Purification of an extracellular thiol-dependent hemolysin from Bacillus alvei]

Alveolysin a sulfhydryl-dependent cytolytic extracellular protein released by Bacillus alvei has been purified by salting-out by ammonium sulfate, gel filtration, isoelectric focusing on pH gradient and chromatography on DEAE-cellulose. The purified protein after reduction by thiols (active hemolytic form) proved homogeneous by disc polyacrylamide gel electrophoresis and by gel immunodiffusion. The molecular weight was 60,000 daltons, Two molecular forms of pI 5.1 and 7.0 were detected by gel isoelectrofocusing. The toxin was lethal to the mouse. Lytic activity was inhibited by cholesterol and antistreptolysin O anstisera. Immunological cross-reaction was observed between alveolysin and streptolysin O.     

The identification and purification of multiple forms of theta-haemolysin (theta-toxin) of Clostridium perfringens type A.

The theta-haemolysin of Clostridium perfringens was purified from culture supernatant fluids of type A strains by fractional ammonium sulphate precipitation and isoelectric focusing in narrow pH 5 to 8 gradients. Four components detected on electrofocusing were designated theta-1(pI6-8to6-9),theta-2(pI6-5to6-6),theta-3(pI6-1to6-3) and theta-4(pI5-7to5-9). Specific activities ranged from 0-4 x 10-6 to 1-2 x 10-6 haemolytic units/mg protein and 2950 to 3600 LD-50/mg protein. Each haemolytic component was activated by cysteine hydrochloride, and inactivated by cholesterol, by addition of sheep erythrocyte ghosts and by heating at 60 degrees C for 10 min; mouse erythrocytes were more resistant than sheep erythrocytes to haemolysis. A reaction of identity was obtained between components in gel diffusion. Sodium dodecyl sulphate polyacrylamide discgel electrophoresis gave molecular weights in the range 59,000 to 62,000 for each component. A similar value was obtained for theta-1 on density gradient ultracentrifugation. Although the multiple forms were free of 11 factors present in culture supernatants, crossed immunoelectrophoresis and disc gel electrophoresis revealed minor contaminants. These studies reveal that theta-haemolysin has physical properties in common with other oxygen-labile haemolysins.     

Purification of RNA-core induced streptolysin S, and isolation and haemolytic characteristics of the carrier-free toxin

RNA-core (RNAase-resistant fraction of yeast RNA) induced streptolysin S (SLS) was purified (40% recovery) to apparent electrophoretic homogeneity by hydroxylapatite chromatography followed by gel filtration on Sephadex G-100 in the presence of 6 M-guanidine. HCl. The specific activity of the purified toxin was 3 X 10(6) haemolytic units (mg protein)-1. The Mr of the toxin was below 4000 on the basis of SDS-PAGE and 20 000 by gel filtration in guanidine. HCl. High-voltage isoelectric focusing of the purified toxin allowed the isolation of the carrier-free SLS peptide for the first time. This peptide was basic (pI 9.2) as compared to native SLS (pI 3.6). The native toxin and the peptide had similar haemolytic properties except for the high lability of the peptide, which was stabilized by RNA-core. The Mr of the denatured peptide was about 1800, as estimated by gel filtration.     

Characterization of staphylococcal gamma-lysin.

gamma-Lysin was purified from Staphylococcus aureus strains Smith 5R and PG23 (a toxic shock syndrome isolate) by a combination of heparin-agarose and hydroxylapatite chromatography. Both strains produced two haemolytic components, designated gamma 1 and gamma 2. Though each component was weakly haemolytic they acted synergistically to potentiate haemolysis on rabbit, sheep and human blood. Rabbit and sheep erythrocytes were more sensitive to lysis by gamma-lysin than human erythrocytes. The molecular mass of gamma 1 was 32 kDa and its pI value was 9.4. gamma 2 had a molecular mass of 36 kDa and a pI value of 9.3. While both trypsin and papain acted synergistically with gamma 2 to induce increased haemolysis, no such synergism was seen with gamma 1. Also, protease inhibitors acted to inhibit synergism between gamma 1 and gamma 2. These findings suggest that gamma 1 could be a protease.     

A serine protease-associated lectin in the cytolytic system of blowfly (Chrysomya megacephala) larvae: Evidence and characterization

Cytolytic activity against invading microorganisms is one of the innate forms of immunity in invertebrates. A serine protease-associated sialic acid-specific cytolytic lectin was purified using glutaraldehyde-fixed ox erythrocytes from the larval extract of blowfly (Chrysomya megacephala). The purified lectin lysed vertebrate erythrocytes with effective haemolysis of ox red blood cells (RBCs) in an isotonic medium. The degree of haemolytic (HL) activity of the purified cytolytic lectin depended on its concentration, pH, temperature, and calcium ions. It was sensitive to ethylenediaminetetraacetic acid. The native molecular mass of the C-type lectin was 260 ± 26 kDa, comprising four different polypeptide subunits of 75 kDa (pI ~8), 69 kDa (pI ~7.0), 61 kDa (pI ~5.3), and 55 kDa (pI ~4.6). The association between the C-type lectin and serine protease was confirmed by MALDI-TOF-MS analysis that revealed its homology in the same spectral peak as well as the proteases and phenylmethylsulphonyl fluoride inhibition of HL activity. Haemolysis inhibition by N-acetylneuraminic acid and other sugars revealed the properties of the lectin. The purified lectin distorted the integrity of ox RBCs and Paenalcaligenes hermetiae. This in vitro study documents the presence of a cytolytic system in blowfly (C. megacephala) larvae for the clearance of invading microbial pathogens in their feeding niche.     

Alveolysin, the thiol-activated toxin of Bacillus alvei, is homologous to listeriolysin O, perfringolysin O, pneumolysin, and streptolysin O and contains a single cysteine.

The gene coding for alveolysin, the thiol-activated toxin produced by Bacillus alvei, has been cloned by means of an oligonucleotide based on the known N-terminal sequence of the secreted protein. The complete nucleotide sequence of the gene has been determined. The deduced amino acid sequence of alveolysin shows that alveolysin shares homologies with listeriolysin O, perfringolysin O, pneumolysin, and streptolysin O. Alveolysin, like the other members of the family, contains a single cysteine in the conserved peptide sequence ECTGLA WEWWR.     

The hydrophobic character of thiol-activated cytolysins

Hydrophobic chromatography on phenyl–Sepharose has revealed the decidedly hydrophobic character of several members of the group of cytolytic proteins termed `thiol-activated'. Pneumolysin, alveolysin, cereolysin, and streptolysin O were found to be equally hydrophobic, as were the oxidized and reduced forms of alveolysin. Hydrophobic chromatography has been utilized in the development of an improved procedure for the purification of pneumolysin.     

Detection, prevalence, purification and characterization of lecithinase of Klebsiella pneumoniae

Lecithinase activity in Klebsiella is a rare trait as out of 208 strains of Klebsiella belonging to 3 species, viz. K. pneumoniae (168), K. planticola (29) and K. oxytoca (11), only 4 strains of K. pneumoniae produced lecithinase positive colonies on egg-yolk-agar. Although cell lysates of 16 K. pneumoniae yielded positive results for lecithinase assay on egg-yolk-agar, 19 strains were detected positive for lecithinase with ELISA using anti-lecithinase serum. Release of up to 52.12% cell-bound lecithinase could be achieved with polymyxin-B treatment at 100 micrograms/ml concentration. Purified lecithinase was determined to be a high molecular weight (70 kDa), crystalizable, anionic (pI, 3.5) protein. It possessed cytolytic, haemolytic and dermonecrotic activities but did not induce fluid accumulation in rabbit ileal loop or infant mouse guts. It was inactivated by boiling, trypsin and chymotrypsin treatment and alkaline pH. Serologically, it was related to lecithinase of Aeromonas caviae and phospholipase-C of Salmonella.     

Inactivation of haemolysin production in Escherichia coli by transposon insertion results in loss of virulence

A haemolytic E. coli strain is nephropathogenic for mice. Mutagenesis by transposon insertion resulted in mutants with altered haemolytic activity.Reduction or elimination of the haemolytic activity is accompanied with loss of virulence. It is shown that this loss of virulence is due to altered haemolytic activity and not caused by the transposon insertion itself.     

Haemolytic effect of the scorpion Heterometrus fulvipes venom

The haemolytic effect of the venom from scorpion Heterometrus fulvipes was studied using sheep erythrocytes. While there was no haemolytic effect seen directly by the erythrocytes, erythrocytes sensitized with homologous haemolysin were lysed by the venom. This factor in Heterometrus fulvipes venom having a 'complement like' haemolytic effect was found to be thermolabile and dialysable and sensitive to the action of 2-mercaptoethanol. It was identified as a low molecular weight peptide containing disulphide groups.     

Purification and characterization of phospholipase C of Salmonella gallinarum

Phospholipase C was isolated from an outbreak strain of Salmonella gallinarum with ciprofloxacin extraction, dialysis, gel filtration, ion exchange chromatography and chromatofocussing. Purified phospholipase C (mol wt. 65 KDa; isoelectric point, pI 3.5) was resistant to pasteurization, stomach enzyme (pepsin), bacterial protease and lipase but lost its activity on trypsin and chymotrypsin treatment. It was sensitive to pH > or = 8.0. It was haemolytic, embryotoxic, enterohaemorrhagic, lethal to birds, cytotoxic to Vero and MDBK cells, dermonecrotoxic in rabbit and antigenically active protein. Antisera raised against purified phospholipase C neutralized its all biological activities and agglutinated the producer Salmonella strains. Serologically it was proved similar to phospholipase C of Klebsiella pneumoniae and S. weltevreden. Fluorescent antibody technique (FAT) was standardized to detect phospholipase producer strains.     

Haemolytic anaemia as initial manifestation of Wilson's disease

Common manifestations of Wilson's disease are disorders of the liver and brain. A rare complication of this inherited disease is acute intravascular haemolytic anaemia. We report the case of a 33-year old female patient who was admitted to the hospital with acute haemolysis as the initial symptom of Wilson's disease. The haemolysis preceded the definitive diagnosis by 20 months. It is concluded that in any case of unclear haemolytic anaemia, especially in adolescents or in young adults, Wilson's disease should be considered.     

Haemolytic factor from the crop of Rhodnius prolixus: Evidence and partial characterization

A haemolytic factor, which lysed sheep red cells in an isotonic buffer, was found in the crop of all larval stages and adult Rhodnius prolixus. Little or no haemolytic factor occurred in unfed insects but haemolytic activity increased for 2–4 days after feeding. From the 4th day on, the activity declined gradually. Fifth-instar larvae fed on whole blood, erythrocytes and haemoglobin produced large quantities of haemolytic factor, while those fed on plasma and erythrocyte stroma did not. The haemolytic factor was purified approximately 1200-fold by a two-step procedure: (1) Bio-Gel P-6 Gel-Filtration and (2) SP-Sephadex chromatography. Purified haemolytic factor was heatstable (100°C, 10 min), dialysable, inactivated by trypsin treatment, and could be recovered in the supernatant after addition of ethanol. It was concluded that the haemolytic factor is a peptide displaying a basic character.     

Damaging effects of Clostridium perfringens delta toxin on blood platelets and their relevance to ganglioside GM2

The lytic effect of Clostridium perfringens delta toxin was investigated on goat, human, rabbit, and guinea pig platelets. In contrast to erythrocytes from the latter three species, which are insensitive to the toxin, the platelets were equally lysed by the same amount of toxin. These results suggest the presence of GM2 or GM2-like ganglioside(s) as a specific recognition site of the toxin on platelet plasmic membrane as previously established for sensitive erythrocytes. Plasmic membrane damage of human platelets was evidenced by the release of entrapped alpha-[14C]aminoisobutyric acid used as a cytoplasmic marker. The specific binding of hemolytically active 125I-delta toxin by human and rabbit platelets was practically identical, dose dependent, and inhibitable by GM2. Labeled toxin was also bound by various subcellular organelles separated from rabbit platelets except the 5-hydroxytryptamine (5-HT)-containing dense bodies, suggesting the absence or inaccessibility of GM2 on the surface of the latter organelles. This result correlates with the low amounts of 5-[3H]HT liberated after platelet challenge with delta toxin whereas this mediator was massively liberated upon lysis by the sulfhydryl-activated toxin alveolysin. The levels of M and P forms of phenol sulfotransferase (PST), involved in 5-HT catabolism, were determined in human platelet lysates after challenge with delta toxin, alveolysin, and other disruptive treatments. The low PST-M activities detected after lysis by delta toxin suggest that this isoenzyme is very likely associated to dense bodies in contrast to PST-P which is cytoplasmic. Platelet lysis by the toxin allows easy separation of these organelles.     

Activity of radiation degradation products of vitamins A and E to haemolyse erythrocyte

Exposure of vitamin A acetate in freely dissolved state to γ-radiationin vitro caused a dose dependent degradation accompanied by the formation of new products. The radiation degradation products were separated by chromatography using step gradient elution. The parent molecule, vitamin A acetate, induced negligible haemolysis of erythrocytes. In contrast, the polar products formed by irradiation were found to be potent haemolysing agents. A highly polar product, eluted with methanol revealed maximum haemolytic activity. Acetylation of these products resulted in loss of their haemolytic properties. Similarly, vitamin E acetate, a known stabilizer of the biomembranes, after irradiation yielded products which caused haemolysis of erythrocytes. It was demonstrated that irradiation introduces hydroxyl groups which impart haemolytic properties to the radiation degradation products of vitamin A     

Bacterial toxins induce heat shock proteins in human neutrophils

We studied the influence of different bacterial toxins (alveolysin; toxic shock syndrome toxin 1, TSST-1 and erythrogenic toxin A, ETA) on the expression of heat shock proteins (hsps) in isolated human polymorphonuclear granulocytes (PMNs). As was shown by Western blotting (anti-hsp72) ETA and TSST-1 were potent inducers of hsps at low toxin concentrations (10 ng/ml). Alveolysin led to the expression of hsps at hemolytic concentrations (1 HU; 700 ng/ml) whereas at subhemolytic concentrations (7 ng/ml) no heat shock response was observed. The induction of heat shock proteins was also accompanied by increased mRNA levels for hsp70 as was determined by PCR-analysis.     

Changes in glycolytic enzyme levels and isozyme expression in human lymphocytes during blast transformation.

The levels of each of the glycolytic enzymes were observed to exhibit a parallel increase of 200 to 300% when human lymphocytes were stimulated to undergo blast transformation. A series of electrofocusing and electrophoretic studies was utilized to assess the isozyme distribution of the glycolytic enzymes during blastogenesis. Hexokinase (pI = 7.40), glucosephosphate isomerase (pI = 9.35), and enolase (pI = 8.30) existed as single electrophoretic components and were unchanged during blast transformation. Phosphoglycerate mutase was observed to exist as two isozymes (pI = 5.80 and 6.63), which were also unchanged by blastogenesis. Aldolase, which was present as two electrophoretic forms in lymphocytes (pI = 9.25 and 8.75), exhibited a shift in the relative content of each. In addition to the lactate dehydrogenase isozymes at pI 9.50 and 7.60 found in lymphocytes, lymphoblasts contained isozymes with pI values of 7.30, 7.05, and 5.85. Although glyceraldehyde 3-phosphate dehydrogenase was present as a single electrophoretic form (pI ? 8.0) in both lymphocytes and lymphoblasts, the association of the enzyme with actin produced electrophoretic artifacts with lower pI values. Phosphoglycerate kinase, which appeared as a single form in lymphocytes (pI = 9.00), was present as two isozymes (9.00 and 8.74) in lymphoblasts. Similarly, pyruvate kinase (pI = 8.73 and 8.50 in lymphocytes) exhibited additional isozymes (pyruvate kinase, pI = 7.60 and 5.85, and triosephosphate isomerase, pI = 5.20) as a result of cell transformation.     

Isolation and Properties of Major Components of Penicillium canescens Extracellular Enzyme Complex

The composition of the enzyme complex secreted by Penicillium canescens was investigated. A scheme for purification of the main components of the complex by chromatofocusing on a Mono P column was developed. It was found that along with beta-galactosidase, the major components of the complex were endo-beta-1,4-xylanase (31 kD, pI 8.2-9.3), alpha-L-arabinofuranosidase (60 kD, pI 7.6), arabinoxylan-arabinofuranohydrolase (70 kD, pI 3.8-4.0), and endo-beta-1,3/1,4-glucanase (40 kD, pI 4.4). The substrate specificity, pH and temperature activity optima, adsorbability, thermal stability, and ability for synergic interaction of the isolated enzymes were studied.     

Comparison between muscle and liver enolases and their behavior during differentiation and growth.

1. Rabbit liver enolase (EC 4.2.1.11) was purified about 200-fold and the enzyme was distinguished from crystalline muscle enolase by column isoelectrofocusing. It was found that the pI of muscle enolase was at about pH 8.8 and the pI of liver enolase was at about pH 6.7. Liver enolase was more liable to heat than muscle enolase. Anti-muscle enolase antibody did not react with liver enolase in double diffusion and immunoprecipitation tests. No substantial difference seemed to exist between muscle and liver enolases in pH optima, kinetic constants, and gel filtration. 2. It was observed by electrofocusing that the pI of rat muscle enolase was pH 7.2 to 7.9 and that of liver enolase was about pH 5.9. The main component of muscle enolase was designated as type A enolase, and liver enolase as type B enolase. Type A enolase was present in skeletal muscle and heart muscle. Type B enolase was widely distributed and present in liver, kidney, spleen, brain, lung, small intestine, and heart muscle. More acidic isozyme than type B enolase coexisted in the brain, and more basic isozyme than type A enolase, coexisted in the small intestine. A prototype of enolase in the early stage of differentiation was found to be type B enolase and, as differentiation progressed, type B decreased in muscle, while type A increased. On the other hand, liver enolase was retained as type B during differentiation. The enolase in regenerating liver was the same as in normal liver.     

An isozyme of the NADP-malic enzyme of a CAM plant, Aloe arborescens, with variation on conservative amino acid residues

In Aloe arborescens, an obligate CAM plant, Western analysis detected three major isoforms of NADP-malic enzyme (NADP-ME), 72kDa with a pI of 6.0, 65kDa with a pI of 5.6 and 65kDa with a pI of 5.5. Among them, the 65kDa protein with a pI of 5.5 was leaf-specific, and the 65kDa protein with a pI of 5.6 was found only in roots, whereas the 72kDa protein was uniformly detected in both organs. Activity staining indicated enzyme activity of both 65kDa NADP-MEs but little activity of the 72kDa protein. A cDNA clone encoding a leaf-abundant NADP-ME, AME1, was isolated. Deduced amino acid sequence of AME1 showed a high degree of homology to known NADP-MEs, but it was also found that AME1 contained substitutions on five conservative amino acid residues, some of which have been predicted to be important for their enzyme activity. Transgenic rice carrying the aloe AME1 gene efficiently produced an additional 65kDa protein with a pI of 5.5 as an active NADP-ME. These results indicate that AME1 corresponds to the leaf-specific 65kDa NADP-ME, which may be involved in CAM photosynthesis. It was also shown that substitutions of these conservative amino acid residues identified in AME1 still allowed it to give enzyme activity.