Difficulties of molecular dissociation of Clostridium botulinum type G progenitor toxin

Clostridium botulinum type G progenitor toxin was chromatographed on DEAE-Sephadex and Q-Sepharose equilibrated with 0.05 M Tris-HCl buffer, pH 8.0, containing 0.2 M urea. The toxin was eluted in a single protein peak from DEAE-Sephadex, but it was eluted in four protein peaks from Q-Sepharose; the third peak was toxic and the others were nontoxic. The third peak, appearing to be the toxic component, had a molecular mass of 150,000. In SDS-polyacrylamide gel electrophoresis, purified type G progenitor toxin migrated in six bands, with molecular masses of 150,000, 140,000, 58,000, 10,800, 10,600, and 10,400. Type G progenitor toxin may be composed of a toxin component with a molecular mass of 150,000 and a nontoxic component in a manner similar to progenitor toxins of other types. Type G toxic component, whether it was reduced or not, migrated in a single band to the same relative positions in SDS-PAGE; type A toxic component reduced with 2-mercaptoethanol migrated in two bands.     

Purification and properties of mammalian toxins from the venom of the Brazilian scorpion Tityus serrulatus Lutz and Mello

The water-soluble part of the dried venom from the scorpion, Tityus serrulatus Lutz and Mello (range, Southeastern Brazil), showed 16 polypeptide bands on polyacrylamide gel electrophoresis. This material exhibited toxic and hyaluronidase activity but no phospholipase, phosphodiesterase, protease, or fibrinolytic activity. Fractionation on glycinamide-treated Sephadex G-50 afforded three protein fractions, which were non-toxic, equitoxic, and three times more toxic than the water-soluble venom. Subsequent separation of the toxic fractions on carboxymethyl-cellulose with phosphate buffers furnished five toxic components, which were further purified on carboxymethyl-cellulose with a salt gradient in acetate buffer. Toxin γ, the major and most basic toxin, is a 62-residue protein that, unlike other scorpion toxins, contains methionine. Automated Edman degradation showed the amino-terminal sequence to be H-Lys-Glu-Gly-Tyr-Leu-Met-Asp-His-Glu-Gly-Cys-Lys-Leu-Ser-Cys-Phe-Ile-Arg-Pro-Ser-Gly-Tyr-Cys-Gly-Arg-Glu-Cys-Gly-Ile-. Toxin γ is the first example of a fifth structural type of mammalian toxin from scorpion venom. Its amino-terminal sequence shows greater homology with toxins similar to Centruroides suffusus suffusus toxin III and Androctonus australis toxin II than with toxins similar to A. australis toxin I or Bhutus occitanus tunetanus toxin I.     

Basic nuclear proteins in the Oömycete fungus Achlya bisexualis

Summary A comparison was made of the basic proteins extracted from the chromatin and nuclei of Achlya bisexualis, Blastocladiella emersonii, and Pisum sativum. Extraction of purified chromatin and nuclei of the Chytridiomycete, B. emersonii followed by gel electrophoresis produced no detectable protein bands. Extractions of purified nuclei and chromatin by either mineral acid or CaCl₂ from the Oömycete A. bisexualis resulted in several protein bands following separation by disc gel electrophoresis. Monitoring the nucleic acids during the nuclear isolation procedure as well as comparing electropherograms of basic nuclear proteins with basic ribosomal proteins suggests no significant contamination of the nuclear preparations with ribosomes. Carboxymethyl cellulose chromatography of the A. bisexualis nuclear basic proteins resolved three distinct fractions. Gel electrophoresis of the CM cellulose fractions indicated heterogeneity of each fraction. Amino acid analysis of the CM fractions showed that they were all lysine-rich and meet the basisity requirements of histones.     

The electrophoresis and detection of transketolase

After electrophoresis impure transketolase preparations stained readily with an activity stain based on a published method, but pure preparations gave no reaction. The bands first obtained were due to the presence of (i) a transketolase-d-glyceraldehyde-3-phosphate dehydrogenase complex, (ii) alcohol dehydrogenases, and (iii) a zone of high local pH at the buffer front. Lyophilization of the reagents eliminated artifacts due to alcohol dehydrogenase. An external starch indicator gel was developed that gave colored bands with both pure and impure transketolase preparations and a similar indicator gel was developed for transaldolase.     

Immunological properties of isolated protein fractions from Artemia ribosomes

Acid-soluble ribosomal proteins from cysts of Artemia salina were separated by high-resolution polyacrylamide gel electrophoresis at pH 4.3. Three distinct protein bands, occurring in different parts of the electrophoretic pattern, were used for immunization in rabbits, and the γ-globulin fractions of the antisera were prepared. These preparations produced precipitation lines in agarose gel with protein extracted from whole 80S ribosomes and from 60S and 40S ribosomal subunits. With γ-globulin preparations from non-immune or anti-ovalbumin sera no reactions were obtained.     

NADP+-linked malic enzymes from pig heart mitochondria--apparent selective inhibition of one enzyme form.

NADP⁺-linked malic enzyme activity was fractionated from mitochondria using sonication, freeze-thawing, ammonium sulfate precipitation, and calcium phosphate gel elution. Three bands of activity could be shown on disc gel electrophoresis using a stain specific for the enzyme. An inhibitor extracted from the calcium phosphate elution fraction was partially effective against freshly prepared enzyme but not against enzyme preparations stored for five days. During the five days there was a drop in enzyme activity nearly equal to the amount of original inhibition observed, and one of the fractions was no longer demonstrable by gel electrophoresis.     

Purification and characterization of a novel high molecular weight exotoxin produced by red tide phytoplankton,Alexandrium tamarense

Our recent studies have demonstrated that the aqueous extract prepared from Alexandrium tamarense, a harmful red tide phytoplankton, showed cytotoxicity on Vero cells. In this study, the toxic substance was purified from the culture supernatant of A. tamarense. Based on the gel‐filtration profile, the molecular mass of a purified toxin was estimated to be about 1,000 kDa. On sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis, a main band with molecular mass of 1,000 kDa was detected with periodic acid‐Schiff (PAS) staining, but no protein bands were detected by Coomassie brilliant blue (CBB) protein staining. Sugar composition analysis of the toxin suggested that the toxin contains galactose, fucose, mannose, N‐acetylglucosamine, xylose, and other minor saccharides, whereas no significant levels of amino acids were detected by amino acid analysis. These results suggest that the toxin is a polysaccharide‐based compound. The toxin showed cytotoxic effects on various cell lines in a concentration‐dependent manner. Among the cell lines tested, U937 cells were the most susceptible to the toxin. In U937 cells treated with the toxin, a typical apoptotic nuclear morphological change and DNA fragmentation were observed. This is the first report demonstrating that a polysaccharide‐based toxin isolated from red tide phytoplankton can induce apoptotic cell death. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:405–415, 2008; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20253     

Cell surface constituents of sarcoma 180 ascites tumor cells

The cell surface protein components of Sarcoma 180 ascites tumor cells have been investigated by a combination of plasma membrane isolation techniques and lactoperoxidase iodination. For plasma membrane isolation cells were homogenized in the presence or absence of Zn²⁺ and fractionated by sucrose density gradient centrifugation or a two-phase partition to give large membrane fragments or membrane envelopes. Membrane purification was monitored by phase contrast microscopy and chemical and enzyme marker assays. The membrane preparations were analyzed by acrylamide gel electrophoresis in sodium dodecylsulfate. Each preparation showed a common protein pattern of about 15 bands ranging in molecular weights from 33 000 to >300000. Two carbohydrate-containing bands were also present in all preparations. Membranes prepared with Zn²⁺ were much less fragmented and showed much greater amounts of three high molecular weight components than those prepared in the absence of Zn²⁺. This might suggest a role for these components in membrane stabilization.The tumor cells were also subjected to iodination with lactoperoxidase, followed by membrane isolation and acrylamide gel electrophoresis in sodium dodecylsulfate in order to identify polypeptides accessible to the cell surface. The major radioactive band coincided with the major carbohydrate-containing band, presumably a surface glycoprotein. A second carbohydrate-containing band showed variable labeling behavior between different cell preparations. This material had a high molecular weight, as indicated by both acrylamide gel electrophoresis and gel permeation chromatography in dodecylsulfate. Several other components are labeled to a lesser extent in the intact cell.     

Zein of maize grain: II — the charge heterogeneity of free subunits

The subunits present as monomers in unreduced zein and isolated as fraction M by gel filtration, were chromatographed on sulfoethyl-cellulose. Three major subfractions were detected and characterized. Each of them, submitted to electrophoresis at pH 3.5, migrated as a single band corresponding to each of the three major electrophoretic forms seen in fraction M at the same pH. The presence of lysine in some polypeptides, suggested by amino acid composition data, was confirmed by electrophoretic analysis of carbamylated subfractions at pH 4.5. At pH 8.9 each subfractions was further resolved into three cationic bands in starch gel and three (or more) anionic bands in polyacrylamide gel. The same fractionation was also obtained by submitting the major electroforms of fraction M, as isolated at pH 3.5, to isoelectric focusing. Based on these observations, the most probable distributions of basic amino acids in subunits detected by electrophoresis at pH 8.9 were specified and compared to those recently published for several zein clones. The presence per polypeptide chain of three carboxyl groups and occasionally of one lysine would be a feature of zein originating from maize hybrid Inra 260.     

Further studies on bovine serum amylase. 2. Affinity electrophoresis

The polymorphism of bovine serum amylase, which is controlled by the Ami locus, has previously only been demonstrated by starch gel electrophoresis. The addition of maltose to starch gels has been demonstrated to inhibit any subsequent separation of the Ami isozymes by starch gel electrophoresis. When electrophoresis was conducted in a support medium in the absence of starch no polymorphic variation was detected amongst samples from animals of different Ami phenotypes. The addition of starch to agarose gels has been shown to facilitate the subsequent detection of the Ami polymorphism by agarose/starch gel electrophoresis. The electrophoretic resolution of the Ami isozymes has been demonstrated to depend upon differences in affinity for starch rather than differences in net charge. The starch gel electrophoretic separation of the Ami isozymes is. therefore, another example of affinity electrophoresis. All the Ami amylases have been shown to share a common isoelectric point of pH 3.5.     

The peptides of chloroplast membranes: I. The soluble coupling factor (Ca2+-ATPase)

The chloroplast coupling factor (CF₁) was analyzed by gel electrophoresis in SDS and found to contain two major bands in equal amounts with mobilities corresponding to molecular weights of 62,000 and 57,000 and three minor bands of molecular weights 38,000, 21,000, and 14,000. The peptides were present in comparable amounts in many different preparations of the protein and, therefore, were thought not to be tightly bound contaminants. The interaction between these five peptides was shown to be noncovalent.Incubation of the enzyme with trypsin, under conditions which activate the latent ATPase, was found to cause selective digestion of the five peptides; the 62,000 M_r peptide was the most susceptible to digestion, while the 57,000 M_r peptide was most stable to trypsin. When chloroplast membranes were exposed to trypsin in the light to activate the postillumination Mg²⁺-dependent ATPase activity, EDTA extraction solubilized a protein fraction which contained the normal CF₁ peptide pattern. Also, the membranes, when solubilized and chromatographed on SDS-gels did not show the disappearance of any band.The ATPase activity of the protein was highly susceptible to ionic strength, being 50% inhibited by monovalent salts at a concentration of 0.05 m.     

Studies of water soluble lipoproteins in rat brain

—The occurrencc of water soluble lipoprotein in the 105,000 g supernatant of rat brain homogenate has been demonstrated. The lipoproteins of brain differed from those of the serum with respect to lipid composition, density and reaction to serum lipoprotein antibodies. Acrylamide gel electrophoresis resolved the brain supernatant into four zones which stained with lipid dye. It was possible to demonstrate that these zones also contained protein by double staining of the gel for lipid and protein. Two of the zones differed in their mobility from serum lipoprotein. Preparative acrylamide gel electrophoresis isolated protein fractions in supematant which contained both lipid and protein and permitted preliminary identification of the lipoprotein bands amongst all the protein bands resolved by acrylamide gel electrophoresis.     

Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated from human plasma

The protein heterogeneity of fractions isolated by immunoaffinity chromatography on anti-apolipoprotein A-I and anti-apolipoprotein A-II affinity columns was analyzed by high resolution two-dimensional gel electrophoresis. The two-dimensional gel electrophoresis profiles of the fractions were analyzed and automatically compared by the computer system MELANIE. Fractions containing apolipoproteins A-I + A-II and only A-I as the major protein components have been isolated from plasma and from high density lipoproteins prepared by ultracentrifugation. Similarities between the profiles of the fractions, as indicated by two-dimensional gel electrophoresis, suggested that those derived from plasma were equivalent to those from high density lipoproteins (HDL), which are particulate in nature. The established apolipoproteins (A-I, A-II, A-IV, C, D, and E) were visible and enriched in fractions from both plasma and HDL. However, plasma-derived fractions showed a much greater degree of protein heterogeneity due largely to enrichment in bands corresponding to six additional proteins. They were present in trace amounts in fractions isolated from HDL and certain of the proteins were visible in two-dimensional gel electrophoresis profiles of the plasma. These proteins are considered to be specifically associated with the immunoaffinity-isolated particles. They have been characterized in terms of Mr and pI. Computer-assisted measurements of protein spot-staining intensities suggest an asymmetric distribution of the proteins (as well as the established apolipoproteins), with four showing greater prominence in particles containing apolipoprotein A-I but no apolipoprotein A-II.     

Protein reagent modification of cholera toxin: characterization of effects on antigenic, receptor-binding and toxic properties.

The effects of protein modification procedures on the biologically most important properties of cholera toxin, i.e. the toxic activity, the GM1 receptor-binding capacity and the antigenic (antibody-fixing) properties, have been studied quantitatively using microgram amounts or less of toxin protein. Most of the 24 group-specific reagents used had either no inhibitory effect on the toxic or the combination of GM1-binding and antibody-fixing properties of cholera toxin, or they had a concomitant inhibitory effect on these activities. Separate testing of GM1- and antibody-binding revealed a close, but not absolute, structural association between these properties, Amino group reactive substances were particularly effective in decreasing the GM1-binding activity, while leucine aminopeptidase had no effect. This suggests that lysine residues may be involved in binding toxin to the acidic GM1 receptor. Sodium dodecylsulphate and mercaptoethanol, which caused dissociation of the subunits of cholera toxin as indicated by polyacrylamide gel electrophoresis, abolished toxicity without inhibiting the concomitant GM1- and antibody-binding properties of the toxin. Similar differential effects were also obtained with three reagents which did not seem to change the aggregation state of the toxin. These substances all had specificity for arginine, suggesting that arginyl residues of the toxin molecule may be involved in a 'toxic site' distinct from the receptor-binding site(s). A selective effect on the toxic site was also found by treating the toxin with carboxypeptidase or trypsin in the presence of urea; in the absence of urea no enzymic effect on any toxin property was noted.     

Chromosomal proteins of Drosophila melanogaster and an approach for their localisation on polytene chromosomes

Chromosomal proteins have been prepared from embryos of Drosophila melanogaster and separated into histone and nonhistone fractions by a procedure which completely avoids exposure to extremes of pH. These fractions have been characterised by amino acid analysis and gel electrophoresis. Antisera have been prepared against whole chromatin and against the two chromosomal protein fractions. — A new method is described for the preparation of Drosophila salivary chromosomes. This method employs microdissection techniques and completely avoids the use of acid fixatives. Preservation of fine structure in these preparations is comparable to, if not better than, that in classical acid-fixed preparations. Antisera against embryo chromatin and chromosomal protein fractions react with the salivary chromosome preparations. These reactions exhibit selectivity with different chromosomal structures. Evidence is presented suggesting a specific distribution of protein antigens along the chromosome.     

Proteins of cytochrome oxidase in fractions prepared with deoxycholate from bovine heart submitochondrial particles

The hemea content of cytochrome oxidase-containing fractions of varying purity obtained from bovine heart submitochondrial particles with deoxycholate was correlated with the intensity of protein bands obtained on polyacrylamide gel electrophoresis of the preparations dissolved in phenol: acetic acid: water. It is concluded that cytochrome oxidase contains two noncovalently associated proteins. Neither of these proteins is electrophoretically identifiable with the noncatalytic protein of Greenet al. [12].Supported by a grant (AM-09762) from the United States Public Health Service.     

Enzyme preparations fromAspergillus flavus for structural studies of the peach gum polysaccharide

Extra- and intracellular glycanohydrolases were isolated fromAspergillus flavus and partially characterized. Both preparations exhibited β-galactosidase activity. Gel chromatography of the extracellular enzyme preparation on Sephadex revealed one protein fraction containing β-galactosidase activity and a second one exhibiting mainly β-xylosidase activity. Electrophoresis in starch gel and disc electrophoresis in polyacrylamide gel showed that the preparation obtained from the cultivation broth contained five protein fractions, whereas two protein fractions could be detected in the intracellular preparation. Hydrolysis of a partially degraded polysaccharide of peach gum by the above preparations yieldedd-galactose as the main product and traces ofd-mannose,l-arabinose,d-xylose and a number of oligosaccharides.     

Phosphohexose isomerase polymorphism of pigs

Using starch gel electrophoresis, polymorphism of phosphohexose isomerase (PHI) was found in haemolysed swine erythrocytes.
Three different phenotypes A, AB and B were observed. They are determined by two autosomal codominant alleles A and B. Homozygous types have three fractions; a strong middle band flanked by two weaker ones. The heterozygous types have five fractions, three strong bands in the middle with weaker anodic and catodic bands.
Segregation data in the family material support this hypothesis. The age does not influence the pattern of the PHI types.     

Separation and Identification of Exo- and Endoinulinase from <Emphasis Type="Italic">Aspergillus ficuum</Emphasis>

A new and convenient method was developed to separate and identify exo- and endoinulinase from Aspergillus ficuum by native polyacrylamide gel electrophoresis. Eight protein bands were obtained. Three bands were identified as exoinulinase, and two bands were endoinulinase, by using TLC and HPLC. The five bands were all detected as glycoproteins with the sensitive periodic acid-silver stain. Received: 3 July 2002 / Accepted: 25 September 2002     

An electrophoretic karyotype of Aspergillus niger

Summary An electrophoretic karyotype of Aspergillus niger was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Chromosomesized DNA was separated into four bands. Seven of the eight linkage groups could be correlated with specific chromosomal bands. For this purpose DNA preparations from seven transformant strains of A. niger each carrying the heterologous amdS gene of Aspergillus nidulans on a different chromosome were analysed. Some of the assignments were confirmed with linkage groupspecific A. niger probes. The estimated sizes of the A. niger chromosome range from 3.5 to 6.6 Mb, based on gel migration relative to the chromosomes of Schizosaccharomyces pombe strains, Saccharomyces cerevisiae and A. nidulans. The total genome size of A. niger significantly exceeds that of A. nidulans and is estimated to be about 35.5–38.5 Mb. Electrophoretic karyotyping was used to allocate non-mutant rRNA genes and to estimate the number of plasmids integrated in a high copy number transformant.