modified as evidenced by digestion with endoglycosidase H. Using a deletion mutant, the presence of a hydrophobic peptide after the initiation signal is established as a signal peptide critical to post translational processing by the canine pancreatic membranes but not to binding thyrotropin.