Zymogram Technique Research Articles

An Autosomal Glucose-6-Phosphate Dehydrogenase (Hexose-6-Phosphate Dehydrogenase) Polymorphism in Human Saliva

Glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase) from human saliva has been demonstrated by the zymogram technique. Three phenotypes were found. Family and population studies suggested that these phenotypes are the products of an autosomal locus with two alleles Sgd-1 and Sgd-2.

Extracellular Enzyme System Utilized by the Fungus Sporotrichum pulverulentum (Chrysosporium lignorum) for the Breakdown of Cellulose

Five endo-1,4-beta-glucanases (EC 3.2.1.4) have been separated from culture solutions OF THE ROT FUNGUS Sporotrichum pulverulentum (formerly called Chrysosporium lignorum) grown on powder cellulose as the sole carbon source. They have been extensively purified and characterized with regard to some physicochemical properties. The purifications have been carried out on a quantitative basis, the purity of the enzymes being tested in several ways. After purification they all showed one single protein band in analytical polyacrylamide electrophoresis, on dodecyl-sulphate gels and in analytical isoelectric focusing on flat-bed polyacrylamide gels. One exo-1,4-beta-glucanase has also been identified in the culture solution and separated from the endo-1,4-beta-glucanases. From the data obtained during the quantitative purification it has been possible to calculate that the ratio of activity between the five endoglucanases T1, T2a, T2b, T3a, and T3b in the culture solutions is 4:1:1:1:1. It has also been calculated that the weight ratio endoglucanase protein to exoglucanase protein is approximately 1:1. Flat-bed isoelectric focusing has been used for the identification of the individual endoglucanases and a new zymogram technique, useful for studies of carbohydrases in general, has been developed. The molecular weights, determined by ultracentrifugation, and calculated on the basis of a knowledge of the amino acid composition and carbohydrate content vary between 28200 and 37500. Small but significant differences in the amino acid compositions of the different endoglucanases have been found. The carbohydrate content varies between 0 and 10.5%, all but one of the enzymes being glycoproteins. For two of these the exact carbohydrate composition has been determined. Enzyme T1 contains 2 glucose and 19 mannose units per enzyme molecule while enzyme T2b contains 5 mannose, 7 galactose, 1 glucose and 1 arabinose unit per molecule.

Examination of Brevibacterium linens by an electrophoretic zymogram technique.

SUMMARY: Fifteen isolates of Brevibacterium linens were tested by polyacrylamide gel electrophoresis. Protein patterns and zymograms of 18 intracellular enzyme activities (α-naphthyl acetate and β-naphthyl butyrate esterases, tributyrinase, alcohol-, lactate-, malate-, succinate-, glucose-6-phosphate-, 6-phosphogluconate-, isocitrate-, alanine- and glutamate-dehydrogenases, tetrazolium oxidase, catalase, peroxidase, arylamidase, proteinase and amylase) and four extracellular enzyme activities (α-naphthyl acetate esterase, arylamidase, proteinase and amylase) were examined. Enzyme activities were demonstrated by either specific staining reactions or by the subsequent diffusion of the proteins into a substrate layer. Striking variations of the enzyme patterns occurred among the strains. The variation, however, depended largely on the activity under consideration. Eight different enzymes were selected to calculate a matrix of similarity. The results indicated a separation of the strains into two major subdivisions or biotypes. Only one strain (BLI07) differed markedly from the others.

A zymogram technique for the detection of carbohydrases

A zymogram technique for the detection of carbohydrases

An esterase zymogram of Escherichia coli.

SUMMARY: The intracellular esterases of 25 strains of Escherichia coli, growing exponentially on a minimal medium, were analysed by the acrylamide-agarose zymogram technique. Five kinds of esterase bands were defined: three major bands (A, B and C) and two minor ones. The A and B esterase bands hydrolysed α-naphthyl, β-naphythl and indoxyl acetates; they were inhibited by di-iso-fluoropropyl phosphate (DFP). Esterase band B also hydrolysed the α- and β-naphthyl butyrates and was stable at 60°C. Esterase band C hydrolysed only β-naphthyl acetate and it resisted DFP. The A, B and C esterase bands showed variations in electrophoretic mobility which seemed to indicate an intraspecific differentiation of molecular structures of the esterase that could have arisen during microbial evolution.

Proteolytic and Lipolytic Enzymes from Larvae of Oedemagena Tarandi (L.)

Some biological properties of proteolytic and lipolytic enzymes of the larval stages of Oedemagena tarandi (L.) were examined. While the proteinase titres seemed to vary only slightly from one larval stage to another, the lipase titres of the first larval stage seemed to be much greater than those of the second and third larval stages. The zymogram technique used showed only one proteinase, which was inhibited by all the biological inhibitors tested. Bacteriological examinations of the external surfaces and internal organs of the different larval stages showed only a very sparse flora.

The zymogram technique for mutagenicity testing

The zymogram technique for mutagenicity testing

The Effects of Gamma Irradiation on Some Properties of Two Aeromonas Proteinases

The proteinases A and B of Aeromonas liquefaciens and proteinase B of Aeromonas salmonicida have, in the crude and purified state, been exposed to various doses of γ-irradiation from a Co60 source, and the D37 values indicate that the proteinase A is the most resistant. Casein was shown to have a marked protective effect on both proteinases during the irradiation, and while solutions of the purified enzymes were inactivated by doses usually used for food pasteurization, the crude enzymes, or solutions of purified enzymes to which casein was added, required doses usually used for food sterilization before being inactivated. Only minor effects of the environmental pH were observed. The antigenic properties of the enzymes seemed to be qualitatively unchanged in solutions exposed to 150 krad as observed using the casein precipitation inhibition test, and the irradiated proteinases were also inhibited by the naturally occurring proteinase inhibitors in the immune sera. The enzymoserological properties were not influenced by the changes in electrophoretic migration which were demonstrated by the zymogram technique. These proteinases are suitable as models for the examination of the physical properties of food spoiling enzymes and also for taxonomical work.

Detection of fibrinolytic activity by a zymogram technique after isoelectric focusing

Fibrin plates containing small quantities of plasminogen have been used to detect protein zones containing staphylokinase after isoelectric focusing in polyacrylamide gels. Staphylokinase promotes the conversion of plasminogen to plasmin, which in turn degrades fibrin so that clear zones are produced in an opaque fibrin plate. The result is conveniently recorded by photography. General aspects of this procedure and conventional zymogram techniques in connection with isoelectric focusing are discussed. It is suggested that studies of this type should be well fitted for investigations on multiple molecular forms of proteins.

Identification of Crude and Purified Bacterial Proteinases by Agar Gel Diffusion and Agar Gel Electrophoretic Procedures

The double diffusion technique, immunoelectrophoresis, the zymogram technique for proteinases and the casein precipitation inhibition test (GPI-test) were employed for identification of specific bacterial proteinases produced by Aeromonas liquefaciens, Aeromonas salmonicida and Pseudomonas aeruginosa. The precipitation lines observed with both homologous and heterologous proteinase-antiproteinase systems in the double diffusion technique and in immunoelectrophoresis, supported in certain respects previous findings regarding the Aeromonas proteinases, but the reactions were not sufficiently specific to give a confirmation of the real relationships between the proteinases. It is concluded that the double diffusion technique and Immunoelectrophoresis are less specific than the GPI-test for the identification of both crude, and purified, proteinase-antiproteinase systems. The zymograms, in combination with the immunoelectrophoretic patterns, could under certain conditions give useful information about the identity of lines representing the proteinase-antiproteinase precipitates in the double diffusion systems. The number of precipitation lines caused by the crude proteinase solutions in Immunoelectrophoresis decreased during storage of the crude antigens, and the solutions could finally behave like the solution of purified antigen. It was shown that the GPI-test was at least 66 to 225 times more sensitive with respect to antigens, and two to three times more sensitive with respect to antisera than the double diffusion technique, for the three systems examined. This is of methodological importance, as high functional activity may be present in a proteinase solution although the structural conditions are unsuitable or the amount of enzyme protein is too small to allow the development of any precipitation lines in the double diffusion technique.

Comparative electrophoretic and serological analyses of Vibrio comma and Aeromonas liquefaciens proteinases.

The occurrence of one, or two, proteinase fractions was demonstrated for 5 strains of Vibrio comma using the zymogram technique for proteinases. Most of the proteinase fractions moved more or less rapidly towards the cathode, although some were shown to migrate in the opposite direction at pH 6.2. Serological cross reactions were observed between proteinase fractions produced by a strain of Aeromonas liquefaciens and 2 of the V. comma stvains examined. The proteinase A of Ae. liquefaciens was shown to be enzymoserologically identical, or closely related, to one of the proteinase fractions of V. comma, while no relationship was observed between the proteinase B of Ae. liquefaciens and any of the V. comma proteinases. In order to remove the naturally occurring proteinase inhibitors from the antiproteinase‐containing γ‐globulins in immune serum, precipitation with sodium sulphate was used, with success, as demonstrated by disc electrophoresis and the Casein Precipitation Inhibition test (CPI‐test). The need for suitable separation techniques when using enzymes as a basis for the taxonomical differentiation of the corresponding organisms is emphasized.

Genetic control of alcohol dehydrogenase isozymes in Triticum dicoccum.

By means of the zymogram technique, two types of electrophoretic patterns were found for the enzyme alcohol dehydrogenase (ADH) in Triticum dicoccum, a tetraploid species of wheat. Each strain examined showed three bands of ADH activity. The two types of patterns found differed with respect to the relative electrophoretic mobilities and staining intensities of the bands. Evidence that the variation between the patterns is controlled at a single gene locus by two codominant alleles was obtained from appropriate genetic crosses. Heterozygotes for the allelic difference have five bands of ADH activity. These probably represent six different forms of the enzyme, two of which have coincident electrophoretic mobilities. These observations support the hypothesis that ADH exists as a dimer in T. dicoccum. It is probable that the enzyme subunits are coded for by duplicated ADH gene loci, each of which was contributed to the original tetraploid wheats by one of the two diploid progenitors of that group.

A Partial Survey of the Genus Cucurbita for Electrophoretic Variants of Esterase and Leucine Aminopeptidase

Starch gel electrophoretic analysis of crude seed extracts of six species of Cucurbita, including five cultivars of two species and one species hybrid, reveals six distinguishable leucine aminopeptidase mobilities. Two cultivars of C. maxima were found to be polymorphic for this enzyme. Certain species were monomorphic and others polymorphic for alpha-naphthyl acetate esterases. With one possible exception, the species could be identified on the basis of their electrophoretic phenotypes. The systematic and evolutionary significance of the reported enzymatic variants are discussed in terms of the genetic differences they might reflect. Electrophoretic analysis of serum proteins in supporting media is a technique finding increasing application among zoologists for aid in the solution of phyletic problems (Dessauer, 1966; Coates and Twitty, 1967). Plant systematists have to a more limited extent utilized this approach to evolutionary and systematic problems; three such applications using plant proteins have been those of Hall and Johnson (1963), Johnson and Hall (1965), and Boulter et al. (1967). An important extension of the electrophoretic analysis of general proteins, i.e., proteins revealed by staining with Buffalo Black, is the zymogram technique (Hunter and Markert, 1957). This technique reveals specific enzymes following treatment of the gel by appropriate histochemical methods. Only limited application has been made of the zymogram technique to plant taxonomic problems. Yang and Turner (1968) have surveyed four genera (including 17 species) of the Lemnaceae for glutamate, malate and lactate dehydrogenases and betaesterases. These authors concluded that isozyme patterns may prove useful at the species level but not above the generic level for the evaluation of taxonomic relationships. Thurman et al. (1967) have surveyed 103 species of the Fabaceae for formic and glutamic dehydrogenases. These authors were unable to draw any taxonomic conclusions from 1 Present address, Department of Biology, George Mason College of The University of Virginia, Fairfax, Virginia.

Sex difference and gonadal hormone influence on Syrian hamster kidney esterase isozymes.

Thirteen esterase isozymes were separated from kidney extracts of normal adult Syrian hamsters ( Mesocricetus auratus) using the zymogram technique with vertical starch gel electrophoresis and naphthyl esters as substrates. The activities of two esterases were altered by gonadectomy and by treatment with testosterone or estradiol-17β but not with progesterone. One of these esterases showed negligible activity in castrated males and in normal and spayed females. The activity of this esterase increased appreciably in castrated males treated with testosterone or estradiol and in testosterone-treated spayed females. This enhanced activity, however, was not consistent in spayed females treated with estradiol. No sex differences in renal esterases were observed in immature hamsters but a variation was detected in normal adults. The difference appears to be of normal enzyme levels rather than the absence of any esterase. Under the influence of estradiol and progesterone, separately or in combination, distinct alterations in kidney esterase profiles were observed. Certain other quantitative hormonal effects were found. These hormone-dependent esterases do not appear to be cholinesterases, as they were unaffected by eserine. The marked sensitivity of various hamster kidney esterases to estrogen appears to be a unique response and may be related to the estrogenic induction of renal adenocarcinoma, initiated under similar conditions, but not found with androgen-treated or other estrogenized animals. These findings indicate that gonadal effects on the hamster kidney may be of a more complex nature than has been considered heretofore.

ISOZYMES OF A POLYPLOID SERIES OF WHEAT

portion of genetic loci showing variation in outbreeding species (SHAW 1965; HARRIS 1966; and LEWONTIN and HUBBY 1966). Such estimates predict that at least 30 to 40% of genetic loci in*Drosophila, Peromyscus, and man are polymorphic. These predictions have led to a flurry of papers presenting mathematical models which show that, contrary to earlier dogma (discussed by KIMURA and CROW 1964; VAN VALEN 1963), populations could theoretically sustain this degree of variation through balanced polymorphism (KING 1967; MILKMAN 1967; SVED, REED, and BODMER 1967). If heterozygote superiority is the responsible factor for maintaining a high degree of genetic polymorphism, one presumes that the increase in number of proteins associated with heterozygosity offers, in general, the selective advantage. In contrast to the attention which polymorphism has received as a mechanism for maintaining an increased number of proteins in heterozygotes, a second type of protein multiplicity, while widely recognized, has received much less attention in terms of its evolutionary significance. It is illustrated by isozyme patterns showing multiplicity of enzymes shared by every individual in the mating population (the lactic dehydrogenase isozymes of the human are an example). Preliminary estimates indicate that this type of protein diversity, probably arising primarily from gene duplication, is very common. For example, we have recently reported (BREWER and SING 1968a) that ten of sixteen randomly selected human red cell enzymes show isozyme multiplicity common to all individuals. We suggest that this type of protein diversity may make a significant contribution to the total protein diversity available to the individual. In a cross fertilizing species such as man, this could be an alternative strategy to allelic variation (FINCHAM 1966), but it may be of prime importance in a species characterized by obligatory inbreeding, as for example, in the self-pollinator, wheat. This paper presents initial studies of the correlation between protein multiplicity, as measured by the zymogram technique, and certain biological characteristics of hexaploid wheat and its tetraploid and diploid progenitor species. The isozyme data reported here were collected to obtain information on three This investigation was supported by contract AT(11 1) 1152 Atomic Energy Commission, USPHS grant AM 09381,

Multiple forms of serum cholinesterase

Two additional molecular forms of serum cholinesterase have been demonstrated in one of two purified enzyme sources. The other preparation revealed five components and occasionally revealed the extra bands. All components were converted to one major form by concentration. All forms hydrolyzed four substrates by the zymogram technique. Each molecular component was completely susceptible to eserine (10 −5 m) inhibition by spectrophotometric assay. The determination of inhibitor susceptibility in starch gels revealed partial susceptibility of the major form to eserine. A hypothesis was presented that described the multiple components of serum cholinesterase as a series of polymers and/or conformational isomers. The evidence indicated that the multiple forms of serum cholinesterase were isozymes as demonstrated by their source (serum or plasma), similar substrate specificity, complete susceptibility to eserine, and interconvertibility.

Multimolecular forms of profibrinolysin revealed by a zymogram technique

Multimolecular forms of profibrinolysin revealed by a zymogram technique

Genetic control of alcohol dehydrogenase isozymes in maize.

By means of horizontal gel electrophoresis and the zymogram technique, genetic variants and the formation of a hybrid molecule of the enzyme alcohol dehydrogenase (ADH) have been found in Zea mays. Each inbred homozygous stock examined showed two types of ADH isozyme patterns: a fast faint zone and a slower deeply staining zone, both anode-migrating at pH 8.5. The variants found differed in that each of the ADH zones varied in its electrophoretic mobility when compared to its counterpart in the other strain. When appropriate genetic crosses were made, the resulting heterozygotes showed the parental ADH zones, and, in addition, a band of intermediate mobility was formed between the deep-staining ADH bands. However, in the fast-moving zone only the parental isozymes were represented in the heterozygote. The formation of the hybrid molecule and the apparent gene dosage effects support the hypothesis that ADH-2 in maize exists as a dimer, whereas ADH-1 may exist as a monomer.

Observations on the origin of pregnancy-associated plasma proteins

1. The “alpha-2 globulin of pregnancy” has been found in the sera of nonpregnant females and in increased amounts in trophoblastic disease, during pregnancy, and during the administration of Enovid. The placenta, therefore, is not essential to the synthesis of this protein.2. The pregnancy-associated phosphatase and two cystine aminopeptidases, not being observed in patients with trophoblastic disease or during the administration of Enovid. seem to be specific for pregnancy. Since they are also absent from fetal blood, the placenta is the probable site of origin of these enzymes.3. Both Enovid administration and trophoblastic disease elicit the appearance of other aminopeptidase bands. one of which is similar in mobility to one of the pregnancy-associated cystine aminopeptidases. It can be differentiated from it by means of incubation with neuraminidase.4. The possibility of further development of the zymogram technique for the differentiation of trophoblastic disease and pregnancy is discussed.

Sex-associated quantitative differences in the plasma esterases of inbred mice.

The presence of sex-associated quantitative differences in 4 of the plasma •esterases of C57BL/6 mice was determined using the zymogram technique with disc •electrophoresis in acrylamide gel. The levels •of 4 of the esterases are controlled by testos- terone and may be manipulated by hormone treatment, gonadectomy, or a combination of both. A testosterone-like effect of estradiol was also found in one of the esterase bands. No quantitative plasma esterase differences be- tween sexes were observed in prepubescent mice and no qualitative esterase differences were found between the sexes of adult mice of the same strain. (Endocrinology 78: 655, 1966) V ARIOUS reports have appeared in the literature concerning the protein and enzyme differences between the sexes in various species of animals. Dimopoullos and Schrader (1) have shown that male cattle have more /3-globulin and 7-globulin and less a-globulin glycoprotein than females. Espi- nosa et at. (2) have reported sex-associated differences in the alpha-1, alpha-2 and beta serum globulins of mice, while Bond (3) has demonstrated a protein in rat liver that is quantitatively under the control of testos- terone. Allen and Hunter (4) have shown that 5 esterases in the mouse epididymis are affected by testosterone. We (5) have re- cently shown that marked quantitative differences exist between the sexes of various strains of inbred mice in several of the ester- ases in mouse plasma separated by disc electrophoresis in acrylamide gel. Since these differences were not apparent in mice less than 3 weeks of age, a study of the effects of age and sex hormones was carried out in in- tact gonadectomized mice.