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.
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).
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