Zymogram Technique Research Articles

Isozyme differentiation among three pathotypes of the entomogenous fungus Nomuraea rileyi.

The zymogram technique has been applied to three pathotypes of the entomopathogenic fungus Nomuraea rileyi. Isozyme profiles of isolates from Heliothis zea, Pseudoplusia includens, and Anticarsia gemmatalis were compared for 17 enzymes of known metabolic function. Electrophoretic data supported the taxonomic differences inferred for the three pathotypes based on host specificity. The isolate from A. gemmatalis was found consistently to be the most distinct. This study demonstrates that isozyme analysis may be used to distinguish closely related fungal isolates of N. rileyi.

Induction and Characterization of a Cellobiose Dehydrogenase Produced by a Species of Monilia

A species of the imperfect fungus Monilia produced cellobiose dehydrogenase extracellularly when grown on cellulose. The inducible enzyme was both bound to the mycelium and released into the growth medium. The enzyme showed a high degree of specificity for cellobiose, but also oxidized lactose and 4-β-glucosylmannose. The specificity of the electron acceptor was restricted to those compounds having a redox potential of +0·22 V. p-Benzoquinone and several other quinones, however, were not reduced. Oxygen was not consumed nor was hydrogen peroxide produced by cellobiose dehydrogenase oxidation of cellobiose. The enzyme had a molecular weight of 48000 and an isoelectric point of 5·3 to 5·5. A new zymogram technique was developed for the detection of cellobiose dehydrogenase in polyacrylamide gels following electrophoresis and isoelectric focusing.

Purification of glycoside hydrolases from Bacteroides fragilis.

Six glycoside hydrolases in the culture medium of Bacteroides fragilis--alpha-glucosidase, beta-glucosidase, alpha-galactosidase, beta-galactosidase, beta-N-acetylglucosaminidase, and alpha-L-fucosidase-were systematically purified by ammonium sulfate precipitation, gel filtration chromatography, and density gradient isoelectric focusing. The isoelectric focusing resolved the glycosidases into distinct, well-separated fractions and revealed three differently charged forms of beta-N-acetylglucosaminidase and of alpha-L-fucosidase. Furthermore, alpha-glucosidase and beta-N-acetylglucosaminidase were shown to possess dual affinities for the respective galactoside substrates, and beta-galactosidase also hydrolyzed beta-D-fucoside. alpha-Glucosidase was purified to homogeneity, as indicated by a thin-layer isoelectric focusing zymogram technique. The glycosidases, with exception of beta-glucosidase and the acid alpha-L-fucosidase, were each separated from other glycosidic activities to 99%. The molecular weights varied between 58,000 and 125,000. The pH optima ranged from 4.8 to 6.9.

Human alpha-2-macroglobulin. Studies on the electrophoretic heterogeneity

Purified alpha-2-macroglobulin may be resolved into as many as five electrophoretic bands on selected polyacrylamide gel systems. The microheterogeneity does not result from prior proteolytic attack but appears to correspond to different conformational states of the inhibitor. Trypsin binding capacity and the extent of subunit cleavage into 120,000 and 70,000 dalton fragments by mild alkaline treatment are related to the proportion of fast and slow electrophoretic forms. Study of proteinase binding after electrophoretic separation by special zymogram techniques confirms that the fastest electrophoretic form has very low binding capacity. No electrophoretic differences conld be observed in alpha-2-macroglobulin derived from cystic fibrosis plasma relative to control alpha-2-macroglobulin. Alpha-2-macroglobulin appears to exist as a simple, slow electrophoretic form in fresh plasma but converts into faster forms upon aging the plasma or during purification. Characterization of the electrophoretic microhetergeneity of alpha-2-macroglobulin preparations should be a prerequisite for the study of its proteinase binding properties.

Genetic variability in natural populations, evidence in support of the selectionist view.

Genetic variability in 6 neotropical anopheline species, analyzed, by zymogram technique, has been described. The results support the 'selectionist' theory of the evolutionary significance of high levels of molecular diversity in natural populations.

Quantitative Immunoelectrophoretic Analysis of Streptococcus pyogenes Membrane

The antigenic composition and molecular structure of the plasma membrane of Streptococcus pyogenes (group A; M type 6) were studied by crossed immunoelectrophoresis (XIE) and other related quantitative immunoelectrophoretic techniques. After establishment of a reference pattern of 29 immunoprecipitates, the relative differences in amounts of individual antigens contained in membranes isolated from cells that were harvested during the exponential or stationary phase of growth were examined. Relative increases and decreases in amounts of individual antigens were estimated from the areas subtended by immunoprecipitates after XIE of Triton X-100 extracts. The asymmetric distribution of antigens on the inner and outer surfaces of the membrane was established in absorption experiments with intact, stable protoplasts. Of the 29 immunoprecipitates, 8 appeared to contain antigens exposed on the outer surface of the membrane, whereas 11 appeared to contain antigens either located on the inner surface or unexposed. Six antigens appeared to have limited exposure on the outer surface, and four others remain to be assigned. Certain immunoprecipitates were characterized with respect to enzymatic activity or interaction with the lectin concanavalin A. Reduced nicotinamide adenine dinucleotide dehydrogenase (EC 1.6.99.3), adenosine triphosphatase (EC 3.6.1.3), and polynucleotide phosphorylase (EC 2.3.7.8) were demonstrated by zymogram techniques. The latter two activities were present within the same immunoprecipitate, suggesting the occurrence of a multienzyme complex. In addition, the areas under the immunoprecipitates containing the three enzymatic activities were not affected by absorption of antimembrane immunoglobulin with intact protoplasts and thus appeared to be located on the inner surface of the membrane. The results from absorption experiments also suggested that the exposure of outer protoplast surface antigens was greater on protoplasts from exponential-phase cells than on those from stationary-phase cells, even when found in increased amounts in the latter.

Chromosomal location and evolution of isozyme structural genes in hexaploid wheat

The genetic control of the production of multiple forms of lipoxygenase, endopeptidase, acid phosphatase, aminopeptidase, and alcohol dehydrogenase in hexaploid wheat has been studied using the zymogram technique. Aneuploid strains which encompass a range of from zero to four doses of each chromosome in the complement were analysed. The localisation of 22 structural genes to specific chromosome arms is reported. Extensive inter-genomic variation between homoeologous isozyme structural genes has been detected. The results of this study are consistent with the hypothesis that many of the duplicated structural genes of this species have diverged in structure and function subsequent to the origin of the polyploid wheats.

Immunochemical studies on a phenobarbital-inducible esterase in rat liver microsomes

Detergent extracts of liver microsomes from drug-treated rats were analyzed semiquantitatively in crossed immunoelectrophoresis in combination with a zymogram technique for nonspecific esterase activity. One esterase-active antigen was quantitatively induced by phenobarbital as demonstrated with both antimicrosomal antiserum and a monospecific antiserum prepared against this antigen. No similar inducation of this or any other esterase-active antigen was detected after 3-methylcholanthrene treatment. With the monospecific antiserum, the antigen was also detected in other organs, capable of carrying out hydroxylation reactions, such as lung and kidney. It was, however, not induced by phenobarbital in these organs. Quantitative enzyme assays with acetanilide as substrate indicated that the phenobarbital-inducible esterase could also function as an amidase. Furthermore, from absorption studies, it was concluded that this antigen is located on the cytoplasmic side of the microsomal vesicles. Crossed immunoelectrofocusing experiments revealed that this esterase-active antigen consists of five subcomponents with different charge properties.

Interaction of mucopolysaccharides with chymotrypsin

Chondroitin sulphates A, B and C form complexes with chymotrypsin which enhance the hydrolytic activity of the enzyme towards glutaryl- l-phenylalanine-p-nitroanilide as much as 270%, as in the case of chondroitin sulphate A. Hyaluronic acid, a non-sulphated mucopolysaccharide, at a weight ratio of 6.67:1 to the enzyme, does not influence the rate of cleavage of the substrate by chymotrypsin. Proflavin difference spectra and ultra-violet spectra suggest that a complex is formed between chymotrypsin and hyaluronic acid, as well as chondroitin sulphates A, B and C. The proflavin molecule is not displaced from the active site of the enzyme upon association with the mucopolysaccharides. When the mucopolysaccharide—chymotrypsin complexes are electrophoresed on analytical polyacrylamide disc gels at pH 8.9, the complexes are visualized by toluidine blue (for mucopolysaccharide), coomassie brilliant blue (for protein) and by the zymogram technique (with glutaryl- l-phenylalanine-β-naphthylamide), at a position between that for chymotrypsin alone and that for the mucopolysaccharide alone.

Arylamidases of rat liver and chemically induced hepatomas 1. Subcellular distribution of l-leucine 2. Naphthylamidase-active antigens

The subcellular distribution of arylamidase-active antigens in rat liver and in two chemically induced hepatomas (D23 and D33) was investigated. Soluble antigens or detergent-solubilized membrane antigens from isolated subcellular fractions were tested in fused rocket immunoelectrophoresis against antisera prepared against each of the fractions. The arylamidase active antigens were identified by means of a zymogram technique using l-leucine 2-naphthylamide as substrate. Two arylamidase-active antigens were shown to be shared between plasma membranes, microsomes, lysosomal membranes and lysosomal content of the hepatocytes. One of these occurred predominantly in the plasma membranes (the plasma membrane arylamidase) while the other was preferentially found in the lysosomal content (the lysosomal content arylamidase). Also a third arylamidase-active antigen was identified and was shown to be restricted to the microsomes and the lysosomal membranes (the microsomal/lysosomal arylamidase). The rat liver plasma membrane arylamidase-active antigen was also present in plasma membrane, microsomal an cell-sap fractions of both the hepatomas. However, in the hepatomas this antigen occurred predominantly in the microsomal fraction. The plasma membrane arylamidase was the only arylamidase-active antigen found in the hepatoma D33 while the plasma membrane and microsomal fractions of hepatoma D23 also contained another antigen with this activity. Neither the lysosomal content arylamidase nor the microsomal/lysosomal arylamidase could be detected in any of the hepatoma fractions.

Gel electrofocusing combined with zymogram techniques for the characterization of β-lactamases from gram negative bacteria

Gel electrofocusing combined with zymogram techniques for the characterization of β-lactamases from gram negative bacteria

Gel electrofocusing combined with zymogram techniques for the characterization of β-lactamases from gram negative bacteria

Gel electrofocusing combined with zymogram techniques for the characterization of β-lactamases from gram negative bacteria

Location of chromosomal control of GA-induced enzyme release by distal half-grains of wheat.

The three enzymes, alpha-amylase, peroxidase, and acid phosphatase, are among those released by the aleurone of bread wheat, Triticum aestivum, as a direct response to gibberellins (GA) from the embryo during germination. Aneuploid genotypes are used to investigate the chromosomal location, nature, and extent of genetic control of the release of these enzymes in endosperms after induction by exogenous GA. Ditelosomics demonstrate the effect of removal of known chromosome arms, and tetrasomics show the effect of duplication of chromosome pairs. Quantitative analysis demonstrates complex control systems over and above those previously found using zymogram techniques. For alpha-amylase, chromosome arms with net promoter effects and chromosomes with net inhibitor effects were found. These effects were not chromosome-dosage responsive, unlike the promoter-inhibitor system found for acid phosphatase control. Peroxidase levels in the endosperms were generally high in both types of aneuploids. With one exception, all chromosomes showing involvement as ditelosomics showed a similar effect as tetrasomics, indicating that in the euploid a balanced state restricting, rather than promoting, peroxidase levels existed.

Relationships between electrophoretic patterns of esterases from Salmonella.

Esterases of 85strains of the four biochemically-defined subgenera of Salmonella, when analysed by the acrylamide-agarose zymogram technique using several synthetic substrates, gave four principal bands (E1, E2, E3, E4) and two minor ones. The E1 esterase band hydrolysed alphs-naphthyl acetate, whereas the E2 band hydrolysed beta-naphthyl acetate. These bands were resistant to di-isofluoropropyl phosphate (DFP) and their electrophoretic distribution among the strains occurred within a relatively small MF range, MF being the distance moved by the esterase band as a percentage of the distance moved by the dye front. The E3 band hydrolysed alpha-naphthyl acetate and alpha-naphthyl napbuyyrate and, to a lesser degree, beta-naphthyl esters, whereas the E4 band hydrolysed alpha-naphthyl acetate. These bands were sensitive to DFP and their electrophoretic distribution among the strains occurred in a wide MF range. ALL Salmonella strains were closely related in terms of their esterase profiles. However, the divergences in electrophoretic distribution of bands E3 and E4 were sufficient to recognize the subgenera of most of the Salmonella strains analysed.

Isoelectric focusing of milk-clotting enzymes

SummaryIsoelectric focusing in thin-layer polyacrylamide gel has been applied successfully to the characterization and identification of calf rennet and its substitutes. The use of acidic pH gradients (pH range 2·5–6) allows the identification of calf and microbial rennets and bovine and pig pepsins.A new, very rapid and sensitive zymogram technique has been developed by overlaying the gel with a highly porous, transparent cellulose acetate film impregnated with casein.

Developmental specificity and evolution of the acid phosphatase isozymes of Triticum aestivum and its progenitor species.

The tissue and developmental specificities of the acid phosphatase (ACPH) isozymes of Triticum aestivum and its progenitor species T. turgidum and T. tauschii have been determined and compared using the zymogram technique. Tissue and/or developmental variation in relative staining intensity, suggestive of variation in the quantity of active enzyme present, was observed for each of the seven major isozymes expressed. Isozymes homologous to each of the major isozymes of the hexaploid were detected in one or the other of the progenitor species. No difference in the pattern of developmental or tissue specificity was observed between the species for any isozyme. However, ACPH-4, encoded by ACph4, a structural gene linked to chromosome 4A, differs in electrophoretic mobility between T. aestivum and T. turgidum, indicating that divergence has occurred between these species at the Acph4 locus since the origin of the hexaploid. The molecular weight of each of five ACPH isozymes of the hexaploid was determined to be approximately 58,000. This finding, plus the results of the developmental study and the earlier demonstration that the structural genes for six isozymes (includinomosomes, provides evidence in support of the suggestion that the ACPH structural genes of hexaploid wheat are homoeologous related.

The Applicability of Agar-Diffusion Test in Measuring Deoxyribonucleases in Pig Intestinal Content

The suitability of an agar-diffusion test (ADT) using toluidine blue deoxyribonucleic acid agar (TDA) for measuring DNase activity in pig intestinal contents was investigated. The ADT was compared with a spectrophotometrical method. Distinct metachromatic zones around wells in the DNA-containing agar, into which the intestinal content was applied, indicated DNase activity. The DNase activity was determined semiquantitatively by making serial twofold dilutions of the intestinal content. The spectrophotometrical method was optimal at pH 7.2. The ADT proved to be most sensitive at pH 5.6. The ability of the 2 methods employed to measure low concentrations of DNases was equal. However, the ADT was considered more suitable than the spectrophotometrical method because ADT measured reduced amounts of enzyme. DNase activity was demonstrated throughout the small intestine and in the large intestine. By the zymogram technique, at least 3 different DNases could be demonstrated in the lower parts of the small intestine, 1 of which could be of extrapancreatic origin.

Preparative capillary isotachophoresis : Principle and some applications

Capillary isotachophoresis has been developed and modified for use as a preparative method. The sample zones are pushed out of the capillary under voltage by a counter-flow of leading electrolyte through a small T-tube connection immediately after the UV detector. The zones are collected on a moving cellulose acetate strip without loss of high resolution. This permits simple detection and identification by methods new to capillary isotachophoresis, e.g., immunological and radioactive methods. The zymogram technique can be used directly on the strip. The preparative procedure also makes capillary isotachophoresis a powerful purification method.

Saliva acid phosphatases: genetic studies.

Acid phosphatases from human saliva have been demonstrated by the zymogram technique. Six phenotypes were found. Family and population studies suggested that the phenotypes are the product of two loci, Sap-A with three alleles A, A' and O, and Sap-B with two alleles B and O.

Human saliva esterases: genetic studies.

Esterases from human saliva have been demonstrated by the zymogram technique. Three phenotypes were found in region 1. Family and population studies suggested that these phenotypes are the products of an autosomal locus with two alleles, Set-1F and Set-1S. Region 1 esterases are carboxylesterases.