Spleen, acid phosphatase

In 1966, Chersi et al. (Cl) submitted a procedure for isolating a highly purified preparation of acid phosphatase from hog spleen. The starting material was a crude spleen nuclease II which contained 110-120 units of acid phosphatase per kilogram of ground spleen and had a specific activity of 0.2-0.3. Chromatography on DEAE-Sephadex A-50 yielded 

The third step consisted in loading the acid phosphatase peak on a Sephadex G-lOO column equilibrated and then eluted with 0.1 M acetate buffer, pH 5.6. The acid phosphatase came off before the main protein peak and had a specific activity of 78.4. For the fourth and final step, the active fraction was applied to a CM-Sephadex C-50 column equilibrated with 0.1 M acetate buffer, pH 5.6, and was eluted by a gradient, 0.1 to 0.3 M, of acetate buffer at a molarity of about 0.26 M. It was again applied to the same column and eluted at 0.26 M acetate buffer. 

This final acid phosphatase preparation had a specific activity of 468 and represented an approximately 1900-fold purification of the acid phosphatase in the starting crude spleen nuclease II. It contained no acid deoxyribonuclease, acid ribonuclease, exonuclease, and phosphodiesterase activities that could be detected in a 0.1-ml sample after 2 hours of incubation with the appropriate substrate. The relative rates of hydrolysis of various substrates were as follows p-nitrophenyl phosphate, 100 5 -AMP, 63 j8-glycerophosphate, 60 ATP, 0. With p-nitrophenyl phosphate as substrate, the pH optimum was broad and lay between pH 3.0 and pH 4.8. The Michaelis constant at 37°C was 7.25 X 10 mM. Phosphate and chloride ions acted as competitive inhibitors. 

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Chersi et al. 103) have carried out extensive purification of spleen acid phosphatase. Spleen was fractionated to yield crude spleen nuclease II 104). This preparation was found to contain large quantities of non-... 

Various kinetic studies of the bovine spleen acid phosphatase have been reported (Vincent etal., 1991). The results are consistent with a picture in which the oxyanions bind in a non-competitive fashion by bridging the two iron atoms in the PAP s dinuclear centre, with the smaller anions also able to bind in a competitive manner at a second site (Fig. 5-21). 

Fig. 1 A, B. Resonance Raman spectra of 5 mM purple (A) and 2.7 mM pink (B) bovine spleen acid phosphatase at pH 5.0 and 5°C. Data were collected with use of 514.5 nm excitation, 100 mW incident power, and 140° back-scattering geometry. The spectra, representing an average of 3 (A) and 8 (A) scans were subjected to a 13-point smoothing procedure. (Adapted from Ref. 42)...

In contrast to uteroferrin, the reaction of phosphate with pink (reduced) bovine spleen acid phosphatase has been reported to show a parallel loss of enzymic activity, shift in visible absorption maximum, and loss of EPR signal intensity These results suggest that phosphate binding is coupled to oxidation of the protein s binudear iron center and do not provide evidence for a paramagnetic enzyme-phosphate intermedi-ate Whether this is indicative of differences between the pordne and splenic enzymes or variabilities in experimental design is uncertain. 

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. 

The adult male prostate contains abundant acid phosphatase which it secretes into the semen. The production of this enzyme is governed by the circulating levels of androgenic hormones. Castration or estrogen administration markedly reduces the prostatic urinary acid phosphatase of males. Other organs such as the liver, kidney, spleen, red cells and platelets also contain significant amounts of acid phosphatase. 

Figure 1. Characteristic EPR signals of Fe(II)Fe(III) sites in semimethemerythrinj (a), semimethemerythrinQ (b), reduced uteroferrin (c), reduced uteroferrin-molybdate complex (d), reduced bovine spleen purple acid phosphatase (e), reduced component A of methane monooxygenase (f). (Reproduced with permission from ref. 26. Copyright 1987 Elsevier.)...

Milk acid phosphatase has been purified to homogeneity by various forms of chromaotgraphy, including affinity chromatography purification up to 40 000-fold has been claimed. The enzyme shows broad specificity on phosphate esters, including the phosphoseryl residues of casein. It has a molecular mass of about 42 kDa and an isoelectric point of 7.9. Many forms of inorganic phosphate are competitive inhibitors, while fluoride is a powerful non-competitive inhibitor. The enzyme is a glycoprotein and its amino acid composition is known. Milk acid phosphatase shows some similarity to the phosphoprotein phosphatase of spleen but differs from it in a number of characteristics. 

Acid phosphatases are produced by erythrocytes, the liver, kidney, spleen, and prostate gland. The enzyme of the prostate gland is clinically important, because its increased activity in the blood can be an indication of prostate cancer. The phosphatase from the prostate gland is strongly inhibited by tartrate ion, but acid phosphatases from other tissues are not. How can this information be used to develop a specific procedure for measuring the activity of the acid phosphatase of the prostate gland in human blood serum ... 

Hog spleen acid DNase, as obtained by the above procedure, is completely free of contaminating phosphatase, exonuclease, and adenosine deaminase activities. The enzyme has a weak intrinsic hydrolytic activity on bis(p-nitrophenyl) phosphate and the p-nitrophenyl derivatives of deoxyribonucleoside 3 -phosphates (see Section III,D,3). 

A number of purple acid phosphatases821 have been isolated from animal sources, including bovine spleen, rat bone and the enamel organ of rat molars. Other phosphatases may belong to this class but the identification is not yet certain. Purple acid phosphatase from the sweet potato, as noted in Section 62.1.3.6.1, contains manganese. 

Little is known about the mechanism of action of the purple acid phosphatase. The beef spleen enzyme as isolated contains one tightly bound phosphate,821 but it is not certain whether this corresponds to a phosphorylated amino acid residue as found for other phosphatases. Addition of phosphate causes a shift in the visible spectrum of the enzyme. 

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