Bovine liver, acid phosphatase

Anagnostopoulos found that the amino group reagents ketene, nitrous acid, formaldehyde, and phenyl isocyanate all inactivated bovine liver and kidney phosphatases 102). On the other hand, acetylation of chicken intestinal phosphatase with acetic anhydride gave an active product with optimum activity more alkaline than normal 103). The enzyme preparation was impure and acetylation only 70% complete... 

The purification of acid phosphatase from the human liver and the description of its properties do not appear to have been accomplished. Partly, this may be due to the inherent difiBculty of obtaining normal, fresh human material in amounts substantial enough for purification. However, because of the cellular and physiological importance of acid phosphatase, it is advisable to describe in the present section the purifications of the enzyme from rat and bovine liver. Moreover, since these purifications were accomplished with the awareness that acid phosphatase from this source might be present in multiple molecular forms, the descriptions will naturally involve a consideration of the isoenzymes and their properties. 

The solution was divided into two 40-ml portions, and each portion was added to a column of Sephadex G-75 that had been equilibrated with 0.01 M sodium acetate, 1 mM EDTA, pH 4.8. Elution was continued in the same buffer. Gel filtration of a crude extract of bovine liver on Sephadex-75 had previously given two small peaks and a third large peak of acid phosphatase activity. Elution of the purified 35-50% ammonium sulfate fraction now gave a small peak of about 10% of the enzyme activity, no second peak, and a third peak that contained 90% of the enzyme. The third peak (acid phosphatase III) represented the low molecular weight component and constituted 30% of the total acid phosphatase present in the 15,000g supernatant starting material the degree of purification was 54-foId. 

H3. Heinrikson, R. L., Purification and characterization of a low molecular weight acid phosphatase from bovine liver. J. Biol. Chem. 244, 299-307 (1969). 

These enzymes differ from APs in the absence of metal ions. The bovine liver enzyme was used to carry out the transphosphorylation from phenyl (i )-[ 0, 0, 0] phosphate to (6)-propane-l,2-diol, and demonstrated that the reaction proceeds with net retention of stereochemistry. This indicates that the catalytic reaction proceeds via formation of an intermediate, with two inversions of configuration at phosphorus. The nucleophilic residue was identified as histidine by trapping experiments with nitrophenyl [ P]-phosphate followed by denaturation. The nucleophilic histidine residue is part of a characteristic amino acid sequence RHGXRXP (using the amino acid single-letter codes where X represents amino acid residues that are not conserved). The acid (or histidine) phosphatases have not been subjected to as much study as APs, but some further mechanistic information has been obtained from X-ray structures, " mutagenesis studies, and LEER analyses. ... 

Both uteroferrin and bovine purple spleen acid phosphatase are competent to catalyze production of hydroxyl radical in vitro when superoxide anion is present . The generation of hydroxyl radical probably entails a Fenton-like reaction sequence dependent upon the easily reducible iron atom of the solvent-accessible binuclear cluster. No evidence is available to suggest that this reaction also occurs in vivo. However, the high mannose content of uteroferrin promotes uptake of the protein by reticuloendothehal cells of the fetal liver, ultimately directing the protein to lysosomes. Since these multifunctional organelles function in antimicrobial defense, it may be that the redox properties of uteroferrin are also exploited by the fetus in guarding against infection. 

DDT inhibits carbonic anhydrase and has been made the basis of a quantitative method which can determine as httle as 0.2 /xg of DDT 11). More recently, Guilbault et al. 20) published a method for the determination of methyl parathion, aldrin, and heptachlor based on the inhibition of acid and alkaline phosphatases by these substances. The substrate was umbelliferone phosphate which was cleaved by the phosphatases to the fluorescent compound umbelliferone. Decreased fluorescence was used as a direct measure of the inhibitor, and the sensitivity was 5 ppm for methyl parathion and aldrin and 50 ppm for heptachlor. Geike 48) reported that some organochlorine insecticides will inhibit bovine esterase after exposure to UV irradiation. Ordinarily, these compounds activate liver esterase in vitro. 

Incubate a solution of polymer 25 (22 mg, approx 20 pmol of substrate), cytidine-5 -monophospho-N-acetyl neuraminic acid (CMP-NeuAc, Sigma C 8271) (15 mg, 24.4 xmol), a-(2—>3)-sialyltransferase (from rat liver, Sigma S 2769) (0.3 unit), bovine serum albumin (BSA, 4 mg), and calf intestinal alkaline phosphatase (CIAP, Sigma P 7923) (20 unit) in 50 mM sodium cacodylate buffer (pH 7.4, 2.0 mL) containing manganese(II) chloride (0.62 mg) and Triton CF-54 (10 xL) at 37°C for 72 h. 

---