1. A sensitive new method is described for the rapid optical assay of polynucleotide phosphorylase in crude bacterial extracts of low specific activity. 2. The assay is non-linear with time and a quick method for determining the approximate activity by using a nomograph is given. 3. The dependence of the assay on Mg(2+) concentration was investigated and the conditions that gave first-order kinetics were determined. 4. The theoretical behaviour of the system was examined mathematically and shown to tend to a simple dynamic steady state, under certain conditions. 5. The potential uses of such cyclic systems are discussed.

Adenine Nucleotides↗

PubMed1968

Some changes in the reactivity of enzymes resulting from their chemical attachment to water-insoluble derivatives of cellulose.

1. Purified ficin was chemically attached to CM-cellulose, and partially purified ATP-creatine phosphotransferase was chemically attached to both CM-cellulose and p-aminobenzylcellulose. 2. The apparent K(m) with respect to ATP and Mg(2+) of ATP-creatine phosphotransferase was observed to increase about tenfold on attachment of the enzyme to CM-cellulose, and to increase by only 23% on its attachment to p-aminobenzylcellulose. 3. The reactivity of both ficin and ATP-creatine phosphotransferase with 5,5'-dithiobis-(2-nitrobenzoic acid) was observed to decrease on chemical attachment of these enzymes to water-insoluble derivatives of cellulose. With derivatives prepared from CM-cellulose, the extent of the reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) was dependent on ionic strength, but with similar derivatives prepared from p-aminobenzylcellulose the extent of this reaction was independent of ionic strength. 4. The effect of diffusion and electrostatic interaction of charged enzyme substrates and charged enzyme supports on the apparent K(m) of a water-insoluble derivative of an enzyme is discussed. An equation is derived that satisfactorily describes the observed effects of these factors on the apparent K(m).

Journal Article↗

PubMed1968

The preparation and kinetics of lactate dehydrogenase attached to water-insoluble particles and sheets.

1. The preparation of lactate dehydrogenase covalently attached to anion-exchange cellulose particles and sheets by use of a dichloro-sym-triazinyl dyestuff, Procion brilliant orange MGS, is described. 2. The stability and kinetic properties of these preparations were investigated. 3. An equation is derived to describe the change in concentration of a substrate when passed through a uniform bed of a substrate-inhibited enzyme. A number of theoretical curves are shown to illustrate the system. 4. A titrimetric assay for lactate dehydrogenase is described, and shown to be stoicheiometric over the range pH5.0-9.2. 5. The results are discussed in relation to previous work, and the effects of charged groups on the support, and of the diffusion film surrounding any particle in suspension, are treated qualitatively.

Cellulose↗

PubMed1968

The preparation and properties of pyruvate kinase attached to porous sheets, and the operation of a two-enzyme continuous-feed reactor.

1. The covalent attachment of pyruvate kinase to filter-paper disks and some kinetic properties of the resultant enzymically active porous sheets are described. 2. A two-enzyme reactor was constructed by using water-insoluble sheets of lactate dehydrogenease in conjunction with the above-mentioned sheets possessing pyruvate kinase activity.

Adenine Nucleotides↗

PubMed1968

Catabolite repression and the induction of amidase synthesis by Pseudomonas aeruginosa 8602 in continuous culture.

Explore the source record for details and available documents.

Amidohydrolases↗

PubMed1966

The preparation and properties of ficin chemically attached to carboxymethylcellulose.

1. Purified ficin has been coupled to four CM-celluloses by reaction with their acid azide derivatives. Insoluble products containing 1.8-4.7mg. of ficin/100mg. of product and retaining 8.0-12.0% of the free enzyme's esterase activity have been obtained. 2. The amount of bound ficin in these preparations is dependent on the degree of carboxymethyl substitution of the CM-cellulose to which the ficin is attached. 3. A shift of the alkaline limb of the pH-activity curve of ficin when chemically attached to CM-cellulose has been shown. 4. Only a small loss has been observed in the enzymic activity of these products when stored at 2 degrees for 4 months. They are more resistant than free enzyme to heat denaturation. 5. Columns of CM-cellulose-ficin have been packed. The degree of hydrolysis of perfused substrate has been measured for different flow rates through the column. 6. The properties of these derivatives have been discussed.

Chemical Phenomena↗

PubMed1966

The kinetics of carboxymethylcellulose--ficin in packed beds.

1. The kinetics of the hydrolysis of benzoylarginine ethyl ester in packed columns of CM-cellulose-70-ficin and CM-cellulose-90-ficin were studied. 2. The apparent Michaelis constant, K'(m), of these preparations was calculated and shown to be dependent on the flow rate at low rates of perfusion through the columns. 3. The values for k(3) of these preparations were calculated and shown to be nearly independent of flow rate. 4. A modified form of the integrated Michaelis rate equation was used to describe the action of these materials and its limitations are discussed. 5. The hydrolysis of solutions of casein by these columns was studied.

Arginine↗