Structural requirements of pancreatic polypeptide receptor binding.

Pancreatic polypeptide (PP) receptors have been identified and characterized on the basolateral membranes (BLM) of canine intestinal mucosa. The present study was designed to ascertain the structural requirements of the PP molecule for binding to its receptor. A radioreceptor assay using purified BLM was employed to elucidate receptors specific to PPs of various mammalian species and to modified bovine PP (bPP) fragments. Receptor cross-reactivities (CR) to various PPs and bPP fragments were established. Results show that percent receptor CR by PPs of various species was as follows: bPP (100%) greater than human PP (68%) greater than porcine PP (50%) greater than canine PP (45%) greater than ovine PP (36%) greater than rat PP (3%). The fragments bPP-(1-15), bPP-(1-17), bPP-(1-26), bPP-(16-23), bPP-(18-30), bPP-(24-36), bPP-(27-35), and bPP-(31-36) at 500 nM did not significantly displace tracer from receptor (less than 0.1% CR). Des-COOH-terminal tyrosinamide [bPP-(1-35)] produced less than 0.1% CR. Oxidation of bPP methionine-30 residue to methionine sulfoxide decreased displacement to 67%. Modification of native amidated tyrosinamide to the free acid abolished receptor binding, whereas esterification to the methyl ester of COOH-terminal tyrosine restored binding to 60%. Additionally, percent CR decreased progressively as amino acid residues were deleted from the NH2-terminal region. We conclude that the molecular homologue of PP primary structure is necessary for full receptor binding. Both the NH2- and COOH-terminal residues are required for recognition, and the COOH-terminal tyrosinamide must be intact for PP binding to its receptor.

Patterns of immunoreactive pancreatic polypeptide in human plasma.

Pancreatic polypeptide (PP) is synthesized as an amino-terminal moiety of a precursor peptide and is released into plasma during stimulation as an amidated hormone (PP1-36). The purpose of this investigation was to ascertain the immunoreactive forms of PP in human plasma using HPLC chromatographic technique. Plasma was obtained from five normal volunteers under various postprandial intervals and from the Blood Bank. PP in each plasma sample was processed for HPLC analysis by immunoprecipitation and/or immunoaffinity extractions. Migration patterns of PP-forms were identified under isocratic elution. This study shows that human plasma contains four distinct immunoreactive (IR) forms of PP during stimulation by a protein-rich meal. These forms are PP1-36 (peak 4), PP3-36 (peak 3) and unidentified material migrating as peak 2 and peak 1. The corresponding migration constants were Kav 0.828 +/- 0.04, Kav 0.790 +/- 0.003, Kav 0.570 +/- 0.009 and Kav 0.409 +/- 0.007, respectively. The predominant fasting from of IR PP chromatographed as peak 1, while peaks 2 and 4 were reduced in amplitude. The 1 h and 3 h postprandial chromatograms of HPLC profiles of plasma PP were similar in shape but lower in relative magnitude and amplitude. The authenticity of peak 4 as the migration of native PP1-36 was confirmed using purified IR native PP1-36 extracted from human pancreas. Partial amino acid sequence analysis of PP peak 3 revealed deletions of two N-terminal amino acid residues. The chemical identities of peaks 1 and 2 are unknown but appear to differ from PP in peaks 3 and 4 by virtue of their migration profiles. It is concluded that there are at least four distinct IR forms of PP in human plasma. Native PP1-36 accounts for less than 1% of total PP after an overnight fast and is about 1/3 of total postprandial IR plasma PP. Discernment of the nature and etiology of forms of PP in plasma may provide a new understanding of the role of PP in mammalian physiology.

Characterization of specific pancreatic polypeptide receptors on basolateral membranes of the canine small intestine.

We have identified specific binding sites for pancreatic polypeptide (PP) on the mucosal lining of canine small intestine. The present study was undertaken to further characterize these binding sites (receptors) on purified intestinal membranes and to establish their location on the brush border or basolateral surface of the intestinal enterocyte. Basolateral and brush border membranes were prepared by sorbitol density centrifugation. PP receptors were localized predominantly to the vascular surface, and thus binding of PP 125I-labeled on Tyr-27 to the basolateral preparation was used to evaluate receptor characteristics. Binding of PP was calcium, time, temperature, and pH dependent. Maximum specific binding of labeled PP occurred after an 8-hr incubation at 4 degrees C with 5 mM calcium at pH 6.8. Data analysis by Scatchard plot showed high- and low-affinity binding sites with relative affinities of 1.5 x 10(-9) M and 2.6 x 10(-8) M and with corresponding binding capacities of 0.23 pmol/mg and 0.84 pmol/mg of protein, respectively. This receptor was specific for PP since peptide YY and neuropeptide Y, peptides of the PP family, cross-reacted by less than 3%, as judged from comparisons of half-maximal displacement of label. Structurally dissimilar peptides, insulin and glucagon, did not compete for binding. Specific 125I-labeled PP binding was localized primarily to basolateral membranes (9.8 +/- 0.8%) with little binding by brush border membranes (0.8 +/- 0.2%). Thus, we have identified highly specific receptors for PP, located predominantly on the vascular surface of the small intestinal mucosa. These data suggest that the mucosal lining of the small intestine is a target tissue for PP and that PP participates in the hormonal regulation of fuel metabolism and substrate transport in the small intestinal mucosa.

Identification and characterization of insulin receptors in basolateral membranes of dog intestinal mucosa.

Little is known about hormonal regulation of substrate transport and metabolism in the mucosal lining of the small intestine. Because insulin regulates these functions in other tissues by binding to its receptor, we have investigated the presence of insulin receptors in canine small intestinal mucosa with basolateral membranes (BLM) and brush border membranes (BBM) prepared by sorbitol density centrifugation. A14-[125I]iodoinsulin was used to study binding and structural characteristics of specific insulin receptors in BLM. Analysis of receptors in BLM identified binding sites with high affinity (Kd 88 pM) and low capacity (0.4 pmol/mg protein) as well as with low affinity (Kd 36 nM) and high capacity (4.7 pmol/mg protein). Binding was time, temperature, and pH dependent, and 125I-labeled insulin dissociation was enhanced in the presence of unlabeled insulin. Cross-reactivity of these receptors to proinsulin, IGF-II, and IGF-I was 4, 1.8, and less than 1%, respectively. Covalent cross-linking of labeled insulin to BLM insulin receptors with disuccinimidyl suberate revealed a single 135,000-Mr band that was completely inhibited by unlabeled insulin. There was a 16-fold greater specific binding of insulin to BLM (39.0 +/- 2.4%) than to BBM (2.5 +/- 0.6%). These results demonstrate the presence of a highly specific receptor for insulin on the vascular, but not the luminal, surface of the small intestinal mucosa in dogs, and suggest that insulin may play an important role in the regulation of gastrointestinal physiology.

Intestinal mucosa is a target tissue for pancreatic polypeptide.

Studies were carried out to identify mammalian tissues capable of specifically binding mammalian pancreatic polypeptide (PP). Bovine PP (bPP) radiolabeled with 125I was purified by HPLC to yield [125I]iodo-(Tyr-27) bPP. The label was injected into three pairs of fasted littermate dogs and allowed to circulate for 5 min. One of the dogs was a control which received an excess of unlabeled porcine PP to provide competition for receptor binding. Unbound bPP was removed by perfusion with Krebs-Ringer bicarbonate and the tissue fixed in situ with Karnovsky's fixative. Tissue samples from various organs were removed, weighed, and counted. The entire gastrointestinal tract demonstrated high levels of 125I after injection of the labeled peptide. The duodenum, jejunum, ileum, and colon were the only tissues to exhibit specific binding of bPP. These tissues (mucosal and muscle layers) from experimental animals exhibited 31-76% higher binding than the corresponding tissues from the control animals. Sections of the gastrointestinal tract were scraped to separate the mucosal layer from the underlying muscle layer. The mucosal layer of the duodenum, jejunum, and ileum exhibited 145-162% increases in binding compared to the control animals. The muscle layer of these tissues demonstrated no significant increase. These findings demonstrate that mucosal layer of the small intestine is a target tissue for mammalian PP.

In vivo acylation of proteolipid protein and DM-20 in myelin and myelin subfractions of developing rat brain: immunoblot identification of acylated PLP and DM-20.

The acylation of proteolipid protein (PLP) was examined in myelin and myelin subfractions from rat brain during the active period of myelination. Proteolipid protein and DM-20 in myelin and myelin subfractions were readily acylated in developing rat brain 22 hours after intracerebral injection of [3H]palmitic acid. No differences in the relative specific activity of PLP in myelin from 9-, 15-, and 30-day-old rat brains was observed; however, the relative specific activity of PLP in the heavy myelin subfraction tended to be higher than that in the light myelin subfraction. The acylation of PLP was confirmed by fluorography of immuno-stained cellulose nitrate sheets, clearly establishing that the acylated protein is in fact the oligodendroglial cell- and myelin-specific protein, PLP. Since PLP is acylated in the 9-day-old animal, when little compact myelin is present, it is possible that the acylation of PLP is a prerequisite for the incorporation of this protein into the myelin membrane.

Regulation of transcription and DNA replication of bacteriophage phi 80.

Transcriptional mapping and DNA replication measurements have been used to characterize a series of phi 80 suppressor-sensitive mutants which are defective in genes 15, 14, 16, and 17. These genes are localized within the inner right arm of the vegetative phi 80 DNA genome. The sus326 mutation in gene 15 leads to a decrease in major leftward (pL-att80) RNA levels and to a marked pleiotropic reduction in major rightward RNA synthesis; however, phi 80 DNA synthesis is reduced only moderately (about two-fold). These findings are consistent with the gene 15 product being a positive control regulator that is essential for normal transcriptional development, in particular, beyond a termination signal(s) (tR) located between genes 16 and 17. The sus8 and sus258 mutations (in genes 14 and 16, respectively) lead to severe blockage of both major rightward RNA transcription and phi 80 DNA synthesis. The products of genes 14 and 16 appear to be required for both autonomous phi 80 DNA replication and the "late" transcriptional development. The sus121 mutation in gene 17 reduces the level of "late" major rightward transcription (gene 17-1-13-att'80 segment) by about 10-fold but does not have any apparent effect on the levels of phi 80 DNA synthesis. These profiles identify the product of gene 17 as a "Q-type" positive control regulator for the "late" major rightward RNA. These studies reveal the functional characterization of four genes, the products of which are necessary for the efficient expression of the "early" RNA transcribed segments, autonomous DNA replication, and the production of normal levels of "late" (17-1-13-att'80) RNA synthesis.

Immunoblot identification of phosphorylated basic proteins of rat and rabbit CNS and PNS myelin: evidence for four phosphorylated basic proteins and P2 in rat PNS myelin.

The immunoblot technique permits the visualization of proteins following their separation on acrylamide gels, transfer to cellulose nitrate sheets and subsequent staining with antiserum. We have utilized this technique to demonstrate the presence of four basic proteins in rat PNS myelin with molecular weights of 21K, 18K, 17K, and 14K. Similarly, we demonstrated the presence of two basic proteins in rabbit PNS myelin (molecular weights of 21K and 18K). Exposure of the immunostained cellulose nitrate strips to X-ray film revealed the phosphorylation of four and two basic proteins in rat and rabbit PNS myelin, respectively. These basic proteins were present in the CNS myelin of the two species and were also phosphorylated. Because of the sensitivity of the immunoblot technique, it was also possible for us to visualize the P2 protein in both rat and rabbit PNS myelin.

Diffuse unilateral subacute neuroretinitis.

Thirty-six young healthy patients developed a peculiar clinical syndrome affecting only one eye. The early stage of the disease was characterized by visual loss, vitritis, mild papilledema, and successive crops of multiple, evanescent, gray-white, deep, retinal lesions. Over a period of many months there developed widespread, diffuse and focal depigmentation of the pigment epithelium, retinal arterial narrowing, optic atrophy, severe visual loss, and electroretinographic changes. A motile, subretinal round worm, probably a Toxocara, was observed in two patients.