An optical biosensor employing tiron-immobilised polypyrrole films for estimating monophenolase activity in apple juice

A method is described for the incorporation of tiron as a substrate for tyrosinase enzyme into a polypyrrole film deposited on indium titanium oxide (ITO) glass. The presence of tiron in the polypyrrole film is verified by cyclic voltammetry (CV). The enzyme activity using the polypyrrole-tiron film is confirmed by the catalytic conversion of immobilised substrate to quinones by the enzyme. The use of both potentiometric and optical methods for the detection of the catalytic activity of the polypyrrole-tiron film and their potential use for the determination of monophenolase activity of apple polyphenol oxidase is described. This is the first report of this kind whereby tiron has been immobilised in a polypyrrole matrix for the enzyme activity determination.     

Identification of denatured enzyme proteins in sodium dodecyl sulfate polyacrylamide gels

A simple modification of the immunological sandwich method of Muilerman et al. for the identification of denatured enzyme proteins in sodium dodecyl sulfate-polyacrylamide gels is described, enabling the method to be used in principle for any enzyme whose activity is not inhibited by binding to antibodies. An immunological sandwich consisting of denatured enzyme, antibodies, and native enzyme is formed on a nitrocellulose filter blot of the gel, the filter is divided into strips, and each strip is tested for enzyme activity. The presence of enzyme activity serves to identify the region in the gel containing denatured enzyme protein. Experiments with human lysosomal alpha-glucosidase as a model system are described. The method was applied to identify a protein of Mr 125,000 as the main component with UDPgalactose pyrophosphatase activity in a partially purified preparation of the enzyme from rat liver.     

Oxaloacetate decarboxylases of rat liver

1. Two oxaloacetate decarboxylases of rat liver are described; one is mitochondrial with no metal ion requirement and activity in the alkaline pH region; the other is cytoplasmic with Mn(2+) or Mg(2+) requirement and activity around pH5. 2. A method for the partial purification of the mitochondrial enzyme is described. 3. The apparent K(m) of the mitochondrial enzyme is 0.23mm. 4. Inhibition of the mitochondrial enzyme by substrate, CoA, acetyl-CoA, citrate and phosphoenolpyruvate is described.     

Disulfide-disulfide interchange catalyzed by a liver supernatant enzyme.

An enzyme widely distributed in rabbit tissues which catalyzes an interchange between N,N-di-dinitrophenyl-L-cystine and oxidized glutathione to form the mixed disulfide is described. D-Penicillamine disulfide can be substituted for oxidized glutathione and the mixed disulfide of cysteine and glutathione can serve as the sole substrate giving as one product of interchange, oxidized glutathione. The enzyme is very labile and only limited purification of it has been achieved. The activity increases with increasing pH above 6.6, the Km for N,N-di-dinitrophenyl-L-cystine is 0.2 mM and for oxidized glutathione 0.8 mM. The enzyme is inhibited by SH reagents with protection against iodoacetamide inactivation provided by N,N-di-dinitrophenyl-L-cystine. Evidence is presented that disulfide-disulfide interchange enzyme is a different activity from the previously described protein disulfide isomerase and thiol transferase.     

A simple procedure for isolating adenosine triphosphatase from mitochondria

A simple method for isolation of adenosine triphosphatase (EC 3.6.1.3) from mitochondria is described. The enzyme is released from mitochondrial Lubrol particles by drastic sonication and purified by gel filtration on Sepharose 6-B. The described procedure is effective in isolating adenosine triphosphatase from rat liver as it is from beef heart mitochondria. The enzyme isolated from beef heart has a specific activity of 120 μmol P/min per mg protein and enzyme isolated from rat liver has a specific activity of 70 μmol P/min per mg protein when measured as a release of inorganic phosphate.     

The two alcohol dehydrogenases of Zymomonas mobilis. Purification by differential dye ligand chromatography, molecular characterisation and physiological roles

The two alcohol dehydrogenases found in Zymomonas mobilis have each been purified using dye-ligand chromatography and affinity elution with nucleotides. The isoenzyme with lower electrophoretic mobility (ZADH-1) is a zinc enzyme with properties essentially similar to preparations described elsewhere. The faster isoenzyme (ZADH-2) accounted for some 90% of the ethanol-oxidizing activity in freshly prepared extracts and corresponded to the iron-activated enzyme previously described. This enzyme was inactivated by zinc; activity could only be retained during purification by including either ferrous ions or cobaltous ions in the buffers. ZADH-2 has relatively low acetaldehyde reductase activity; consequently ZADH-1 is responsible for about half of the physiological activity (acetaldehyde reduction) in Zymomonas cells. Kinetic studies showed that ZADH-2 is activated by ethanol in both reaction directions; a hypothesis for the mechanism of activation is presented. Metal ion analyses of ZADH-2 prepared in the presence of iron or cobalt indicated one atom of the relevant metal per subunit, with no significant zinc content. N-terminal sequence analyses showed that the ZADH-1 has some homology with the Bacillus stearothermophilus enzyme, whereas ZADH-2 resembles the yeast enzyme more closely.     

Enzyme activity dot blots: a rapid and convenient assay for acetyltransferase or protein kinase activity immobilized on nitrocellulose

Methods are described for assaying (Tetrahymena) histone acetyltransferase activity and (Drosophila) casein kinase II activity by spotting extracts on nitrocellulose filters. The methods are quantitative over a wide range of enzyme concentrations and are almost as sensitive as liquid assays. Examples are presented for illustrating the use of these methods for enzyme purification, concentration, and desalting, as well as for electrophoretic blotting from agarose gels. A simple method for autoradiographic enhancement of nitrocellulose filters is also described.     

An ultrafiltration assay for lysyl oxidase

A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware.     

A versatile assay for total cellulase activity using U-[14C]-labelled bacterial cellulose

Summary A versatile and sensitive assay for total cellulase activity and not just carboxymethylcellulase activity, is described. The method is equally suitable for studying the kinetics of solubilization of cellulose by growing cells or isolated enzyme fractions.     

The detection of hyaluronidase on electrophoresis membranes

A method is described for the detection of hyaluronidase enzyme activity following electrophoresis on cellulose acetate membranes prior to elution of the enzyme from the support medium. It appears to be specific for endo-N-acetylhexosaminidase activity. It detects 0.005 of the least amount of enzyme detected by a miniaturized turbidity reduction test or by a standard viscosity reduction test.     

Dipeptidyl peptidase IV. Purification for use in peptide sequencing

Isolation and purification of dipeptidyl peptidase IV from pig kidney are described. The specific activity of the enzyme is 24.9 U/mg protein. Chromatography on Gly-Pro-AH-Sepharose is the most important procedure for the separation from other enzyme activities. The enzyme preparation is free of aminopeptidase, dipeptidase and prolyl endopeptidase activity; it can be used for peptide-sequence analysis.     

Monophenolase activity of latent Terfezia claveryi tyrosinase: Characterization and histochemical localization

The monophenolase activity of Terfezia claveryi tyrosinase (EC 1.14.18.1) is described for the first time. This enzyme is fully latent and can only be detected if SDS is present in the reaction medium. Monophenolase activity was localized within the ascocarp using histochemical techniques. A detailed kinetic study of the parameters affecting this activity has been carried out. Both the characteristic lag period and the steady-state rate are affected by pH and the enzyme and substrate concentrations. The presence of catalytic concentrations of o -diphenols affected the lag period but not the steady-state rate. By increasing the concentration of o- diphenols, it was possible to evaluate the enzyme activation constant, K_act, which showed a value of 7.2 μ M . The experimental results are compatible with the mechanism previously described for tyrosinases from other sources.     

Short-term regulation of carnitine palmitoyltransferase activity in isolated rat hepatocytes

An assay procedure for carnitine palmitoyltransferase is described which allows rapid measurement of the overt activity of this enzyme in isolated rat hepatocytes. In a one-step procedure digitonin permeabilizes the plasma membrane and at the same time carnitine palmitoyltransferase activity is measured. The use of the present procedure shows that carnitine palmitoyltransferase activity is regulated on the short term by different types of agonists. Thus, insulin, epidermal growth factor, vasopressin and the phorbol ester PMA inhibit carnitine palmitoyltransferase activity, whereas glucagon treatment renders the enzyme more active. These changes in enzyme activity coincide with corresponding changes in the rate of fatty acid oxidation.     

Evidence that the cytosolic activity of 3-hydroxybutyrate dehydrogenase in chicken liver isL-3-hydroxyacid dehydrogenase

Classical fractionation studies showed that chicken liver contains two enzymes which can oxidize DL-3-hydroxybutyrate. The cytosolic enzyme is specific for the L-(+) isomer and accounts for 60% of the total activity. The mitochondrial activity is specific for the D-(?) isomer and accounts for 40% of the total activity. Kinetic studies showed that L-gulonic acid is a competitive inhibitor of the enzyme. We conclude that the cytosolic enzyme is the previously described L-3-hydroxyacid dehydrogenase.     

The incorporation of homologous and heterologous hypoxanthine-guanine phosphoriboxyltransferase into mutant cells

Experiments are described leading to partial compensation of a deficiency in the enzyme hypoxanthine-guanine phosphoribosyltransferase in mutant cells by supplying the cells with exogenous purified enzymes. DEAE-dextran is an effective helper agent, whereas poly (L-lysine), lysolecithin and amphotericin B seem to inhibit the entry of the enzymes of their activity. Enzyme preparation from Chinese hamster was found to have different effects in different mutant cell lines. In mutant Chinese hamster cells, the electrophoretic activity pattern remains unchanged for the Chinese hamster enzyme, but changes progressively to faster-moving activity peaks for the human enzyme after several hours. The metabolic effect of the incorporated enzyme is in the range between 3 and 4% of the normal cellular enzyme activity which corresponds to a 10--20 fold increase of hypoxanthine-guanine phosphoribosyltransferase activity in the mutant cells.     

Cytochemical model system for microsomal rat liver glucose-6-phosphate.

A method is described for the incorporation of a microsomal rat liver fraction into polyacrylamide films without significant loss of its glucose-6-phosphatase activity. The enzymatic activity was completely lost when the films were prepared with ammonium persulfate as initiator of the polymerization as previously described for alkaline phosphatase, but modification of this method showed that about 90% of the glucose-6-phosphatase activity could be retained. The enzyme in the films prepared with the new method was completely inhibited by alloxan, HgCl2, and preincubation in 0.05 M acetate buffer (pH 5.0) at 37 degrees C, as determined biochemically. Similar results were obtained for the enzyme in films determined histochemically according to the lead method of Wachstein and Meisel. In this respect the behavior of the incorporated enzyme is similar to that in suspension. Films fixed with 1.5% glutaraldehyde showed rapid inactivation of glucose-6-phosphatase. There was good correlation between the biochemical and histochemical activity determined after fixation. A method to embed polyacrylamide films in Epon for electron-microscopical investigation is also described. Dimethyl sulfoxide was used as the dehydrating agent instead of ethanol/acetone.     

Purification of the enzyme NADPH: protochlorophyllide oxidoreductase.

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.     

Localisation of a particulate luliberin hydrolysing activity in microsomal membranes of guinea pig brain

A particulate luliberin hydrolysing enzyme has been described for guinea pig brain. Examination of subcellular fractions generated under different conditions indicated that particulate luliberin hydrolysing activity was most closely associated with the microsomal marker, rotenone-insensitive NADH cytochrome C reductase. The results obtained indicate that luliberin hydrolysing activity is not associated with synaptosomal membrane preparations and that such luliberin hydrolysing activity as is observed in synaptosomal membranes is probably the result of contamination by microsomes. The enzyme could be released from microsomes by Triton X-100 treatment and the solubilised enzyme was found to be inhibited by puromycin and sulphydryl reagents but to be unaffected by phosphoramidon, captopril, phenylmethyl sulphonyl fluoride and by chelating agents except 1,10-phenanthroline.     

A simple and sensitive fluorometric assay for tyrosine hydroxylase.

A simple, rapid, and sensitive fluorometric assay for measuring the activity of tyrosine hydroxylase is described. The enzyme activity is detected by converting tyrosine to 3,4-dihydroxyphenylalanine (dopa), which is then subjected to conversion to the highly fluorescent product by the trihydroxyindole method. The assay method is very reproducible, more sensitive than a radiochemical method for the determination of tyrosine hydroxylase activity using the isolation of [³H]water commonly used, and linear from 0.2 to 12 nmol of dopa. The method should be applicable for the assay of the enzyme with a wide range of activity.     

Phospholipase A1 of human blood platelets (Short Communication)

Substantial phospholipase A(1) activity has been demonstrated in human blood platelets, and a rapid method for its measurement is described. The enzyme requires taurocholate for full activity and in these conditions the pH optimum is 4.8. The phospholipase activity is released from platelets by incubation with thrombin.