The complete primary structure of phospholipase A2 from human pancreas

The complete amino acid sequence of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from human pancreas was determined. The protein consists of a single polypeptide chain of 125 amino acids and has a molecular weight of 14003. The chain is cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digestion of the reduced and thialaminated derivative of the protein with clostripain, yielding three fragments. The largest fragment (residues 7-100) was further degraded both with staphylococcal proteinase and chymotrypsin. The sequence was determined by automated Edman degradation of the intact protein and of several large peptide fragments. Phospholipase A2 from human pancreas contains the same number of amino acids (125) as the enzyme from horse, while the enzymes from pig and ox contain 124 and 123 residues, respectively. The enzymes show a high degree of homology; human phospholipase differs from the other enzymes by substitutions of 26 (porcine), 28 (bovine) and 32 (equine) residues, respectively.     

Mapping of the microvillar 110K-calmodulin complex (brush border myosin I). Identification of fragments containing the catalytic and F-actin-binding sites and demonstration of a calcium ion dependent conformational change

In intestinal microvilli, the 110K-calmodulin complex is the major component of the cross-bridges which connect the core bundle of actin filaments to the membrane. Our previous work showed that the 110-kDa polypeptide can be divided into three functional domains: a 78-kDa fragment that contains the ATPase activity and the ATP-reversible F-actin-binding site, a 12-kDa fragment required for binding calmodulin molecules, and a terminal 20-kDa domain of unknown function [Coluccio, L. M., & Bretscher, A. (1988) J. Cell Biol. 106, 367-374]. By analysis of limited alpha-chymotryptic cleavage products, we now show that the molecular organization is very similar to that described for the S1 fragment of myosin. The catalytic site was identified by photoaffinity labeling with [5,6-3H]UTP, and fragments binding F-actin were identified by cosedimentation assays. Cleavage of the 78-kDa fragment yielded major fragments of 32 and 45 kDa, followed by cleavage of the 45-kDa fragment to a 40-kDa fragment. Of these, only the 32-kDa fragment was labeled by [5,6-3H]UTP. Physical characterization revealed that the 45- and 32-kDa fragments exist as a complex that can bind F-actin, whereas the 40-kDa/32-kDa complex cannot bind actin. We conclude that the catalytic site is located in the 32-kDa fragment and the F-actin-binding site is present in the 45-kDa fragment; the ability to bind actin is lost upon further cleavage of the 45-kDa fragment to 40 kDa. Peptide sequence analysis revealed that the 45-kDa fragment lies within the molecule and suggests that the 32-kDa fragment is the amino terminus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of a DNA- and heparin-binding domain (Domain III) in human plasma fibronectin

The complete amino acid sequence of a DNA- and heparin-binding domain isolated by limited thermolysin digestion of human plasma fibronectin has been obtained. The domain contains 90 amino acids with a calculated molecular weight of 10,225. The apparent molecular mass of this domain is 14 kDa when analyzed by sodium dodecyl sulfate-gel electrophoresis. The anomalously high molecular size estimation may be due to the inaccuracy of this method in the low range. The structure was established from microsequence analysis of the chymotryptic, tryptic, and Staphylococcus aureus protease peptides. The molecular ion of each of the chymotryptic peptides was obtained by fast atom bombardment mass spectrometry. The domain has a preponderance of basic residues with a net charge of +5 at neutral pH. The basic nature of the domain may account for its affinity for the polyanions, DNA and heparin. The predicted secondary structure is beta-sheet, in common with all of the type III internal sequence homology structures obtained for fibronectin so far. The location of the domain in fibronectin was made possible by limited thermolysin digestion and identification of the fragments and by comparison of the sequence of the 14-kDa fragment with the partial structure of bovine plasma fibronectin. The domain comprises residues 585-675 and defines a region immediately adjacent to the collagen-binding domain. Numbering domains beginning at the amino terminus, this domain is Domain III after the fibrin/heparin/actin/S. aureus binding Domain I and the collagen-binding Domain II. The domain was obtained from a larger precursor (56 kDa) which bound heparin, DNA, and gelatin. Further digestion of the 56-kDa fragment gave rise to a 40-kDa fragment which only bound gelatin, and a 14-kDa fragment which only bound heparin or DNA. The 14-kDa fragment (Domain III) marks the beginning of the type III homology region in fibronectin, for there may be up to 15 repeats of 90 amino acids. The size of this domain corresponds to one repeat of 90 amino acids and it has some sequence homology to the other type III sequences found thus far in fibronectin.     

Amino acid sequence studies on cyanogen bromide peptides of chicken caldesmon which bind to calmodulin

Three major calmodulin-binding cyanogen bromide peptides (fragments A, B, and D) were isolated from chicken gizzard muscle caldesmon and their amino acid sequences were determined. The molecular masses of fragments A, B, and D were estimated to 16, 12, and 9 kDa, respectively, by SDS-urea polyacrylamide gel electrophoresis. Fragment A was composed of 102 amino acid residues and contained homoserine at the C terminus. The amino acid sequence from the 37th residue of fragment A corresponds to the N-terminal sequence of the 15 kDa peptide which was obtained by thrombin digestion [Mornet, D., Audemard, E., & Derancourt, J. (1988) Biochem. Biophys. Res. Commun. 154, 564-571]. Thrombin 15 kDa peptide binds to F-actin but does not bind to calmodulin. Thus the N-terminal 36 residues and the C-terminal part from the 37th residue of fragment A are supposed to bind to calmodulin and F-actin, respectively. The sequences of fragments B and D were identical, but fragment D was composed of 64 amino acid residues and ended with tryptophan, whereas fragment B was of 98 or 99 amino acid residues and ended with proline. Both fragments B and D are supposed to be the C-terminal peptides of chicken caldesmon. Fragment B had heterogeneous sequences at the C-terminal region. These results can explain the reported heterogeneity of chicken caldesmon in charge and molecular mass.     

The amino acid sequences of two 13 kDa polypeptides and partial amino acid sequence of 30 kDa polypeptide of complex I from bovine heart mitochondria: possible location of iron-sulfur clusters

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is the most complicated system in the respiratory chain. It consists of many subunits, some of which hold iron-sulfur clusters, but structural information is still limited. The amino acid sequences of two 13 kDa polypeptides, 13 kDa-A and 13 kDa-B polypeptides, of iron-sulfur protein fraction (IP) of bovine heart mitochondrial complex I were determined by a combination of protease digestion, Edman degradation, and carboxypeptidase digestion. The 13 kDa-A polypeptide was composed of 96 amino acids with a molecular weight of 10,536. The 13 kDa-B polypeptide consisted of 114 amino acids and had an acetylated amino terminus. The molecular weight of this protein was calculated to be 13,130 including the acetyl group. These proteins had no obvious sequence similarity to other known proteins. The partial amino acid sequence of 30 kDa-B polypeptide of IP was also determined to reveal a characteristic arrangement of cysteine residues that could be involved in iron-sulfur cluster formation.     

Identification of suberization-associated anionic peroxidase as a possible allergenic protein from tomato

A 45 kDa protein, which is recognized by IgE antibodies in sera of food-allergic patients, was purified and characterized as an allergenic protein from the tomato. The IgE-binding protein purified from tomato extract was found to be a glycoprotein with a molecular weight of approximately 45,000, an isoelectric point of 4.2, and no free N-terminal amino group. Furthermore, it was shown that the purified protein had peroxidase activity. From the amino acid sequence of a peptide fragment prepared by lysylendopeptidase digestion, the allergenic protein was identified to be the tomato suberization-associated anionic peroxidase 1 known as one of the pathogenesis-related proteins widely distributed in plants. These properties suggested the protein isolated from tomato to be a new allergenic protein in plant foodstuffs.     

Structural analysis of rod GTP-binding protein, Gt. Limited proteolytic digestion pattern of Gt with four proteases defines monoclonal antibody epitope.

The epitope of monoclonal antibody (mAb 4A), which recognizes the alpha subunit of the rod G protein, Gt, has been suggested to be both at the carboxyl terminus (Deretic, D., and Hamm, H.E. (1987) J. Biol. Chem. 262, 10839-10847) and the amino terminus (Navon, S.E., and Fung, B.K.-K. (1988) J. Biol. Chem. 263, 489-496) of the molecule. To characterize further the mAb 4A binding site on alpha t and to resolve the discrepancy between these results limited proteolytic digestion of Gt or alpha t using four proteases with different substrate specificities has been performed. Endoproteinase Arg-C, which cleaves the peptide bond at the carboxylic side of arginine residues, cleaved the majority of alpha t into two fragments of 34 and 5 kDa. The alpha t 34-kDa fragment in the holoprotein, but not alpha t-guanosine 5'-O-(3-thiotriphosphate), was converted further to a 23-kDa fragment. A small fraction of alpha t-GDP was cleaved into 23- and 15-kDa fragments. Endoproteinase Lys-C, which selectively cleaves at lysine residues, progressively removed 17 and then 8 residues from the amino terminus, forming 38- and 36-kDa fragments. Staphylococcus aureus V8 protease is known to remove 21 amino acid residues from the amino-terminal region of alpha t, with the formation of a 38-kDa fragment. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin cleaved alpha t progressively into fragments of known amino acid sequences (38, then 32 and 5, then 21 and 12 kDa) and a transient 34 kDa fragment. The binding of mAb 4A to proteolytic fragments was analyzed by Western blot and immunoprecipitation. The major fragments recognized by mAb 4A on Western blots were the 34- and 23-kDa fragments obtained by endoproteinase Arg-C and tryptic digestion. Under conditions that allowed sequencing of the 15- and 5-kDa fragments neither the 34- nor the 23-kDa fragments could be sequenced by Edman degradation, indicating that they contained a blocked amino terminus. The smallest fragment that retained mAb 4A binding was the 23-kDa fragment containing Met1 to Arg204. Thus the main portion of the mAb 4A antigenic site was located within this fragment, indicating that the carboxyl-terminal residues from Lys205 to Phe350 were not required for recognition by the antibody. Additionally, the antibody did not bind the 38- and 36-kDa or other fragments containing the carboxyl terminus, showing that the amino-terminal residues from Met1 to Lys17 were essential for antibody binding to alpha t.     

Identification and purification of GTP-binding regulatory proteins from plasma membranes of swine heart

A 23 kDa GTP-binding protein was purified from pig heart sarcolemma. This protein was not ADP-ribosylated by cholera, pertussis and botulinum C3 toxins. In pig heart sarcolemma pertussis toxin ADP-ribosylated 40 kDa subunit of Gi-protein, cholera toxin--45 kDa subunit of Gs-protein, botulinum C3 toxin ADP-ribosylated a group of proteins with Mr 22, 26 and 29 kDa. Antiserum generated against the peptide common for all alpha-subunits of G-proteins did not react with purified 23 kDa protein. Trypsin cleaved the 23 kDa protein in the presence of guanyl nucleotides into a 22 kDa fragment. Proteolysis of the 39 kDa alpha 0-subunit from bovine brain plasma membranes and ADP-ribosylated 40 kDa alpha i-subunit from pig heart sarcolemma in the presence of GTP gamma S yielded the 37 and 38 kDa fragments, respectively. In the presence of GTP and GDP the proteolysis of alpha 0 yielded the 24 and 15 kDa fragments, while the proteolysis of ADP-ribosylated alpha i-subunit yielded a labelled 16 kDa peptide. Irrespective of nucleotides trypsin cleaved the ADP-ribosylated 26 kDa substrate of botulinum C3 toxin into two labelled peptides with Mr 24 and 17 kDa. The data obtained indicate the existence in pig heart sarcolemma of a new 23 kDa GTP-binding protein with partial homology to the alpha-subunits of "classical" G-proteins.     

Phosphopeptide analysis of phenylalanine hydroxylase isolated from liver cells exposed to hormonal stimuli.

Hormonal control of the phosphorylation of phenylalanine hydroxylase was studied by using rat liver cells incubated with [32P]Pi. After immunoprecipitation from cell extracts, the hydroxylase was subjected to proteinase digestion and subsequent sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. V8-proteinase digestion yielded one major 32P-labelled fragment, of approx. 9 kDa. Chymotrypsin digestion gave five 32P-labelled fragments ranging from approx. 39 kDa to approx. 10 kDa. Noradrenaline (10 microM) and glucagon (0.1 microM) enhanced the 32P content of all peptide fragments uniformly. Phorbol ester, in contrast with ionophore A23187, did not stimulate enzyme phosphorylation or enhance phenylalanine metabolism in liver cells. These results are discussed in relation to the nature of the protein kinase(s) that mediate phosphorylation of phenylalanine hydroxylase in liver cells.     

Affinity purification and characterization of an anti-PEG IgM

Anti-PEG IgM was purified by affinity chromatography using variable length PEG chains (5, 10, 20 and 30 kDa) as affinity ligands. Maximal binding of anti-PEG IgM was observed using the 30 kDa PEG-derivatized NuGel (single passage). Purified anti-PEG IgM was characterized for binding to PEG functionalized proteins/peptides by surface plasmon resonance, western blotting and ELISA. Anti-PEG IgM, in solution and adsorbed on 20 kDa PEG-derivatized NuGel, was subjected to pepsin digestion followed by affinity chromatography. SDS-PAGE analysis of eluates in both preparations yielded one fragment that was similar in size. However, an additional lower molecular weight band was observed in solution-digested affinity purified material that was not present in the eluate from the material subjected to pepsin digestion on the affinity matrix. The lower MW fragment could be eluted under milder conditions, suggesting loss of binding multiplicity. Analysis by mass spectrometry yielded molecular weights of 132 kDa (both) and 82 kDa (solution) for the respective fragments. N-terminal sequencing of both fragments resulted in primary sequences (heavy and light chains) that were not only identical to each other but also to those of native IgM. The anti-PEG IgM fragments were characterized for binding to pegylated interferon alfa-2a by ELISA. The results from these studies suggest that affinity purified anti-PEG IgM and fragments can be used as probes in detection assays for PEG functionalized biotherapeutics in pre-clinical and clinical studies.     

Characterization of a flagellar sheath component, PF60, and its structural gene in marine Vibrio

The Polar flagella (Pof) of Vibrio alginolyticus are surrounded by a membrane structure called a sheath. Five major proteins, whose molecular masses are 60, 47, 45, 44, and 18 kDa (named PF60, PF47, PF45, PF44, and PF18, respectively), have been detected in polar flagella. PF47 and PF45 have been identified as flagellins while the other proteins are thought to be sheath-associated ones. In this study, we isolated and partially characterized a major sheath protein, PF60. We found that PF60 can be solubilized by Triton X-100 treatment, but not by heat or acid treatment. After digestion with a peptidase, the N-terminal sequences of several fragments were determined and the N-terminus of intact PF60 seemed to be blocked. Through PCR in conjunction with oligonucleotide primers deduced from the peptide sequences, a DNA fragment of PF60 was amplified. A 4.5 kb HindIII restriction fragment was cloned by colony hybridization using the PCR fragment. Subsequent sequence analysis revealed three complete and one partial open reading frame (ORFs). The three ORFs, which exhibit sequence homology, correspond to PF60 (named pfsA), an amino acid transport ATP-binding protein, and an amino acid binding periplasmic protein. The single pfsA gene constitutes an operon and encodes a protein of 491 amino acids containing a putative signal peptide sequence at the N-terminal. A sequence database search revealed no homologous protein. However, PfsA seems to resemble lipoproteins in the N-terminal signal sequence and the biochemical data obtained in this study are consistent with that PfsA is a lipoprotein. The expression of the pfsA gene may be coordinately regulated with flagellar formation and similarly regulated to PF47 flagellin.     

Evidence that platelet and skeletal sarcoplasmic reticulum Ca2+-ATPase are structurally distinct

Proteolytic digestion and indirect immunostaining were used to compare the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins. When the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins were digested in the native state with trypsin, the platelet Ca2+-ATPase, which had an apparent undigested molecular mass of 103 kDa, yielded 78-kDa and 25-kDa fragments. Calcium transport activity depended on the integrity of the 103-kDa protein, while the digested protein had residual ATPase activity. Tryptic digestion of the sarcoplasmic reticulum pump protein, which also had an undigested molecular mass of 103 kDa, yielded products with apparent molecular masses of 55 kDa, 36 kDa, and 26 kDa. Distinct patterns were also observed when the platelet and sarcoplasmic reticulum calcium pump proteins were digested with chymotrypsin and Staphylococcus aureus protease in the presence of sodium dodecyl sulfate. Chymotrypsin digestion of the platelet protein resulted in the appearance of products with apparent molecular masses of 70 kDa, 39 kDa, and 31 kDa, while a similar digestion of the sarcoplasmic reticulum calcium pump protein yielded 54-kDa, 52.5-kDa, 46-kDa, 41-kDa, and 36-kDa fragments. Exposure of the sarcoplasmic reticulum and platelet Ca2+-ATPase proteins to S. aureus protease also yielded dissimilar fragmentation patterns. These results indicate that the Ca2+-ATPases from platelets and sarcoplasmic reticulum are distinct proteins.     

The archaebacterial hypusine-containing protein. Structural features suggest common ancestry with eukaryotic translation initiation factor 5A.

The amino acid hypusine is formed by post-translational modification of a lysine residue in eukaryotes and archaebacteria but up to now only the eukaryotic translation initiation factor eIF-5A has been known to contain this unique component. We isolated and purified a hypusine-containing protein from the thermophilic archaebacterium Sulfolobus acidocaldarius. The mainly cytosolic protein comprised about 0.03% of the post-ribosomal supernatant protein. No other hypusine-containing protein could be detected in S. acidocaldarius. The molar ratio of hypusine/hypusine-containing protein was 1:1. SDS/PAGE showed a molecular mass of 16.8 kDa; a pI of 7.8 for the native protein resulted from IEF. The N-terminus was blocked. Four cyanogen bromide fragments were partially sequenced and used to derive two 17-base oligonucleotide probes. A 3-kb HindIII fragment of genomic DNA hybridizing with both probes was cloned. By sequencing of exonuclease III deletion clones an open reading frame of 405 nucleotides was found coding for a protein of 135 amino acids with a molecular mass of 15 kDa. It contained all cyanogen bromide sequences analysed. Sequence alignment revealed that seven of eight residues around Lys40 in the Sulfolobus hypusine-containing protein were identical to the nonapeptides centered by hypusine in the three eIF-5A proteins sequenced so far. The Edman procedure gave no phenylthiohydantoin derivative for this position. For a central region of 44 residues a sequence similarity of 54% between the archaebacterial and eukaryotic proteins was calculated; for the total sequence about 33% similarity resulted. In addition, there were a number of conservative changes. The unique lysine modification surrounded by a conserved sequence strongly suggests a common ancestry of archaebacterial hypusine-containing protein and eIF-5A. Together with similarities in molecular mass and intracellular localization, it may point to an analogous biochemical function.     

In vitro cytotoxicity of non-cyt inclusion proteins of a Bacillus thuringiensis isolate against human cells, including cancer cells

A soil isolate designated 90-F-45-14, belonging to Bacillus thuringiensis serovar dakota (H15), was examined for characterization of in vitro cytotoxicity, associated with parasporal inclusion proteins, against human cells. When activated with proteolytic processing, inclusion proteins of the isolate 90-F-45-14 exhibited a moderate cytotoxicity against the human uterus cervix cancer cells (HeLa) with an EC(50) value of 60.8 microg ml(-1), while showing extremely high activities on the human leukaemic T cells (MOLT-4) and the normal T cells with EC(50) values of 0.27 and 0.20 microg ml(-1), respectively. Anti-leukaemic cell activity of the 90-F-45-14 proteins was eight to nine times greater than that of the B. thuringiensis serovar israelensis proteins containing the Cyt1 protein, a broad-spectrum cytolysin. The cytopathy by the 90-F-45-14 proteins was characterized by marked cell-ballooning, while the israelensis proteins induced early breakdown of the cells due to cytolysis. Inclusions of the isolate consisted of five major polypeptides of 170, 103, 73, 40 and 32 kDa. A 100% homology was observed in the sequence of 15 N-terminal amino acids between the proteins of 170 and 103 kDa. There was no N-terminal sequence homology between 90-F-45-14 proteins and the existing Cry/Cyt proteins of B. thuringiensis. Proteolytic processing by proteinase K yielded several proteins with molecular masses ranging from 40 to 28 kDa.     

The Ch21 protein, developmentally regulated in chick embryo, belongs to the superfamily of lipophilic molecule carrier proteins

Ch21, a developmentally regulated low molecular weight protein observed in chick embryo skeletal tissues, is expressed "in vitro" by differentiating chondrocytes at a late stage of development. Here we report the complete amino acid sequence of the protein. 86% of the total amino acid sequence was deduced by sequences of 17 high performance liquid chromatography-separated proteolytic fragments and 33 amino acid residues at the amino-terminal end of protein purified from spent culture medium of hypertrophic chondrocytes. Furthermore we isolated by molecular cloning the corresponding cDNA and determined its nucleotide sequence. By combining protein and nucleotide sequence data we determined the primary structure of the entire Ch21. It consists of 158 amino acids and has a molecular mass of 18.065 kDa. Computer-assisted analysis showed that the Ch21 belongs to the superfamily of low molecular weight proteins sharing a basic framework for binding and transport of small hydrophobic molecules.     

Molecular cloning of a full-length cDNA for human alpha-N-acetylgalactosaminidase (alpha-galactosidase B)

In the process of molecular cloning of cDNA for proteins associated with a purified human placental sialidase fraction, we discovered one of the proteins with apparent molecular weight of 46 kDa is in reality alpha-N-acetylgalactosaminidase. The full length cDNA, pcD-HS1204, codes for 358 amino acids with the first 17 residues representing a putative signal peptide. The predicted amino acid sequence shows striking homology with human alpha-galactosidase A and yeast alpha-galactosidase. The substrate specificities as well as the behavior of the 46 kDa protein on hydroxylapatite chromatography confirmed that the 46 kDa protein is in reality alpha-N-acetylgalactosaminidase.     

Plant protein hydrolysates as fish fry feed in aquaculture. Hydrolysis of rapeseed proteins by an enzyme complex from king crab hepatopancreas

Rapeseed proteins were processed by an enzyme complex isolated from king crab hepatopancreas in order to obtain a hydrolysate for use as fish fry feed. The amino acid composition of the obtained protein preparation was close to the amino acid composition of fishmeal traditionally used in the production of fish feed. SDS-PAGE, HPLC, and mass spectrometric analysis of the products of enzymatic hydrolysis of rapeseed proteins showed that the proteins were hydrolyzed to a high degree. The composition of the hydrolysates depended on the hydrolysis time and included free amino acids (27% of the total weight of the protein mix after 3 h of hydrolysis and 56% after 21 h of hydrolysis), short peptides (2 to 20 amino acid residues), and small amounts of protein fragments with a molecular weight of approximately 14 kDa, as shown by by SDS-PAG electrophoresis.     

Isolation and NH2-terminal amino acid sequences of rat serum carrier proteins for insulin-like growth factors

Three N-glycosylated carrier proteins (CP) for insulin-like growth factors (apparent molecular weights 30-32, 42 and 45 kDa) were isolated from adult rat serum. They share the same amino terminus (up to amino acid 31) and are constituents of the growth hormone-dependent native 150-200 kDa IGF carrier complex. Residues 12-31 display 60 and 50% sequence homology, respectively, to residues 2-21 of fetal rat and to residues 4-22 of a human amniotic fluid IGF carrier protein. No homology exists with the type I or II IGF receptors. Adult rat serum also contains a fourth IGF CP (24 kDa) whose 9 NH2-terminal amino acids are identical to those of the fetal form. Our findings suggest that the three N-glycosylated components originate from the same IGF carrier protein (adult form) and that the 24 kDa protein is a separate (fetal) species.     

Identification and characterization of major bovine serum tyrosine-O-sulfate-binding protein as a complement factor H

A major tyrosine-O-sulfate (TyrS)-binding protein present in bovine serum was purified to electrophoretic homogeneity using a combination of TyrS-Affi-Gel 10 affinity chromatographyy, DEAE-Bio-Gel A ion-exchange chromatography, and hydroxylapatite chromatography. The purified TyrS-binding protein migrated as doublet protein bands with apparent molecular weights of ca. 160, 000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. N-termini of the two forms of purified TyrS-binding protein contain most likely identical sequence for the first fifteen amino acids residues, which displays a high degree of homology to those of human and mouse complement factor H. Furthermore, the purified TyrS-binding protein exhibited immunologic cross-reactivity with anti-human complement factor H. These results indicate the identity of the purified TyrS-binding protein being bovine complement factor H. The two forms of the purified bovine factor H were investigated with respect to the sensitivity to limited trypsin digestion. The high-molecular weight form was cleaved into two fragments with apparent molecular masses of, respectively, 45 kD and 125 kD. The low-molecular weight form was cleaved in a different manner to generate three major fragments with molecular masses of 25 kD, 45 kD and 100 kD, respectively. Limited V8 protease mapping of the two forms yielded similar, yet unidentical, peptide band patterns. Purified bovine factor H appeared to bind agarose-bonded heparin through its anion-binding domain and the binding was inhibited by the presence of free heparin or dextran sulfate.Abbreviations HEPES N-2-hydroxylpiperazine-N-2-ethanesulfonic acid - NP-40 Nonidet P-40 - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TyrS tyrosine-O-sulfate     

Purification and characterization of S layer proteins from Clostridium difficile GAI 0714

The S layer of Clostridium difficile GAI0714 was shown to be composed of two proteins, of 32 kDa and 45 kDa, as determined by SDS-PAGE. The two proteins were extracted with 8 M-urea (pH 8.3) from a cell wall preparation and purified by DEAE-Sepharose CL-6B chromatography followed by HPLC gel filtration. When solubilized in 0.1 M-urea, both proteins appeared to exhibit dimeric forms, with respective molecular masses of about 61 kDa and 99 kDa, upon HPLC. Although the amino acid compositions of the two proteins differed from each other, both proteins had a high content of acidic amino acids, very low contents of histidine and methionine, and no cysteine. The 32 kDa protein exhibited multiple isoelectric forms (pI 3.7-3.9), whereas the 45 kDa protein had a single form (pI 3.3). Radioiodination and immunogold labelling revealed that both proteins were exposed evenly over the entire cell surface. Based on immunodiffusion analysis using monospecific antiserum raised to the individual proteins, there was no antigenic relationship between the two proteins. Furthermore, immunoblot analysis showed that the antigenicity of the 32 kDa protein appeared to be strain specific, whereas that of the 45 kDa protein appeared to be group specific.