Abstract
Abstract Several mammalian tissues have been shown to contain as many as three acid phosphatases which may be separated on the basis of differences in size by gel filtration on Sephadex G-75. The smallest of these enzymes from bovine liver, termed acid phosphatase III, has been purified to apparent homogeneity. The purified enzyme migrates as a single band during electrophoresis on polyacrylamide gel and yields arginine as the only detectable amino terminal residue. Acid phosphatase III has been characterized with respect to its substrate specificity, molecular weight, and amino acid composition. The Km value determined with p-nitrophenyl phosphate, the most readily hydrolyzed substrate tested, is 7.5 x 10-4 m. Optimal rates of hydrolysis were observed at pH 5.5. The pure enzyme is stabilized by ethylenediaminetetraacetate and phosphate ion, but is rapidly inactivated in the presence of Mg++ or mercaptoethanol. The molecular weight of acid phosphatase III determined by sedimentation equilibrium analysis is 16,590. This figure is in close agreement with the value of 16,296 obtained from the amino acid composition.
Highlights
To facilitate comparison of these results with those obtained in a similar fashion from other tissues, the peaks of acid phosphatase activity depicted in Fig. 1 have been labeled I, in molecular weight (II), and III in the order of elution
The relative amounts of activity eluted in the positions of Peaks I to III depend upon the source of enzyme but
Inspection of the chromatogram under ultraviolet light revealed a single spot corresponding to dimethylaminonaphthalene-5sulfonyl arginine. These results indicate that the amino-terminal residue in acid phosphatase III is arginine, and in addition provide further evidence in support of the homogeneity of the Step VII preparation
Summary
Protein Determinations-Protein concentrations were determined calorimetrically by a modification of the biuret reaction [17] with cryst,allized, lyophilized bovine albumin as a referenceBovine Liver Acid Phosphatase Vol 244, No 2standard. Bovine Liver Acid Phosphatase Vol 244, No 2. Effluent fractions from column chromatographic procedures were monitored spectrophotometrically for protein content by measuring the absorbance of the solutions at 280 rnp. Enzyme Assays-Acid phosphatase activities were routinely measured at 37” and at pH 5.5 by the rate of liberation of p-nitrophenol from p-nitrophenyl phosphate (Sigma 104). Assay mixtures contained 500 pmoles of sodium acetate, 5 pmoles of EDTA, 20 pmoles of p-nitrophenyl phosphate, and enzyme in a total volume of 5.0 ml. One-millilit.er portions were removed at intervals and mixed with 4.0 ml of 0.1 M KOH and the absorbance of these solutions was measured at 400 rnp against a blank prepared as described above but lacking enzyme. Effluent fractions from preparative and analytical columns were assayed by adding
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