Pairs Of Basic Amino Acid Residues Research Articles

Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities.

Aeromonas sobria serine protease (ASP) is secreted from Aeromonas sobria, a pathogen causing gastroenteritis and sepsis. ASP resembles Saccharomyces cerevisiae Kex2, a member of the subtilisin family, and preferentially cleaves peptide bonds at the C-terminal side of paired basic amino acid residues; also accepting unpaired arginine at the P1 site. Unlike Kex2, however, ASP lacks an intramolecular chaperone N-terminal propeptide, instead utilizes the external chaperone ORF2 for proper folding, therefore, ASP and its homologues constitute a new subfamily in the subtilisin family. Through activation of the kallikrein/kinin system, ASP induces vascular leakage, and presumably causes edema and septic shock. ASP accelerates plasma clotting by α-thrombin generation from prothrombin, whereas it impairs plasma clottability by fibrinogen degradation, together bringing about blood coagulation disorder that occurs in disseminated intravascular coagulation, a major complication of sepsis. From complement C5 ASP liberates C5a that induces neutrophil recruitment and superoxide release, and mast cell degranulation, which are associated with pus formation, tissue injury and diarrhea, respectively. Nicked two-chain ASP also secreted from A. sobria is more resistant to inactivation by α2-macroglobulin than single-chain ASP, thereby raising virulence activities. Thus, ASP is a potent virulence factor and may participate in the pathogenesis of A. sobria infection.

Cancer cells release anaphylatoxin C5a from C5 by serine protease to enhance invasiveness.

Anaphylatoxin C5a indirectly fosters cancer cells through recruitment of myeloid-derived suppressor cells (MDS) for inhibiting antitumor CD8+ T cells and induction of neovascularization. We recently found activation of cancer cells by C5a directly via the C5a-receptor (C5aR; CD88) to enhance invasiveness. Thus, C5a possibly contributes to cancer progression rather than elimination. C5a generation in cancer tissues has been reported; however, the mechanism is not fully elucidated. Cancer cell expression of complement regulatory molecules suggests inefficient C5a generation through activation of the complement system in response to cancer cells. To explore another C5a generation mechanism in cancer tissues, we examined cancer cells for C5a-releasing activity from C5. C5a was present in C5-supplemented culture media of cancer cells including C5aR-expressing cells, and the media enhanced C5aR-expressing cancer cell invasion, which was abolished by anti-C5a antibody. The C5a-releasing activity was absent in the supernatants of the media and was inhibited by aprotinin, a serine protease inhibitor, and decanoyl-Arg-Val-Lys-Arg-chloromethylketone but not by inhibitors specific for cysteine, acid, or metal proteases. These results indicated C5a release from C5 by a cancer cell membrane-bound serine protease that can cleave peptide bonds at the carboxy-terminal site of paired basic amino acid residues. Cancer cell C5a release from the complement-immobilized plasma supported feasibility of this cancer cell protease-dependent C5a generation in cancer tissues. The new mechanism of C5a generation suggests self-activation of C5aR-expressing cancer cells to enhance invasiveness and induction of MDS recruitment and neovascularization to create a microenvironment favorable for cancer progression.

Genetics of the First Seven Proprotein Convertase Enzymes in Health and Disease

Members of the substilisin/kexin like proprotein convertase (PCSK) protease family cleave and convert immature pro-proteins into their biologically active forms. By cleaving for example prohormones, cytokines and cell membrane proteins, PCSKs participate in maintaining the homeostasis in a healthy human body. Conversely, erratic enzymatic function is thought to contribute to the pathogenesis of a wide variety of diseases, including obesity and hypercholestrolemia. The first characterized seven PCSK enzymes (PCSK1-2, FURIN, PCSK4-7) process their substrates at a motif made up of paired basic amino acid residues. This feature results in a variable degree of biochemical redundancy in vitro, and consequently, shared substrate molecules between the different PCSK enzymes. This redundancy has confounded our understanding of the specific biological functions of PCSKs. The physiological roles of these enzymes have been best illustrated by the phenotypes of genetically engineered mice and patients that carry mutations in the PCSK genes. Recent developments in genome-wide methodology have generated a large amount of novel information on the genetics of the first seven proprotein convertases. In this review we summarize the reported genetic alterations and their associated phenotypes.

MSPL/TMPRSS13

Two variant cDNAs, mosaic seine protease large-form (MSPL) and transmembrane protease serine 13 (TMPRSS13), have been identified from a human lung cDNA library by polymerase chain reaction. The deduced amino acid sequences of these proteins show a type II transmembrane protein structure with a unique and long cytoplasmic tail containing tandem repeat phosphorylation motifs of protein kinases, a transmembrane domain, and a trypsin-like serine protease domain at the extracellular C-terminal side. These proteins have an identical serine protease sequence except for the C-terminal ends, and the consensus protease domain exhibits 42, 39 and 43% sequence identity with those of plasma kallikrein, hepsin and transmembrane protease serine 2 (TMPRSS2), respectively. Although both genes are widely expressed in various tissues, they are predominantly expressed in human lung, placenta, pancreas and prostate. TMPRSS13 is expressed higher than MSPL in thymus, spleen and peripheral blood lymphocytes, particularly in CD8+ cells and CD19+ cells. Enzymatic properties of the recombinant soluble MSPL and TMPRSS13 show that these enzymes preferentially recognize the sites consisting of paired basic amino acid residues, and are strongly inhibited by aprotinin, benzamidine and Bowman-Birk trypsin inhibitor, but poorly inhibited by alpha 1-antitrypsin and leupeptin. These properties raise the possibility that MSPL and TMPRSS13 play roles in the proteolytic processing of prohormones, precursors of growth factors, and also play roles in the pathogenicity of many viruses and bacteria in vivo.

On the Processing of Proghrelin to Ghrelin

The orexigenic hormone ghrelin is a 28-amino-acid peptide derived from a 99-amino-acid precursor and acylated at Ser-3, which was initially isolated from rat stomach. In addition to stimulating appetite and growth, it also plays various important roles in energy homeostasis and in the cardiovascular and immune systems. Although analysis of its physiological effects has yielded many significant results, much less information is available on its biosynthesis and the mechanism of its acylation. In this report, we have studied the endoproteolytic processing of this molecule from its precursor (proghrelin) into mature ghrelin in various prohormone convertase null mouse strains generated in our laboratory and have identified the convertase responsible for this event. Using Western blotting, mass spectrometry, and immunocytochemical methods, we have demonstrated that (a) in mouse stomach, prohormone convertase 1/3 (PC1/3) is the endoprotease responsible for the conversion of proghrelin to ghrelin, (b) the acylation of this peptide is processing-independent, and (c) the expression of proghrelin mRNA is increased in the processing-deficient (PC1/3 null) mouse.

Prediction of proprotein convertase cleavage sites.

Many secretory proteins and peptides are synthesized as inactive precursors that in addition to signal peptide cleavage undergo post-translational processing to become biologically active polypeptides. Precursors are usually cleaved at sites composed of single or paired basic amino acid residues by members of the subtilisin/kexin-like proprotein convertase (PC) family. In mammals, seven members have been identified, with furin being the one first discovered and best characterized. Recently, the involvement of furin in diseases ranging from Alzheimer's disease and cancer to anthrax and Ebola fever has created additional focus on proprotein processing. We have developed a method for prediction of cleavage sites for PCs based on artificial neural networks. Two different types of neural networks have been constructed: a furin-specific network based on experimental results derived from the literature, and a general PC-specific network trained on data from the Swiss-Prot protein database. The method predicts cleavage sites in independent sequences with a sensitivity of 95% for the furin neural network and 62% for the general PC network. The ProP method is made publicly available at http://www.cbs.dtu.dk/services/ProP.

VGF: A Novel Role for This Neuronal and Neuroendocrine Polypeptide in the Regulation of Energy Balance

Insight into the mechanisms of action of neurotrophic growth factors has been obtained through the identification and characterization of gene products that are regulated or modified at the transcriptional, translational, and/or posttranslational level in response to neurotrophin treatment. VGF (non-acronymic) was identified approximately 15 years ago as a nerve growth factor (NGF)-regulated transcript in rat PC12 pheochromocytoma cells. Subsequent studies have demonstrated that neurotrophins such as NGF and brain-derived neurotrophic factor induce vgf gene expression relatively rapidly in PC12 cells and cultured cortical neurons, respectively, in comparison to less robust regulation by epidermal growth factor (EGF) and insulin, growth factors which do not trigger the neuronal differentiation of PC12 cells. vgf gene expression is stimulated in vitro by NGF and the ras/map kinase signaling cascade through a CREB-dependant mechanism, while in vivo, VGF mRNA levels are regulated by neuronal activity, including long-term potentiation, seizure, and injury. Both the mRNA and encoded ∼68-kDa protein (VGF) are selectively synthesized in neuroendocrine and neuronal cells. The predicted VGF sequence is rich in paired basic amino acid residues that are potential sites for proteolytic processing, and VGF undergoes regulated release from dense core secretory vesicles. Although VGF mRNA is synthesized widely, by neurons in the brain, spinal cord, and peripheral nervous system, its expression is particularly abundant in the hypothalamus. In addition, VGF peptides are found in hypophysial, adrenal medullary, gastrointestinal, and pancreatic endocrine cells, suggesting important neuroendocrine functions. Recent analysis of VGF knockout mice indeed demonstrates that VGF plays a critical role in the control of energy homeostasis. VGF knockout mice are thin, small, hypermetabolic, hyperactive, and relatively infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic pro-opiomelanocortin, neuropeptide Y, and agouti-related peptide expression. Coupled with the demonstration that VGF mRNA levels are induced in the normal mouse hypothalamic arcuate nuclei in response to fasting, important central and peripheral roles for VGF in the regulation of metabolism are suggested. Here we review previous studies of VGF in the broader context of its newly recognized role in the control of energy balance and propose several models and experimental approaches that may better define the mechanisms of action of VGF.

Canine prosomatostatin: Isolation of a cDNA, regulation of gene expression, and characterization of post-translational processing intermediates

Somatostatin is a tetradecapeptide (SS-14) initially isolated from the hypothalamus that is also found in D cells of the stomach and pancreas where it exerts an inhibitory action on a variety of gastrointestinal functions. Since many of concepts important to an understanding of gastrointestinal physiology are derived from experiments in the dog we examined somatostatin gene expression and post-translational processing in the canine fundus, antrum and pancreas. The canine somatostatin cDNA which is highly homologous to other known mammalian somatostatins was used to examine somatostatin expression in isolated canine fundic D-cells. Somatostatin expression induced by cholecystokinin (10 −8 M) was inhibited by the somatostatin analog, octreotide (10 −7 M). To examine somatostatin processing in the canine gut we noted that synthesis of SS-14 and somatostatin octacosapeptide (SS-28) involves endoproteolytic cleavage of prosomatostatin (proSS) at both paired and single basic amino-acid residues, respectively. Antisera capable of recognizing the amino-terminal residues of SS-28, SS-28 (1 – 14) and SS-28 (1 – 12) were characterized and identified concentrations of SS-28 (1 – 12) but not SS-28 (1 – 14) in the fundus, antrum and pancreas equivalent to those of SS-14. Since previous biosynthetic studies in canine fundic D-cells showed that SS-14 was synthesized without the appearance of a SS-28 intermediate, we hypothesize that proSS is sequentially cleaved at a dibasic site to produce SS-14 followed by monobasic cleavage that results in the formation of SS-28 (1 – 12). Furthermore, equivalent amounts of SS-14 and SS-28 (1 – 12) were co-released from canine fundic D-cells by CCK (10 − 8 M) suggesting that the generation of these products occurs within the same regulated pathway of secretion.

Purification and characterization of a novel membrane-bound arginine-specific serine proteinase from porcine intestinal mucosa

A novel membrane-bound serine proteinase has been purified from the microsomal membranes of porcine intestinal mucosa. It was solubilized from the microsomal membrane fraction with 1% sodium deoxycholate, then purified by a series of column chromatographic steps on DE52, butyl-Toyopearl, Bio-Gel P-150, Mono Q, and benzamidine-Sepharose in the presence of 0.02% Lubrol PX. Its molecular mass was estimated to be 50 kDa both by SDS-polyacrylamide gel electrophoresis under non-reducing conditions and by gel filtration, and to be 32 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions, suggesting that the enzyme may exist as a homodimer in which two subunits are linked by disulfide bond(s). It had a pH optimum at around 9 and did not require Ca2+ for activity. It cleaved several peptide 4-methylcoumaryl-7-amide substrates almost exclusively after arginine residues, the best substrate among those tested being t-butyloxycarbonyl-Gln-Ala-Arg-4-methylcoumaryl-7-amide. Various neuropeptides were also cleaved by this enzyme after arginine, mainly between paired basic amino acid residues, Arg-Arg or Arg-Lys. Activity toward protein substrates was scarcely detected. Further, its partial amino acid sequences were highly homologous, but not identical, with those of trypsin-type serine proteinases. These results indicate that the present enzyme is a novel arginine-specific trypsin-like endopeptidase, possibly involved as a processing proteinase in the production of certain gastrointestinal neuropeptides or peptide hormones from their precursors, or their specific degradation.

Furin-mediated proprotein processing activity: Involvement of negatively charged amino acid residues in the substrate binding region

Furin, which is encoded by the recently discovered FUR gene, appears to be the first known mammalian member of the subtilisin family of serine proteases with cleavage selectivity for paired or multiple basic residues. A consensus cleavage sequence, Arg-X-Lys/Arg-Arg has been proposed. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Homology modelling of the catalytic domain of this protein suggested that negatively charged amino acid residues near or in the substrate binding region might contribute to the observed specificity for substrate segments with paired and multiple basic amino acid residues. To investigate this hypothesis, furin mutants were generated in which negatively charged residues, predicted to be located near or in the substrate binding pockets and involved in interactions with basic residues of the substrate, were replaced by neural residues. Analysis of processing by these furin mutants of wild-type and cleavage mutants of pro-von Willebrand factor (pro-vWF) revealed that particular negatively charged residues are critical for specific cleavage activity.

Cloning and functional expression of Dfurin2, a subtilisin-like proprotein processing enzyme of Drosophila melanogaster with multiple repeats of a cysteine motif.

Production of a variety of regulatory eukaryotic proteins, such as growth factors and polypeptide hormones, often involves endoproteolytic processing of proproteins at cleavage sites consisting of paired basic residues. The first known mammalian proprotein processing enzyme with such specificity is the human fur gene product furin. Structurally and functionally, furin is related to the subtilisin-like serine endoprotease kexin (EC 3.4.21.61) of yeast Saccharomyces cerevisiae; unlike kexin, it contains a cysteine-rich region with an unknown function. Here, we describe cloning and sequencing of a 5.8-kbp cDNA of the Dfur2 gene, a fur-like gene of Drosophila melanogaster, which we found expressed during various stages of development. This Dfur2 cDNA has an open reading frame for a 1680-residue protein, called Dfurin2. Dfurin2 contains similar protein domains as mammalian furin, however, it has an extended amino-terminal region and its cysteine-rich region is much larger than that of mammalian furin. Because of this latter phenomenon, we were able to identify a particular cysteine motif that was repeated multiple times in Dfurin2 but present only twice in mammalian furin. Furthermore, we show that Dfur2 encodes an endoproteolytic enzyme with specificity for paired basic amino acid residues as, in cotransfection experiments, correct cleavage was demonstrated of the precursor of the von Willebrand factor but not of a cleavage mutant. Finally, Dfur2 was mapped to region 14C of the X chromosome of D. melanogaster.

Purification and characterization of a putative proenkephalin cleaving enzyme

A putative proenkephalin-cleaving enzyme (PCE) extracted from bovine adrenal chromaffin granules was purified with soybean trypsin inhibitor high-performance affinity chromatography. The 12,600-fold purified enzyme was maximally active at pH 8.0. The enzyme was completely inhibited with lima bean trypsin inhibitor (0.1 mg/ml), soybean trypsin inhibitor (0.1 mg/ml), and p-(chloromercuri)benzenesulfonic acid (1.0 m m), indicating PCE is a serine protease with cysteine residues likely to be involved in its structure or activity. It exhibited significant autoproteolysis without specific substrates present. The substrate specificity and kinetic constants with the enkephalin-containing (EC) peptides Leu-9 and proenkephalin Peptides B, E, and F as substrates were studied. The cleavage patterns were substantially different than with trypsin digestion. PCE specifically recognized the paired basic amino acid residues and predominantly cleaved the peptide bonds between Lys and Arg sites and peptide bonds after Lys-Lys and Arg-Arg sites. Different K m and V max values for the different Lys-Arg sites indicate sequences in addition to the paired basic residues can affect enzyme activity. Also, the lower K m and V max of Peptide E suggest a higher affinity for this peptide but much slower cleavage. The C-terminally located Lys-Arg site appears responsible for this high affinity. Based on these observations, we propose the following: (a) the primary structure of these peptides contains enough information to be processed correctly by PCE and (b) PCE may be regulated by pH and Peptide E to prevent extensive processing of the intermediate EC peptides which are the major opioid peptides found in the adrenal chromaffin granules.

Proteolysis in Rat Hypothalamic Neurosecretory Granules: Characterization of an a-Chymotrypsin-Like Activity in the Pathway of Intracellular Processing of Prohormones*

To yield biologically active hormones, prohomones are processed by cleavages at paired basic amino acid residues. In this study we report that a chymotrypsin-like activity has been colocalized with trypsin-like and carboxypeptidase-B-like proteases in neurosecretory granules, the site of intracellular processing of prohomones. Using a peptide of 11 amino acids as a simple model system for the study of prohomone processing, we have identified in neurosecretory granule membranes a novel protease that specifically recognizes and cleaves peptide bonds at aromatic residues. Studies were also performed with the fluorogenic peptide substrate N-succinyl-leucyl-leucyl-valyl-tyrosine-7-amino-4-methyl-coumarine . The identified protease activity is inactivated by a cloromethyl ketone derivative (Tos-Phe-CH2-Cl) by Trasylol, and markedly by phenylmethylsulfonylfluoride and diisopropylfluorophosphate. This activity is colocalized with other prohomone-processing enzymes in neurosecretory granules, which indicates that this activity is involved in the prohomone processing. Alternatively, this activity may function in the degradation of homones and neuropeptides when they are released in synapses.

Specific cleavages of arginyl peptide bonds at basic amino acid pairs by a serine proteinase from the microsomal membranes of rat liver

The specificity of action of a serine proteinase from the microsomal membranes of rat liver was investigated at pH 7.5 and 37°C using various peptides as substrates. HPLC analyses of the peptides produced followed by their amino acid analyses have revealed that the enzyme is a unique endopeptidase specifically cleaving arginyl peptide bonds at paired basic amino acid residues. Thus, the enzyme is suggested to be a kind of processing proteinase involved in the conversion of proproteins to their mature forms. Indeed, the enzyme cleaved specifically the NH 2-terminal 20-residue peptide of proalbumin at the Arg-Arg sequence.

Furin: The Prototype Mammalian Subtilisin-Like Proprotein-Processing Enzyme

Furin, the translational product of the recently discovered fur gene, appears to be the first known mammalian member of the subtilisin family of serine proteases and the first known mammalian proprotein-processing enzyme with cleavage selectivity for paired basic amino acid residues. Structurally and functionally, it resembles the prohormone-processing enzyme, kexin (EC 3.4.21.61), which is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Here, we review the discovery of the fur gene and describe the isolation of cDNA clones corresponding to human and mouse fur and to two fur-like genes of Drosophila melanogaster, Dfur1 and Dfur2. We also compare the structural organization of the various deduced furin proteins to that of yeast kexin, and of other members of the subtilisin family of serine proteases. Furthermore, the biosynthesis of biologically active human and mouse furin is evaluated. Finally, the cleavage specificity for paired basic amino acid residues of human and mouse furin is demonstrated by the correct processing of the precursor for von Willebrand factor.

Proopiomelanocortin gene and gene products: A comparative study

The beneficial effects of morphine, mainly euphoria and analgesia, have been known for several centuries. However, it is only twenty years ago that the story of endogenous opioid peptides started. In 1971, Goldstein and collaborators established the existence of receptors to morphine within the brain (Goldstein et al., 1971). The discovery of the numerous endogenous ligands of these receptors began in 1974 (Terenius and Wahlstrom, 1974). After these first and somewhat hesitating steps, the tale of opioids has been written extremely quickly. The main opioid peptides, beta-endorphin, Met-enkephalin, Leu-enkephalin, and the dynorphins, are today established to derive from three large precursors. Proopiomelanocortin (POMC), proenkephalin and prodynorphin were cloned and sequenced within a few years (Nakanishi et al., 1979; Noda et al., 1982; Kakidani et al., 1982). These three precursors are all large prohormones processed into several biologically active peptides, these being separated by pairs of basic amino-acid residues. Proopiomelanocortin, precursor of betaendorphin, is probably the most studied of these three molecules. It was first shown to be expressed in the pituitary, both in the corticotroph cells of the anterior lobe, where it is mainly processed into adrenocorticotropin (ACTH) and beta-lipotropin (BLPH), and in the melanotroph cells of the intermediate lobe, where it is cleaved into alphamelanocyte stimulating hormone (AMSH), corticotropin-like intermediate lobe peptide (CLIP), gamma-lipotropin (GLPH) or betamelanocyte stimulating hormone (BMSH) and beta-endorphin (BEnd). Although they were first supposed to be only expressed in nervous tissues, POMCderived peptides have been observed in many non-pituitary tissues such as the reproductive tract (Tsong et al., 1982), gastrointestinal tract, kidney, liver and heart (Saito and Odell, 1983; DeBold et al., 1988). They are also synthesized in several cells of the immune system (DiAugustine et al., 1980; Smith and Blalock, 1981; LacazeMasmonteil et al., 1987; Lolait et al., 1986) and constitute for this reason important common signal molecules of the neuroendocrine and the immune systems.

Expression of a human proprotein processing enzyme: correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site.

Intracellular proteolytic processing of precursor polypeptides is an essential step in the maturation of many proteins, including plasma proteins, hormones, neuropeptides, and growth factors. Most frequently, propeptide cleavage occurs after paired basic amino acid residues. To date, no mammalian propeptide processing enzyme with such specificity has been purified or cloned and functionally characterized. We report the isolation and functional expression of a cDNA encoding a propeptide-cleaving enzyme from a human liver cell line. The encoded protein, called PACE (paired basic amino acid cleaving enzyme), has structural homology to the well-characterized subtilisin-like protease Kex2 from yeast. The functional specificity of PACE for mediating propeptide cleavage at paired basic amino acid residues was demonstrated by the enhancement of propeptide processing of human von Willebrand factor when coexpressed with PACE in COS-1 cells.

Distribution and characterization of VIP-related peptides in the rat spinal cord

The possible existence in the rat spinal cord of a peptide related to VIP, VIP(22–28), has been evaluated. VIP contains paired basic aminoacid residues at which postrranslational cleavage of these peptides might occur. The lumbo-sacral region of rat spinal cord had the most VIP(22–28)-like immunoreactivity (ir-VIP(22–28)). Chromatographic analysis of spinal extracts showed that ir-VIP(22–28) consisted of two major peaks, one eluting as authentic VIP(1–28) and the other as VIP(22–28). HPLC confirmed these results, revealing the presence of intact VIP(1–28) and two or more less hydrophobic peptides, one of which corresponded to authentic VIP(22–28). The other two components found have not yet been identified. Further studies are necessary to provide information on the biological significance of VIP(22–28).

A processing enzyme for prorenin in mouse submandibular gland. Purification and characterization.

Renin is produced from an inactive precursor, prorenin, through proteolytic cleavage at paired basic amino acid residues. In this study, an enzyme which specifically cleaves mouse Ren 2 prorenin at the paired basic residues has been purified from mouse submandibular gland by CM-Toyopearl chromatography, antipain-Sepharose chromatography, and isoelectric focusing. This enzyme, named prorenin converting enzyme, consists of two polypeptide chains of 17 and 10 kDa. The enzyme has an isoelectric point of 9.5-9.8, and its pH optimum is between 7.5 and 8.5. It specifically cleaves the peptide bond on the carboxyl side of the Arg at the Lys-Arg pair of mouse Ren 2 prorenin to yield mature renin but does not cleave mouse Ren 1 and human prorenins. Studies on the effects of inhibitors indicate that this enzyme is a serine protease that differs from the enzymes processing other prohormones at paired basic amino acid residues.

Vasoactive intestinal polypeptide carboxy-terminal fragment, VIP(22–28), and other fragments of VIP, in the central nervous system of the rat

The possible existence in rat brain tissues of shorter peptides related to VIP has been examined. VIP and PHI both contain paired basic amino acid residues at which posttranslational cleavage of these peptides might occur. Antiserum to VIP(22–28) was raised in rabbits. The antiserum was carboxy-terminus directed, showing cross-reactivity with all tested peptides containing the VIP carboxy-terminus sequences. Chromatographic analysis of rat brain extracts demonstrated that recovered VIP(22–28) immunoreactivity [VIP(22–28)-ir] was heterogeneous, consisting of a major fraction [60–70% of total VIP(22–28)-ir] which eluted as authentic VIP(1–28) on gel filtration and on reversed phase high performance liquid chromatography (HPLC) columns. A second fraction (30–35% of total VIP(22–28)-ir] eluted from gel filtration columns in the position of VIP(22–28). HPLC analysis of this fraction from extracts of rat cortex, hippocampus, and midbrain indicated that it was heterogeneous. One component corresponded to authentic VIP(22–28). The other two components have not been identified; one appears to be a VIP fragment intermediate in size between VIP(1–28) and VIP(22–28).