Cooperative kinetics of human prostatic acid phosphatase.

The steady-state kinetics of hydrolysis reaction catalysed by human prostatic acid phosphatase (PAP) by using 1-naphthyl phosphate, phenyl phosphate and phosphotyrosine as substrates has been studied at pH 5.5. The substrate binding curves were sigmoidal and Hill cooperation coefficient h was higher than 1 for each of the examined compounds. Thus, human prostatic acid phosphatase kinetics exhibits positive cooperativity towards the studied substrates. The extent of cooperativity was found to depend on the substrate used and on enzyme concentration. The highest cooperativity of PAP was observed for 1-naphthyl phosphate and the lowest for phosphotyrosine. When prostatic phosphatase concentration increased, Hill cooperation coefficient (h) and half saturation constant (K(0.5)) both grew, but the catalytic constant (k(cat)) remained constant, for each of the substrates studied. Ligand-induced association-dissociation equilibrium of the active oligomeric species (monomer-dimer-tetramer-oligomers) is suggested.

Continuous assay for acid phosphatase using 1-naphthyl phosphate as a substrate.

The described continuous acid phosphatase assay is based on kinetics of the release of 1-naphthol in the course of the enzyme-catalyzed hydrolysis of 1-naphthyl phosphate, measured at 320 nm in aqueous solution and at 322 nm in sodium-bis(2-ethylhexyl)sulfosuccinate isooctane-water reverse micelles in a broad pH range (1.0-8.2). The method allows precise determination of the initial rate of the reaction and therefore may be used in the steady-state and pre-steady-state studies on the phosphatase-catalyzed reaction. The kinetic parameters (Km and kcat) for human prostatic acid phosphatase in aqueous solution and in reverse micelles, at pH 3.8, 4.5 and 5.7, by the proposed 1-naphthyl phosphate assay have been determined.

Continuous assay for acid phosphatase using phenyl phosphate.

A continuous spectrophotometric assay for the determination of the initial rate of an acid phosphatase-catalyzed reaction in an acidic environment, using phenyl phosphate as a substrate, is presented. The method is based on the continuous determination of phenol, a product of the enzymatic hydrolysis, by the kinetic measurement of its absorbance. The method allows for the direct estimation of acid phosphatase activity in an acidic solution. This is possible without interrupting the reaction by alkalization or precipitation required for commonly used end-point colorimetric detection procedures for phosphate or phenol, and without the use of any coupled assays. The method has been developed for acid phosphatase activity determination in an aqueous solution and in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)-isooctane-water reverse micelles in a broad pH range (pH 3.8 to 8.8). The proposed procedure has been used for the determination of kinetic constants (K(m) and kcat) for human prostatic acid phosphatase in aqueous solutions, and in AOT-isooctane-water reverse micelles, at pH 3.8, 4.5, and 5.7.

Stabilization of human prostatic acid phosphatase by coupling with chondroitin sulfate.

Human prostatic acid phosphatase (PAP) (EC 3.1.3.2) was covalently linked to chondroitin sulfate A from whale cartilage. In order to bind the protein amino groups with the preactivated carboxyl groups of chondroitin sulfate, 1-ethyl-3-(3'-dimethylaminepropyl)carbodiimide and N-hydroxysulfosuccinimide were used as coupling agents. The product was soluble and enzymatically active. The activity was on average 25% higher than that of the free enzyme. The product was heterogeneous in respect to charge and Mr (50-1500) kDa, as determined by chromatography on Sephacryl S 300 and polyacrylamide gel electrophoresis. The resulting polymers contained covalently bound chondroitin sulfate, as shown by the biotin-avidin test. The modified enzyme is more resistant against various denaturing agents, e.g., urea, ethanol, and heat. Thus covalent modification of PAP by cross-linking to chondroitin sulfate could be the preferred method for stabilization of its biological activity.