Analytical isoelectric focusing of secreted dermatophyte proteins applied to taxonomic differentiation of Microsporum and Trichophyton species (preliminary studies)

Culture filtrates were prepared from dermatophytes under standard conditions and adapted for analytical isoelectric focusing in thin layer polyacrylamide gels over the pH range 3.5-9.5. Dermatophytes grown in trypticase soy broth secreted a large number of proteins displaying a wide range of isoelectric points (pIs). Trichophyton megninii extracts contained a triplet of proteins focusing in the pH 8.0-8.5 range that were absent in taxonomically related T. kuryangei isolates. Single ascospore isolates and standard tester strains of Nannizzia otae (+) mating type were differentiated from the (-) mating type by proteins focusing at pH 6.5 and 8.4. These were markedly reduced in the (+) type. The isofocused pattern of Microsporum canis conformed closely to the (-) mating type of N. otae. The protein patterns of T. megninii and T. kuryangei were distinct from those obtained with M. canis and M. equinum because of an intense-staining broad protein band, pI 7.2, and three periodic acid-Schiff-positive glycoproteins focusing in the acidic range which were absent in the Microsporum species. A characteristic protein or doublet (pI 8.7) was present in the Microsporum species and absent in the Trichophyton species. Analytical isoelectric focusing is a potentially useful method to distinguish inter- and intra-species differences in the pattern of secreted dermatophyte proteins present in culture filtrates and in trichophytins. The information derived may be useful in the classification of species.     

Chemical heterogeneity of major outer membrane pore proteins of Escherichia coli.

Peptide mapping and isoelectric focusing were used to compare the major outer membrane pore proteins from various strains of Escherichia coli K-12, including strains carrying mutations in the nmpA, nmpB, and nmpC genes which result in the production of new membrane proteins. Proteins 1a, 1b, and 2 and the NmpA proteins each gave unique peptide and isoelectric focusing profiles, indicating that these are different polypeptides. The NmpA protein and the NmpB protein appeared to be identical by these criteria. The NmpC protein and protein 2 were nearly identical, although one different peptide was observed in comparing the proteolytic peptide maps of these proteins and there were slight differences in their isoelectric focusing profiles. Antiserum against protein 2 showed partial cross-reactivity with the NmpC protein. These results indicate that the various pore proteins of E. coli K-12 fall into four different classes.     

Bioinformatic analysis of outer membrane proteome of Neisseria meningitidis and Neisseria lactamica.

Two-dimensional electrophoresis (isoelectric focusing/SDS-PAGE) and Western-blotting techniques were used to analyze and compare common and/or specific outer-membrane proteins and antigens from Neisseria meningitidis and Neisseria lactamica. Bioinformatic image analyses of proteome and immunoproteome maps indicated the presence of numerous proteins and several antigens shared by N. meningitidis and N. lactamica, although the inter-strain variation in the maps was of similar magnitude to the inter-species variation, and digital comparison of the maps did not reveal proteins found to be identical by MALDI-TOF fingerprinting analysis. PorA and RmpM, two relevant outer-membrane antigens, manifested as various spots at several different positions. While some of these were common to all the strains analyzed, others were exclusive to N. meningitidis and their electrophoretic mobilities were different than expected. One such spot, with a molecular mass of 19 kDa, may be the C-terminal fragment of RmpM (RmpM-Cter). The results demonstrate that computer-driven analysis based exclusively on spot positions in the proteome or immunoproteome maps is not a reliable approach to predict the identity of proteins or antigens; rather, other identification techniques are necessary to obtain accurate comparisons.     

The preparation and properties of gonococcal pili.

Pili have been isolated from Neisseria gonorrhoeae by controlled homogenization followed by selective disaggregation in sucrose and purification by CsCl density gradient centrifugation. Pili from six gonococcal strains had buoyant densities of 1-30 to 1-31 g ml-1 on CsCl. The pili were immunologically distinct when tested with rabbit antisera to purified pili. The amino acid composition of pilin from strains P9 and 201 was very similar, consisting of 208 and 212 amino acid residues respectively giving molecular weights of 22 600 and 22352. The pili contained a high proportion (46%) of non-polar amino acids. Further analysis of strain P9 pili revealed the presence of 1 to 2 phosphate groups and 1 to 2 hexose groups per pilin subunit; no amino sugars were detected. Pili from strain P9 were resolved into two bands by equilibrium density gradient centrifugation or column isoelectric focusing, suggesting the presence of more than one kind of pilus.     

Electrophoretic Differences in the Capsid Proteins of Simian Virus 40 Plaque Mutants

The structural proteins of three mutants of simian virus 40 (SV40) which differ in plaque size, temperature sensitivity, oncogenicity, host cell restriction, and immunological properties were studied. The polypeptide components of these SV40 strains could not be distinguished by their polyacrylamide gel electrophoretic patterns. When the dissociated virions of two of the mutants were analyzed by the isoelectric focusing technique in a urea gradient, the capsid protein peaks were found to differ significantly in their isoelectric points. The capsid protein of the small-plaque mutant had an isoelectric point of pH 6.51 as compared with pH 6.28 for the large-plaque strain. Isoelectric focusing of the isolated capsid protein revealed three components, a single major subunit and two minor forms. The coat proteins of two of the mutants, small-plaque and minute-plaque strains, were indistinguishable by this technique. The capsid protein peaks obtained by isoelectric focusing were further analyzed by polyacryalmide gel electrophoresis.     

Isolation and partial characterization of a genus common antigen and species specific antigen of Capnocytophaga

Sixteen strains of Capnocytophaga were isolated from the pocket of a localized juvenile periodontitis patient. These strains were divided into four groups on the basis of morphological and physiological traits. Strains from group I and group III were identified as C. ochracea and group II as C. sputigena. An antigen common to genus Capnocytophaga was purified utilizing immunoabsorbent chromatography from lysates obtained by sodium dodecyl sulfate treatment of C. ochracea strain S1. An antigen specific to C. ochracea was prepared by sequential gel filtration and preparative isoelectric focusing. The genus common and species specific antigens isolated were immunologically unique and pure when tested by immunoelectrophoresis and immunodiffusion against rabbit antisera prepared to Capnocytophaga and other gram-negative rods. The genus common antigen was susceptible to trypsin and pronase digestion, was soluble in chloroform-methanol, but was unaltered by ribonuclease and deoxyribonuclease treatments and periodate oxidation. Antigenicity of the species specific antigen was destroyed by periodate oxidation. The genus common antigen appeared to be lipid-associated protein, while the species specific antigen consisted mainly of carbohydrate. These specific immunological reagents would be valuable in diagnosing and monitoring diseases.     

Characterization of porins from the outer membrane of Salmonella typhimurium. 1. Chemical analysis.

The three species of channel-forming outer membrane proteins, porins, have been purified to homogeneity from mutant strains of Salmonella typhimurium which produce single species of porin. Purification was by stepwise solubilization with dodecylsulfate or guanidine thiocyanate, gel filtration, and preparative gel electrophoresis. Amino acid compositions and tryptic peptide maps of the three species of porins showed close resemblance, but at the same time clear differences among them. The number of amino acid residues in the porins purified from the strains SH5551, SH6377 and SH6017 were 361, 354 and 345, and their calculated molecular weights 39800, 39300 and 38000, respectively. Amino-terminal and carboxyl-terminal amino acids in all three species of porins appeared to be alanine and phenylalanine, respectively. Neither half-cystine nor hexosamine was found in these preparation of porins. The isoelectric points of porins from the strains SH5551, SH6377 and SH6017, determined by isoelectric focusing, showed slight differences from each other. These results, and the genetic experiments from another laboratory, suggest that the three species of porins in Salmonella typhimurium are distinct polypeptides, probably coded for by distinct structural genes, which might have been derived from the same ancestral gene.     

Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, and Erwinia carotovora subsp. atroseptica

Erwinia spp. that cause soft-rot diseases in plants produce a variety of extracellular pectic enzymes. To assess the correlation between patterns of pectic enzyme production and taxonomic classification, we compared the enzymes from representative strains. Supernatants obtained from polygalacturonate-grown cultures of nine strains of Erwinia chrysanthemi, three strains of E. carotovora subsp. carotovora, and three strains of E. carotovora subsp. atroseptica were concentrated and subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. Pectate lyase, polygalacturonase, and exo-poly-alpha-D-galacturonosidase activities were visualized by staining diagnostically buffered pectate-agarose overlays with ruthenium red after incubation of the overlays with the isoelectric focusing gels. The isoelectric focusing profiles of pectate lyase and polygalacturonase were nearly identical for strains of E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica, showing three pectate lyase isozymes with isoelectric points higher than 8.7 and a polygalacturonase with pI of ca. 10.2. Isoelectric focusing profiles of the E. chrysanthemi pectic enzymes were substantially different. Although there was considerable intraspecific heterogeneity, all strains produced at least four isozymes of pectate lyase, which could be divided into three groups: basic (pI, ca. 9.0 to 10.0), slightly basic (pI, ca. 7.0 to 8.5), and acidic (pI, ca. 4.0 to 5.0). Several strains of E. chrysanthemi also produced a single form of exo-poly-alpha-D-galacturonosidase (pI, ca. 8.0).(ABSTRACT TRUNCATED AT 250 WORDS)     

Isoelectric focusing of ten strains of Giardia duodenalis

Ten strains of human- and animal-source Giardia duodenalis were evaluated using an isoelectric focusing technique. Banding patterns obtained from total cell proteins of trophozoites demonstrated both similarities and differences between strains. This confirms the heterogeneity of this morphological group of Giardia sp. demonstrated by others. Heterogeneity was demonstrated among the strains retrieved from human and animal hosts and from hosts within the same geographical region.     

Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1.

The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.     

Comparative study of sturgeon oocyte soluble proteins by isoelectric focusing

1. Soluble caviar (oocyte) proteins of four sturgeon species from the Caspian sea, Acipenser stellatus (Sevrouga), Acipenser guldenstadti (Ossietre), Acipenser nudiventris (Chipe) and Huso huso (Belouga) were studied by isoelectric focusing. 2. Isoelectric focusing patterns of these proteins differ from one species to another and allow the identification of the specific origin of caviar. 3. Moreover, this technique allowed the discrimination of a subspecies, Acipenser guldenstadti persicus from the species. 4. This biochemical characterization of caviar proteins allowing identification of sturgeon species producing caviar could be added to data used in fraud tests.     

Selective depletion of small basic non-glycosylated proteins in diabetes.

Degradative rates of small basic non-glycosylated proteins are preferentially enhanced in rat liver cytosol during severe streptozotocin-induced diabetes. Synthetic rates of these classes of proteins are not selectively enhanced in diabetes, so small basic non-glycosylated proteins should be depleted from liver cytosol as a consequence of this disease. To test this hypothesis, proteins were analysed from normal animals, from diabetic animals receiving insulin and from diabetic animals after insulin withdrawal for 3 days. The proteins were separated according to subunit molecular weight by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, according to isoelectric point by isoelectric focusing and according to carbohydrate content by affinity chromatography with concanavalin A linked to agarose. Severe uncontrolled diabetes is associated with the predicted depletion of small basic non-glycosylated proteins from liver cytosol. The preferential degradation and loss of these protein classes may be of considerable physiological importance to the animal.     

Genetic markers for identification of Patagonian toothfish and Antarctic toothfish

Fillet samples of the toothfish Dissostichus eleginoides and D. mawsoni can be distinguished readily by muscle proteins revealed by isoelectric focusing and mitochondrial DNA markers. The proteins also distinguish toothfish from other species marketed under similar trade names.     

Genetic relationships among Neisseria species assessed by comparative enzyme electrophoresis

The electrophoretic mobilities of 12 enzymes from 19 Neisseria species (including 6 strains of N. perflava), Gemella haemolysans, Escherichia coli and Branhamella catarrhalis were characterized by polyacrylamide slab gel electrophoresis. All strains and species tested exhibited qualitatively different zymogram patterns. Species and strain relationships were quantified by pairwise comparisons of all 12 enzyme systems to obtain similarity indices; these data were subjected to numerical clustering methods to obtain groups and a phenogram. The electrophoretic classification compared favourably with those obtained by other criteria. In addition, the quantitative clustering data indicated that N. ovis and N. caviae are sufficiently different from the other Neisseria species to warrant their separation into a distinct group. These two species also lacked the characteristic NADPH-diaphorase zymogram pattern found in all the other Neisseria species. Intra-species similarity indices were generally greater than the inter-species index values. However, certain species such as N. meningitidis and N. gonorrhoeae had similarity index values in the range of inter-strain index values.     

High-Resolution Polyacrylamide Gradient Gel Electrophoresis (PGGE) of Isoenzymes from Five Naegleria Species

ABSTRACT. High-resolution polyacrylamide gradient gel electrophoresis (PGGE) was used to separate isoenzymes of 12 Naegleria strains: one N. australiensis , two N. lovaniensis , one N. jadini , two N. gruberi isolated from environmental samples, and six N. fowleri strains isolated from patients with primary amoebic meningoencephalitis. Of the eight enzymes studied, seven showed zymograms with interspecific variation that identified all the species tested. Although the six N. fowleri strains were biochemically the most homogeneous, they showed intraspecific isoenzyme variation that allowed them to be grouped into four zymodemes. The PGGE technique, which separates isoenzymes by their molecular shape, is both sensitive and economical. It offers an addition or an attractive alternative to isoelectric focusing which has commonly been used to aid species identification of Naegleria by separating isoenzymes by their isoelectric point.     

A new analytical procedure for two-dimensional electrophoresis of cellular proteins: comparison of protein compositions of parent strain and a K+-accumulation mutant of E. coli.

An improved two-dimensional analytical electrophoretic technique fractionates according to molecular weight in the presence of dedecyl sulfate in the first dimension, then fractionates according to isoelectric point in a perpendicular dimension. Electrofocusing in the second dimension achieves nearly complete removal of most protein components while providing true estimates of their isoelectric points. Because not all proteins penetrate isoelectric focusing gels, some proteins may go unrecognized using conventional two-dimensional systems where isoelectric focusing precedes electrophoresis. However, such components do enter dodecyl sulfate gels; hence the presence and molecular weight of those components can be established by the new procedure. A concurrent finding was that, in general, penetration of isoelectric focusing gels by discrete protein subunits dissolved in 9 M urea is an all-or-none phenomenon depending on the solubility of the specific subunit. The procedure was applied to comparison of the protein compositions of a parental strain (CBH) of Escherichia coli and a derived mutant strain (RD-2) deficient in ability to accumulate K+. The strains showed similar two-dimensional patterns except for one discrete isoelectric component absent in the parent strain but present in the mutant.     

High-resolution polyacrylamide gradient gel electrophoresis (PGGE) of isoenzymes from five Naegleria species

High-resolution polyacrylamide gradient gel electrophoresis (PGGE) was used to separate isoenzymes of 12 Naegleria strains: one N. australiensis, two N. lovaniensis, one N. jadini, two N. gruberi isolated from environmental samples, and six N. fowleri strains isolated from patients with primary amoebic meningoencephalitis. Of the eight enzymes studied, seven showed zymograms with interspecific variation that identified all the species tested. Although the six N. fowleri strains were biochemically the most homogeneous, they showed intraspecific isoenzyme variation that allowed them to be grouped into four zymodemes. The PGGE technique, which separates isoenzymes by their molecular shape, is both sensitive and economical. It offers an addition or an attractive alternative to isoelectric focusing which has commonly been used to aid species identification of Naegleria by separating isoenzymes by their isoelectric point.     

The effect of the nature of the strain on the character of the production of iron-dependent proteins by meningococci]

The comparative study of three Neisseria meningitidis strains (15, 125, 2394) was carried out by the method of electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate and by the method of immunoblotting. The intensive expression of 8 iron-regulated proteins (IRP) was shown to occur in iron-deficient culture medium. The major IRP with a molecular weight of 35 kD was expressed by all above-mentioned N. meningitidis strains under the conditions of iron deficiency and cross-reacted with 10 mouse and rabbit antisera to N. meningitidis of different groups, i.e. it was common to all Neisseria species. The antigenic activity of various IRP essentially differed with respect to antisera of animals and sera of patients with meningococcal infection.     

Identity of SDS-insoluble Proteins with Purified Glutenin from Wheat Flour

Sodium dodecyl sulfate (SDS)-insoluble proteins from wheat flour were solubilized by the reduction of their disulfide linkages with 2-meracaptoethanol. The polypeptide compositions of the reduced SDS-insoluble proteins were compared with those of the reduced glutenin by SDS-polyacrylamide gel electrophoresis, isoelectric focusing and amino acid analysis. SDS-polyacrylamide gel electrophoretic patterns of the reduced SDS-insoluble proteins almost coincided with those of the reduced glutenin. Seven major bands (Band 1–7) were obtained from both samples of the reduced proteins. These protein bands were subjected to analysis of amino acid compositions and isoelectric focusing, and similarities between polypeptides of the SDS-insoluble proteins and the glutenin were observed in their amino acid compositions and isoelectric focusing patterns. The results obtained suggested that the preparation of the reduced SDS-insoluble proteins might be used as a simple and rapid method to obtain the glutenin subunits.     

Isoenzyme and Total Protein Analysis by Agarose Isoelectric Focusing, and Taxonomy of the Genus Acanthamoeba

Thirty axenically grown reference strains belonging to 15 different Acanthamoeba spp. were investigated for isoenzyme patterns by agarose isoelectric focusing in the pH range 3–10. Zymograms of acid phosphatase, leucine amino peptidase, malate dehydrogenase, propionyl esterase, glucose phosphate isomerase, phosphoglucomutase, and alcohol dehydrogenase were compared. The same strains were also analyzed for protein patterns separated by agarose isoelectric focusing in a pH gradient of 5–8. The results suggested changes in taxonomy within morphology group II of Pussard & Pons. Acanthamoeba paradivionensis becomes a synonym of A. divionensis. Although this species seems to be related to A. rhysodes, it could not be concluded that the species names are synonyms since the type strain of A. rhysodes was not available for comparison. In the subgroup A. polyphaga–A. quina–A. lugdunensis, A. lugdunensis becomes the species name for pathogenic strains of this subroup, A. quina for the nonpathogenic strains, while A. polyphaga is the species name for an atypical strain. Two strains of A. castellanii showed different zymograms from strain Neff of this species, but related protein patterns. In group III, A. pustulosa is found to be a synonym of A. palestinensis, while one strain of A. lenticulata is also found to belong to the A. palestinensis species. All other species names in both morphology groups could be retained as valuable, on the basis of the techniques used. Group I was not investigated, as axenic cultures could not be obtained.