Enzyme Activity Staining Research Articles

Genetic evidence for a role of hexokinase isozyme PII in carbon catabolite repression in Saccharomyces cerevisiae.

A mutant of Saccharomyces cerevisiae that was selected for resistance to carbon catabolite repression also had reduced hexokinase activity. Hexokinase isoenzymes were purified from mutant and wild type cells. The specific glucokinase and hexokinase isozyme PI were present at normal levels in mutant and wild type, but no hexokinase isozyme PII activity was detected in the mutant. Staining for enzyme activity after electrophoresis of crude extracts also indicated that hexokinase PII was absent in the mutant. Mutant and wild type segregants gained by tetrad analysis were investigated electrophoretically. Staining for enzyme activity confirmed that catalytically inactive hexokinase PII and the defect in carbon catabolite repression always co-segregated. The results support the hypothesis that hexokinase PII might mediate carbon catabolite repression.

Oxidoreductase activity in the pulp of replanted and autotransplanted teeth in young dogs

The purpose of the present investigation was to assess the vitality of the pulp of replanted and autotransplanted teeth with incomplete root formation by means of histochemical demonstration of oxidoreductase activity during a 180-day period. Staining for enzyme activity was seen in the whole pulp during the first postoperative days. After 10 days, staining was observed only in the foraminal area of the pulp and in blood vessels in apparently necrotic tissue. Thirty days postoperatively staining was observed in the whole pulp; 180 days after replantation or autotransplantation six out of eight teeth exhibited enzyme activity in the entire pulp. Thus, it appeared that the pulp of the replanted and autotransplanted teeth with partly formed roots first underwent necrosis and that the dead tissue then was replaced by new tissue.

Purification and characterization of uricase, a nitrogen-regulated enzyme, from Neurospora crassa

Uricase, a purine catabolic enzyme, is controlled by induction and by nitrogen catabolite repression in Neurospora. Uricase was purified nearly 1000-fold to homogeneity. Only a single protein band could be detected in analytical gels of the pure enzyme, and the protein band in each case corresponded exactly to the position of in situ staining for enzyme activity. The molecular weight of native uricase was estimated to be 123,000 ± 7000. The enzyme is a tetramer composed of identical or similar-sized subunits. The K m value of uricase for uric acid was estimated to be 4.2 × 10 −5, m. Oxonic acid was shown to be a competitive inhibitor of uricase, with a K i value of 6.7 × 10 −7, m. Uricase is a stable enzyme and is not subject to feedback inhibition by ammonia, glutamate, or glutamine in Neurospora. The regulation of uricase appears to occur primarily at the biosynthesis level. Uricase appears to be a metalloenzyme with no essential sulfhydryl groups.

20α-Hydroxysteroid dehydrogenase activity and the x-zone of the female mouse adrenal

20α-Hydroxysteroid dehydrogenase (20α-OHD) activity can be observed in the adrenal cortex of the young virgin female mouse using a histochemical staining procedure with blue tetrazolium (BT) which corresponds in distribution to the adrenal X-zone. The occurrence of both the 20α-OHD activity and the BT staining cells have been compared with changes in the X-zone after hormonal manipulation. The enzyme activity and BT staining cells can be induced in the male and female adrenal at age 8 days by estrogen administration. Normally the enzyme activity cannot be observed in the male adrenal, and not until the age of 12 days in the female adrenal. Treatment of both the male and the female with testosterone or progesterone causes regression of the X-zone and in the female there is a loss both of BT staining (qualitative) and of 20α-OHD enzyme activity, as well. The adrenal X-zone of the female mouse, which heretofore has been characterized only on the basis of morphology, can now be assessed by a biochemical parameter, the presence of 20α-OHD activity, an enzyme affected by sex steroid administration.

酵素結合性免疫グロブリン検出への免疫固定法の応用

The combination of both the electrophoresis and the immunofixation and of the thin layer gel filtration and the immunofixation followed by enzyme activity staining on antigen-antibody complexes were applied to detect and identify the enzyme-linked immunoglobulins which were observed as macromolecular enzyme complexes in human sera. For any given antigen-antibody system, the optimum concentration of antigen in the serum to be analysed must be given experimentally. Moreover, removing of unreacted proteins must be carefully performed with the solution which will protect the activity of the enzyme to be detected.In the cases of LDH- and amylase-linked immunoglobulins the detection of minimum enzyme activities on Cellogel membrane were limited by staining sensitivity. Thus, two to ten times concentration of commercially available antibodies was required to give the optimum concentration of antigen-antibody complexes. In the cases of alkaline-phosphatase-linked immunoglobulins, the detection of the enzyme activity was easily amplified by the elongation of the incubation period. Thus, the optimum concentration of antigen was given without concentration of commercial antibodies.In the studies of macromolecular enzyme complexes in human sera, the immunoprecipitation technique and enzymo-immunoelectrophoresis were conventionally carried out to identify the enzyme-linked immunoglobulins. Some cases, however, still ramain ambiguous because of unskilled and unstable techniques used for analysing these macromolecular enzyme complexes. Thus, these immunofixation techniques followed by enzyme activity staining (enzyme immunofixation), described in this paper can facilitate the detection and identification of such macromolecular complexes as enzyme-linked immunoglobulins.

Purification and partial characterization of three forms of alpha-glucosidase from the fruit fly Drosophila melanogaster.

Three forms of alpha-glucosidase, I, II, and III, have been purified from the whole body extract of adult flies of Drosophila melanogaster in yields of 2.1, 5.3, and 6.7%, respectively. The purification procedures involved ammonium sulfate fractionation, Con A-Sepharose 4B affinity chromatography, DEAE-Sepharose CL-6B ion exchange chromatography, Sephacryl S-200 gel filtration, and preparative gel electrophoresis. Each purified enzyme showed a single band on polyacrylamide gel on both protein and enzyme activity staining. The molecular weights of alpha-glucosidases I, II, and III were estimated to be 200,000, 56,000, and 76,000, respectively, by gel filtration. SDS gels indicated that alpha-glucosidases II and III were each composed of a single polypeptide chain, whereas alpha-glucosidase I was composed of two identical subunits. Both alpha-glucosidases II and III hydrolyzed sucrose and p-nitrophenyl-alpha-D-glucoside (PNPG), but alpha-glucosidase I hydrolyzed PNPG to a much lesser extent than sucrose. For sucrose the pH optima of alpha-glucosidases I, II, and III were pH 6.0, 5.0, and 6.0 and the Km values were 13.1, 8.9, and 10 mM, respectively. For PNPG the pH optima of alpha-glucosidases II and III were pH 5.5 and 6.5 and the Km values were 0.77 and 0.21 mM, respectively.

Les phénomènes immunitaires de la syngamose expérimentale: 1. Structure antigénique deSyngamus trachea1

The soluble somatic antigens of Syngamus trachea were studied by gel electrophoresis and by immunoelectrophoresis, including staining for enzyme activity. Rabbit antiserum prepared by repeated injections of parasite material was used to demonstrate the antigens. Serum obtained after 12-15 weekly injections produced up to 16 precipitation arcs with the S. trachea preparation. Two of the arcs represented reactions against contaminating host material, and enzyme activity was detected in seven arcs. Cross reactions occurred with extracts of Trichinella spiralis (two arcs) and Ascaris lumbricoides (one arc).

Electrophoretic separation of γ-glutamyl transpeptidase isoenzymes on cellulose acetate “Cellogel”

A method was described for separation of γ-glutamyl transpeptidase (γ-GTP) isoenzymes by cellulose acetate membrane (Cellogel). Enzyme activity staining was carried out by use of γ-L-glutamyl-α-naphthylamide as substrate and fast blue B as color developing agent. Zymogram on Cellogel membrane was treated with 5% trichloroacetic acid solution and applied for densitometry.The conditions for this method (concentration of substrate, diazo reagent, glycylglycine, pH of buffer, incubation time, etc.) were examined and optimal conditions were proposed.This method was simple and sensitive enough to detect γ-GTP isoenzyme patterns from the normal serum containing 30mU/ml activity. Linear relationship between color development of zymogram and enzyme activity was also obtained from 30mU/ml to 230mU/ml in serum sample and this method, therefore, was available for the routine work as semi-quantitative method.

15-Hydroxyprostaglandin-Dehydrogenase aus Humanplacenta, l. Isolierung und Charakterisierung

15-Hydroxyprostaglandin dehydrogenase was isolated from human term placenta up to a final purification of 380-fold. A spec. act. of 2000 mU/mg of protein was reached. The preparation was not homogeneous as judged by analytical disc electrophoresis. The enzyme could be stored in the presence of 50% glycerol and 10mM 2-mercaptoethanol without any loss of activity for at least one year. A distinct single protein band stained after discontinuous polyacrylamide gel electrophoresis was shown by enzymatic activity staining to correspond to 15-hydroxyprostaglandin dehydrogenase activity. Thus no evidence for the exitstence of isoenzymes was obtained. The protein in the final preparation steps showed neither alcohol dehydrogenase, NAD reductase, nor NADH oxidase activity, nor enzymatic conversion of prostaglandin or 15-oxoprostaglandin in the absence of NAD and NADH. No spontaneous reactions between NAD and prostaglandin or NADH and 15-oxoprostaglandin were detectable in the absence of the enzyme. Ethanol and glycerol slightly inhibited the reaction. Various buffers (Tris/HC1, potassium phosphate, HEPES, and triethanolamine) and salts (ammonium chloride, ammonium sulfate, potassium chloride, and sodium chloride) had different effects on the reaction rate. The pH profile of the reaction shows a plateau between pH 7.0 and 7.8 and a steep maximum at pH 9.5. A linear Arrhenius plot was obtained for the temperature dependence of the reaction from 20 to 37 degrees C. The molar activation enthalpy of the reaction was calculated to be 13.1 kcal/mole. The molecular weight of 15-hydroxyprostaglandin dehydrogenase was estimated to be 32000 -/+ 3000 by gel filtration on Sephadex G-150 in the presence of 10mM mercaptoethanol.

HISTOCHEMICAL DISTRIBUTION OF THE SECONDARY ALCOHOL DEHYDROGENASE IN THE RAT, ESPECIALLY IN THE ADRENAL CORTEX

In the site of steroid production, the histochemical activity of secondary alcohol dehydrogenase on isopropanol as its substrate with NAD as its coenzyme will reflect the inversion of 20ƒ?-22R-dihydroxycholesterol in the biosynthesis of steroid hormone, We applied this method on organs from all parts of the body of Wistar strain rats and observed positive reaction in the adrenal cortex, ovary and testis. In the adrenal cortex of mature rats, the zona glomerulosa exhibited a weakly positive activity, the outer to middle portions of the zona fasciculata an intensely positive activity, the inner portion of the zona fasciculata a weakly positive or negative activity, and the zona reticularis a positive activity. A proportional relationship between secondary alcohol dehydrogenase activity and the amount of lipid was present in the adrenal cortex of mature rats. In addition, the specific distribution of young and old rats was described. The secondary alcohol dehydrogenase is the enzyme of dehydration on the secondary alcohol. In 1965, Hardonkl' noted the similarity between the enzymes participating in the oxidative cleavage of the side chain of cholesterol as a precursor of steroid and secondary alcohol dehydrogenase. Using various secondary alcohols, the distribution of activity of the dehydrogenase in the human body was studied. Among them an enzyme acting on isopropanol as its substrate with nicotinamideadenine dinucleotide (NAD) as its coenzyme had a distribution of activity in the human body in agreement with the site of steroid production such as the adrenal cortex, ovary and testis, according to the description of Hardonkl'. The histochemical detection of various enzymes participating in the biosynthesis and metabolism of steroid hormones in the adrenal cortex and other tissues has been carried out on steroid-3/9-o1 dehydrogenase6', glucose-6-phosphate dehydrogenase and other enzymes2>''4). However, when no specific significance has yet been established, the secondary alcohol dehydrogenase activity will be important, too. We applied this method on organs from all parts of the body, particulary the site of steroid production of male and female Wistar rats, considering the relationship between this enzyme activity and lipid stain.

AXENIC CULTURE STUDIES OF ENTAMOEBA SPECIES.

Summary Axenic Entamoeba invadens could be grown at room temperatures in the diphasic types of medium used for E. histolytica. Conversely, axenic E. histolytica from diphasic cultures could at ca. 37°C, within limits, be adapted into variations of the monophasic media originally developed for E. invadens. Only occasional E. histolytica inocula grew for more than one or two weekly subcultures in such monophasic media, but several series have been transferred up to seven times. Of the various natural ingredients in E. histolytica media, only serum was required at consistently high levels (ca. 10%). E. invadens has been maintained for several weeks in chemically defined and partially defined media, but population increases were not observed. The survival of E. invadens but not E. histolytica was significantly extended by helping to preserve anaerobic conditions in cultures with a petroleum jelly seal. Zone electrophoresis in starch gel combined with chemically specific staining for enzymatic activity was used in an attempt to compare the esterase enzymes of the two axenically cultured species. However, only esterases associated with the culture media and not with the amebae themselves were demonstrated.

Zymograms of the Parasitic Nematodes, Neoaplectana glaseri and N. carpocapsae, Grown Axenically

The application of a technique for separating different molecules (zone electrophoresis in starch gel) combined with identification techniques (chemically specific staining for enzymatic activity), i.e., the zymogram, was used to show molecular similarities and differences among enzymes of two parasitic nematodes grown axenically. The esterases of Neoaplectana glaseri Steiner, 1929, were multiple and varied in activity to substrates. They were similar for worms grown on media from natural sources or in a medium that was in part chemically defined. They were insensitive to eserine and activated by taurocholate. In addition, they were qualitatively similar for populations of worms composed almost exclusively of third-stage larvae as compared to populations of worms which contained adult and larval stages. The esterases of N. carpocapsae Weiser, 1955, differed from those of N. glaseri. Neither species showed alkaline phosphatase or lactic dehydrogenase activity. Acid phosphatase, present in multiple molecular forms in N. glaseri, differed from that of N. carpocapsae. Working with parasites in culture, one must consider many criteria of adaptation or change. Morphological changes might be the last and least apparent. Subtler criteria such as infectivity, antigenicity, metabolic activity, and genetic transformability may correspond to molecular differences. The quality and/or quantity of these can mirror not only adaptations from nature to culture, or from one type of culture to another, but also differences between related species and changes associated with development. The evidence so far is that Neoaplectana glaseri Steiner, 1929, a nematode of certain insects (Glaser and Fox, 1930), has changed little under axenic conditions compared to protozoa or cultured mammalian cells. No changes in morphology or infectivity have appeared during 17 years or ca. 216 cultured generations (Stoll, 1953, and personal communication). The present work is a study of molecular similarities and differences among selected enzymes of N. glaseri at different stages of development and grown under different axenic conditions. Comparison is made with enzymes of another nematode of insects, Received for publication 19 March 1963. * Present address: Division of Life Sciences, The University of California, Riverside. N. carpocapsae Weiser, 1955. The major tool used, the zymogram, combines a technique for separating molecules according to differences of size and charge (zone electrophoresis in starch gel) and identification techniques (chemically specific staining for enzymatic