Purification of the Atlantic salmon hepatic 21 kDa protein and classification as a high mobility group chromatin protein.

The 21 kDa protein of liver from Atlantic salmon (Salmo salar) has been purified. Hepatic nuclei were extracted with 0.75 M HClO4. The extracted proteins were fractionated using reversed phase high performance liquid chromatography. The purity of the protein was analysed by isoelectric focusing in the first, and SDS-polyacrylamide gel electrophoresis in the 2nd dimension. Isoelectric focusing separated the protein into 5 spots. In gel trypsin digestion after isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis resulted in identical migration of the tryptic peptides. The amino acid composition of the 21 kDa protein was similar to that of high mobility group (HMG) proteins C and D from rainbow trout (Oncorhynchus mykiss). The N-terminal sequence of the amino acids 1-19 revealed a conserved region characteristic for HMG 14/17 proteins of mammals and avians, and their equivalents in rainbow trout. Considering the electrophoretic mobility, amino acid composition and N-terminal amino acid sequence it is concluded that the 21 kDa protein of Atlantic salmon is a member of the HMG protein family resembling the HMG D protein of rainbow trout.     

Chromogranin from normal human adrenal glands: purification by monoclonal antibody affinity chromatography and partial N-terminal amino acid sequence

A method is presented for the purification of human chromogranin from adrenal glands obtained at autopsy. The procedure involved homogenization of whole glands in aqueous buffer, salt precipitation, affinity chromatography using a highly specific monoclonal antibody (LK2H10) and reverse-phase high-pressure liquid chromatography. Chromogranin purified from autopsy adrenal glands revealed a high degree of polypeptide heterogeneity when analyzed by silver-stained SDS polyacrylamide gels. Greater than 90% of the protein was represented by a cluster of polypeptides with an Mr = 70 000 (i.e. chromogranin A), while the remaining protein was highly disperse in molecular weight. That these various polypeptides were in fact chromogranin was shown by Western blotting using monoclonal antibody LK2H10. About 6 nmol of chromogranin were obtained from 97 g of starting adrenals which was estimated to be a 25% yield and a 250-fold enrichment from adrenal homogenates. Critical to achieving reasonable yields of this protein was the need for particular low pH buffers for resuspension of chromogranin after solvent removal steps. Chromogranin purified from human adrenal glands was similar in amino acid composition, and identical in the N-terminal amino acid sequence (24 residues) to bovine chromogranin A. A secondary sequence representing 25% of the total protein and missing the first three residues of the N-terminus suggested the possibility of N-terminal processing of chromogranin in situ. The conservation of the N-terminal amino acid sequence of human and bovine chromogranin contrasts with the strong sequence variability predicted by antisera cross-reactivity and suggests that the N-terminus of chromogranin may be critical for its biological activity.     

Purification and partial characterization of a major outer-membrane protein of Fusobacterium nucleatum

The major outer-membrane proteins of 40-41 kDa were identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in Fusobacterium nucleatum strains ATCC 10953, ATCC 25586, F3, F6 and Fev1. The proteins were purified by preparative gel electrophoresis. Their behaviour in gel filtration and gel electrophoresis, their sensitivity to proteolytic enzymes, and their amino acid composition were investigated. The purified proteins were partly sequenced from the N-terminal end. A 36.5 kDa portion was protected against extrinsic proteolytic (trypsin, chymotrypsin or pronase) digestion of whole cells. This polypeptide was isolated and partially sequenced from the N-terminal end. From these data and data from extrinsic iodination it was concluded that the N-terminal end of the protein is probably exposed on the surface of the cell. A database search revealed amino acid sequence similarity in an Ala-Pro-rich region of outer-membrane protein A (OmpA) in other Gram-negative bacteria.     

Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase.

cDNA clones encoding mammalian poly(A) polymerase were isolated with probes generated by the polymerase chain reaction based on amino acid sequences derived from the purified enzyme. A bovine cDNA clone was obtained encoding a protein of 82 kDa. Expression in Escherichia coli resulted in the appearance of a poly(A) polymerase activity that was dependent on the addition of the purified specificity factor CPF and the presence of the polyadenylation signal AAUAAA in the RNA substrate. The activity copurified with a polypeptide of the expected size. A second class of cDNAs encoded a polypeptide of 43 kDa which was closely related to the N-terminal half of the 82 kDa protein. Northern blots showed two mRNAs of 4.2 and 2.4 kb that probably correspond to the two classes of cDNAs, as well as a third band of 1.3 kb. The sequence of the N-terminal half of bovine poly(A) polymerase is 47% identical with the amino acid sequence of the corresponding part of yeast poly(A) polymerase. Homologies to other proteins are of uncertain significance.     

Biosynthesis of betagranin in pancreatic beta-cells. Identification of a chromogranin A-like precursor and its parallel processing with proinsulin.

The biosynthesis of insulin and betagranin, a 20-21 kDa co-secreted chromogranin A-related protein, were investigated in isolated insulinoma cells and islets. The insulinoma tissue processed proinsulin to insulin with kinetics similar to those reported in islet tissue. Unlike islets, however, the insulinoma released almost one-quarter of the newly synthesized proinsulin into the medium 10-40 min after its formation. Betagranin was initially immunoprecipitated as a 100 kDa precursor form, which was indistinguishable from chromogranin A in size and immunoreactivity and by peptide mapping. After an initial lag of 10-20 min, the precursor was converted progressively into betagranin, which appeared to be a stable end product. Formation of betagranin and insulin from their respective precursors followed a parallel course and could likewise be inhibited by NH4+, chloroquine and monensin, added either before labelling or at any point of time up to 15 min after labelling. As with proinsulin, approximately one-quarter of the betagranin precursor was released 10-40 min after synthesis. It is concluded that betagranin is produced by limited proteolysis from a chromogranin A precursor in pancreatic beta-cells by a cellular pathway indistinguishable from that of insulin from proinsulin. Chromogranin A is highly conserved in the N-terminal region represented by betagranin, further suggesting that the biological activity of chromogranin A may reside in a derived peptide rather than in the parent molecule.     

The periplasmic flagella of Serpulina (Treponema) hyodysenteriae are composed of two sheath proteins and three core proteins.

The major components of the periplasmic flagella of the spirochaete Serpulina (Treponema) hyodysenteriae strain C5 were purified and characterized. We demonstrate that the periplasmic flagella are composed of five major proteins (molecular masses 44, 37, 35, 34 and 32 kDa) and present their location, N-terminal amino acid sequence and immunological relationship. The 44 kDa and the 35 kDa protein are on the sheath of the periplasmic flagellum, whereas the 37, 34 and 32 kDa protein reside in the periplasmic flagellar core. The two sheath flagellar proteins are immunologically related but have different N-terminal amino acid sequences. The N-terminus of the 44 kDa protein shows homology with the sheath flagellins of other spirochaetes, but the 35 kDa protein does not. The three core proteins are immunologically cross-reactive and their N-terminal amino acid sequences are almost, but not completely, identical, indicating that the core proteins are encoded by three distinct genes. The core proteins show extensive N-terminal sequence similarities and an immunological relationship with periplasmic flagellar core proteins of other spirochaetes.     

cDNAs encoding large venom proteins from the parasitoid wasp Pimpla hypochondriaca identified by random sequence analysis

Venom from the parasitoid wasp Pimpla hypochondriaca contains numerous proteins, has potent in vitro anti-haemocytic properties, and disrupts host encapsulation responses. By sequencing 500 cDNAs randomly isolated from a venom gland library, we have identified 60 clones that encode proteins containing potential secretory signal sequences. To identify cDNAs encoding particular venom proteins, N-terminal amino acid sequences were determined for large (>30 kDa) venom proteins that had been separated using a combination of gel filtration and SDS-PAGE. We describe five of these cDNAs, which encoded residues that matched with the N-terminal sequences of previously undescribed venom proteins. cDNAs vpr1 and vpr3 encoded related proteins of approximately 32 kDa that were found in widely different fractions of gel filtration-separated venom. Neither vpr1 nor vpr3 were closely related to any other protein in the GenBank database, suggesting that they are highly specialised venom components. vpr2 encoded a 57-kDa polypeptide that was similar to a Drosophila protein, of unknown function, which lacks a signal sequence. A fourth clone, tre1, encoded a 61-kDa protein with extensive sequence similarity to trehalases. The 76-kDa sequence encoded by lac1 contained three regions which were very similar to histidine-rich copper-binding motifs, and could be aligned with the laccase from the fungus Coprinus cinereus. This study represents a significant step towards a holistic view of the molecular composition of a parasitoid wasp venom.     

Intergeneric and intrageneric conjugal transfer of plasmids pAMβ1, pIL205 and pIP501 in Lactobacillus sake

Abstract Three membrane-bound acid-stable cytochromes c with molecular masses of 46, 30 and 21 kDa were characterized from a new Thiobacillus ferrooxidans strain. They were solubilized with high concentrations of dodecylmaltoside at pH 8. The 30 kDa cytochrome c was purified to a homogeneous state as established by SDS-PAGE analysis. It showed an absorption peak at 410 nm in the oxidized form and at 418, 523 and 552 nm in the reduced form. The 46 kDa cytochrome c co-purified with a non-heme protein of 36 kDa. The amino acid composition and the N-terminal amino acid sequence of the 46 kDa cytochrome c were determined and compared with those of the soluble 14 kDa and the membrane-bound 21, 22.3 and 68 kDa cytochromes c isolated from two different strains. The results clearly show that this cytochrome is distinct from both the 22.3, 21 and 14 kDa cytochrome species, and exhibits some similarities with the 68 kDa cytochrome c as regards its amino acid composition.     

Isoforms of corneal keratan sulfate proteoglycan

Bovine corneal keratan sulfate proteoglycan was found to contain three major protein components. Two proteins (37 and 25 kDa) were released from the proteoglycan by endo-beta-galactosidase, N-glycanase, or chemical deglycosylation. A smaller protein (20 kDa), not covalently linked to keratan sulfate, co-purified with the proteoglycan by conventional and high performance ion exchange chromatography, by ethanol precipitation, and by affinity purification on columns of monoclonal antibody to keratan sulfate, but could be separated from the proteoglycan by gel filtration chromatography in dissociative agents. The three proteins produced different fragmentation patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after digestion with V8 protease, and each had unique two-dimensional tryptic peptide maps. The N-terminal amino acid sequence of the core proteins differed. In addition, the proteoglycans containing these proteins differed in molecular size, suggesting different levels of glycosylation of the two core proteins. Similarity of the core proteins was suggested by similar amino acid composition, similarities in tryptic maps, and antigenic cross-reactivity. Corneal keratan sulfate proteoglycan, therefore, seems to occur in two different, but related, forms whose core proteins may represent members of a homologous family.     

Chromaffin granule and PC12 cell chondroitin sulfate proteoglycans and their relation to chromogranin A

Two major proteoglycans, which appear to be structurally closely related, were isolated from bovine chromaffin granule matrix proteins by ion-exchange chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis they have apparent average molecular sizes of 35-40 kDa (range of 23-75 kDa) and generate a 14-kDa core glycoprotein after chondroitinase treatment. Previous studies demonstrated that these two major chromaffin granule proteoglycans are very similar in terms of their peptide mapping patterns and carbohydrate composition (having a high proportion of tri- and tetraantennary N-glycosidic oligosaccharides, and O-glycosidic oligosaccharides consisting predominantly of disialyl derivatives of galactosyl(beta 1-3)N-acetylgalactosamine), and that they differed in these respects from the chromogranins. By using antisera to five synthetic peptide fragments of chromogranin A to stain immunoblots of purified chromaffin granule proteoglycans before and after chondroitinase treatment, we have now shown that these major proteoglycans are not immunochemically related to chromogranin A. However, it has recently been reported that some chromogranin A-immunoreactive material disappears after chondroitinase treatment, and our studies demonstrate that approximately 1-2% of the chromogranin A occurs in the form of a 110-kDa proteoglycan, which is converted to a 95-kDa core glycoprotein after chondroitinase treatment. Similar chromogranin A proteoglycans could be detected in rat PC12 pheochromocytoma cells, where they have a molecular size of 115-145 kDa and yield a 105-kDa core protein after chondroitinase treatment. Studies using antibodies to synthetic peptide fragments of chromogranin B (secretogranin I) did not provide any evidence that this related protein occurs in a proteoglycan form.     

The histone-like H protein of Escherichia coli is ribosomal protein S3.

We report the purification of four proteins from Escherichia coli that stimulate or inhibit inter- and/or intramolecular recombination promoted by the yeast plasmid-encoded FLP protein. The proteins are identified as the ribosomal proteins S3 (27 kDa), L2 (26 kDa), S4 (24 kDa), and S5 (16 kDa), on the basis of N-terminal sequence analysis. The S3 protein is found to be identical to H protein, an E. coli histone-like protein that is related to histone H2A immunologically and by virtue of amino acid content. The H protein/S3 identity is based on co-migration on polyacrylamide gels, heat stability, amino acid analysis, and effects on FLP-promoted recombination. These results are relevant to current studies on the structure of the E. coli nucleoid. Since the H protein has previously been found associated with the E. coli nucleoid, the results indicate that either (a) some ribosomal proteins serve a dual function in E. coli, or, more likely, (b) ribosomal proteins can and are being mis-identified as nucleoid constituents.     

Isolation, characterization, and N-terminal sequence studies of cuticular proteins from the migratory locust, Locusta migratoria

The cuticle of the migratory locust, Locusta migratoria, contains more than a hundred different structural proteins, which can be extracted before but not after the cuticle is sclerotized. Fourteen of the proteins have been purified, covering a pI range of 6.4-10.6 and a molecular mass range of 15.2-36.8 kDa. The amino acid sequence from the N-terminal, ranging in length over 10-59 residues, have been obtained for eight of the proteins. A number of similarities, both in amino acid composition and in sequences, indicate that the proteins belong to a new protein family, characterized by an N-terminal part which is rich either in glycine, tyrosine and leucine or in hydrophilic amino acids, followed by a very alanine-rich portion. Similarities between this family of proteins and other structural proteins from insects are discussed.     

A second form of collagenous lectin from the tunicate, Styela plicata

This study characterised a 90 kDa lectin from an invertebrate chordate, the tunicate Styela plicata. One- and two-dimensional electrophoresis showed that the apparent molecular weight of this protein is maintained under both reducing and non-reducing conditions, suggesting that its native form is a monomer. The 90 kDa lectin was localised within a single type of hemocyte (morula cells), but was secreted from those cells when tunicates were challenged with the inflammatory elicitor, zymosan. Functional studies showed that the 90 kDa protein binds to galactose-based sugars in a divalent cation-dependent manner. Amino acid composition analysis and N-terminal amino acid sequencing indicated that the 90 kDa lectin is related to a previously characterised, collagenous lectin from S. plicata, splic43. However, peptide mass fingerprinting identified numerous differences between the two proteins. This suggests that the 90 kDa molecule represents a novel protein that is involved in host defence.     

Purification and characterization of a DnaK-like and a GroEL-like protein from Porphyromonas gingivalis

Heat-shock proteins of Porphyromonas gingivalis were demonstrated and two of them were purified and further characterized. The amplified de novo synthesis of two different proteins, with apparent molecular weights of 75 kDa and 68 kDa, was observed by autofluorography when a P. gingivalis culture incubated in a 14C-labeled amino acid mixture was shifted from 37 degrees C to 44 degrees C. Both proteins possessed ATP-binding abilities and were purified to almost homogeneity employing affinity chromatography on ATP-agarose followed by preparative SDS-PAGE. Purified 75 kDa and 68 kDa proteins had isoelectric points of 4.4 and 4.6, respectively. They were shown to be immunoreactive with commercial anti-DnaK and anti-GroEL polyclonal antibodies, respectively. Immunoblotting analysis of whole cells using antiserum raised against each purified protein from P. gingivalis, confirmed elevated synthesis of both proteins during thermal shock. A GroEL protein reacted strongly with antiserum against the 68 kDa protein. However, a DnaK protein reacted weakly with antiserum to the 75 kDa protein. Analysis of the N-terminal amino acid sequence of the DnaK-like protein (75 kDa) showed a high degree of homology with those of the HSP70 family including both prokaryotic and eukaryotic cells. The N-terminal amino acid analysis of the GroEL-like protein (68 kDa) indicated that it was identical to those of cloned GroEL homologues from P. gingivalis.     

Chromogranin B: isolation from pheochromocytoma, N-terminal sequence, tissue distribution and secretory vesicle processing

The chromogranins/secretogranins are a family of neuroendocrine vesicle secretory proteins. Immunohistology and immunoblotting have suggested that a major soluble protein in human chromaffin granules may be chromogranin B (CgB). We purified from pheochromocytoma chromaffin granules an SDS-PAGE 110-120 kDa protein whose N-terminal sequence matched that previously deduced from a human CgB cDNA. An antibody directed against a synthetic human CgB N-terminal region specifically recognized the CgB N-terminus, though not the chromogranin A (CgA) N-terminus or the CgB C-terminus on immunoblots. An antiserum directed against CgB's C-terminus also visualized CgB but not CgA. By immunoblotting, CgB was a quantitatively major protein in human pheochromocytoma chromaffin granules, but a relatively minor in normal bovine adrenal medullary chromaffin granules. In a variety of normal bovine neuroendocrine tissues, the relative abundance of CgB immunoreactivity on immunoblots was: adrenal medulla greater than anterior pituitary greater than pancreas greater than small intestine, hypothalamus. Immunoblotting of neuroendocrine tissues (or their hormone storage vesicle cores) with both anti N-terminal and anti C-terminal CgB antisera suggested bidirectional cleavage or processing of CgB; in the anterior pituitary, a unique 40 kDa C-terminal fragment was observed. Bidirectional CgB cleavage was also suggested on immunoblots of chromaffin tissue from three species (human, bovine, rat). C-terminal processing of CgB was also confirmed by amino acid sequencing of SDS-PAGE-separated, polyvinylidene difluoride membrane-immobilized CgB fragments from pheochromocytoma chromaffin granules. Whether such fragments possess biological activity remains to be investigated.     

The parasporal inclusion of Bacillus thuringiensis subsp. shandongiensis: characterization and screening for insecticidal activity.

The parasporal body of Bacillus thuringiensis subsp. shandongiensis was characterized in terms of its structure, protein composition, and toxicological properties against several types of insects. The crystals of B. thuringiensis shandongiensis appear to consist of a major protein of 144 kDa present in an spherical inclusion, as determined by transmission electron microscopy, titration curve analysis, and SDS-PAGE of the solubilized crystals. A second protein of ca. 60 kDa is present in trace amounts and appears to be associated with a small bar-shaped inclusion. The 144-kDa protein has been characterized by isoelectric point determination, N-terminal amino acid sequence analysis, amino acid analysis, and immunological cross reactivity. Its N-terminal amino acid sequence differed from that of other B. thuringiensis crystal proteins. The 144-kDa protein was not immunologically related to the crystal proteins of two toxic serovars (B. thuringiensis israelensis and B. thuringiensis kurstaki HD-1) and one nontoxic serovar (B. thuringiensis indiana), as shown in immunoblots probed with antiserum raised against the 144-kDa B. thuringiensis shandongiensis protein, the B. thuringiensis israelensis crystal proteins, and the trypsin resistant fragment of B. thuringiensis kurstaki P1 proteins. In contrast to most B. thuringiensis serovars, B. thuringiensis shandongiensis crystals did not dissolve at pH 12. Solubilization was achieved in sodium bicarbonate at pH 8.3 and in the presence of 25 mM dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and cDNA cloning of ovarian cortical rod protein in kuruma prawn Marsupenaeus japonicus (Crustacea: Decapoda: Penaeidae)

Two cortical rod proteins having molecular weights of 28.6 kDa and 30.5 kDa were isolated from the mature ovary of Marsupenaeus japonicus using gel filtration and reversed-phase HPLC. Analysis of the N-terminal amino acid sequence of the 28.6 kDa molecule revealed that amino acid residues 1-21 corresponded to residues 9-29 of the 30.5 kDa molecule. Examination of homology using BLAST showed that 21 amino acids out of 29 residues of the 28.6 kDa molecule, and 14 out of 29 residues of the 30.5 kDa molecule were identical to that of the ovarian cortical rod proteins of Penaeus semisulcatus. Positive immunohistochemical reaction to antiserum raised against the 28.6 kDa protein was observed on cortical rods forming around the periphery of oocytes at the maturation stages. Western blotting analysis revealed that both the 28.6 kDa and 30.5 kDa molecules stained with the anti-28.6 kDa antiserum. Furthermore, the 28.6 kDa and 30.5 kDa proteins were both glycosylated, as evidenced by positive carbohydrate staining using Concanavalin A and production of positive PAS reaction. These results indicate that the cortical rods are comprised of the 28.6 kDa and 30.5 kDa molecules. We subsequently cloned two full-length cDNAs based on the N-terminal sequences of the 28.6 kDa and 30.5 kDa molecules. The open reading frame of 28.6 kDa and 30.5 kDa encoded 276 amino acid residues. Comparison analysis of the two cDNAs revealed that the location of the processing site and sequence of signal peptides differed, indicating that the two cDNAs are products of two separate genes and encode the 28.6 kDa molecule and 30.5 kDa molecule, respectively. Both proteins possessed one potential N-linked glycosylation site. It is considered that both molecules are components of the cortical rods, forming a jelly layer after fertilization.