Antisera to synthetic peptide recognize high molecular weight enkephalin-containing proteins

Antisera to a synthetic peptide corresponding to the 95-117 sequence of proenkephalin were used to develop a sensitive radioimmunoassay. Gel-filtration of acid extracts of bovine adrenal medulla and purified chromaffin granules revealed that the antisera recognized high molecular weight material (Mr approximately 5,000-30,000). The material in peak I ( Mr 20 ,000-30,000) and peak II (Mr 10,000-20,000) was further purified by immunoaffinity chromatography. Sequential digestion of each of these fractions with trypsin and carboxypeptidase B generated immunoreactive Met-enkephalin. This study demonstrates that antisera against a synthetic peptide cross-react with high molecular weight enkephalin-containing precursors, validating the use of these antisera in studies of enkephalin biosynthesis.     

Neuronal cell lines expressing PC5, but not PC1 or PC2, process Pro-CCK into glycine-extended CCK 12 and 22.

Endocrine tumor cells in culture and in vitro cleavage assays have shown that PC1 and PC2 are capable of processing pro-CCK into smaller, intermediate and final, bioactive forms. Similar studies have shown that PC5 has the ability to process a number of propeptides. Here, we use GT1-7 (mouse hypothalamic) and SK-N-MC and SK-N-SH (human neuroblastoma) tumor cell lines to study the ability of PC5 to process pro-CCK. RT-PCR and Western blot analysis showed that the cells express PC5 mRNA and protein, but not PC1 or PC2. They were engineered to stably overexpress CCK and cell media was analyzed for pro-CCK expression and cleavage of the prohormone. Radioimmunoassays showed that pro-CCK was expressed, but no amidated CCK was detected. Lack of production of amidated CCK may be due to the lack of the appropriate carboxypeptidase and amidating enzymes. Production of glycine-extended CCK processing products was evaluated by treatment of media with carboxypeptidase B followed by analysis with a CCK Gly RIA. Glycine-extended forms of the peptide were found in the media. The predominant forms co-eluted with CCK 12 Gly and CCK 22 Gly on gel filtration chromatography. The results demonstrate that these cell lines which express PC5 and not PC1 or PC2 have the ability to process pro-CCK into intermediate, glycine-extended forms more closely resembling pro-CCK products in intestine than in brain.     

Cloning and expression of a new rat procarboxypeptidase B gene in Escherichia coli and purification of recombination carboxypeptidase B

A new coding sequence of the procarboxypeptidase B gene was obtained from SD rat fresh pancreas by RT-PCR and highly expressed in Escherichia coli in inclusion bodies. The folded procarboxypeptidase B was subjected to trypsin enzymatic cleavage to produce active carboxypeptidase B, subsequently, carboxypeptidase B was effectively purified with anion exchange chromatography DEAE-FF and hydrophobic interaction chromatography Octyl FF, as a result, 40 mg carboxypeptidase B per litre cell culture with specific activity 7.42 u/mg was achieved. Further research showed that the obtained recombinant carboxypeptidase B could substitute carboxypeptidase B isolated from pancreas.     

Evidence for the occurrence of the opioid octapeptide dynorphin-(1–8) in the neurointermediate pituitary of rats

Evidence is provided for the existence of the opioid peptide dynorphin-(1–8) in the neurointermediate pituitary of rats. The octapeptide was isolated by immunoadsorption to antibodies directed against porcine dynorphin-(1–13) followed by a variety of chromatographic separation procedures. The identity of the purified material with dynorphin-(1–8) was indicated by the following criteria: comigration with synthetic dynorphin-(1–8) on gelfiltration chromatography and high-performance liquid chromatography systems and liberation of a peptide with the same chromatographic behavior as leucine-enkephalin after sequential cleavage with trypsin and carboxypeptidase B.Radioimmunological estimations revealed that dynorphin-(1–8) is a major dynorphin-related opioid peptide in the pituitary of rats.     

Identification and characterization of glycine-extended post-translational processing intermediates of progastrin in porcine stomach

We developed a radioimmunoassay specific for glycine-extended progastrin processing intermediates (G-Gly) using antisera generated against the synthetic peptide Tyr-Gly-Trp-Met-Asp-Phe-Gly. Distribution of immunoreactivity in the porcine gastrointestinal tract obtained with this antibody paralleled that of gastrin with the mucosa containing the highest quantity, 116 +/- 22 pmol/g, wet weight (mean +/- S.E., n = 5), or roughly 4% of gastrin concentration. This immunoreactivity was localized specifically to antral mucosal G-cells by immunohistochemistry. On Sephadex G-50 column chromatography of porcine antral mucosal extracts glycine-extended progastrin processing intermediates were separated into three principal molecular forms, each corresponding to known molecular forms of gastrin, component I, tetratriacontagastrin (G34) and heptadecagastrin (G17). Following purification by antibody-coupled affinity chromatography, one molecular form corresponding to G17 in size was shown to have an amino terminus identical to that of G17. Another molecular form corresponding to G34 in size could be converted to the molecular form corresponding to G17 by tryptic digestion. Our findings indicate that glycine-extended progastrin processing intermediates may serve as immediate precursors for each molecular form of gastrin, thus suggesting an alternative pathway for gastrin biosynthesis more complex than that previously conceived.     

paH gradients in isoelectric focusing experiments at subzero temperatures

We have developed a simple enzymatic procedure for evaluation of antisera reactivity against the large molecular forms of gastrin and cholecystokinin (CCK). The procedure can be used for radioimmunochemical quantitation of the precursor molecules. The different molecular forms of gastrin or CCK in tissue extracts or plasma were separated by gel chromatography. The concentration of each form was then measured with 17 different antisera before and after tryptic cleavage. The ratio between the molar concentrations before and after tryptic cleavage varied from 0.32 to 1.00. Such variation can explain the variable hormone concentrations in serum and tissue measured with different radioimmunoassays. The present procedure can be performed with any biological fluid containing the precursor forms. It does not require the large molecular forms in pure state. In principle the procedure can be used for quantitation of all peptide precursors.     

Three-dimensional structure of porcine procarboxypeptidase B: a structural basis of its inactivity.

Procarboxypeptidase B is converted to enzymatically active carboxypeptidase B by limited proteolysis catalysed by trypsin, removing the long N-terminal activation segment of 95 amino acids. The three-dimensional crystal structure of procarboxypeptidase B from porcine pancreas has been determined at 2.3 A resolution and refined to a crystallographic R-factor of 0.169. The functional determinants of its enzymatic inactivity and of its activation by limited proteolysis have thus been unveiled. The activation segment folds in a globular region with an open sandwich antiparallel-alpha antiparallel-beta topology and in a C terminal alpha-helix which connects it to the enzyme moiety. The globular region (A7-A82) shields the preformed active site, and establishes specific interactions with residues important for substrate recognition. AspA41 forms a salt bridge with Arg145, which in active carboxypeptidase binds the C-terminal carboxyl group of substrate molecules. The connecting region occupies the putative extended substrate binding site. The scissile peptide bond cleaved by trypsin during activation is very exposed. Its cleavage leads to the release of the activation segment and to exposure of the substrate binding site. An open-sandwich folding has been observed in a number of other proteins and protein domains. One of them is the C-terminal fragment of L7/L12, a ribosomal protein from Escherichia coli that displays a topology similar to the activation domain of procarboxypeptidase.     

Alpha-carboxyamidation of antral progastrin. Relation to other post-translational modifications

Using radioimmunoassays for amidated and glycine-extended gastrin before and after trypsin-carboxypeptidase B cleavage and chromatography, alpha-carboxyamidation of porcine antral progastrin has been related to tyrosine-O-sulfation and proteolytic cleavages. Corresponding to the sequence at the proteolysis and amidation site, -Gly-Arg-Arg-, antrum contained three COOH-terminally extended precursor types. The glycine-extended gastrins were present in the highest concentrations (241 +/- 58 pmol/g). The degree of tyrosine-O-sulfation was identical for amidated and precursor gastrins irrespective of component size, whereas the component size differed for glycine-extended and amidated forms. For instance, gastrin-34-Gly constituted 54% of the glycine-extended gastrins, while gastrin-34 comprised 8% of the amidated gastrins. The results indicate that tyrosine-O-sulfation occurs prior to NH2-terminal cleavages, which again precede carboxyamidation; but a significant correlation between tyrosine-O-sulfation and proteolytic cleavages or alpha-carboxy-amidation of antral gastrin could not be demonstrated. Furthermore, our results suggest that the immediate precursor of the principal hormonal form, gastrin-17, is gastrin-17-Gly rather than gastrin-34 as previously believed.     

Primary structure of ascidian trypsin inhibitors in the hemolymph of a solitary ascidian, Halocynthia roretzi

The amino acid sequences of trypsin inhibitors I and II from the hemolymph of a solitary ascidian, Halocynthia roretzi, were determined after reduction and S-pyridylethylation. The results indicated that inhibitor I consists of a single polypeptide chain with 55 amino acid residues and four intramolecular disulfide bridges, whereas inhibitor II is composed of two polypeptide chains corresponding to a form derived from inhibitor I by cleavage at the Lys16-Met17 bond. Lys16 may be the reactive-site residue of these inhibitors, because carboxypeptidase B treatment destroys most of the inhibitory activity of inhibitor II but not that of inhibitor I.     

Brain endopeptidase generates enkephalin from striatal precursors

An enzyme capable of converting putative opioid peptide intermediates to free enkephalin has been purified 300-fold from washed rat brain membranes. The action of this enzyme, an enkephalin-generating endopeptidase (EGE), was compared with the action of carboxypeptidase B after trypsin treatment on enkephalin precursor peptides present in rat striata. After Sephadex G-100 gel filtration of striatal material, fractions were radioimmunoassayed for enkephalin content using an antiserum specific for the carboxyl terminal of enkephalin. Additionally, aliquots of the column fractions were treated with either trypsin and carboxypeptidase B, trypsin and EGE, or EGE alone. The peak of enkephalin immunoreactivity increased with the enzymes' treatment indicating the conversion of the low molecular weight proenkephalin precursor peptides to enkephalin. Trypsin and EGE generated almost as much enkephalin as trypsin and carboxypeptidase B in the conditions of the experiment. Thus EGE is capable of processing precursors to enkephalin after the action of trypsin-like enzyme(s) in the brain. The gel filtration fractions containing enkephalin and its low molecular weight precursors were pooled and one-half treated with EGE. The contents were analyzed by HPLC and the increase in immunoreactivity co-eluted with enkephalin and Leu-enkephalin. Small peptides found to be the most potent competitive inhibitors of this enzyme are Met-Arg-Phe-Ala, and Met-Arg-Phe.     

Seasonal changes in photosynthetic capacity of leaves of kiwifruit (Actinidia deliciosa) vines

A simplified procedure for the assay and purification of an enzyme which activates a galactosyltransferase (EC 2.4.1.96) involved in volume regulation of the unicellular alga Poterioochromonas malhamensis (Peterfi) is described. The enzyme was extracted with water from membranes, followed by chromatography on DEAE-Sephacel, phenyl-Sepharose and fetuin-agarose. Its proteinase activity was demonstrated by cleavage of oxidized insulin A- and B-Chains. The predominant cleavage site of the oxidized A-chain is the peptide bond between ¹³Leu and ¹⁴Tyr whereas ¹⁶Leu-¹⁷Glu is also hydrolyzed with minor activity. Besides this chymotrypsin-like endopeptidase activity some carboxypeptidase activity was also observed.     

Purification of carboxypeptidase B from human pancreas.

Carboxypeptidase B of the human pancreas was purified by chromatography on DEAE-cellulose and CM-cellulose columns. Two forms of the enzyme, named carboxypeptidase B1 and B2, were separated. They have similar mol.wts. (34250 +/- 590) as established by polyacrylamide-gel disc electrophoresis and by gel filtration. Carboxypeptidase B2 migrates further towards the anode in disc electrophoresis. When the amino acid content of the enzymes was analysed, carboxypeptidase B2 had four more glycine and three more aspartic acid residues than had form B1. The amino acid sequence of the human carboxypeptidase B1 differs from that of the bovine enzyme only in two places in the N-terminal 20-amino-acid sequence. The N-terminal amino acid in carboxypeptidase B1 and B2 is alanine. The peptide 'map' of the tryptic digest of carboxypeptidase B1 contained more peptides than did that of form B2. The Km, the Vmax. and the pH optimum of the cleavage of the peptide substrate hippurylarginine and the ester substrate hippurylargininic acid were similar for both enzymes. CoCl2 accelerated the peptidase activity, and cadmium acetate enhanced the esterase activity, of human carboxypeptidases B1 and B2. Urea and sodium dodecyl sulphate inhibited the enzymes.     

Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma

A novel plasminogen-binding protein has been isolated from human plasma utilizing plasminogen-Sepharose affinity chromatography. This protein copurified with alpha 2 antiplasmin when the plasminogen affinity column was eluted with high concentrations of epsilon-aminocaproic acid (greater than 20 mM). Analysis by sodium dodecyl sulfate suggests this protein has an apparent Mr of 60,000. The amino-terminal amino acid sequence showed no similarity to other protein sequences. Based on the amino-terminal amino acid sequence, oligonucleotide probes were designed for polymerase chain reaction primers, and an approximately 1,800 base pair cDNA was isolated that encodes this Mr 60,000 protein. The deduced amino acid sequence reveals a primary translation product of 423 amino acids that is very similar to carboxypeptidase A and B and consists of a 22-amino acid signal peptide, a 92-amino acid activation peptide, and a 309-amino acid catalytic domain. This protein shows 44 and 40% similarity to rat procarboxypeptidase B and human mast cell procarboxypeptidase A, respectively. The residues critical for catalysis and zinc and substrate binding of carboxypeptidase A and B are conserved in the Mr 60,000 plasminogen-binding protein. The presence of aspartic acid at position 257 of the catalytic domain suggests that this protein is a basic carboxypeptidase. When activated by trypsin, it hydrolyzes carboxypeptidase B substrates, hippuryl-Arg and hippuryl-Lys, but not carboxypeptidase A substrates, and it is inhibited by the specific carboxypeptidase B inhibitor (DL-5-guanidinoethyl)mercaptosuccinic acid. We propose that the Mr 60,000 plasminogen-binding protein isolated here is a novel human plasma carboxypeptidase B and that it be designated pCPB.     

Dynorphin-Related Opioid Peptides in the Neurointermediate Pituitary of Rats Are Not α-N-Acetylated

Immunoreactive dynorphin in the neurointermediate pituitary of rats was found to consist of four different molecular weight forms. The three larger molecular weight forms, with apparent molecular weights of 4800, 3200, and 1700, constituted more than 80% of the total dynorphin immunoreactivity, and each liberated leucine-enkephalin but not alpha-N-acetyl-leucine-enkephalin upon enzymatic treatment with trypsin followed by carboxypeptidase B. Only a minor portion of the smallest dynorphin-related molecular weight form, dynorphin-(1-8), released alpha-N-acetyl-leucine-enkephalin upon enzymatic cleavage. This suggests that the vast majority of dynorphin-related peptides in the rat neurointermediate pituitary is not alpha-N-acetylated. The exceptionally high opiate-like activity of the molecular weight 1700 dynorphin suggests that this dynorphin-related opioid peptide may constitute the major part of opioid activity in the neurointermediate pituitary of rats.     

Specific inactivation of lysosomal glycosidases in living fibroblasts by the corresponding glycosylmethyl-p-nitrophenyltriazenes.

Glicentin (a highly purified 100-amino acid peptide with glucagon-like immunoreactivity from porcine gut) was subjected to limited digestion with trypsin and carboxypeptidase B, and the resulting peptides were studied by gel filtration and region-specific glucagon radioimmunoassays. Similar digests of glucagon and purified fragments of glucagon were studied in parallel. Glicentin gave rise to peptides that corresponded closely to the 1-17 and 19-29 fragments of glucagon. Also, 125I-labelled glicentin and 125I-labelled glucagon gave rise to identical fragments after trypsin treatment. On the basis of this and other evidence [Jacobsen, Demandt, Moody & Sundby (1977) Biochim. Biophys. Acta 493, 452-459] it is concluded that glicentin contains the entire glucagon sequence at residues number 64-92 and thus fulfills one of the requirements for being a 'proglucagon'.     

Covalent structure of protein A. A low molecular weight protein degraded during germination of Bacillus megaterium spores.

The complete covalent structure of Protein A, a protein degraded during bacterial spore germination, has been determined. The intact protein was cleaved with a highly specific spore protease into two peptides, residues 1 to 21 and 22 to 61. The larger peptide was further cleaved into two fragments with either cyanogen bromide or by trypsin cleavage following arginine modification with cyclohexanedione. The peptides derived from cyanogen bromide fragmentation encompassed residues 22 to 53 and 54 to 61 while trypsin hydrolysis yielded overlapping fragments comprising residues 22 to 48 and 49 to 61. Automated sequenator analysis together with carboxypeptidase Y digestion of the intact protein and the peptide fragments provided data from which the following unique amino acid sequence was deduced. NH2-Ala-Asn-Thr-Asn-Lys-Leu-Val-Ala-Pro-Gly10-Ser-Ala-Ala-Ala-Ile-Asp-Gln-Met-Lys-Tyr20-Glu-Ile-Ala-Ser-Glu-Phe-Gly-Val-Asn-Leu30-Gly-Pro-Glu-Ala-Thr-Ala-Arg-Ala-Asn-Gly40-Ser-Val-Gly-Gly-Glu-Ile-Thr-Lys-Arg-Leu50-Val-Gln-Met-Ala-Glu-Gln-Gln-Leu-Gly-Gly60-Lys-COOH.     

Immunoreactive dynorphin in the rat adenohypophysis consists exclusively of 6000 dalton species

Immunoreactive dynorphin in the rat adenohypophysis exhibited an apparent molecular size of approximately 6 kilodaltons (6 kDal) upon characterization by gelfiltration. Essentially no dynorphin-related peptides with a molecular size of dynorphin-(1–17) or dynorphin-(1–8), which constitute the great majority of ir-dynorphin in the rat neurointermediate pituitary and brain, could be detected in the adenohypophysis. The possible presence of putative aggregations of smaller peptides were largely excluded by rechromatography under denaturating conditions in 4 M guanidine-HCL. SDS-gelelectrophoresis revealed that 6 kDal dynorphin consisted of at least three components of similar molecular size. From the predominant form of 6 kDal dynorphin, leucine-enkephalin could be liberated by sequential enzymatic cleavage with trypsin and carboxypeptidase B.     

Rat mast cell carboxypeptidase: amino acid sequence and evidence of enzyme activity within mast cell granules

The amino acid sequence of rat mast cell carboxypeptidase has been determined. The major form has 308 residues; a minor form has an additional (glutamyl) residue at the amino terminus that may indicate an alternate cleavage site during zymogen activation. The enzyme is homologous to pancreatic carboxypeptidases A and B, with conservation of the functional amino acid residues of the active site. The putative substrate binding site resembles that of carboxypeptidase A, although other structural features bear more similarity to carboxypeptidase B. Mast cell carboxypeptidase retains enzymatic activity toward a peptide substrate (angiotensin I) while bound within the granular matrix of the rat connective tissue mast cells. Evidence is presented to suggest that a cluster of positively charged lysyl and arginyl residues binds the enzyme to the negatively charged heparin of the granular matrix but leaves the active site exposed to bind and cleave peptide substrates.     

Alternative processing pathways for preprovasoactive intestinal peptide in the enteric nervous system of the rat

In order to study biosynthetic processing of preprovasoactive intestinal peptide (prepro VIP) we have raised antisera to sequences that flank the biologically active peptides VIP and PHI (peptide with N-terminal His and C-terminal Ile). We have used these antisera in radioimmunoassays to identify the N-terminal flanking peptide (NFP) and C-terminal flanking peptide (CFP)-like immunoreactivities in rat brain and gastrointestinal tract. Concentrations of NFP-LI were similar to those of VIP in brain and throughout the gut. Concentrations of CFP-LI were 10-20% those of VIP-LI but could be increased 5-fold by digestion with carboxypeptidase B, suggesting that the C-terminal lysine residue of prepro VIP is not normally removed during processing. In rat stomach the NFP-LI was of higher molecular weight and greater hydrophobicity than the intestinal component. The data are consistent with alternative processing pathways for prepro VIP in enteric nerves of rat stomach and intestine.     

Characterization of preprocholecystokinin products in the porcine cerebral cortex. Evidence of different processing pathways

Using gel, ion-exchange, and reverse-phase chromatography monitored by radioimmunoassays specific for five sequences of preprocholecystokinin (prepro-CCK), its processing products were measured in neutral and acid extracts of porcine cerebral cortex before and after incubation with trypsin, carboxypeptidase B, and arylsulfatase. Three categories of peptides were found: biologically active peptides, i.e. peptides with the alpha-amidated COOH terminus Trp-Met-Asp-Phe-NH2, comprising large CCKs, i.e. peptides larger than CCK-58 and peptides eluting like CCK-58, CCK-33, and CCK-22; CCK-octapeptides in sulfated and traces of nonsulfated forms; and small CCKs, i.e. traces of CCK-7, large amounts of CCK-5, and modest concentrations of CCK-4 (the structures of CCK-5 and -4 were confirmed by sequence analysis); four NH2-terminal fragments, of which the two predominant ones correspond to the desnonapeptide fragments of CCK-58 and CCK-33; and COOH-terminal extended peptides corresponding to glycine-extended CCK-58, CCK-33, and CCK-8 in small but significant amounts. Thus, in addition to CCK-8 the porcine cerebral cortex synthesizes larger and smaller active CCK peptides in quantities of an order similar to those of CCK-8. The occurrence of these together with the NH2-terminal fragments and glycine-extended peptides can be explained only by the existence of different processing pathways for preproCCK. Consequently, the results suggest that cerebral CCK neurons are heterogeneous and comprise at least three populations with different biosynthetic machineries.