Acid phosphatase preparation

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. 

The analysis of substrate dose-response curves for alkaline phosphatase (ALP), immobilized in methanol-containing polymerization mixture, revealed at least two kinetically different forms of the enzyme (27), The high affinity enzyme component had the Michaelis constant = 0.8 mM, which was also measured for the soluble ALP. About 90% of the enzyme activity, however, had an average of 7 mM. The pH maximum of immobilized ALP was about one pH unit higher than for the native enzyme. However, trypsin and acid phosphatase, prepared in the presence of PEG, demonstrated single form kinetics with close to that of the soluble trypsin (30),... 

The binuclear iron unit consisting of a (p,-oxo(or hydroxo))bis(p.-carboxylato)diiron core is a potential common structural feature of the active sites of hemerythrin, ribonucleotide reductase, and the purple acid phosphatases. Synthetic complexes having such a binuclear core have recently been prepared their characterization has greatly facilitated the comparison of the active sites of the various proteins. The extent of structural analogy among the different forms of the proteins is discussed in light of their spectroscopic and magnetic properties. It is clear that this binuclear core represents yet another stractural motif with the versatility to participate in different protein functions. 

Although detailed structural as well as mechanistic knowledge of an enzyme is desirable, it is by no means necessary in order to design a suicide substrate. This has been shown by Myers and Widlanski (1993) who have designed a simple inhibitor of human prostatic acid phosphatase (PAP), an enzyme that is believed to be involved in the regulation of androgen receptor activity in prostate cells. Since the enzyme shows a preference for hydrolysis of aryl phosphates, the 4-(fluoromethyl)-phenyl phosphate (FMPP) was prepared as a substrate that would, on hydrolysis by the... 

Fia. 7. Inhibition of prostatic acid phosphatase by D-(+)-tartaric acid. The reaction mixtures all contained equivalent amounts of the enzyme preparation, the indicated concentration of substrate (pH 5.0), 0.05 iff acetate buffer (pH 5.0), and tartaric acid (pH 5.0) total volume, 4.5 ml. Each point represents average values of determinations made with 5 X 10-5 iff and 10 X 10"5 iff tartaric acid except in the case of /3-glycerophosphate for which 1 X 10 iff and 2 X 10 iff tartaric acid was used. From Kilsheimer and Axelrod (4 ). 

Table XI (73) shows the Stokes radii and frictional ratio obtained by the study of purified acid phosphatase. The preparations show molecular homogeneity during filtration on Sephadex G-100, in the analytical ultracentrifuge, and during immunolectrophoresis. These data obtained by chromatography on Sephadex G-200 indicate that human prostatic acid phosphatase has an effective Stokes radius of 47.1 A and a frictional ratio of 1.56, suggesting considerable molecular asymmetry.

Scott (80) purified red cell acid phosphatase of homozygous types A and B by using ammonium sulfate and DEAE-cellulose chromatography. The relative activity of these isozyme preparations was the same when tested with a number of substrates. Type B enzyme showed small kinetic... 

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-... 

Resonance Raman spectra of one-iron preparations of uteroferrin and the splenic acid phosphatase show that tyrosine is a ligand.826 The intense visible spectra of these proteins is due to tyrosine - Fem charge-transfer transitions. Two or three tyrosine residues are implicated. 

Figure 2. Erythrocytic acid phosphatase schematic. A schematic drawing illustrating typical results of an EAP determination in a 13%, 1mm starch gel prepared in 0.24M Nall TO, 0.15M trisodium citrate tannic acid buffer diluted 1 100. The electrophoresis is carried out for 41/2 hr at approximately 410 V. The gels are stained by the fluorescence produced after enzymatic hydrolysis by methylumbellifertjl phosphate at 37° C for 1V2 hr.

Application and Principle This procedure is used to determine acid phosphatase activity in preparations derived from Aspergillus niger var. The test is based on the enzymatic hydrolysis of p-nitrophenyl phosphate, followed by the measurement of the released inorganic phosphate. 

Stor e characteristics of the native enzyme are generally good, with activities maintained over years. Alkaline phosphatase conjugates are usually prepared via amino or carboxylic acid side chains and purified by gel filtration chromatography. Conjugates are very stable, but the enzyme is costly due to the limited supply of calf intestine. Alkaline (and also neutral and acid) phosphatase enzymes in biological samples are a potential problem with the use of this enzyme label. Careful washing of solid phases may be required to ensure no interference in assays. 

This procedure is applicable to most proteins which can be obtained in a highly purified form. Therefore, any of a variety of enzyme proteins may be used in place of avidin. Acid phosphatase is a very good choice for this purpose because it is easily assayed (see Chapter 10). It is advisable to prepare antisera against at least two unrelated proteins. These two types of sera will be used as part of the experiment as described below. 

Add 0.2 ml supernatant VI that has been appropriately diluted. The enzyme preparation must be diluted because at the high concentrations of acid phosphatase present in supernatant VI all of the substrate would be expended immediately. The proper degree of dilution must be determined by trial and error using reaction mixtures with a volume of 0.5 ml. The proper dilution should yield an absorbance of 0.3 to 0.4 for the assay mixture after 5 minutes incubation. If extremely high dilutions are needed it may be necessary to include serum albumin (1% final concentration) in the diluent to protect the enzyme against inactivation. 

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