Extracellular chitinase production by wild-type B-10 and mutant M-1 strains of Serratia marcescens

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, while M-10 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused overproduction of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the production of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).     

High-yield purification of a pp60c-src related protein-tyrosine kinase from human platelets

A protein-tyrosine kinase (PTK, EC 2.7.1.112) from human platelets was purified with high yield. Purification of the enzyme involved sequential chromatography on casein-agarose, tyrosine-agarose, heparin-Sepharose and hydroxylapatite. The procedure resulted in substantially enriched 54/52 kDa polypeptides on SDS-polyacrylamide gel electrophoresis and a yield of about 25% in PTK activity. About 250 micrograms of purified protein could be obtained from 1 g of cell protein. The purification factor varied between 1000 and 1500. Determination of the molecular mass of the purified PTK under nondenaturating conditions by molecular sieve chromatography revealed that the enzyme is a monomer of about 50 kDa. Among various protein substrates tested, casein was most prominently phosphorylated. All substrates were exclusively phosphorylated at tyrosine residues. Autophosphorylation at tyrosine residues of the 54/52 kDa proteins was observed in the presence of Mg2+ or Mn2+. At each purification step, the 54/52 kDa proteins were precipitated by sera from tumor-bearing rabbits immunoprecipitating pp60src, but not by control sera. The amount of the immunoprecipitated purified 54/52 kDa phosphoproteins was directly proportional to the amount of antiserum used. Partial peptide mapping by V8 proteinase showed a 26 kDa tyrosine-phosphorylated fragment for the 54 and the 52 kDa proteins as well as for the pp60c-src molecules of intact platelets. All these data indicated that purified PTK is closely related to pp60c-src of human platelets. Using casein as a substrate for the purified enzyme, the Km for ATP was 4 microM and the Vmax for the reaction was 2.0 nmol/min per mg.     

Characterization of the Pseudomonas aeruginosa alginate lyase gene (algL): cloning, sequencing, and expression in Escherichia coli.

Mucoid strains of Pseudomonas aeruginosa produce a viscous exopolysaccharide called alginate and also express alginate lyase activity which can degrade this polymer. By transposon mutagenesis and gene replacement techniques, the algL gene encoding a P. aeruginosa alginate lyase enzyme was found to reside between algG and algA within the alginate biosynthetic gene cluster at 35 min on the P. aeruginosa chromosome. DNA sequencing data for algL predicted a protein product of ca. 41 kDa, including a 27-amino-acid signal sequence, which would be consistent with its possible localization in the periplasmic space. Expression of the algL gene in Escherichia coli cells resulted in the expression of alginate lyase activity and the appearance of a new protein of ca. 39 kDa detected on sodium dodecyl sulfate-polyacrylamide gels. In mucoid P. aeruginosa strains, expression of algL was regulated by AlgB, which also controls expression of other genes within the alginate gene cluster. Since alginate lyase activity is associated with the ability to produce and secrete alginate polymers, alginate lyase may play a role in alginate production.     

Synthesis of Extracellular Chitinase by Wild-Type B-10 and Mutant M-1 Strains of Serratia marcescens

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, whereas M-1 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused oversynthesis of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the synthesis of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).     

Suppression of phaseolin and lectin in seeds of common bean, Phaseolus vulgaris L.: increased accumulation of 54 kDa polypeptides is not associated with higher seed methionine concentrations

Suppression of phaseolin and lectin accumulation in common bean resulted in higher concentrations of bean seed polypeptides with apparent molecular weights of 54 kDa and from 70 to 84 kDa on SDS-polyacrylamide gel electrophoresis. Polypeptides of 54 and 56 kDa segregated as products of different alleles. Genes for the 54/56 kDa bands and phaseolin were estimated to be 26.2±3.7 map units apart. The 54 kDa band phenotype manifested by SDS-PAGE consisted of from one to three polypeptides of 54 kDa MW on 2D gels, and the 56 kDa phenotype consisted of one polypeptide of 56 kDa plus two minor polypeptides of 54-54.5 kDa molecular weight. The pK_I of these polypeptides was approximately 5.25. The methionine content of the 54 kDa polypeptides of the cultivar Great Northern Star was 1.6±0.1 g/100 g protein, which was not statistically different from the value (1.5±0.1%) obtained for phaseolin isolated by the same procedure. F₂ seeds deficient for phaseolin and lectin contained as much total N per g as wild-type seeds and were not shrunken, but contained 50% more free amino acids. F₂ seeds from two of the three populations contained from 8 to 13% less methionine per mg total N.     

Suppression of phaseolin and lectin in seeds of common bean,Phaseolus vulgaris L.: increased accumulation of 54 kDa polypeptides is not associated with higher seed methionine concentrations

Suppression of phaseolin and lectin accumulation in common bean resulted in higher concentrations of bean seed polypeptides with apparent molecular weights of 54 kDa and from 70 to 84 kDa on SDS-polyacrylamide gel electrophoresis. Polypeptides of 54 and 56 kDa segregated as products of different alleles. Genes for the 54/56 kDa bands and phaseolin were estimated to be 26.2±3.7 map units apart. The 54 kDa band phenotype manifested by SDS-PAGE consisted of from one to three polypeptides of 54 kDa MW on 2D gels, and the 56 kDa phenotype consisted of one polypeptide of 56 kDa plus two minor polypeptides of 54-54.5 kDa molecular weight. The pK_I of these polypeptides was approximately 5.25. The methionine content of the 54 kDa polypeptides of the cultivar Great Northern Star was 1.6±0.1 g/100 g protein, which was not statistically different from the value (1.5±0.1%) obtained for phaseolin isolated by the same procedure. F₂ seeds deficient for phaseolin and lectin contained as much total N per g as wild-type seeds and were not shrunken, but contained 50% more free amino acids. F₂ seeds from two of the three populations contained from 8 to 13% less methionine per mg total N.     

Comparison of Ca2+ -dependent phosphorylation in viable dispersed brain cells with calmodulin-dependent protein kinase activity in cell-free preparations of rat brain.

Using two depolarizing agents, veratrine and high concentrations of extracellular KCl, we studied depolarization-stimulated phosphorylations in 32P-labelled dispersed brain tissue in order to identify phosphoprotein substrates for Ca2+ - and calmodulin-dependent protein kinase activity at the cellular level, for comparison with findings in cell-free preparations. In intact brain cells, the only prominent depolarization-stimulated phosphorylation was a 77 kDa protein separated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This phosphorylation was dependent on external Ca2+, since chelation of Ca2+ in media with 6 mM-EGTA or the presence of verapamil (a Ca2+ -channel blocker) in the incubation media inhibited depolarization-stimulated phosphorylation of the 77 kDa protein. Phosphorylation of the 77 kDa protein also appeared to be dependent on calmodulin, because depolarization-stimulated phosphorylation was significantly decreased (P less than 0.05) when 100 microM-trifluoperazine was present in the incubation media. Polymyxin B, an inhibitor of Ca2+- and phospholipid-dependent phosphorylation, and 12-O-tetradecanoylphorbol 13-acetate, the phorbol ester enhancing Ca2+- and phospholipid-dependent phosphorylation, had no effect on the phosphorylation of the 77 kDa protein. The 77 kDa phosphoprotein was identified as a protein previously named synapsin I [Ueda, Maeno & Greengard (1973) J. Biol. Chem 248, 8295-8305] on the basis of similar migration of native and proteolytic fragments of the 77 kDa protein with those of authentic synapsin I on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Whereas several studies with cell-free preparations showed that 57 kDa and 54 kDa endogenous phosphoproteins were the most prominent species phosphorylated in a Ca2+ and calmodulin-dependent manner, these results indicate that synapsin is the most prominent Ca2+-and calmodulin-dependent phosphorylation in intact cells. The phosphorylations of 54 kDa and 57 kDa proteins may not be as important in vivo, but instead occur as a result of the disruption of cellular integrity inherent in preparation of cell-free subfractions of brain tissue.     

An effective method for isolating alginate lyase-producing Bacillus sp. ATB-1015 strain and purification and characterization of the lyase

A new alginate lyase-producing micro-organism, designated as Bacillus sp. strain ATB-1015, was effectively isolated from soil samples pretreated for 3 months with a substrate of the enzyme, sodium alginate. Alginate lyase activity was assayed by the degrading activity of biofilm on Teflon sheet discs, which was formed by a mucoid strain of Pseudomonas aeruginosa PAM3 selected from clinical isolates. The extracellular alginate lyase was precipitated with ammonium sulphate from the culture broth, and purified by gel filtration and anion exchange chromatography. The molecular weight of the lyase was estimated to be 41 kDa by SDS polyacrylamide gel electrophoresis and Sephacryl S-200 HR column chromatography. The optimum pH and temperature for the enzyme activity were around 7·5 and 37 °C, respectively, and the Km value was 0·17% with the substrate, sodium alginate. The lyase activity was completely inhibited by treatment with 1 mmol l⁻¹ of EDTA and the decreased activity was almost completely recovered by the addition of 2 mmol l⁻¹ of CaCl₂. The activity was not affected by treatment with the protein denaturants, 0·01 mol l⁻¹ of SDS or 1 mmol l⁻¹ of urea. The lyase had substrate specificity for both the poly-guluronate and poly-mannuronate units in the alginate molecule.     

A pool of beta-tubulin is hyperphosphorylated at serine residues in Alzheimer disease brain.

In Alzheimer disease (AD) brain, activities of protein phosphatase (PP)-2A/PP-1 which are known to be associated with microtubules are compromised and are probably a cause of neurofibrillary degeneration through hyperphosphorylation of microtubule proteins. In the present study, an increase of approximately 11 pmol phosphate/microg protein in 100,000 x g pellet from AD compared with age-matched control brains was found. Tau protein, which is hyperphosphorylated in AD can only account for approximately 4 pmol phosphate/microg protein, suggesting the presence of non-tau hyperphosphorylated proteins in the diseased brain. Western blot analysis with phosphoserine antibodies revealed a approximately 54 kDa non-tau protein to be significantly hyperphosphorylated in AD compared with age-matched control cases in the particulate fraction. The approximately 54 kDa protein was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified as beta-tubulin by immunolabeling with specific antibodies, mass spectrometry analysis and by N-terminal amino acid sequencing. The purified protein was hyperphosphorylated at serine residues in AD.     

A comparative study of the extracellular glucosyl- and fructosyltransferases from cariogenic and non-cariogenic Streptococcus mutans strains of two different serotypes

Extracellular proteins from continuous cultures of serotype c and g Streptococcus mutans strains were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Gels stained with raffinose after electrophoresis revealed that although serotype c strains secrete two fructosyltransferases of molecular mass 68 kDa and 79 kDa, no fructosyltransferase was secreted by the serotype g strain K1. A sucrose activity stain was used to detect two glucosyltransferases (GTF) of molecular mass 162 kDa (bifunctional 1,6-alpha-D-glucan 3-alpha- and 6-alpha GTF or 'dextransucrase') and 153 kDa (a 1,3-alpha-D-glucan 3-alpha-GTF) in samples from cariogenic serotype c strains. Neither the 153 kDa protein nor the corresponding GTF activity was secreted by the non-cariogenic mutant C 67-25. The molecular masses of the corresponding 1,3-alpha and 1,6-alpha-GTF proteins from the serotype g strain K1 were 164 kDa and 158 kDa, respectively. All of the GTF proteins were degraded to discrete bands of lower molecular mass on storage at 4 degrees C even after extensive purification. The results provide an explanation for several outstanding controversies in the GTF literature.     

Kinetics of inhibition of platelet calpain II by human kininogens.

Organophosphate-resistant and -susceptible strains of Culex quinquefasciatus (mosquito) have been compared on the basis of their esterase activities. The homozygous resistant strain (Dar) shows two highly active esterases after starch-gel electrophoresis, of Rm 0.2 and 0.4, which are absent from susceptible strains (Apo, Mon), and which previous selection studies have shown to be inseparable from organophosphate resistance. After SDS/polyacrylamide-gel electrophoresis and silver staining of total C. quinquefasciatus proteins, a 62 kDa band is observed in strain Dar at high concentrations, and in susceptible strains in trace amounts. After Western blotting, this 62 kDa protein is recognized by antisera raised against the two esterases eluted from starch gels. After chromatofocusing of Dar proteins, the 62 kDa protein is seen to be associated with esterase activity, and of a similar pI to that observed for esterases after isoelectric focusing. Post-translational modification is not required for recognition of the 62 kDa putative esterase, since the protein is immunoprecipitated by the anti-esterase serum from products of translation of Dar mRNA in vitro.     

A Pseudomonas aeruginosa alginate-exotoxin A conjugate that elicits anti-alginate and exotoxin A-neutralizing antibodies

Pseudomonas aeruginosa alginate was covalently coupled to exotoxin A by reductive amination using adipic acid dihydrazide as spacer. The conjugate was composed of 25% alginate and 75% exotoxin A and possessed an average molecular mass higher than 700 kDa as determined by polyacrylamide gel electrophoresis. The conjugate had virtually no ADP-ribosyltransferase activity and a reduced cytotoxicity for TSA8 murine cells, derived from Friend erythroleukemia cells, as indicated by a greater than 50-fold increased LD50. Anti-conjugate antibodies recognized exotoxin A and alginate. A booster injection resulted in markedly increased antibody ELISA titers to both exotoxin A and alginate. The antibodies neutralized the exotoxin A toxicity.     

Integration of the DNA of a novel filamentous bacteriophage VSK from Vibrio cholerae 0139 into the host chromosomal DNA

Abstract The surface of three strains of enteroaggregative Escherichia coli (EAggEC) and three strains of enteropathogenic E. coli (EPEC) were examined using the freeze-substitution technique of electron microscopy and as a result an electron dense surface layer was found only on EAggEC strains but not on EPEC strains. The analysis of the outer membrane proteins by polyacrylamide gel electrophoresis revealed the existence of a unique 38 kDa protein in EAggEC strains. The protein could be easily extracted from the bacterial surface with 5 M LiCl treatment at room temperature. The antiserum raised in mice with 38 kDa protein extracted from the electrophoresed gel was immunoreacted with the surface of the bacteria of EAggEC by immunoelectron microscopy. The hydrophobic surface character of the EAggEC strains was lost after the extraction of the protein layer by LiCl. We thus conclude that the surface protein layer therefore plays an important role in the expression of the aggregative phenotype in EAggEC strains.     

Purification of wheat germ amylase by precipitation

alpha-Amylase from various sources was found to bind alginate in free solution. The alginate-enzyme complex could be precipitated with Ca(2+). The enzyme activity could be recovered by dissolving the precipitate in 1 M maltose and precipitating alginate alone by addition of Ca(2+). Based upon these observations, alpha-amylase from wheat germ was purified with 68-fold purification and 72% recovery. The molecular weight estimated by SDS-PAGE was 18 kDa. The method also worked equally well with alpha-amylase for the whole wheat seed. The latter enzyme could be purified 54-fold with 70% activity recovery. The molecular weight of this second enzyme was estimated to be 45 kDa by SDS-PAGE.     

Ethanol-mediated variations in cellular fatty acid composition and protein profiles of two genotypically different strains of Escherichia coli O157:H7

Two strains of Escherichia coli O157:H7 were grown in tryptic soy broth (TSB, pH 7.1) supplemented with 0, 2.5, 5.0, 7.5, and 10% ethanol at 30 degrees C for up to 54 h. Growth rates in TSB supplemented with 0, 2.5, and 5.0% ethanol decreased with an increase in ethanol concentration. Growth was not observed in TSB supplemented with 7.5 or 10% ethanol. The pH of TSB containing 5.0% ethanol decreased to 5.8 within 12 h and then increased to 7.0 at 54 h. The ethanol content in TSB supplemented with 2.5 or 5.0% ethanol did not change substantially during the first 36 h of incubation but decreased slightly thereafter, indicating utilization or degradation of ethanol by both strains. Glucose was depleted in TSB supplemented with 0, 2.5, or 5.0% ethanol within 12 h. Cells grown under ethanol stress contained a higher amount of fatty acids. With the exceptions of cis-oleic acid and nonadecanoic acid, larger amounts of fatty acid were present in stationary-phase cells of the two strains grown in TSB supplemented with 5.0% ethanol for 30 h than in cells grown in TSB without ethanol for 22 h. The trans-oleic acid content was 10-fold higher in the cells grown in TSB with 5.0% ethanol than those grown in TSB without ethanol. In contrast, cis-oleic acid was not detected in ethanol-stressed cells but was present at concentrations of 0.32 and 0.36 mg/g of cells of the two strains grown in TSB without ethanol. Protein content was higher in ethanol-stressed cells than in nonstressed cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles varied qualitatively as affected by the strain and the presence of ethanol in TSB. An ethanol-mediated protein (28 kDa) was observed in the ethanol-stressed cells but not in control cells. It is concluded that the two test strains of E. coli O157:H7 underwent phenotypic modifications in cellular fatty acid composition and protein profiles in response to ethanol stress. The potential for cross protection against subsequent stresses applied in food preservation technologies as a result of these changes is under investigation.     

Heat Shock Protein Produced by Helicobacter pylori

The cells of Helicobacter pylori were suspended in the medium containing³⁵S-methionine. After a heat shock of the cells at 42 C for 5, 10, and 30 min, the production of proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Out of many proteins produced by the cells, only 66 kDa protein production was dramatically increased by heat treatment. The N-terminal amino acid sequence of 66 kDa protein was quite similar to that of 62 kDa and 54 kDa proteins previously suggested as heat shock protein (HSP) of H. pylori based on the reaction with polyclonal and monoclonal antibodies against HSP 60 family proteins produced by other bacteria. Therefore, it was concluded that H. pylori produces the 66 kDa protein as its major heat shock protein which belongs to HSP 60 family.     

Alg44, a unique protein required for alginate biosynthesis in Pseudomonas aeruginosa

Here the putative alginate biosynthesis gene alg44 of Pseudomonas aeruginosa was functionally assigned. Non-polar isogenic alg44 deletion mutants of P. aeruginosa were generated and did neither produce alginate nor released free uronic acids. No evidence for alginate enrichment in the periplasm was obtained. Alginate production was restored by introducing only the gene alg44. PhoA fusion protein analyses suggested that Alg44 is a soluble protein localized in the periplasm. Hexahistidine-tagged Alg44 was detected by immunoblotting. The corresponding 42.6 kDa protein was purified and identified by MALDI/TOF-MS analysis. Alg44 might be directly involved in alginate polymerization presumably by exerting a regulatory function.     

Generation of monoclonal antibodies specific to Hepatopancreatic parvovirus (HPV) from Penaeus monodon

Hepatopancreatic parvovirus (HPV) was isolated from the hepatopancreas (HP) of slow growth Penaeus monodon by urografin gradient centrifugation. The presence of HPV in the fraction was monitored by PCR and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Only 1 major 54 kDa protein band was observed in the strong PCR-positive fractions used to immunize mice for monoclonal antibody production. After cell fusion, the first step in selecting specific antibodies was performed by dot-blot assay with purified HPV viral particles. The second screening step was carried out using Western blots of purified HPV proteins and immunohistochemistry of HPV-infected HP tissue. Four monoclonal antibodies were isolated; these bound to the 54 kDa protein in Western blots and to intranuclear inclusion bodies in tubule epithelial cells of HPV-infected prawn tissue by immunohistochemistry. None of the antibodies showed cross-reactivity either to uninfected shrimp tissue or to other shrimp viruses tested. These reagents have potential for use in developing a highly sensitive immunoassay such as sandwich ELISA or a convenient kit for detection of HPV infection.     

Characterization of the Renibacterium salmoninarum haemagglutinin

Water-extracted proteins from nine geographically diverse strains of Renibacterium salmoninarum, all of which agglutinated rabbit erythrocytes and rainbow trout spermatozoa, were compared by SDS-PAGE. Extracts from eight strains, including the type strain, ATCC 33209, were similar, containing a major protein of 57 kDa and a minor protein of 58 kDa. The SDS-PAGE protein profile of the Char strain did not contain the 58 kDa protein. A non-agglutinating strain, MT-239, which was also non-hydrophobic, did not produce any water-extractable protein. Immunoblot reactions with rabbit antiserum prepared against whole cells of the type strain demonstrated that the water-extracted haemagglutinins from the various strains were antigenically related. When purified by polyacrylamide gel zone electrophoresis, the haemagglutinin from R. salmoninarum ATCC 33209 formed a doublet band with molecular masses of 57 and 58 kDa, similar to the previously described F antigen. The water-extracted haemagglutinin agglutinated salmonid spermatozoa, was degraded by protease K and trypsin, and was shown to self-assemble onto the cell surface.     

Purification and partial characterization of a beta-1,3-glucan-binding-protein membrane receptor from blood cells of the crayfish Pacifastacus leniusculus.

A receptor for the 100 kDa beta-1,3-glucan-binding protein [Duvic, B. and S?derh?ll, K. (1990) J. Biol. Chem. 265, 9327-9332] has been purified from hemocyte membranes of the crayfish Pacifastacus leniusculus. The purification was achieved by DEAE-cellulose chromatography of detergent-solubilized membranes. The receptor had an apparent molecular mass of 350 kDa when subjected to native polyacrylamide-gel electrophoresis and was composed of two non-covalently associated subunits of about 230 kDa and 90 kDa, as judged by SDS/polyacrylamide-gel electrophoresis or two-dimensional electrophoresis. The receptor could only bind the beta-1,3-glucan-binding protein if this protein had previously reacted with a beta-1,3-glucan, laminarin, and the binding site was located on the 230 kDa subunit. The binding of laminarin-treated beta-1,3-glucan-binding protein to its receptor was a saturable process and binding data indicated a single high-affinity-binding site with a Kd of 0.35 +/- 0.15 microM as determined by Scatchard analysis. The receptor had a requirement for divalent cations and a pH optimum of 6.5 for binding the laminarin-treated beta-1,3-glucan-binding protein. Laminarin, as well as oligosaccharides such as D-glucose, sialic acid, N-acetyl glucosamine or methyl-alpha-D-mannoside, could not affect the binding of the beta-1,3-glucan-binding protein to its receptor.