Acyl-coenzyme A: cholesterol acyltransferase assay: silica gel column separation of reaction products

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) assays are usually performed by incubation of the enzyme with a labeled substrate followed by thin-layer chromatography separation and subsequent quantification of cholesteryl esters (CE) formed. Herein, a method is described for rapid separation of CE from other lipids, by elution from a silica gel column with a solvent mixture of petroleum ether/diethyl ether (98:2, v/v). Silica gel column chromatography is reliable and more rapid and safer than TLC. The best results were obtained when the reaction was stopped by Dole extraction followed by CE separation on a silica gel column. Assays for ACAT from rat intestinal microsomes showed that the specific activity values obtained using this method were reproducible and in good agreement with those obtained by conventional TLC method.     

Southern blotting

This protocol describes a basic method to perform the Southern blot. Blotting allows the detection of specific molecules among a mixture separated by gel electrophoresis. Molecules are transferred from the gel to a porous membrane by capillary action using absorbent paper to soak solution through the gel and the membrane. For DNA, specific sequences are detected in the membrane by molecular hybridization with labeled nucleic acid probes. The original method, on which this protocol is based, used labeled RNAs to detect specific DNA fragments in genomic DNA that had been digested with restriction endonucleases. This protocol can be completed in 1-5 d and is inexpensive to carry out, as it requires only basic laboratory equipment.     

Phthalate metabolism in Pseudomonas fluorescens PHK: purification and properties of 4,5-dihydroxyphthalate decarboxylase.

Pseudomonas fluorescens PHK uses 4,5-dihydroxyphthalate as the sole carbon source for o-phthalate catabolism. This intermediate is the substrate for a decarboxylase of the pathway yielding protocatechuate. The decarboxylase was purified to homogeneity by an affinity chromatography procedure in which the reaction product, protocatechuate, was used as a ligand. We describe some properties of the enzyme, including its apparent molecular weight of 420,000 as determined by gel filtration and of 66,000 after sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, consistent with a hexameric functional protein. The apparent Km for the substrate 4,5-dihydroxyphthalate was 10.4 microM. The characteristics of this enzyme are compared with those described for the isofunctional enzyme from P. testosteroni.     

Crystalline desoxyribonuclease; digestion of thymus nucleic acid; the kinetics of the reaction

A study was made of the enzymatic properties of crystalline desoxyribonuclease. The general effect of the crystalline enzyme on its specific substrate, thymus nucleic acid, was found to be essentially the same as described by previous workers for the digestive action of crude preparations of the enzyme. The digestive action consists mainly in splitting thymus nucleic acid into fragments approaching the size of tetranucleotides. The digested nucleic acid is diffusible through collodion or cellophane membranes and is non-precipitable with strong acid, alcohol, or proteins. The digestion of thymus nucleic acid by desoxyribonuclease is accompanied by the liberation of one atom equivalent of free acid per four atoms of nucleic acid phosphorus. Crystalline desoxyribonuclease acts very slowly, if at all, in the absence of magnesium (or manganese) ions. The optimal concentration of magnesium ion required increases with the increase in concentration of the substrate but is independent of the enzyme concentration. The optimal pH range for the action of crystalline desoxyribonuclease is 6.0 to 7.0. A study was made of the kinetics of the digestion of thymus nucleic acid as manifested mainly by the gradual formation of acid-soluble split products. At low concentrations of nucleic acid, the process approximates closely a reaction of the first order, the unimolecular constant being independent of the concentration of desoxyribonuclease in the digestion mixture. At relatively higher concentrations of substrate, however, the initial rate of reaction decreases rapidly with the increase in concentration of substrate, and the reaction as a whole is represented by non-symmetric S-shaped curves apparently too complicated for a simple rational interpretation.     

A quantitative Western Blot method for protein measurement

A radioimmunologic assay method for the quantitation of small amounts of protein in recombinant vaccines at the level of 20-150 ng is evaluated which uses the techniques of SDS-PAGE and electrophoretic protein transfer ("Western Blot'). Known amounts of the protein being determined are included on the same gel as the unknown. After protein blotting, the nitrocellulose membrane is treated with antibody specific for the protein being determined and subsequently with [125I] Protein A. An autoradiogram is produced which corresponds directly to the nitrocellulose blot. It can, therefore, serve as a template to locate the labeled protein which is excised from the blot and measured in a gamma counter. The technique is found especially useful for evaluating cell lysates of recombinant bacteria and yeast for the percentage of the recombinant protein in the total protein mixture.     

A radioisotopic assay for polyamine oxidase

A new radioisotopic assay for polyamine oxidase with N1-acetylspermine as substrate is presented. A modified method for the chemical synthesis of radioactive N1-acetylspermine, which gave a good yield, is also described. The reaction mixture, containing N1-[14C]acetylspermine and tissue homogenate, was incubated for the enzyme reaction and applied to a minicolumn of Amberlite CG-50. The reaction product 3-[14C]-acetamidopropanal did not adsorb to the column, but passed through it; thus the eluate could be directly subjected to liquid scintillation counting. The blank levels were low and relatively constant even with crude tissue homogenates. The detection limit obtained was 0.05 nmol per tube. This method is simple, highly sensitive, and highly specific.     

Studies on an Endonuclease Activity Associated with the Bacteriophage T7 DNA-Membrane Complex

Infection of Escherichia coli with bacteriophage T7 results in the formation of an endonuclease which is selectively associated with the T7 DNA-membrane complex. A specificity of association with the complex is indicated by the finding that the enzyme is completely resolved from a previously described T7 endonuclease I. When membrane complexes containing (3)H-labeled in vivo synthesized DNA are incubated in the standard reaction mixture a specific cleavage product is formed which is about one-fourth the size of T7 DNA. The endonuclease associated with the complex produces a similar cleavage product after extensive incubation with native T7 DNA or T7 concatemers. Degradation of concatemers occurs by a mechanism in which the DNA is converted to molecules one-half the size of T7. This product is in turn converted to fragments one-fourth the size of mature phage DNA. The endonuclease is not present in membrane complexes from uninfected cells or cells infected with gene 1 mutants. The enzyme activity is, however, present in cells infected with mutants defective in T7 DNA synthesis or maturation.     

Rapid and specific assay of collagen glucosyltransferase with a Sepharose-bound substrate

A simple and specific assay has been developed for collagen glucosyltransferase in which a solid-phase substrate, consisting of a mixture of Type II collagen peptides linked to CNBr-activated Sepharose 4B, is used. Following incubation with enzyme, the substrate is simply washed, and its radioactivity is determined. The specificity of the procedure has been validated by the isolation of radiolabeled Glc-Gal-Hyl from the reaction products after incubation with a crude enzyme preparation from embryonic chick cartilage. Under the conditions described, product formation was linear for 6 h and was proportional to the concentration of enzyme protein up to 180 μg/ml. The K_m was 33 μm for UDP-glucose and 48 μm for the gel-bound collagen substrate, expressed as Gal-Hyl. The V_max for the Sepharose-bound substrate was 30% of the value observed for the identical substrate in soluble form, suggesting that the solid matrix exerted some sterical hindrance on the bound substrate.     

Trimethylamine dehydrogenase from a methylotrophic bacterium. I. Isolation and steady-state kinetics.

1. The isolation of trimethylamine dehydrogenase (EC 1.5.99.7) from a restricted facultative methylotroph to electrophoretic homogeneity is described. 2. The molecular weight and subunit molecular weights were found to be 146800 for the enzyme by sedimentation equilibrium ultracentrifugation and 70000-80000 for the two non-identical subunits by sodium dodecyl sulphate gel electrophoresis. 3. Initial velocity studies indicate that the enzymatic reaction proceeds by a Ping-Pong mechanism. 4. Further kinetic evidence was obtained by analysis of product inhibition patterns using the alternate substrate diethylamine and the products acetaldehyde and ethylamine as product inhibitors, for the release of ethylamine before the addition of phenazine methosulphate and for the existence of an enzyme-two-carbon unit complex as a stable form of the enzyme. 5. Some properties of the unusual prosthetic group of trimethylamine dehydrogenase and its photodegradation product are described in preliminary form.     

Fluorogenic substrate turnover in single living cells

Synopsis Intracellular diffusion properties and enzyme activities in single living cells can be analysed by means of fluorogenic substrates that diffuse into the cells where they are converted into a fluorescent product by an enzymic reaction. The reaction-kinetic analysis of this process as a system of consecutive reactions provides information on the diffusion of the substrate into the cells, on intracellular enzyme activities and on the efflux of the fluorescent product. Separation of diffusion and enzyme-mediated processes is obtained by inducing specific changes of the cellular membrane using gramicidin D. A model for the functional interpretation of the experimental findings is proposed. Application of this method as a viability test for freshly prepared and frozen platelets is discussed.Paper given at the Royal Microscopical Society's European Histochemistry Meeting at Nottingham in September 1975.     

Enzyme reactions at the surface of living cells. I. Electric repulsion of charged ligands and recognition of signals from the external milieu

The dynamic behaviour of a polyelectrolyte-bound enzyme is studied when diffusion of substrate or diffusion of product is coupled to electric repulsion and to Michaelis-Menten enzyme reaction. The definition of the classical concepts of electric partition coefficients and Donnan potential of a polyelectrolyte membrane has been extended under global non-equilibrium conditions. This extension is permissible when a strong repulsion exists of substrate and product by the fixed negative charges of the membrane. Coupling between product diffusion, electric repulsion and enzyme reaction at constant advancement may result in a hysteresis loop of the partition coefficient as the product concentration is increased in the reservoir. This hysteresis loop vanishes as the rate of product diffusion increases. No hysteresis loop may occur when electric repulsion effects are coupled to substrate diffusion and reaction. The existence of multiple values of the partition coefficient for a fixed concentration of product implies that the membrane may store short-term memory of the former product concentration present in the external milieu. The occurrence of hysteresis generated by coupling enzyme reaction, product diffusion, electric partition effects at constant advancement of the reaction may be viewed as a sensing device of product concentration in the external milieu. Surprisingly, non-linearities required to generate this sensing device come from electrostatic effects and not from enzyme kinetics.     

Radiochemical assays for adenylate kinase and AMP deaminase using polyethyleneimine-cellulose thin layers

Simple and specific radiochemical asays for adenylate kinase and AMP deaminase activities in crude tissue extracts are described. The radioactive substrate (AMP) is separated from the radioactive product (ADP or IMP) by chromatography on polyethyleneimine-cellulose thin layers. A rapid modification of the adenylate kinase assay is described in which samples of the reaction mixture are transferred directly to the polyethyleneimine-cellulose thin layer.     

Actinomycin synthesis in Streptomyces antibioticus. Purification and properties of a 3-hydroxyanthranilate 4-methyltransferase

A methyltransferase, which utilizes 3-hydroxyanthranilic acid (HAA) as a substrate, has been purified to near homogeneity from 30-36-h mycelium of the bacterium Streptomyces antibioticus. The enzyme was obtained in approximately 20% yield with a purification of 130-fold. Polyacrylamide gel electrophoresis under denaturing conditions indicates that the enzyme is composed of a single subunit with Mr of about 36,000. On chromatography in 0.5 M NaCl, the enzyme displays a molecular weight of about 37,000. The specific activity of the enzyme in S. antibioticus mycelium is maximal between 30 and 36 h following inoculation of galactose/glutamic acid medium and, at those times post-inoculation, the specific activity is essentially the same in extracts of mycelium obtained from cultures grown on glucose rather than galactose as the carbon source. The enzyme activity is stimulated by Na2EDTA (in crude extracts) and by 2-mercaptoethanol and the methyltransferase shows a strong preference for HAA as substrate as compared with a number of HAA analogs. Thin layer chromatography of ethyl acetate extracts of large-scale incubation mixtures confirms that the product of the reaction is 4-methyl-3-hydroxyanthranilic acid. The reaction product was also a substrate for phenoxazinone synthase and was incorporated into actinomycin by S. antibioticus mycelium. Kinetic parameters for the methyltransferase reaction was determined.     

alpha-Glucan synthesis on a protein primer, uridine diphosphoglucose: protein transglucosylase I. Separation from starch synthetase and phosphorylase and a study of its properties

It was found that the DEAE-cellulose-treated UDP-Glc:protein transglucosylase I catalyzing the first step (reaction 1) in the formation of alpha-glucan bound to protein in potato tuber is not only specific for the glucosyl donor but also for the endogenous acceptor. A single radioactive 38-kDa macromolecular component appeared during denaturing polyacrylamide gel electrophoresis of reaction 1 product. The labeled component is probably the polypeptide subunit of the endogenous acceptor which is being glucosylated. The radioactivity incorporated in reaction 1 product was isolated from a protease digest as a low-molecular-mass glucopeptide fraction. A beta-elimination reaction carried out in the presence of a reducing agent demonstrated that only one glucosyl moiety is transferred from UDP-Glc to the aminoacyl residue, thus forming an O-glucosidic linkage. 3H-labeled sodium borohydride showed that serine and threonine are involved in the peptide bond to glucose. Ion-exchange chromatography on DEAE-cellulose, affinity chromatography on concanavalin-A--Sepharose, gel filtration on Sephacryl S-300 and sucrose density gradient centrifugation failed to separate the enzyme catalyzing reaction 1 from the endogenous acceptor.     

Glutamate-Dependent Active-Site Labeling of Brain Glutamate Decarboxylase

A major regulatory feature of brain glutamate decarboxylase (GAD) is a cyclic reaction that controls the relative amounts of holoenzyme and apoenzyme [active and inactive GAD with and without bound pyridoxal 5'-phosphate (pyridoxal-P, the cofactor), respectively]. Previous studies have indicated that progression of the enzyme around the cycle should be stimulated strongly by the substrate, glutamate. To test this prediction, the effect of glutamate on the incorporation of pyridoxal-P into rat-brain GAD was studied by incubating GAD with [32P]pyridoxal-P, followed by reduction with NaBH4 to link irreversibly the cofactor to the enzyme. Adding glutamate to the reaction mixture strongly stimulated labeling of GAD, as expected. 4-Deoxypyridoxine 5'-phosphate (deoxypyridoxine-P), a close structural analogue of pyridoxal-P, was a competitive inhibitor of the activation of glutamate apodecarboxylase by pyridoxal-P (Ki = 0.27 microM) and strongly inhibited glutamate-dependent labeling of GAD. Analysis of labeled GAD by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed two labeled proteins with apparent molecular masses of 59 and 63 kDa. Both proteins could be purified by immunoaffinity chromatography on a column prepared with a monoclonal antibody to GAD, and both were labeled in a glutamate-dependent, deoxypyridoxine-P-sensitive manner, indicating that both were GAD. Three peaks of GAD activity (termed peaks I, II, and III) were separated by chromatography on phenyl-Sepharose, labeled with [32P]pyridoxal-P, purified by immunoaffinity chromatography, and analyzed by SDS-polyacrylamide gel electrophoresis. Peak I contained only the 59-kDa labeled protein. Peaks II and III contained the both the 59- and 63-kDa proteins, but in differing proportions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Theoretical analysis of a translocation-like model with saturable kinetics.

A theoretical analysis of the initial rates of product appearance in both compartments of a specifically designed diffusion cell separated by an asymmetrical enzyme membrane is presented. Variable substrate concentrations and different substrate diffusional limitations were considered. Our analysis shows that, under specific conditions, not only a product accumulation occurs in the compartment opposite to that in which the reaction takes place, but that substrate saturable kinetics can be obtained. These product translocation-like kinetics appear similar to those observed with translocation processes reported for biological situations. For such phenomena, a key role of the diffusion layer surrounding a bioactive surface is proposed.     

Analysis of ping-pong reaction mechanisms by positional isotope exchange. Application to galactose-1-phosphate uridyltransferase

A new positional isotope exchange method has been developed that can be used for the analysis of enzyme-catalyzed reactions which have ping-pong kinetic mechanisms. The technique can be used to measure the relative rates of ligand dissociation from enzyme-product complexes. Enzyme is incubated with the labeled substrate and an excess of the corresponding unlabeled product. The partitioning of the enzyme-product complex back toward free enzyme is determined from the rate of positional isotope exchange within the original labeled substrate. The partitioning of the enzyme-product complex forward toward free enzyme is determined from the rate of formation of totally unlabeled substrate. It has been shown that the ratio of the two rates provides a lower limit for the release of product from the enzyme-product complex. The technique has been applied to the reaction catalyzed by galactose-1-phosphate uridyltransferase. The lower limit for the release of glucose 1-phosphate from the uridyl-enzyme relative to the maximal velocity of the reverse reaction was determined to be 3.4 +/- 0.5.     

Purification and some properties of beta-transglycosylase of Trichoderma longibrachiatum

A beta-transglycosylase was purified to a homogeneous state from the extract of a wheat bran Koji culture of Trichoderma longibrachiatum by column chromatography. The purified enzyme showed a typical disproportionation reaction with cellopentaose as the substrate, producing a high molecular component (a water-insoluble glucan). The enzyme showed neither cellulase nor beta-glucosidase activity. The reaction was optimal at pH 6.0 and 37 degrees C. The molecular weight of the enzyme was estimated to be 11,000 by gel filtration using a TOYOPEARL HW-55F column. The amount of the glucan synthesized by the enzyme increased with prolonged incubation in a reaction with cellopentaose, and soluble cellooligosaccharides, such as cellobiose, cellotriose, cellotetraose, and cellohexaose, were also produced. No glucose was produced in the reaction even when it was carried out for a long time. The total number of molecules (cellooligosaccharides) in the reaction mixture remained at the initial substrate level during the entire reaction. The beta-transglycosylase proved to be a specific transferase showing transfer activity of glucosyl, cellobiosyl, and cellotriosyl moieties from one cellopentaose to an acceptor molecule from cellopentaose upwards with almost 100% efficiency.     

Pancreas acinar cell differentiation. IV. Assay of nanogram levels of chymotrypsin by radioactively labeled synthetic substrate

A procedure for synthesizing ¹⁴C-labeled N-benzoyl-l-tyrosine ethyl ester with the label in the benzoyl group has been described. Using this substrate, which is specific for chymotrypsin, and employing trypsin activation of chymotrypsinogen in an incubation medium containing radiolabeled BTEE, we have presented a method which permits assay of nanogram quantities of chymotrypsin activity in organ-cultured tissue. The radiolabeled product of enzyme hydrolysis, N-benzoyltyrosine, is readily separated by paper chromatography from the unreacted labeled substrate and measured by radioactivity counting.     

Propylamine transferases in chinese cabbage leaves

We have found spermidine synthase and spermine synthase activities in extracts of leaves of Chinese cabbage (Brassica pekinensis var. Pak Choy) and have developed an assay of the former in crude extracts. The method is based on the transfer of the propylamine moiety of decarboxylated S-adenosylmethionine to labeled putrescine, followed by ion-exchange separation of the labeled amine substrate and product, which are then converted to the 5-dimethylamino-1-napthalene sulfonyl (dansyl) derivatives and further purified and identified by thin layer chromatography. The specific radioactivity of putrescine present in the reaction mixture is determined, as is the radioactivity present in dansyl spermidine. The enzyme is also present in extracts of spinach leaves.

Spermidine synthase has been purified about 160-fold from Chinese cabbage leaves. After partial purification, a rapid coupled enzymic assay has been used to study various properties of the enzyme. The plant enzyme shows maximum activity at pH 8.8 in glycine-NaOH buffer and has a molecular weight of 81,000. The K_m values for decarboxylated S-adenosylmethionine and putrescine are 6.7 and 32 micromolar, respectively. The enzyme activity is inhibited strongly by dicyclohexylamine, cyclohexylamine, and S-adenosyl-3-thio-1, 8-diaminoctane. Of these, dicyclohexylamine is the most potent inhibitor with an I₅₀ at 0.24 micromolar.