Purple bovine spleen

Fe—P distance of 3 A, thus providing direct evidence for phosphate interaction with the iron cluster in purple bovine spleen add 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.)...

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. 

PAPs are glycoenzymes of 35-40kDa characterized by their intense purple color due to a tyrosine ligand-to-iron(III) charge transfer transition near 600 nm. The best studied PAPs are those from bovine spleen, porcine uterine fluids, and rat bone, which have been proposed to be involved in iron transport, the immune response, and bone resorption respectively. However, the biological relevance of the phosphatase activity of PAPs has not been unequivocally established. Recently, a bacterial phospholipase with active site properties resembling those of PAPs has also been reported. All PAPs contain a conserved dimetal binding... 

The purple acid phosphatases (PAP) catalyze the hydrolysis of phosphate esters under acidic pH conditions (pH optimum 5) (9, 10). They differ from other acid phosphatases in having a distinct purple color due to the presence of iron or manganese and in being uninhibited by tartrate. Diiron units have been found in the active sites of the enzymes from mammalian spleen (171-173) and uterus (173, 174), while a heterodinu-clear FeZn unit has been characterized for the enzyme from red kidney bean (175). Either the Fe2 or the FeZn unit is catalytically competent in these enzymes, since the enzymes from porcine uterus and bovine spleen can be converted into active FeZn forms and the kidney bean enzyme can be transformed into an active Fe2 form (176). There are also enzymes from other plant sources (particularly sweet potato) that have been reported to have either a mononuclear Mn(III) or Fe(III) active site (177), but these are beyond the scope of the review. This section will focus on the enzymes from porcine uterus (also called uteroferrin), bovine spleen, and red kidney bean. 

Purple acid phosphatases contain a dinuclear Fe " M + centre in their active site (M = Fe or Zn +). To resolve the specific role of the ferric ion in catalysis, a series of metal-substituted forms of bovine spleen purple acid phosphatase (BSPAP) of general formula M Zn°-BSPAP has been prepared, in which the trivalent metal ion was systematically varied (M = Al, Fe, Ga and In). The activity of the AlZn-BSPAP form was only slightly lower kcat 2000 s- ) than that of the previously reported GaZn and FeZn forms (fecat 3000 s- ), The InZn form was inactive. This... 

Averill BA, Davis JC, Bimnan S, Zirino T, Sandersloehr J, Loehr TM, Sage JT, De-bruimer PG. 1987. Spectroscopic and magnetic studies of the purple acid phosphatase from bovine spleen. JAm Chem Soc 109 3760-3767. 

Pinkse MWH, Merkx M, Averill BA. 1999. Fluoride inhibition of bovine spleen purple acid phosphatase characterization of a ternary enzyme-phosphate-fluoride complex as a model for the active enzyme-substrate-hydroxide complex. Biochemistry 38 9926-9936. 

Add phosphatases are ubiquitously distributed enzymes defined by the pH optimum (usually 4.9-6.0) of their hydrolytic activity toward orthophosphate monoesters. A metal-dependent subclass of these enzymes was first recognized in 1973, when iron-bearing acid phosphatases were isolated from porcine uterine fluid and bovine spleen Because of their intense colors they have become known as the purple add phosphatases. Its source, content of iron, and presumed role in iron transport fix>m pregnant sow to fetal pig have earned the pordne protein the euphonious name of uteroferrin. 

In addition to its archetypical members, uteroferrin and bovine spleen add phosphatase, the class of purple add phosphatases includes proteins isolated from rat bone and. spleen spleens of patients with Gaucher s disease or leukemic reticuloen-dotheliosis equine uterine flushings bovine cortical bone giant ceU tumors human placenta and microorganisms . The plant enzymes include an Fe-Zn phosphatase from red kidney beans and an Fe-Fe or Mn(in) protein from sweet potato tubers . Although less well-defined and more heterogeneous than their mammalian counterparts, the color and iron content of the plant enzymes warrant their designation as purple acid phosphatases. 

Unlike uteroferrin, which produces a single band of expected mobility for its molecular size by SDS-FAGE, bovine spleen add phosphatase yields two bands, even when isolated in the continuous presence of proteolytic inhibitors However, the extensive sequence homology between the spleen and uterine enzyme, and the fact that isolation of the spleen enzyme is accomplished by prolonged add extraction of spleen homogenates, make it likely that the purple add phosphatase of spleen is also a single-chain protein ... 

A purple add phosphatase, substantially similar to uteroferrin and bovine spleen add phosphatase in molecular weight, iron content, and binding to ConA-Sepharose, has been isolated from rat spleen. However, the protein is heterogeneous by isoelectric focusing, perhaps because of variations in carbohydrate content. This protein appears identical to a purple add phosphatase found in developing rat bone and also bears some similarities to a less well diaracterized add phosphatase with protein phosphoty-... 

Two iron-bearing purple add phosphatases, uteroferrin and the bovine spleen enzyme, have been extensively studied by laser-Raman spectroscopy (Fig. detec-... 

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)...

To date. X-ray absorption studies have been performed only on the purple add phosphatase fi om bovine spleen Iron K-edge near-edge (XANES) and extended X-ray absorption fine structure (EXAFS) results, comparing the splenic enzyme with oxo-bridged model complexes, support the presence of a binuclear site in which the iron atoms are multiply bridged ... 

Although reduction and activation are synonymous for the vast majority of the purple acid phosphatases, several exceptions exist. The Fe-Zn forms of uteroferrin and bovine spleen phosphatase do not require prior reduction to exhibit enzymatic activity . The Fe-Cu and Fe-Hg derivatives of uteroferrin also do not require activation and in fact, the Fe-Cu preparation is inactivated by reducing agents The recently described high molecular weight pink form of uteroferrin has enzymic properties identical to those of purple uteroferrin treated with 2-mercaptoethanoP. Finally, the sweet potato acid phosphatase, which may exist as an 02 dimer with separate mononuclear iron centers, does not require the addition of reductant to promote its enzymatic activity. ... 

The role of the Zn atom in E. coU alkaline phosphatase, which catalyzes the phosphorylation of serine 99 in the amino add sequence to form the covalent enzyme-phosphate intermediate is well understood. Unfortunately, knowledge of the molecular mechanisms underlying the enzymic activity of the purple add phosphatases is far more rudimentary. Spectroscopic studies utilizing inhibitors and perturbants (Sect. III.B.10), such as phosphate and molybdate, indicate that substrate binds close to the binuclear iron cluster of uteroferrin and bovine spleen phosphatase Most likely, the substrate interacts with the redox active iron of the pair . ... 

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. 

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