Intraspecific polymorphism of glucose-6-phosphate dehydrogenase in Amoeba proteus

Seven Amoeba proteus strains of different origin were studied using a polyacrylamide gel electrophoresis micromethod in the presence of NADP+ in cathode buffer. The strains examined differ in glucose-6-phosphate dehydrogenase (G6PD) zymograms: the majority of them (A, L, F, Da and Bk) display 3 bands, whereas strains B and C show 4 and 2 bands, respectively. All but strain C have a common band of high relative mobility. Strain B shows two fast migrating bands instead of a single band typical of strains A, L, F, Da and Bk. The results obtained testify to a certain intraspecific polymorphism of electrophoretic G6PD patterns in A. proteus. These difference between strains may be used as genetic markers in the nuclear transfer experiments.     

Characterization of Enterobacter cloacae and E. sakazakii by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases

Acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases of 34 strains of Enterobacter cloacae and 22 strains of Enterobacter sakazakii were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. The two species could be separated on the basis of distinct electrophoretic patterns of all enzymes analysed. Glutamate dehydrogenase and acid phosphatase were detected exclusively in E. cloacae, whereas esterase bands were more intensively stained in E. sakazakii. For each species, two zymotypes could be distinguished, on the basis of electrophoretic mobilities of malate dehydrogenase and banding patterns of esterase for E. cloacae, and by both isoelectric point and electrophoretic mobilities of an esterase and of lactate and malate dehydrogenases for E. sakazakii. The high degree of enzyme polymorphism within the two species permitted precise identification of strains. The variations in electrophoretic patterns might therefore provide useful epidemiological markers.     

Enzymes of the phosphogluconate pathway in amebas

Glucose-6-phosphate and 6-phosphogluconate dehydrogenases (G6PD and 6PGD) are revealed in Amoeba proteus by electrophoresis in polyacrylamide gels, thus proving the availability of the phosphogluconic pathway in amoebae. 6PGD is marked as a single band, and G6PD shows multiple banding. When an amoebic homogenate is obtained using Triton-100, a supplementary form of G6PD extracted from membranes of some cell organelles (presumably mitochondria) becomes apparent. Hexose-6-phosphate dehydrogenase seems to be absent and therefore all the G6PD forms found may be specific G6PDs proper.     

Epidemiological typing of Acinetobacter strains by esterase electrophoresis

Fifty-three strains of Acinetobacter, belonging to the species A baumannii, A. haemolyticus and A. johnsonii, were differentiated by electrophoretic typing of their esterases, on the basis of both the enzyme specific activity profiles and their electrophoretic mobilities. Each esterase was defined by its spectrum of hydrolytic activity toward five synthetic substrates and its sensitivity to di-isopropyl fluorophosphate. Since each enzyme was not detected in all strains of a given species, several zymotypes could be defined by the patterns of combinations of esterases. Thus, 24 zymotypes were defined in the 32 A. baumannii strains, 4 were defined in the 10 A. haemolyticus strains and 6 were defined in the 11 A. johnsonii strains. When the electrophoretic mobilities of the various esterases were included, each of the 53 strains of Acinetobacter (with the exception of three A. haemolyticus strains) showed a distinct electrotype.     

Isoenzymes and Taxonomy in Scandinavian Hydroids (Cnidaria, Campanulariidae)

Acid phosphatases, esterases and leucine aminopeptidases were studied in Campanularia integra, Clytia hemisphaerica (the johnstoni form), Laomedea ftexuosa, Gonothyraea loveni, Obelia geniculata, O. longissima and O. dichotoma , using polyacrylamide gel electrophoresis. Species-specific patterns were obtained, notably regarding acid phosphatase, despite strong individual variation. The electrophoretic results combined with differences in morphology and cnidom indicate that O. longissima and O. dichotoma are separate species. The acid phosphatase patterns of the two species and of O. geniculata showed basic similarities, supporting their assignment to a common genus. The profound differences in the acid phosphatase patterns of C. integra and L. flexuosa , may in conjunction with morphological distinction justify their assignment to separate genera. C. integra also included specimens of caliculata morphology: however, these did not deviate in their band patterns. Electrophoretograms of the typical O. dichotoma resembled those of the dubia and plicata forms. Moreover, O. gelatinosa with free Obelia medusae (sensu Hincks 1868) is suggested to be conspecific with O. longissima . The present study indicates that electrophoretic analyses, particularly of acid phosphatases presenting highly reproducible patterns, is a valuable complement to morphological characters when studying the taxonomy in hydroids.     

A Method for Analysis of Protein in Individual Amoebae

SYNOPSIS The microelectrophoresis technic of Hydén and Lange has been adapted for analysis of the proteins of individual Amoeba proteus, A. dubia , and Pelomyxa carolinensis. The method has the sensitivity required for analyzing electrophoretic mobilities of the proteins of these 3 species and is reproducible. The possibility of using this technic to differentiate amoeba species according to their protein composition is discussed.     

Isoenzymatic variability in seeds of some spanish common beans (Phaseolus vulgaris L. Leguminosae): Relation to their domestication centers

Phaseolus vulgaris is an important crop species. The cultivated common bean derives from wild forms in two independent domestication centers in Mesoamerica and South America. We report a study of electrophoretic patterns of seven isoenzymatic systems in 29 samples of P. vulgaris seeds. Nineteen of them are from northern Spain, four are from Mesoamerican and six are South American forms. The isoenzymatic activity of esterases, cytochrome c oxidase, acid phosphatase, alkaline phosphatase, alcohol dehydrogenase, shikimate dehydrogenase and glutamate oxaloacetic transaminase were studied. On the basis of electrophoretic patterns of seed isoenzymes, four groups can be recognized using clustering procedures (UPGMA). Comparing this biochemical information with previous morpho-agronomic studies, the possible primary domestication centers of the cultivars are discussed.     

Characterization of enterobacteria by esterase specific-activity profiles

The spectrum of specific activities and the electrophoretic mobilities of esterases produced by 550 strains of Enterobacteriaceae belonging to 36 species and subclassified into six groups (group 1, Escherichia coli, Shigella and Escherichia hermanii; group 2, genus Salmonella and genus Citrobacter; group 3, genus Klebsiella and genus Enterobacter; group 4, genus Serratia and Serratia fonticola; group 5, genus Proteus, genus Providencia and genus Morganella; and group 6, genus Yersinia) were analysed by acrylamide/agarose gel electrophoresis using standardized methods for staining and mobility comparisons. Nineteen types of esterase were defined by their respective esterase specific-activity profile (ESAP). A multiple correspondence analysis (MCA) of the ESAP data enabled 82% of the strains in the 36 species to be correctly classified. In each group, the species were clearly delineated after MCA on both ESAP and electrophoretic mobility data. In addition, the smallest number of characters providing species identification of Yersinia strains by esterase polymorphism was identified by means of a binary segmentation tree technique.     

Acidification of phagosomes is initiated before lysosomal enzyme activity is detected

We have measured changes of pH in a protein's microenvironment consequent on its binding to the cell surface and incorporation into pinosomes. Changes of pH were measured from single, living cells and selected regions of cells by the fluorescence ratio technique using a photon-counting microspectrofluorimeter. The chemotactic agent and pinocytosis inducer, ribonuclease, labeled with fluorescein (FTC- RNase), adsorbed to the surface of Amoeba proteus, and was pinocytosed by cells in culture media at pH 7.0. The FTC-RNase entered an apparently acidic microenvironment, pH approximately 6.1, upon binding to the surface of amoebae. Once enclosed within pinosomes, this protein's microenvironment became steadily more acidic, reaching a minimum of pH approximately 5.6 in less than 10 min. FTC-RNase pinocytosed by the giant amoeba, Chaos carolinensis, entered pinosomes whose pH was correlated with their cytoplasmic location during the initial 30-40 min after pinocytosis. The majority of pinosomes containing FTC-RNase clustered in the tail ectoplasm of C. carolinensis during this interval and had a pH of approximately 6.5; those released into endoplasm and carried into the tip of cells had a pH below 5.0. As pinosomes became distributed at random in C. carolinensis (1-2 h after initial pinocytosis), differences in pH between tip and tail pinosomes vanished. We have also measured the pH within single phagosomes of A. proteus. Phagosomal pH dropped steadily to approximately 5.4 within 5 min after particle ingestion in 70% of the cells measured, and reached this level of acidity within 10 min in 90% of the cells measured. By contrast, stain for the lysosomal enzyme, acid phosphatase, was evident within only 20% of 5-min-old phagosomes visualized by light microscopy, and within only 40% of 10-min-old phagosomes. A microfluorimetric assay was used to simultaneously record changes in pH, and the initial deposition of lysosomal esterases, within phagosomes of single, living Amoeba proteus. Near complete acidification of the phagosome was recorded from some cells before phagosomal fusion was evident by this microfluorimetric assay. From other cells, however, continued acidification of phagosomes was recorded after lysosomal fusion was initiated. We conclude that acidification of phagosomes by A. proteus is initiated but not necessarily completed prior to phagosome-lysosome formation, and that the two events are closely linked in time. Initial acidification of endosomes is a property intrinsic to the plasma membrane which envelops particles at the cell surface, rather than the result of lysosomal fusion with phagosomes.     

Comparative zone electrophoresis of esterases of Staphylococcus species isolated from mammalian skin.

The electrophoretic mobilities of non-specific esterases in vertical polyacrylamide slab gels were determined for 184 strains of staphylococci, representing a total of 18 proposed species and subspecies. Markedly uniform esterase patterns were seen within species demonstrating a high degree of human host specificity, while those species demonstrating a wide host range were polytypic and often showed considerable polymorphism. The unique banding patterns found in several species indicate that this technique may serve as a valuable aid to existing taxonomic schemes. Starch gel electrophoresis of representative strains usually produced sharper esterase bands than were found with polyacrylamide electrophoresis. However, the additional molecular-sieving effect produced by the polyacrylamide gels differentiated esterases to a greater extent.     

Activity and thermal stability of acid phosphatase in homogenates of Amoeba proteus, acclimated to various temperatures

Activity and thermoresistance of acid phosphatase were determined in supernatant of Amoeba proteus homogenates using 1-naphthyl phosphate (pH 4.0) and p-nitrophenyl phosphate (pH 5.5). Although tartrate-resistant and tartrate-sensitive acid phosphatases hydrolyse both substrates, the former mainly hydrolyses p-nitrophenyl phosphate and the latter 1-naphthyl phosphate. A decrease in the activity of the total and tartrate-sensitive acid phosphatases, when using 1-naphthyl phosphate, and of the total and tartrate-resistant acid phosphatases, when using p-nitrophenyl phosphate, was found in amoebae acclimated to 10 degrees C (10 degrees-amoebae) compared to those acclimated to 25 degrees C (25 degrees-amoebae). Using 1-naphthyl phosphate, the thermoresistance of the total acid phosphatase was lower in 10 degrees-amoebae than in 25 degrees-amoebae, but the thermostability of tartrate-resistant enzyme was the same in both groups of amoebae. Using p-nitrophenyl phosphate, the thermoresistance of the total and tartrate-resistant acid phosphatases was lower (the latter only slightly) in 10 degrees-amoebae than in 25 degrees-amoebae. It is suggested that at least with the use of 1-naphthyl phosphate a decrease in thermostability of the total acid phosphatase may be due to a decrease in thermoresistance of tartrate-sensitive enzyme. The results obtained confirm the author's previous data on the activity and thermostability of electrophoretic forms of acid phosphatase using 2-naphthyl phosphate in 10- and 25 degrees-amoebae (Sopina, 2001). It is the first case of discovering a correlation between changes in primary cell thermoresistance of amoebae cultured at different temperatures and changes in the activity and thermostability of acid phosphatase in their homogenates, with the number of electrophoretic forms of this enzyme and their mobility being permanent.     

Characterization of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate and malate dehydrogenases

Acid phosphatase, esterases, and glutamate and malate dehydrogenases of 192 strains of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gels. The six species were clearly separated from each other by their distinct enzyme electrophoretic polymorphism. For Y. enterocolitica, the strains of biotype 5 were differentiated from the other biotypes by the mobility of glutamate dehydrogenase. For Y. frederiksenii, six zymotypes were delineated by pI and by the mobility of the enzymes. Variation in number or mobility of esterases within each species could represent a marker for epidemiological and ecological analyses. A linear relationship was obtained between the mean genetic diversity coefficient of enzymes and the mean percentage DNA-DNA relatedness of Y. intermedia, Y. aldovae, Y. enterocolitica and Y. frederiksenii.     

The electrophoretic polymorphism of bacterial esterases

Abstract: The specific activities of esterases and certain other molecular properties including immunospecificity indicate that the electrophoretic variations of these enzymes in bacterial populations are the result of allelic variations at specific gene loci. The esterase polymorphism of Enterobacteriaceae and some other species isolated from man or animals demonstrates that esterases can distinguish between bacteria at the species or subspecies level, both by their biochemical properties and by their electrophoretic differences. The esterase data complement DNA hybridization studies and agree with ribosomal DNA polymorphism, especially for delineating a phylogenetically distinct group of highly pathogenic strains in Escherichia coli . A two-dimensional electrophoretic profile obtained by establishing a direct correspondence between homologous esterase bands resolved by independent runs of isoelectric focusing and standard electrophoresis considerably improves the detection of allelic variations, whereas protein titration curves (electrophoresis in pH gradient) can be used to demonstrate the real electrophoretic homogeneity of allozymes or evalue their molecular relationship in terms of apparent amino acid substitutions. This overview establishes that esterases, by their significant electrophoretic polymorphism, are reliable molecular markers for systematics and epidemiology, and are suitable enzyme systems for studying population genetics and phylogeny.     

The Protein Amino Acid Compositions of Amoeba proteus,A. discoides,A. dubia and Pelomyxa carolinensis*

SYNOPSIS. A comparison has been made of the protein amino acid compositions of Amoeba proteus, A. discoides, A. dubia and Pelomyxa carolinensis. The protein amino acid compositions of each of these species differed in 1–3 of the following amino acids: arginine, aspartic acid-asparagine, threonine and glycine. The possibility of using these characteristics as acceptable genetic markers is discussed.     

Identification of HeLa cell glucose 6-phosphate dehydrogenase

Although the electrophoretic mobility of HeLa G6PD is similar to that of the common Negro variant G6PD A+, several reports have suggested slight differences between HeLa G6PD and G6PD A+. This study, carried out using the pure homogeneous B+, A+, and HeLa G6PD, showed that (1) the electrophoretic mobility of HeLa G6PD is identical to that of G6PD A+, (2) the enzymatic properties and thermostability of HeLa G6PD are indistinguishable from those of G6PD A+, and (3) the peptide map of the tryptic digest of HeLa G6PD is identical to that of G6PD A+, with one peptide spot of HeLa G6PD different from the corresponding spot of G6PD B+. These results indicate that the structure of HeLa G6PD is identical to that of G6PD A+, and that the amino acid substitution in HeLa G6PD is from one asparagine residue in the wild-type G6PD B+ to an aspartic acid residue in HeLa G6PD.This research was supported by research grant GM 15253 from the National Institutes of Health.     

Isoenzyme patterns: a valuable molecular tool for the differentiation of Zygosaccharomyces species and detection of misidentified isolates

Electrophoretic analysis of esterase, acid phosphatase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and alcohol dehydrogenase isoenzymes was performed in 39 strains classified into six species of the yeast genus Zygosaccharomyces. The electrophoretic profiles obtained allowed the clear separation of Z. bailii, Z. bisporus, Z. florentinus, Z. lentus, Z. mellis and Z. rouxii, strains of the latter species clustering into two subgroups. Furthermore, this methodology enabled the detection of misidentified strains, as subsequently confirmed by DNA-DNA reassociation and sequencing of the D1/D2 domain of the 26S rRNA gene. Cluster analysis of the global electrophoretic data and those obtained using only two of the isoenzyme systems, esterase and lactate dehydrogenase, yielded similar grouping of the strains examined, indicating that these enzymes are good markers for the differentiation of Zygosaccharomyces species.     

Phosphatase activity in Amoeba proteus at pH 9.0

In the free-living amoeba Amoeba proteus (strain B), after PAAG disk-electrophoresis of the homogenate supernatant, at using 1-naphthyl phosphate as a substrate and pH 9.0, three forms of phosphatase activity were revealed; they were arbitrarily called "fast", "intermediate", and "slow" phosphatases. The fast phosphatase has been established to be a fraction of lysosomal acid phosphatase that preserves some low activity at alkaline pH. The question as to which particular class the intermediate phosphatase belongs to has remained unanswered: it can be both acid phosphatase and protein tyrosine phosphatase (PTP). Based on data of inhibitor analysis, large substrate specificity, results of experiments with reactivation by Zn ions after inactivation with EDTA, other than in the fast and intermediate phosphatases localization in the amoeba cell, it is concluded that only slow phosphatase can be classified as alkaline phosphatase (EC 3.1.3.1).     

Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens.

The relationships of 77 aerotolerant Arcobacter strains that were originally identified as Campylobacter cryaerophila (now Arcobacter cryaerophilus [P. Vandamme, E. Falsen, R. Rossau, B. Hoste, P. Segers, R. Tytgat, and J. De Ley, Int. J. Syst. Bacteriol. 41:88-103, 1991]) and 6 reference strains belonging to the taxa Arcobacter nitrofigilis, Arcobacter cryaerophilus, and "Campylobacter butzleri" were studied by using a polyphasic approach, in which we performed DNA-rRNA hybridizations, DNA-DNA hybridizations, a numerical analysis of whole-cell protein patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an analysis of cellular fatty acid compositions, and a phenotypic analysis and determined DNA base ratios. Our results indicate that "C. butzleri" should be transferred to the genus Arcobacter as Arcobacter butzleri comb. nov., as was suggested by Kiehlbauch and coworkers (J. A. Kiehlbauch, D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth, J. Clin. Microbiol. 29:376-385, 1991). A rapid screening of all strains in which we used the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique revealed five major groups, which were identified by using DNA-DNA hybridization data as A. cryaerophilus (two distinct electrophoretic subgroups), A. butzleri, A. nitrofigilis, and a new species, for which we propose the name Arcobacter skirrowii. The phylogenetic position within rRNA superfamily VI was established for each species. A. butzleri strains and strains belonging to one of the electrophoretic subgroups of A. cryaerophilus had similar fatty acid contents. An analysis of fatty acid compositions allowed clear-cut differentiation of all of the other groups. All of the species could be distinguished by using classical phenotypic tests, although erroneous identifications due to a shortage of clear-cut differentiating tests could occur.     

Differentiation of Shigella by esterase electrophoretic polymorphism

The electrophoretic mobilities of four esterases (A, B, C, and I) of 182 strains of Shigella dysenteriae, S. flexneri, S. boydii and S. sonnei were compared to those of 636 strains of Escherichia coli from various origins, including the Alkalescens Dispar group and enteroinvasive strains. Discriminant analysis of the distribution of esterases among the strains revealed that Shigella could be distinguished from E. coli by differences in the distribution of allozymes of esterases C and I. Principal components analysis distinguished four major clusters of Shigella strains corresponding to the following: S. dysenteriae serotype 1; S. flexneri serotypes 1 to 5; S. flexneri serotype 6 and S. boydii serotypes 2 and 4; and S. sonnei. The last three were characterized by distinct electrophoretic variants of carboxylesterase B, as judged by the two-dimensional electrophoretic profile and titration curves. The distinct esterase pattern obtained for the strains of S. boydii serotype 13 substantiates the view that this serotype may constitute a new species.     

Isolation and partial characterization of two plasmid deoxyribonucleic acids from endosymbiotic bacteria of Amoeba proteus.

Obligatory endosymbiotic bacteria in a strain of Amoeba proteus were found to harbor two distinct species of plasmid, pHJ11 and pHJ12. Their molecular weights were 39 x 10(6) and 14 x 10(6), respectively.