Immunological Characterization of Secretory Proteins of Chromaffin Granules: Chromogranins A, Chromogranins B, and Enkephalin-Containing Peptides

The soluble proteins of bovine chromaffin granules can be resolved into about 40 proteins by two-dimensional electrophoresis. Use of several antisera enabled us to characterize most of these proteins with the immune replica technique. An antiserum against dopamine beta-hydroxylase reacted with one protein of Mr 75,000. Met-enkephalin antisera labeled eight proteins of Mr 23,000-14,000. A new method was developed to obtain highly purified chromogranin A for immunization. The antiserum reacted with chromogranin A and several smaller proteins of similar pI. This specific antiserum did not react with a second family of hitherto undescribed proteins, which we propose to call chromogranins B. An antiserum against these proteins was raised. It labeled several proteins ranging in Mr from 100,000 to 24,000 and focusing at pH 5.2. Subcellular fractionation established that chromogranins B are specifically localized in chromaffin granules of several species. They are secreted from the adrenal medulla during cholinergic stimulation. We conclude that apart from dopamine beta-hydroxylase chromaffin granules contain three families of immunologically unrelated proteins.     

Presence of chromogranin A, B and C in bovine endocrine and nervous tissues: a comparative immunohistochemical study

Summary Antisera against chromogranin A, B and C were used to study the distribution of these acidic proteins in bovine endocrine and nervous tissues. The three chromogranins occur together in several endocrine organs (adrenal medulla, anterior pituitary, endocrine pancreas) and in sympathetic ganglion cells. In the posterior pituitary, only chromogranin C and in the intermediate lobe only A and C are found. The parathyroid gland contains only A, and enterochromaffin cells are immunoreactive for A and B. Cells of the thyroid gland and some cells of the anterior pituitary apparently do not contain any chromogranins. It is concluded that the three chromogranins are not always stored together and that they are not present in all endocrine cells. This distinct localization of the chromogranins indicates some special, although still undiscovered, function for these proteins.     

Membranes of chromaffin granules. Isolation and partial characterization of two proteins

Membranes of chromaffin granules were isolated from the adrenal glands of four different species. The solubilized membrane proteins could be resolved into several bands by polyacrylamide-gel electrophoresis (alkaline and acid gel systems). Two major protein components appeared to be common to the chromaffin granule membranes of ox, horse, pig and man. The various membrane proteins of bovine chromaffin granules were separated by filtration on Sephadex G-200 in the presence of sodium dodecyl sulphate. Two major membrane proteins (A and B) were obtained in purified form. Treatment of protein A with 2-mercaptoethanol before electrophoresis resulted in two more rapidly migrating subunits, whereas protein B was unaffected by mercaptoethanol treatment. The amino acid compositions of the two purified proteins were determined. They are very similar to that of the total membrane proteins but significantly different from that of the chromogranins, the soluble proteins of chromaffin granules.     

Isolation and purification of calmodulin from the shrimp, Crangon crangon.

Calmodulin was isolated and purified from shrimp abdominal muscle by heat precipitation, ion exchange and hydrophobic interaction chromatography. The purified calmodulin was homogeneous when evaluated by polyacrylamide gel electrophoresis. A still remaining contaminant was eliminated by high performance liquid chromatography on a phenyl column. The biological and physicochemical properties of shrimp calmodulin such as amino acid composition, molecular weight and the ability to activate calmodulin-deficient bovine heart phosphodiesterase were compared to those of other invertebrate calmodulins.     

Bovine neurophysin lipid complex. Their isolation, characterization and reaggregation

A lipid-containing neurophysin fraction was isolated and purified from bovine posterior pituitary glands by acid extraction and affinity chromatography on a heparin-Sepharose 4B column. This lipid-rich fraction was found to be composed of noncovalent aggregates of neurophysin proteins and phospholipids such as phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and sphingomyelin. The lipid-containing neuophysin was delipidated by treatment with choloform-methanol. The resultant apoproteins were characterized as bovine neuroions were developed for the reaggregation of purified bovine neurophysin-I and -II with lipids extracted from bovine posterior pituitary and hypothalamus and with synthetic lecithin. The resultant neurophysin lipid complexes have been shown to band upon isopycnic centrifugation at densities different from those of the respective purified bovine neurophysins.     

Isolation and amino acid sequence of cyclophilin

Cyclophilin, a specific cyclosporin A-binding protein has been purified to homogeneity from human spleen and bovine thymus cytosol. Purification of bovine and human cyclophilin was achieved by large scale molecular filtrations, Matrex Blue A affinity chromatography, preparative isoelectric focusing, phenyl-Sepharose chromatography, and weak cation exchange high performance liquid chromatography. Major and minor bovine and human cyclophilin isoforms were identified and found to have an apparent molecular weight of 17,000 and very similar amino acid compositions. The complete amino acid sequence of the major bovine cyclophilin isoform (163 residues, Mr 17,737) was determined from analysis of peptides derived by endoproteinase lysine C and cyanogen bromide cleavage and an NH2-terminal sequence of the intact protein. The first 72 NH2-terminal residues of the major human cyclophilin isoform were also determined and found to be identical to bovine cyclophilin. A computer search of cyclophilin with the National Biomedical Research Foundation database (3,182 protein sequences) did not detect any significant homologies. Cyclophilin represents a new class of abundant, highly conserved cytosolic proteins that probably play an important role in the regulation of T lymphocyte activation and proliferation.     

Isolation and characterization of δ-melanotropin,a new peptide from bovine pituitary glands

A new melanotropin (MSH) was isolated from bovine pituitary extract by means of gel filtration, ion exchange chromatography, high performance liquid chromatography and paper electrophoresis. Amino terminal analysis, amino acid composition and tryptic hydrolysis were performed on the purified peptide. The peptide was found to contain the amino acid sequence of γ-MSH, a theoretical segment of the proopiomelanocortin molecule. However, theoretical segment of the proopiomelanocortin molecule. However, the new peptide differs from the γ-MSH in several major ways, thus it is designated a bovine δ-MSH or δ_b-MSH.     

Phylogenetic Distribution of Peptides Related to Chromogranins A and B

The presence of chromogranin-related peptides in a wide range of species was investigated by one and two-dimensional electrophoresis followed by immunoblotting. Antisera against bovine chromogranins A and B and the peptide WE-14 (chromogranin A316-329) were used. Chromogranins were identified by their heat stability, by their electrophoretic behavior, and by immunological cross-reaction with antisera. In all species investigated ranging from mammals to birds, amphibians, fish, and arthropods, chromogranin A- and B-like proteins could be demonstrated. For all species, there was an immunological cross-reaction with antisera against bovine chromogranins. The molecular sizes and isoelectric points of the chromogranins were similar in all species. The antiserum against WE-14 cross-reacted with pig, rat, and chicken chromogranins. It is concluded that the chromogranins A and B have a widespread phylogenetic distribution with a significant conservation of molecular size, isoelectric points, and immunological epitopes. This is consistent with the concept that these peptides have a specific function.     

Arachidonic acid metabolites by cytochrome P-450 dependent monooxygenase pathway in bovine adrenal fasciculata cells

[1-14C]Arachidonic acid was incubated with microsomes of bovine adrenal fasciculata cells in the presence of 1 mM NADPH for 30 min at 37 degrees C. The metabolites were separated and purified by reverse phase high performance liquid chromatography, and identified by gas chromatography-mass spectrometry. Identified metabolites were four dihydroxyeicosatrienoic acids (DHTs) (5,6-, 8,9-, 11,12-, 14,15-DHTs), 20-hydroxyeicosatetraenoic acid and eicosatetradioic acid. The formation of these metabolites was dependent on NADPH and inhibited by SKF-525A. 14,15-DHT was also formed by isolated bovine adrenal fasciculata cells. These results indicate that cytochrome P-450 dependent arachidonate monooxygenase pathway may exist in bovine adrenal fasciculata cells. Addition of the chemically synthesized epoxyeicosatrienoic acids (EETs) to isolated bovine adrenal fasciculata cells stimulated cortisol production. Among four regioisomeric EETs, 14,15-EET was most potent and stimulated steroidogenesis in a dose-related manner over a range of 0.5 to 5.0 microM.     

Chromogranin A, B and C: immunohistochemical localization in ovine pituitary and the relationship with hormone-containing cells

Chromogranins A, B and C, three distinct groups of proteins found in bovine chromaffin granules, were also found to be present in the pituitary using immunoblotting techniques. Their distribution was therefore studied in the normal ram pituitary using an immunoperoxidase technique applied to semithin serial sections and compared with that of some of the hormones of the anterior pituitary. Chromogranin-immunoreactivity was found in gonadotrophs (all three), thyrotrophs (A with some positive for C) and corticotrophs (a fraction with A and fewer with B and C). The mammotrophs and somatotrophs were negative. Chromogranin C was the only one of the three to be located in the pars nervosa, whilst chromogranin B was rarely found in the pars intermedia. The results suggest that chromogranins A, B and C are not always stored together, some hormone-containing cells do not contain immunohistologically detectable levels of the chromogranins.     

Purification and partial sequence analysis of human interleukin-2 derived from peripheral blood leukocytes

Human peripheral blood leukocyte-derived interleukin-2 (IL-2) was resolved by DEAE-cellulose column chromatography into three peaks of activity, IL-2A, B, and C, with isoelectric points of 7.2, 6.6, and 7.9, respectively. IL-2 A, B, and C were further purified by reverse phase high performance liquid chromatography and resolved into two apparently homogeneous peaks each with identical molecular weight: A-1 and A-2 (Mr17000); B-1 and B-2 (Mr17500); and C-1 and C-2 (Mr14400). The amino acid compositions and partial NH2-terminal amino acid sequences of these molecular species were consistent with those predicted from IL-2 cDNA sequences derived from Jurkat and peripheral blood leukocytes.     

A method for systematic purification from bovine plasma of six vitamin K-dependent coagulation factors: prothrombin, factor X, factor IX, protein S, protein C, and protein Z

A systematic purification scheme is presented for the isolation of six vitamin K-dependent coagulation factors from bovine plasma in a functionally and biochemically pure state. The vitamin K-dependent proteins concentrated by the ordinary barium citrate adsorption were first separated into four fractions, fractions A, B, C, and D, by DEAE-Sephadex A-50 chromatography. From the pooled fraction A, protein S, factor IX, and prothrombin were purified by column chromatography on Blue-Sepharose CL-6B. Heparin-Sepharose chromatography of the pooled fraction B provided mainly pure factor IX, in addition to homogeneous prothrombin. A high degree of resolution of protein C and prothrombin from the pooled fraction C was obtained with a Blue-Sepharose column. This dye-ligand chromatographic procedure was also very effective for the separation of protein Z and factor X contained in the pooled fraction D. Thus, these preparative procedures allowed high recovery of milligram and gram quantities of six vitamin K-dependent proteins from 15 liters of plasma in only two chromatographic steps, except for protein S, which required three (the third step was rechromatography on Blue-Sepharose CL-6B).     

Isolation of alpha-lactalbumin,beta-lactoglobulin,and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity

The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Globulins were then precipitated from whey by half-saturated ammonium sulfate and beta-lactoglobulin was purified further using Sephadex G-50 gel filtration. The proteins in the supernatant were also fractionated using diethylaminoethyl cellulose chromatography in which beta-lactoglobulin was separated from alpha-lactalbumin and bovine serum albumin. The latter two proteins that co-eluted in anion-exchange chromatography were then gently isolated from each other by Sephadex G-50 gel filtration. Pure beta-lactoglobulin was also obtained by anion-exchange chromatography of the ammonium sulfate-precipitated globulins. Using enzyme-linked immunosorbent assay (ELISA), Western blotting, and ELISA inhibition assay, antigenicity of the purified proteins was evaluated. Our results showed high purity and well-preserved antigenicity of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin thus purified.     

Antitumor proteins of Streptomyces macromomyceticus: purification and characterization of auromomycin, macromomycin A, and macromomycin D

Macromomycin A and the two related proteins auromomycin and macromomycin D were isolated from the culture filtrates of Streptomyces macromomyceticus by chromatography on columns of DEAE-cellulose, Amberlite XAD-7, and decylagarose. Antibodies prepared against macromomycin A showed antigenic identity by Ouchterlony double diffusion between the three purified proteins. This similarity was further demonstrated by their behavior on disc gel electrophoresis, the amino acid compositions, and comparative peptide mapping of the aminoethylated derivatives. They differed, however, in other chemical and biological properties. Auromomycin and macromomycin A, pI 5.4, have antibiotic activity, which is absent in macromomycin D, pI 5.2. This antibiotic activity was associated with chromophore groups that were extractable by methanol. High-pressure liquid chromatography of the methanol extracts gave difference profiles for each of the purified proteins. The differences in the three proteins extended to their ultraviolet-visible spectra, fluorescence and circular dichroism, and the changes of these properties with heating. The heat denaturation, with auromomycin and macromomycin melting at 70.5 degrees C and macromomycin D at 57.0 degrees C, was reversible. Changes were noted in the spectra both during and following heating at 80 degrees C; the antibacterial activity was lost in auromomycin and only partially reduced in macromomycin A. The properties of the three proteins support the general similarities in their polypeptide structures, modifications in the properties of which are endowed by the differences in the associated nonprotein chromophores.     

Analysis of lysine-rich histones from the unicellular green alga Chlamydomonas reinhardtii

The H1 histones of the unicellular green alga Chlamydomonas reinhardtii were extracted from isolated nuclei, fractionated by high performance liquid chromatography, and analyzed by two-dimensional electrophoresis, peptide mapping, and N-terminal sequencing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 5% perchloric acid extracts of isolated C. reinhardtii nuclei revealed two H1 proteins (H1A and H1B). Two-dimensional gel analysis did not reveal heterogeneity of either algal H1 protein, but did detect differences in the hydrophobic amino acid content of the C. reinhardtii H1A and H1B. Digestion of H1A and H1B with V8 protease revealed two distinctly different peptide maps. C. reinhardtii H1 peptide maps were not at all similar to those of Pisum H1, but algal and pea H2B peptide maps did show some peptides in common. Seventeen amino acid residues were obtained from C. reinhardtii H1A amino terminal sequencing, while the H1B N-terminus was blocked. A search of protein data bases revealed no sequence homology of the H1A N-terminus with any known protein. Chlamydomonas histones fractionated by high performance liquid chromatography revealed minor components (histone variants) for H2A and H2B. The amino acid composition of Chlamydomonas lysine-rich histones was compared to those of various other unicellular algae.     

Reverse phase high performance liquid chromatography of Escherichia coli ribosomal proteins: standardization of 70 S, 50 S, and 30 S protein chromatograms

We recently described the use of reverse phase high performance liquid chromatography for the separation of the proteins of the 30 S subunit of Escherichia coli ribosomes (Kerlavage, A. R., Kahan, L., and Cooperman, B. S. (1982) Anal. Biochem. 123, 342-348). In the present studies we report improvements in the technique and its extension to the separation of the proteins of the 50 S subunit and of 70 S ribosomes. Using an octadecasilyl silica column and a trifluoroacetic acid/acetonitrile solvent system, the 21 proteins of the 30 S subunit have been resolved into 17 peaks, the 33 proteins of the 50 S subunit into 22 peaks, and the 53 proteins of the 70 S ribosome into 31 peaks. The proteins present in each peak have been identified by polyacrylamide gel electrophoresis, by comparison with previously standardized chromatograms, and by calibration with authentic samples of purified proteins. All of the known ribosomal proteins have been identified on the chromatograms with the exception of L31 and its variant, L31'. Three protein peaks, not corresponding to known ribosomal proteins, have been observed in preparations from the total protein from 50 S subunits and 70 S ribosomes, but the significance of these peaks is unclear. The reverse phase high performance liquid chromatography technique has the potential for purifying all ribosomal proteins, as demonstrated by the increase in resolution we obtain when a peak isolated under standard gradient conditions and containing several proteins is reapplied to the column and eluted with a shallower gradient. Its utility in preparing proteins for functional studies is demonstrated by a reconstitution of active 30 S particles using 30 S proteins prepared by reverse phase high performance liquid chromatography.     

Chromogranin B (secretogranin I), a putative precursor of two novel pituitary peptides through processing at paired basic residues

During the course of reversed-phase high-pressure liquid chromatography (RP-HPLC) purification of the 7B2 peptide originally isolated in our laboratory from human pituitary gland extracts, two novel peptides were identified and purified to homogeneity. The complete amino acid sequence of the first one was established in 1985 and recently found to be entirely homologous to positions 420-493 of the just published chromogranin B sequence. This peptide, denoted GAWK, could originate from chromogranin B following specific cleavage at the basic amino acids flanking both termini of GAWK. Moreover, another peptide isolated in our laboratory from the same source and denoted CCB has been discovered and its sequence is also part of the same chromogranin B molecule. Here again, this peptide, occupying positions 597-653 and located at the COOH-terminal region of chromogranin B, could derive from specific processing at basic amino acids, Arg-Lys-Lys, present at positions 594-596. In a manner reminiscent of the relationship between pancreastatin and chromogranin A, it is proposed that both GAWK and CCB are produced from chromogranin B after specific processing at basic amino acids. These data are thus in favor of a putative role of chromogranins as precursors to potentially bioactive peptides.     

Isolation and identification of two protein-protein crosslinks within the two subunits of Bacillus stearothermophilus ribosomes

Bacillus stearothermophilus 30S and 50S ribosomal subunits were isolated and crosslinked with diepoxybutane. The crosslinked proteins were extracted with LiCl or with 67% acetic acid and purified by a combination of different high performance liquid chromatography techniques. The protein fractions were analysed by two-dimensional and diagonal polyacrylamide gel electrophoresis and by immunological methods. Two crosslinked protein pairs, one from the large and one from the small subunit, consisting of proteins L23-L29 and S13-S19 respectively, were isolated in milligram amounts for determination of the crosslinked amino acids.     

Transformation of arachidonic acid by 5- and 15-lipoxygenase pathways in bovine adrenal fasciculata cells

[1-14C]Arachidonic acid was incubated with isolated bovine adrenal fasciculata cells for 15 min at 37gC. The metabolites were separated and purified by reverse- and straight-phase high performance liquid chromatography, and identified by gas chromatography-mass spectrometry or radioimmunoassay. Identified metabolites were 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), leukotriene B4 and 11,14,15-trihydroxy-5,8,12-eicosatrienoic acid (11,14,15-THET). Addition of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), an intermediate metabolite of 15-lipoxygenase pathway to microsomes of bovine adrenal fasciculata cells resulted in the formation of 11,14,15-THET. The formation of 11,14,15-THET by microsomes was not dependent on the presence of NADPH, while it was dose-dependently suppressed by ketoconazole, a potent inhibitor of cytochrome P-450 dependent enzymes. These results indicate that 5- and 15-lipoxygenase pathways of arachidonic acid may exist in bovine adrenal fasciculata cells and that 15-HPETE is further metabolized to 11,14,15-THET by adrenal microsomal cytochrome P-450.     

Physicochemical characterization of A and B subunits of Shiga toxin and reconstitution of holotoxin from isolated subunits

The A and B subunits of Shiga toxin were isolated by high performance liquid chromatography and their physicochemical properties were examined. The A subunit of Shiga toxin purified from culture supernatant was not nicked, but it could be nicked in vitro by trypsin. The isoelectric points of the A and B subunits were determined to be 8.2 and 5.8, respectively. Amino acid compositions of the two subunits were also determined. The isolated A and B subunits were reconstituted to form active holotoxin which showed lethal activity to mice which was similar to that of native Shiga toxin.