Purification and properties of a novel broad substrate specific alcohol oxidase from Aspergillus terreus MTCC 6324

An alcohol oxidase was isolated from the microsome of n-hexadecane grown Aspergillus terreus and purified by ion exchange chromatography. The oxidase was found to act on short chain-, long chain-, secondary-, and aromatic-alcohol substrates with highest affinity for n-heptanol (K(M)=0.498 mM, K(cat)=2.7x10(2) s(-1)). The native protein molecular mass was 269+/-5 kDa and the subunit molecular masses were 85-, 63-, 43-, 27-, and 13-kDa. The isoelectric point of the proteins was within 8.3-8.5. High aggregating property of the protein was demonstrated by AFM, DLS and TEM analyses. Chemical analysis showed the presence of oleic acid and palmitic acid at a ratio of 2:1 in the purified protein. This lipoidic nature of the protein particles was correlated to the high aggregating property. In this flavoenzyme, flavin was non-covalently but avidly associated. Peptide mass fingerprinting studies showed the presence of two FAD binding domains in 63 kDa protein. Among these two FAD binding domain sequences only the YPVIDHEYDAVVVGAGGAGLR peptide shows 45-50% sequence homology with the reported N-terminal sequences of other known alcohol oxidases. Non-redundant database search of 63- and 43-kDa subunits peptide sequences showed no sequence similarity with the other alcohol oxidase protein reported till now.     

Subunit composition of a large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86

A xylanolytic complex (xylanosome) was isolated from Streptomyces olivaceoviridis E-86 grown on corncob xylan. The isolated xylanosome exhibited a high molecular mass of approximately 3.8 x 10(7) Da (weight average) using size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS), and was composed of at least 8 subunits with a mass range from 12 to 60 kDa. When a SDS-polyacrylamide gel zymogram was examined, the subunits of 47, 35, 32, and 23 kDa were found to have xylanase activity, while the 30-kDa subunit had CMCase activity. According to N-terminal sequence analyses, the 47- and 23-kDa subunits were found to be identical to the two reported xylanases, namely FXYN and GXYN, of S. olivaceoviridis E-86. Both the 35- and 32-kDa subunits were found to be truncated forms of the intact FXYN xylanase that possibly resulted from the degradation by proteases. The 15-kDa subunit consisted solely the xylan-binding domain of the FXYN xylanase. The purified xylanosome appeared to bind partially to xylan and poorly to Avicel.     

On the molecular weight and subunit composition of calf thymus ribonuclease H1

We have reinvestigated the molecular weight and subunit composition of calf thymus ribonuclease H1. Earlier studies suggested a variety of molecular weights for the enzyme in the range of 64K-84K and reported that the enzyme either was a single polypeptide of 74 kDa or consisted of from two to four subunits in the range of 21-34 kDa. Although we too find bands in this lower molecular weight range in our highly purified preparations following SDS-PAGE, our data suggest that the native structure of RNase H1 is a dimer of 68-kDa subunits. The evidence includes the following: (1) Western blot analysis of fractions taken at various stages of the purification indicates that the predominant antigenic form of the enzyme in crude extracts has a molecular weight of 68K but that during purification in the absence of sufficient protease inhibitors a variety of lower molecular weight forms appear concomitant with the disappearance of the 68-kDa band. (2) Activity gel analysis of the highly purified enzyme prepared in the presence of a battery of protease inhibitors reveals that the 68-kDa band (as well as several bands of lower molecular weight) possesses RNase H activity. (3) The 68-kDa band recognized by Western blotting with anti-RNase H immune sera is not detected by using preimmune sera. Furthermore, when immune sera are used, a trace of a 140-150-kDa antigenic form can sometimes be detected, consistent with the existence of a dimeric form of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification, Characterization, and Genetic Analysis of Cu-Containing Dissimilatory Nitrite Reductase from a Denitrifying Halophilic Archaeon, Haloarcula marismortui

Cu-containing dissimilatory nitrite reductase (CuNiR) was purified from denitrifying cells of a halophilic archaeon, Haloarcula marismortui. The purified CuNiR appeared blue in the oxidized state, possessing absorption peaks at 600 and 465 nm in the visible region. Electron paramagnetic resonance spectroscopy suggested the presence of type 1 Cu (g(II) = 2.232; A(II) = 4.4 mT) and type 2 Cu centers (g(II) = 2.304; A(II) = 13.3 mT) in the enzyme. The enzyme contained two subunits, whose apparent molecular masses were 46 and 42 kDa, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis indicated that the two subunits were identical, except that the 46-kDa subunit was 16 amino acid residues longer than the 42-kDa subunit in the N-terminal region. A nirK gene encoding the CuNiR was cloned and sequenced, and the deduced amino acid sequence with a residual length of 361 amino acids was homologous (30 to 41%) with bacterial counterparts. Cu-liganding residues His-133, Cys-174, His-182, and Met-187 (for type 1 Cu) and His-138, His-173, and His-332 (for type 2 Cu) were conserved in the enzyme. As generally observed in the halobacterial enzymes, the enzymatic activity of the purified CuNiR was enhanced during increasing salt concentration and reached its maximum in the presence of 2 M NaCl with the value of 960 microM NO(2)(-) x min(-1) x mg(-1).     

Molecular and immunological characterization of plastid and cytosolic pyruvate kinase isozymes from castor-oil-plant endosperm and leaf

1. Monospecific antiserum was raised in rabbits to homogeneous cytosolic pyruvate kinase isolated from 5-day-old germinating endosperm of the castor oil plant, Ricinus communis. An earlier study demonstrated that the purified enzyme is putatively heterotetrameric, composed of two subunits which migrate as 57-kDa and 56-kDa proteins upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis [Plaxton, W. C. (1988) Plant Physiol. (Bethesda) 86, 1065-1069]. Both proteins were detected on Western blots of extracts prepared under denaturing conditions from 4-8-day-old, but not 0-3-day-old, germinating-endosperm tissue. This suggests that both subunits exist in vivo, and that the large increase in pyruvate kinase activity which occurs around the fourth day of germination is due to an increase in pyruvate kinase concentration. 2. The cytosolic and plastidic pyruvate kinase isozymes (termed PKc and PKp, respectively) from castor-oil-plant developing endosperm and expanding leaf tissue were separated by anion-exchange chromatography on Q-Sepharose. The antigenic reaction of the partially purified enzyme preparations to rabbit polyclonal antibodies raised against homogeneous germinating-castor-bean PKc was tested by immunoprecipitation and Western blotting. Although developing-endosperm and leaf PKc appeared to be antigenically very similar to germinating-endosperm PKc, they differed from the heterotetrameric germinating-endosperm enzyme by being composed of a single type of subunit with a molecular mass of about 56 kDa. No cross-reactivity of the PKc antibodies was observed with either developing-endosperm or leaf PKp, nor with rabbit muscle or Bacillus stearothermophilus pyruvate kinase. Conversely, none of the castor-oil-plant pyruvate kinase preparations showed significant cross-reactivity with antibodies raised against purified yeast or rabbit muscle pyruvate kinases. 3. To investigate the structural relationship between the two germinating-endosperm-PKc subunits, each polypeptide was characterized by amino acid composition analysis and peptide mapping by CNBr fragmentation. The amino acid compositions and CNBr cleavage patterns of the two subunits were similar, but not identical, suggesting that these polypeptides are related, but distinct, proteins. Mild tryptic attack of native enzyme led to an approximate 6-kDa reduction in the apparent molecular mass of both subunits, further indicating sequence similarity between the two polypeptides. 4. Native molecular masses of the various castor-oil-plant pyruvate kinases were estimated by Superose-6 gel-filtration chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of Vitronectin-Binding Proteins of Staphylococcus epidermidis

Staphylococcus epidermidis is the most common microorganism that is isolated from the cerebrospinal fluid (CSF) shunt infection patients. Vitronectin adsorbed on the surface of implants may mediate bacterial adhesion and colonization. To characterize vitronectin-binding properties, we analyzed S. epidermidis BD5703 isolated from a CSF shunt infection. Expression of vitronectin-binding protein(s) depended on culture media. Two proteins (60 and 52 kDa) were purified from vitronectin affinity chromatography. Two other vitronectin-binding proteins (21 and 16 kDa) were purified from an ion-exchange column. All purified proteins blocked bacterial binding of immobilized vitronectin significantly except the 16-kDa protein. The N-terminal sequences of the 21- and 16-kDa proteins did not show any appreciable amino acid sequence homology. The 52-kDa protein was sequenced by mass spectrometry and identified as an autolysin. This report demonstrates that interaction of vitronectin with multiple recognition sites on BD5703 surface may contribute to bacterial colonization. Received: 6 September 2000 / Accepted: 6 November 2000     

Assembly of EcoKI DNA methyltransferase requires the C-terminal region of the HsdM modification subunit

The methyltransferase component of type I DNA restriction and modification systems comprises three subunits, one DNA sequence specificity subunit and two DNA modification subunits. Limited proteolysis of the EcoKI methyltransferase shows that a 55-kDa N-terminal fragment of the 59-kDa modification subunit is resistant to degradation. We have purified this fragment and determined by mass spectrometry that proteolysis removes 43 or 44 amino acids from the C-terminus. The fragment fails to interact with the other subunits even though it still possesses secondary and tertiary structure and the ability to bind the S-adenosylmethionine cofactor. We conclude that the C-terminal region of the modification subunit of EcoKI is essential for the assembly of the EcoKI methyltransferase.     

Purification of the larvicidal toxin of Bacillus sphaericus and evidence for high-molecular-weight precursors

Crystals were purified from spore-crystal complexes of Bacillus sphaericus 2362 by disruption in a French pressure cell followed by centrifugation through 48% (wt/vol) NaBr. Crystals from such preparations had a 50% lethal concentration of 6 ng of protein per ml for the larvae of the mosquito Culex pipiens. When subjected to polyacrylamide gel electrophoresis under denaturing conditions, the proteins in B. sphaericus crystals migrated in positions corresponding to 43, 63, 98, 110, and 125 kilodaltons (kDa); solubilization of the crystal at pH 12 with NaOH eliminated all but the bands at 43 and 63 kDa. Since NaOH-solubilized preparations were toxic to mosquito larvae, these proteins were purified to electrophoretic homogeneity and antiserum was obtained to each. Analysis of the two purified proteins indicated that the 43-kDa protein was toxic to mosquito larvae (50% lethal concentration, 35 ng of protein per ml), whereas the 63-kDa protein was not. Further differences between them were their amino acid compositions, their lack of immunological cross-reactivity, their opposite net charges at pH 7.5, and their susceptibility to digestion by larval midgut proteases (the 63-kDa protein was highly susceptible, whereas the 43-kDa protein was not). The sequence of the 40 N-terminal residues of the 43-kDa protein was determined and found to contain a high percentage of hydrophobic amino acids. The sequence of the 63-kDa protein could not be determined, since it had multiple N termini. By electrophoretically separating the crystal proteins and then electroblotting onto nitrocellulose paper and visualizing the bands with antisera to the 43- and 63-kDa proteins in conjunction with an immunoblot assay, it was found that the high-molecular-mass crystal proteins (98 to 125 kDa) contained antigenic determinants of both proteins. These results suggested that the lower-molecular-weight crystal proteins detected in polyacrylamide gels after electrophoresis under denaturing conditions were derivatives of one or more of the higher-molecular-weight crystal proteins. In vivo studies of the products of crystal degradation by larvae of Culex pipiens indicated that the high-molecular-weight proteins and the 63-kDa antigenic determinants were rapidly degraded and that a 40-kDa protein related to the 43-kDa toxin persisted for the duration of the experiment (4 h). Some of the studies performed with B.sphaericus 2362 were extended to strains 1593, 1691, and 2297 of this species with results which indicated a high degree of similarity between the crystal proteins of all these larvicidal strains.     

Molecular characterization of the 28- and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein.

The major outer membrane protein of Legionella pneumophila exhibits an apparent molecular mass of 100 kDa. Previous studies revealed the oligomer to be composed of 28- and 31-kDa subunits; the latter subunit is covalently bound to peptidoglycan. These proteins exhibit cross-reactivity with polyclonal anti-31-kDa protein serum. In this study, we present evidence to confirm that the 31-kDa subunit is a 28-kDa subunit containing a bound fragment of peptidoglycan. Peptide maps of purified proteins were generated following cyanogen bromide cleavage or proteolysis with staphylococcal V8 protease. A comparison of the banding patterns resulting from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a common pattern. Selected peptide fragments were sequenced on a gas phase microsequencer, and the sequence was compared with the sequence obtained for the 28-kDa protein. While the amino terminus of the 31-kDa protein was blocked, peptide fragments generated by cyanogen bromide treatment exhibited a sequence identical to that of the amino terminus of the 28-kDa protein, but beginning at amino acid four (glycine), which is preceded by methionine at the third position. This sequence, (Gly-Thr-Met)-Gly-Pro-Val-Trp-Thr-Pro-Gly-Asn ... , confirms that these proteins have a common amino terminus. An oligonucleotide synthesized from the codons of the common N-terminal amino acid sequence was used to establish by Southern and Northern (RNA) blot analyses that a single gene coded for both proteins. With regard to the putative porin structure, we have identified two major bands at 70 kDa and at approximately 120 kDa by nonreducing SDS-PAGE. The former may represent the typical trimeric motif, while the latter may represent either a double trimer or an aggregate. Analysis of these two forms by two-dimensional SDS-PAGE (first dimensions, nonreducing; second dimensions, reducing) established that both were composed of 31- and 28-kDa subunits cross-linked via interchain disulfide bonds. These studies confirm that the novel L. pneumophila major outer protein is covalently bound to peptidoglycan via a modified 28-kDa subunit (31-kDa anchor protein) and cross-linked to other 28-kDa subunits via interchain disulfide bonds.     

Identification and purification of a nuclease from Zinnia elegans L.: a potential molecular marker for xylogenesis

A single-strand specific nuclease was identified during a particular stage of a defined cellular differentiation pathway characteristic of xylem development. Using a hormone-inducible system in which cultured mesophyll cells of Zinnia elegans differentiated to xylem cells in synchrony, the enzymatic activity on single-stranded (ss) DNA was highest during the maturation phase of differentiation. Nondifferentiating cells contained little of this activity throughout a similar course of culture. After electrophoresis of extracts from differentiating cells, a 43-kilodalton (kDa) polypeptide was detected by its activity in the gels containing either ssDNA or RNA. Lectins specific for mannose residues on glycoproteins bound to the 43-kDa nuclease and were used to distinguish it from several ribonucleases. The nuclease was purified by a two-step chromatographic procedure: a lectin-affinity column followed by a phosphocellulose column. The purified protein was determined to be a single polypeptide with a relative molecular mass of 43000 by the analysis of its mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration of the native enzyme. A sensitive detection system using biotinylated-concanavalin A and avidin was demonstrated to be specific as a probe for the nuclease protein. An N-terminal amino-acid sequence was derived from 5 pmol of the enzyme. The nuclease was most active on ssDNA at pH 5.5 in the presence of Zn²⁺ and dithiothreitol. The purified preparation hydrolyzed RNA and to a lesser extent, native DNA. It digested closed circular duplex DNA by introducing a single endonucleolytic cleavage followed by random hydrolysis. During the induced pathway of synchronous differentiation in Zinnia the 43-kDa nuclease rapidly increased just prior to the onset of visibly differentiated features, and developed to a maximum level during xylem cell maturation. At this time a similar but slightly smaller nuclease appeared and became dominant as differentiation continued, and subsequently both enzymes decayed. After autolysis, a nuclease of about 37 kDa was found together with the 43-kDa enzyme in the culture medium. Complementing these analyses was the examination of the tissue distribution of the 43-kDa enzyme in Zinnia and other dicotyledonous plants, which also indicated an invivo role of the nuclease in autolysis, the terminal stage of vascular differentiation in plants. The Zinnia nuclease is therefore a potential marker for xylogenesis.Abbreviations Con A Canavalia ensiformis (concanavalin) agglutinin - DNase deoxyribonuclease - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - kDa kilodalton - M_r relative molecular mass - RNase ribonuclease - ss single-stranded - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Rat liver phosphoribosyl pyrophosphate synthetase: existence of the purified enzyme as heterogeneous aggregates and identification of the catalytic subunit

Mammalian phosphoribosyl pyrophosphate (PRPP) synthetase has been extensively investigated. However, considerable ambiguity remains concerning its physical and regulatory properties. We purified PRPP synthetase from rat liver and studied some of the physical properties, in parallel with cloning experiments (Taira, M. et. al. [1987] J. Biol. Chem. 262, 14867-14870). 1) The enzyme was purified to a specific activity of 7,280 milliunits/mg, the highest value in the literature for a mammalian PRPP synthetase. The apparent molecular mass was over 1,000 kDa. 2) The final preparation contained 34-kDa, 38-kDa, and 40-kDa protein species, as analyzed by SDS gel electrophoresis. 3) Further attempts at separation using conventional procedures only led to a co-purification of the components. Thus, the purified enzyme appears to exist as complex aggregates composed of heterogeneous components. 4) Gel filtration of the enzyme in the presence of 1 M MgCl2 isolated part of the 34-kDa component, free of other species. The preparation was catalytically active, indicating that this component is the catalytic subunit. 5) The amino acid composition of the 34-kDa subunit and the amino acid sequences of its N-terminal region and of two tryptic peptides were determined. The results are in accord with the results of cDNA analyses.     

Purification and characterization of a soluble beta-fructofuranosidase from Daucus carota.

Soluble beta-fructofuranosidase with an intracellular location and an isoelectric point of 3.8 (isoenzyme I) was purified and characterized from dry seeds and seedlings of carrot (Daucus carota). The enzyme hydrolyzed sucrose with a Km of 5 mM and a broad pH optimum around 5.0. The purified protein, which was N-glycosylated with high-mannose-containing and high-xylose-containing complex glycans, eluted as a monomeric polypeptide with a molecular mass of 68,000 from a gel-filtration column. On SDS/PAGE, the protein separated in the presence of SDS and 2-mercaptoethanol into three polypeptides with molecular masses of 68, 43 and 25 kDa. The amount of the 68-kDa polypeptide was highest in dry seeds and decreased with increasing age of carrot seedlings. Amino acid sequence analysis and immunological studies showed that the 43-kDa and 25-kDa polypeptides were N-terminal and C-terminal proteolytic fragments of the 68-kDa polypeptide. A comparison of partial amino acid sequences of the soluble beta-fructofuranosidase with the complete sequence of carrot cell-wall beta-fructofuranosidase showed that their N-terminal sequences were different, whereas some of the internal tryptic peptide sequences were up to 70% identical.     

Sarcosine reductase of Tissierella creatinophila: purification and characterization of its components

Sarcosine reductase is the only reductase system present in Tissierella creatinophila when grown on creatinine plus formate. The acetyl-phosphate-forming component protein C was purified to homogeneity. SDS-PAGE of the purified protein revealed two protein bands with apparent mol. masses of 62 and 50 kDa. The N-terminal amino acid sequence of the two subunits was determined. Antibodies raised against each of the subunits of protein C from Eubacterium acidaminophilum cross-reacted with the corresponding protein present in T. creatinophila, Clostridium litorale and Clostridium sporogenes. The arsenate-dependent hydrolysis of acetyl phosphate catalyzed by protein C was partly inhibited by antibodies directed against the large subunit. Antibodies raised against the small subunit were twice as effective, which indicates that this subunit is the primary site of acetyl transfer from acetyl phosphate. The protein A component of the sarcosine reductase of T. creatinophila was purified to homogeneity by cochromatography with thioredoxin reductase on DEAE-Sephacel, hydroxylapatite, Q-Sepharose, and Sephacryl 100-HR. Protein A had an apparent mol. mass of 21 kDa. Its N-terminal amino acid sequence showed high similarities to that of other proteins A. Initial steps for the purification and preliminary characterization of the sarcosine-specific, substrate-binding protein B_sarcosine component of T. creatinophila indicated the involvement of a 50-kDa protein. Received: 18 May 1998 / Accepted: 5 August 1998     

Identification of a chitin-binding protein secreted by Pseudomonas aeruginosa

One of the major proteins secreted by Pseudomonas aeruginosa is a 43-kDa protein, which is cleaved by elastase into smaller fragments, including a 30-kDa and a 23-kDa fragment. The N-terminal 23-kDa fragment was previously suggested as corresponding to a staphylolytic protease and was designated LasD (S. Park and D. R. Galloway, Mol. Microbiol. 16:263-270, 1995). However, the sequence of the gene encoding this 43-kDa protein revealed that the N-terminal half of the protein is homologous to the chitin-binding proteins CHB1 of Streptomyces olivaceoviridis and CBP21 of Serratia marcescens and to the cellulose-binding protein p40 of Streptomyces halstedii. Furthermore, a short C-terminal fragment shows homology to a part of chitinase A of Vibrio harveyi. The full-length 43-kDa protein could bind chitin and was thereby protected against the proteolytic activity of elastase, whereas the degradation products did not bind chitin. The purified 43-kDa chitin-binding protein had no staphylolytic activity, and comparison of the enzymatic activities in the extracellular medium of a wild-type strain and a chitin-binding protein-deficient mutant indicated that the 43-kDa protein supports neither chitinolytic nor staphylolytic activity. We conclude that the 43-kDa protein, which was found to be produced by many clinical isolates of P. aeruginosa, is a chitin-binding protein, and we propose to name it CbpD (chitin-binding protein D).     

Particulate methane monooxygenase genes in methanotrophs.

A 45-kDa membrane polypeptide that is associated with activity of the particulate methane monooxygenase (pMMO) has been purified from three methanotrophic bacteria, and the N-terminal amino acid sequence was found to be identical in 17 of 20 positions for all three polypeptides and identical in 14 of 20 positions for the N terminus of AmoB, the 43-kDa subunit of ammonia monooxygenase. DNA from a variety of methanotrophs was screened with two probes, an oligonucleotide designed from the N-terminal sequence of the 45-kDa polypeptide from Methylococcus capsulatus Bath and an internal fragment of amoA, which encodes the 27-kDa subunit of ammonia monooxygenase. In most cases, two hybridizing fragments were identified with each probe. Three overlapping DNA fragments containing one of the copies of the gene encoding the 45-kDa pMMO polypeptide (pmoB) were cloned from Methylococcus capsulatus Bath. A 2.1-kb region was sequenced and found to contain both pmoB and a second gene, pmoA. The predicted amino acid sequences of these genes revealed high identity with those of the gene products of amoB and amoA, respectively. Further hybridization experiments with DNA from Methylococcus capsulatus Bath and Methylobacter albus BG8 confirmed the presence of two copies of pmoB in both strains. These results suggest that the 45- and 27-kDa pMMO-associated polypeptides of methanotrophs are subunits of the pMMO and are present in duplicate gene copies in methanotrophs.     

Novel lectin-like proteins on the surface of human monocytic leukemia cell line THP-1 cells that recognize oxidized cells

Presence of lectin-like receptors on the membranes of human monocytic leukemia cell line THP-1 cells for clustered sialylated poly-N-acetyllactosaminyl sugar chains on the membranes of oxidized erythrocytes and T-lympoid cells was investigated. Membranes of THP-1 cells differentiated into macrophages were solubilized, and the membrane proteins obtained by affinity chromatographies using lactoferrin-Sepharose and band 3-Sepharose were purified by successive DE column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins of 50, 60, and 80 kDa with specificity to bind to sialylated poly-N-acetyllactosaminyl sugar chains were detected in the chromatographic fractions. A 50-kDa protein was isolated in a pure form. N-Terminal amino acid sequence of the protein was Lys-Gln-Lys-Val-Ala-Gly-Lys-Gln-Pro-Val-, which has not been found in the N-terminal regions of the hitherto known proteins. The antibody, raised against the chemially synthesized peptide composed of the N-terminal amino acid sequence, bound to 50-, 60-, and 80-kDa proteins as analyzed by immunoblotting and immunoprecipitation, indicating that these proteins had the same N-terminal amino acid sequence. The results demonstrate that THP-1 cells have novel 50-, 60-, and 80-kDa lectin-like proteins with the same N-terminal amino acid sequence on the cell surface which would bind to clustered sialylated poly-N-acetyllactosaminyl sugar chains generated on oxidized erythrocytes and T-lymphoid cells.     

Antigenic relatedness and N-terminal sequence homology define two classes of periplasmic flagellar proteins of Treponema pallidum subsp. pallidum and Treponema phagedenis

The periplasmic flagella of many spirochetes contain multiple proteins. In this study, two-dimensional electrophoresis, Western blotting (immunoblotting), immunoperoxidase staining, and N-terminal amino acid sequence analysis were used to characterize the individual periplasmic flagellar proteins of Treponema pallidum subsp. pallidum (Nichols strain) and T. phagedenis Kazan 5. Purified T. pallidum periplasmic flagella contained six proteins (Mrs = 37,000, 34,500, 33,000, 30,000, 29,000, and 27,000), whereas T. phagedenis periplasmic flagella contained a major 39,000-Mr protein and a group of two major and two minor 33,000- to 34,000-Mr polypeptide species; 37,000- and 30,000-Mr proteins were also present in some T. phagedenis preparations. Immunoblotting with monospecific antisera and monoclonal antibodies and N-terminal sequence analysis indicated that the major periplasmic flagellar proteins were divided into two distinct classes, designated class A and class B. Class A proteins consisted of the 37-kilodalton (kDa) protein of T. pallidum and the 39-kDa polypeptide of T. phagedenis; class B included the T. pallidum 34.5-, 33-, and 30-kDa proteins and the four 33- and 34-kDa polypeptide species of T. phagedenis. The proteins within each class were immunologically cross-reactive and possessed similar N-terminal sequences (67 to 95% homology); no cross-reactivity or sequence homology was evident between the two classes. Anti-class A or anti-class B antibodies did not react with the 29- or 27-kDa polypeptides of T. pallidum or the 37- and 30-kDa T. phagedenis proteins, indicating that these proteins are antigenically unrelated to the class A and class B proteins. The lack of complete N-terminal sequence homology among the major periplasmic flagellar proteins of each organism indicates that they are most likely encoded by separate structural genes. Furthermore, the N-terminal sequences of T. phagedenis and T. pallidum periplasmic flagellar proteins are highly conserved, despite the genetic dissimilarity of these two species.     

Isolation and characterization of a tumor-associated NADH oxidase (tNOX) from the HeLa cell surface.

Cell-surface-located, drug-responsive and tumor-associated NADH oxidase (tNOX) proteins were purified and characterized from HeLa cells. The proteins isolated exhibited NADH oxidase activity inhibited by capsaicin and were resistant to heating and to protease digestion. The activity was purified 200- to 500-fold to provide apparently homogeneous gel bands for N-terminal sequencing using three different protocols. All three protocols involved heat (50 degrees C) and proteinase K treatment. Recovery of the total NADH oxidase activity was 86% and inhibition by capsaicin was 60 to 80%. After 450-fold purification, a 52-kDa component was obtained as a single gel band that retained the capsaicin-inhibited NADH oxidase activity. Amino acid composition and partial amino acid sequences were obtained. The partial amino acid sequences were used to generate peptide antisera. Both the peptide antisera and polyclonal antisera to the 52-kDa component immunoprecipitated capsaicin-inhibited NADH oxidase activity and reacted with 52-, 34-, and 17-kDa components on Western blots from different steps of the purification. The tNOX protein exhibited immunological cross-reactivity and amino acid sequence identity with tNOX cloned from a HeLa cDNA library using a monoclonal antibody to tNOX from sera of cancer patients. The results provide a direct sequence link between tNOX of the HeLa cell surface and the cloned tNOX representative of patient sera. The tNOX form from the surface of HeLa cells yielded N-terminal sequence consistent with a coidentity of the cell surface and serum forms of the two activities.