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Results for “activity assay”

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At least 19 recordsLinked to original sources
PubMed1979

Pyruvate carboxylase activities in red blood cells and liver of chicks and their dependency on biotin status. First results with activation assays..

Pyruvate carboxylase activities of erythrocytes and liver preparations and their in vitro stimulation by biotin were used for the determination of the biotin status of chicks. Reasonable stability of the enzyme in erythrocytes was achieved when storing the erythrocytes deep-frozen in a glycerol-containing medium. Results of the activation assays in erythrocytes and liver are compared with biotin levels in feed, plasma and liver. The pyruvate carboxylase activation assay appears to be a useful tool for assessing the biotin status of chicks.

Animals↗
PubMed1976

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine and methyl methanesulphonate on liver regenerating after partial hepatectomy. III. Effect on DNA synthesis in vivo and on DNA polymerase activity assayed in vitro.

A single treatment with dimethylnitrosamine (DMN) but not with methyl methanesulphonate (MMS) induces liver cell carcinoma if given during the period of restorative hyperplasia following partial hepatectomy, a higher incidence of tumours being induced if the carcinogen is given during the period of DNA synthesis (24 h after the operation) than if given early in the prereplicative stage (at 6 h). To study the effect of treatment with DMN and with MMS on the regenerating liver, DNA replication was measured in vivo in partially hepatectomised animals treated with the methylating agents, and DNA polymerase activity was assayed in vitro.

Animals↗
PubMed1979

A noncycling activity assay for the omega subunit of Escherichia coli RNA polymerase.

We describe a new method for quantitatively assaying the omega subunit of Escherichia coli RNA polymerase. The assay is based on the ability of RNA polymerase holoenzyme to catalyze the continuous synthesis of the dinucleotide pApU on a poly[d(A-T)] . poly[d(A-T)] template when supplied with AMP and UTP as substrates. Core enzyme, lacking omega subunit, catalyzed this reaction at a rate less than 1% that of holoenzyme. The omega subunit was not released from the enzyme/DNA complex during dinucleotide synthesis. Using this assay, a titration of a fixed concentration of core enzyme was observed with increasing concentrations of added omega subunit. Below a 1:1 omega:core ratio the measured activity increased linearly with omega concentration, whereas above a 1:1 ratio the activity remained constant. An immediate application of the assay is in determining the concentration of active omega, or equivalently of active holoenzyme, in any RNA polymerase preparation.

DNA-Directed RNA Polymerases↗
PubMed1977

Biosynthesis of the yeast cell wall: selective assays and regulation of some mannosyl transferase activities.

Assays have been developed for some transfer reactions involved in the synthesis of Saccharomyces cerevisiae wall mannoproteins, both in a particulate preparation in the presence of EDTA or Triton X-100, and after lipid extraction with chloroform-methanol at -20 C. The mannosyl transferase activities were also studied in cells made permeable to GDP-mannose by toluene-ethanol treatment ("in situ"). In these permeabilized cells, the glycosylating reactions dependent on lipid carriers (dolichol derivatives) did not function, but those independent of them were unaffected. The lipid-independent mannosyl transferase activities were partially inhibited by nucleotide diphosphates probably in a competitive manner. Increase of the nucleotide diphosphate pool "in vivo" might slow down the speed of the transfer reactions carried out by the mannan synthetase system.

Adenosine Diphosphate↗
PubMed1976

Assay for type C virus in mouse sera based on particulate reverse transcriptase activity.

Assay of particulate reverse transcriptase activity in the sera from feral mice naturally infected with type C virus provides a sensitive and rapid procedure for the determination of in vivo virus infection. The results compare well with assays for infectious virus and with complement fixation or competitive radio-immunoassays for the p30 internal antigen of the virus.

Animals↗
PubMed1979

A hemolytic plaque assay for activated murine T cells.

In an earlier report, it was shown that murine spleen cells cultured with concanavalin A (Con A) released into the culture supernatants helper and suppressor substances for antibody production. The present communication describes the production of rabbit antisera against culture supernates from Con A-activated spleen cells and their use in a plaque assay for mitogen-activated T cells. The plaque assay, utilizing SRBC to which Staphylococcal protein A had been coupled, the developing anti-supernatant antiserum and guinea pig complement, readily detected secreting T cells. The T-cell nature of the plaque-forming cells (PFC) was established principally by the following: (a) the majority of lymphocytes in the centers of plaques were Thy-1-positive by fluroescence; (b) spleen cells depleted of B cells by incubation in plastic dishes coated with rabbit anti-mouse Ig antibody gave greatly enriched PFC responses; (c) anti-Thy-1 and anti-Lyt-2.2 treatment of spleen cells almost completely depleted PFC; (d) T-cell mitogens (Con A and phytohemagglutinin) but not B-cell mitogens (lipopolysaccharides) induced PFC responses; (e) T cells maintained in culture for 10 d with Con A and T-cell growth factor yielded PFC. Kinetic and dose response studies showed that high doses of mitogen induced rapidly appearing T-PFC and the responses peaked at day 1--2 of culture. Lower doses of mitogen-induced PFC required longer periods of incubation for detection, indicating that cell activation and secretion may be different dose-dependent activities of mitogens. Another noteworthy finding was that the antiserum reacted with surface antigens of T-PFC, indicating that secreted products are expressed on the membranes of T cells, offering the possibility of isolating populations of cells with specific secretory potential. Although the precise nature of the T-cell products detected by the antiserum used in this assay are unresolved, 10% of the target-cell-adherent population from spleen cells of BALB/c mice sensitized to L929 cells formed plaques. This suggests that the antiserum has significant activity against the products of cytotoxic T cells, a finding which accords with the activity of anti-Lyt-2.2 serum against mitogen-induced T-PFC. The method clearly offers new possibilities for the analysis of T cells and their products and should provide an important approach to the clonal analysis of lymphokine production.

Animals↗
PubMed1977

Biological activities of tritiated endotoxins: correlation of the Limulus lysate assay with rabbit pyrogen and complement-activation assays for endotoxin.

Tritiated endotoxins were prepared by three different methods. The biological activities of the tritiated endotoxins were determined by the Limulus amebocyte lysate assay, a rabbit pyrogen assay, and a complement-activation assay and were compared to native, unlabeled endotoxin. All three tritiated endotoxin preparations manifested adequate biological activity in each of the three assay systems, and all three assays ranked the biological activity of the different endotoxin preparations in the same order. Endotoxin tritiated by the Wilzbach procedure retained most of its biological activity and also had the highest specific radioactivity. The good correlation between the Limulus lysate, rabbit pyrogen, and complement-activation assays suggests that the same active site of the endotoxin molecule is identified by the three different assays.

Animals↗
PubMed1976

On the reliability of plasminogen measurement employing the proactivator-activator converting method.

A simple plasminogen determination method is presented. It is based upon the conversion of plasminogen into activator by large and constant amounts of streptokinase. The activator contained in a standard coagulum consisting of bovine fibrin, streptokinase, and a 1:40 dilution of human plasma converts the plasminogen adsorbed on bovine fibrin into plasmin. Lysis of the test coagulum is hereby induced. The speed of such lysis is limited by the concentration of the activator incorporated in the test coagulum. The variable component of the activator being human plasminogen, the speed of lysis is directly dependent upon the concentration of human plasminogen in the standard coagulum. Using the thromboelastograph according to Hartert in recording the test clot lysis times, this method of plasminogen determination was shown to be a simple and quick procedure. The standard deviation ranged from +/- 13,2 tp 68%, depending upon the plasminogen value to be measured (lower rates of error were attached to high, and higher rates of error to low, plasminogen concentrations). The biological variation of plasminogen values in a group of 26 men aged from 40 to 65 years was calculated to be +/- 21%. Both plasminogen and plasmin, its activated form, were exchangeable in the test, i.e. plasminogen determinations performed by activator assay did not differentiate between plasminogen and plasmin. There was no influence by varying anti-SK titers in the plasma up to a circulating antibody content of 2 million. Furthermore, plasma antiplasmins did not affect the plasminogen measuring system. Plasminogen tested by activator assay displayed values closely related to those achieved by immunochemical methods. Plasminogen measurements were performed in patients undergoing streptokinase and urokinase infusion treatment. 5,000 u streptokinase per hour, as well as 270,000 CTA-u urokinase per hour, infused over a period of 2 days produced a fall in plasminogen down to 30-60% of normal. In contrast, 100,000 u streptokinase per hour lowered the plasminogen concentration down to values of below 1%. The foregoing data indicate that plasminogen measurement, according to the principles outlined here (activator assay), may be regarded as a valuable and reliable method for the routine control of streptokinase and urokinase therapy.

Adult↗
PubMed1977

Enzyme activities in human liver biopsies: assay methods and activities of some lysosomal and membrane-bound enzymes in control tissue and serum.

1. Highly sensitive technique are described for the assay of plasma membrane (5'-nucleotidase, alkaline phosphatase), microsomal (neutral alpha-glucosidase, leucyl-2-naphthylamidase) and biliary canalicular (gamma-glutamyltransferase) enzymes and for nine acid hydrolases (acid phosphatase, phosphodiesterase, beta-glucosidase, alpha-glucosidase, alpha-galactosidase, beta-galactosidase, alpha-mannosidase, N-acetyl-beta-glucosaminidase, beta-glucuronidase) in human liver. 2. Optimum and specific assay systems have been developed which give linear kinetics for all enzymes. 3. The range of enzyme activities in samples of human liver, obtained by closed needle biopsy, and sera have been determined.

Alkaline Phosphatase↗
PubMed1979

In vitro assay for prekallikrein activator (PKA).

The assay measures the action of prekallikrein in generating kallikrein. The prekallikrein activator is the limiting reagent. The product kallikrein is an esterase which can be quantitated using benzoyl arginine ethyl ester. The assay is reproducible and results are comparable with other methods. The large scale preparation of prekallikrein is described.

Blood Proteins↗
PubMed1979

Improved in vitro light activation and assay systems for two spinach chloroplast enzymes.

An improved system for the in vitro light activation of the chloroplast enzymes fructose-1,6-bisphosphatase and NADP-dependent malate dehydrogenase is described. Through the presence of the monothiol beta-mercaptoethanol in the reaction mixtures the activated forms of the enzymes can be stabilized and their activity determined spectrophotometrically.

Chloroplasts↗
PubMed1975

Host response to infection by Coxiella burneti.

A host response to infection by Coxiella burneti was investigated. Infectedyolk sacs were harvested from embryonated eggs and assayed for glycolytic activity. Assays of glycolytic enzymes included glucose isomerase, aldolase, phosphofructokinase,fructose-1,6-diphoshatase, glyceraldehyde-3-phosphate dehydrogenase, enolase, and pyruvate kinase. No significant differences in enzymatic activity between normal and infected tissues through the 12th day was observed. From the 13th day through the 16thday, the glycolytic activity of normal tissues decreased. Glycolytic activity of infected tissues did not decrease, but showed a gradual increase during this same time period. Embryos from infected eggs demonstrated a gradual decrease in total weight fromthe 12th day until death occurred on the 16th day.

Animals↗
PubMed1979

Mitochondrial ATPase of Zajdela hepatoma. VI. Effect of extramitochondrial ATP and pH on uncoupler-sensitivity of mitochondrial ATPase activity.

Coupled Zajdela hepatoma mitochondria were reported to exhibit uncoupler-insensitive ATPase activity. The results of this study show that under specific conditions the ATPase activity of Zajdela hepatoma mitochondria can be stimulated by uncouplers. These conditions include (a) the addition of ATP to the mitochondria before the uncoupler in the ATPase activity assay or (b) elevation of pH (above 8.5) of the ATPase activity assay medium.

Adenosine Triphosphatases↗
PubMed1977

Assay of proteolytic activity by gelatin liquefaction (trypsin assay by gelatin liquefaction).

A new assay for proteolytic enzymes and their inhibitors based on the liquefaction of gelatin gels has been developed. The assay is more sensitive than colorimetric tests, can be carried out upon colored or turbid samples and does not require the use of a spectrophotometer. The procedure consists of incubating the test sample with a fluid gelatin solution, cooling the solution so that it sets to a firm gel and then incubating at a warmer temperature until the gel iquefies. The time taken for liquefaction is several days for a sample of pure buffer, about one minute for a sample containing 0.5 microgram of trypsin per ml and longer for samples containing less trypsin, following an empirical calibration. An appreciable decrease in tryptic activity can be detected in the presence of only 0.1 KIU of Trasylol (bovine pancreatic trypsin inhibitor) by this method.

Aprotinin↗
PubMed2025

OsIDD6, an INDETERMINATE DOMAIN containing transcription factor in rice, plays an essential role in reproductive development.

INDETERMINATE DOMAIN containing proteins (IDD) are plant-specific transcriptional factors with a diverse range of roles in plants. Among the 15 IDD genes in rice, a staple food crop for the world, only about half have been functionally characterized. To elucidate the function of the remaining members, we created loss-of-function mutants using the CRISPR genome editing technique. Although no mutant exhibited obvious growth phenotypes, the Osidd6 mutant was completely sterile. By genetic crossing, we showed that both the male and female gametophytes were defective in the mutant. Histochemical staining and thin sectioning revealed that microspore development was compromised, likely due to a delay in tapetum degeneration. We also showed that meiosis was impaired in the mutant, resulting in defective megaspore development. Through a series of experiments, including transcriptome analysis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), in situ hybridization, β-glucuronidase (GUS) staining with promoter-GUS transgenic plants, yeast one-hybrid method, a dual-visible reporter assay, and transcriptional activity assay, we demonstrated that OsIDD6 is expressed in all cell types in the male and female reproductive organs and that the OsIDD6 protein directly regulates genes potentially having a role in meiosis and tapetum development. Since reproductive development is directly related to crop yield, OsIDD6 could be an important target for genetic manipulation in rice breeding.

Oryza↗
PubMed1979

Improved methods for the assay and activation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

A simple and rapid mixed-phase method for the quantitative assay of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase and a procedure for the efficient reactivation of Mg-ATP-inactivated microsomal HMG-CoA reductase by potato acid phosphatase are described. The mixed-phase assay entails the direct addition of the acidified, deproteinized incubation mixture to a toluene-based scintillation fluor. The enzymatic reaction product [3H]-mevalonolactone partitions into the toluene while unreacted 3H-labeled HMG-CoA substrate remains in the aqueous phase and is not detected on scintillation counting. The accuracy and reproducibility of this method are compared to a thin-layer chromatographic assay for HMG-CoA reductase. Microsomal and solubilized HMG-CoA reductase inactivated by incubation with Mg-ATP is reactivated by purified potato acid phosphatase. Under appropriate conditions quantitative reactivation of HMG-CoA reductase is achieved, indicating that endogenous inhibitory and activating proteins regulate HMG-CoA reductase via a kinase-phosphatase system.

Acid Phosphatase↗