Acid phosphatase fluoride inhibition

Sample Collection and Enzyme Stability. Serum samples are collected with chemically clean, sterile glassware. Blood is allowed to clot at room temperature, the clot is gently separated from the test tube with an applicator stick, and the blood is centrifuged for 10 minutes at 1,000 g. If the red cells are known to contain the enzymes whose activity is being measured, as in the case of LD, even slightly hemolyzed serums must be discarded. When acid phosphatase is to be measured, the serum should be placed immediately in ice and processed as soon as possible, or it should be acidified by the addition of a small amount of sodium citrate. Anticoagulants such as EDTA, fluoride and oxalate inhibit some serum enzymes. However, heparin activates serum lipoprotein lipase. 

Enzymes activities are particularly sensitive to the anticoagulant used in collecting the specimen. Heparin inhibits acid phosphatase (W16) and muramidase (Z5). Amylase activity is inhibited by oxalate or citrate (MIO), and lactic dehydrogenase and acid phosphatase lose activity in oxalate (C2). Alkaline phosphatase is stable in oxalate, oxalate-fluoride, or heparin, but 25 mAf citrate inhibits 50% of the activity, and as little as 50 mlf EDTA is completely inhibitory (B19). Leucine aminopeptidase is inhibited by EDTA, as is creatine phosphokinase (F3). Amylase activity has been reported to be only 83% of that in serum when oxalate or citrate-plasma is used (MIO). Heparin plasma appears to have no inhibitory effect. Despite the fact that clotting factor V is not stable in oxalate or EDTA, these are often used as anticoagulants to obtain plasma for prothrombin determinations (Z2, Z4). 

K.H. Lau, T.K. Freeman, D.J. Baylink, A proposed mechanism of the mitogenic action of fluoride on bone cells. Inhibition of the activity of an osteoblastic acid phosphatase, Metabolism 38 (1989) 858-868. 

Acid phosphomonoesterase (EC 3.1.3.2). Milk contains an acid phosphatase which has a pH optimum at 4.0 and is very heat stable (LTLT pasteurization causes only 10-20% inactivation and 30 min at 88°C is required for full inactivation). Denaturation of acid phosphatase under UHT conditions follows first-order kinetics. When heated in milk at pH 6.7, the enzyme retains significant activity following HTST pasteurization but does not survive in-bottle sterilization or UHT treatment. The enzyme is not activated by Mg2+ (as is alkaline phosphatase), but it is slightly activated by Mn2+ and is very effectively inhibited by fluoride. The level of acid phosphatase activity in milk is only about 2% that of alkaline phosphatase activity reaches a sharp maximum 5-6 days post-partum, then decreases and remains at a low level to the end of lactation. 

Reiner and his colleagues (40) demonstrated that fluoride inhibition of prostatic acid phosphatase showed interesting and unexpectedly complex kinetics. The unusual nature of the inhibition can readily be appreciated from Fig. 4 (40). As the fluoride concentration is increased over a 1000-fold range, the extent of inhibition rises and then subsequently falls with a further increase of inhibitor. At lower fluoride concentration, the inhibition is clearly competitive. Two possibilities were explored for an explanation of these unusual concentration effects of inhibition. There could be two forms of fluoride in the reaction mixtures the inhibitory form and the second which predominates at higher... 

Vescia and Chance Hi) demonstrated that fluoride and tartrate inhibition vide infra) of acid phosphatase showed completely different kinetics when the hydrolysis of phenyl phosphate was compared with transphosphorylation from this substrate to glucose. Figures 5 and 6 (41) show that fluoride inhibition is competitive when the data are plotted according to Lineweaver and Burk. However, the inhibition is noncompetitive with respect to transphosphorylation of the same substrate to glucose. The authors suggested that there are two distinct sites... 

Seedlings are a rich source for nonspecific acid phosphatase. Newmark and Wenger (114) have reported on a 1000-fold purification from lupine seedlings. The purified enzyme hydrolyzes phosphate monoesters and pyrophosphate with p-nitrophenyl phosphate as substrate. The optimal activity was at pH 5.2-5.5, and Km was 3 X 10 4 M. Fluoride inhibition was noncompetitive. 

Shaw (115) reported a 300-fold purification of enzyme from tobacco leaves. Activity of the enzyme was optimal at pH 5.5-5.7, and divalent cations were not required for activity. The enzyme possessed high activity toward ribonucleoside 2 - and 5 -monophosphates and glucose 1-phosphate. There was no activity toward RNA or phosphodiesters. Fluoride acts as a noncompetitive inhibitor for this enzyme. This behavior of fluoride is in contrast to the behavior with prostatic acid phosphatase where the inhibition is strictly competitive. 

Abul-Fadl and King (Al, A2, A3, A4) also investigated the effect of various ions and organic compounds on the acid phosphatase activity of these two tissues. Without describing the results in detail, some of the outstanding effects may be noted. A concentration of 0.5 X 10 M Cu inhibited erythrocytic phosphatase to the extent of 88-96%, but prostatic phosphatase only to the extent of 10-18%. Similarly, 0.5% formaldehyde inhibited completely the erythrocytic phosphatase, but had no effect on prostatic phosphatase. The reverse patterns were shown by 0.5 X 10 M Fe + (ferric) ion, which inhibited erythrocytic phosphatase slightly, about 5-9%, and inhibited the prostatic enzyme to the extent of 80%. Fluoride in 0.01 Af concentration also had comparatively little effect (8% inhibition) on erythrocytic phosphatase but exerted a marked inhibition, 96%, on prostatic phosphate. Of various organic radicals tested, only L-( + )-tartrate (0.01 A/) had a marked differential effect, with 94% inhibition of the prostatic phosphatase and none of the erythrocytic phosphatase. 

Georgatsos (Gl) failed to obtain any fractions upon applying whole hemolysates to Sephadex G-75 or G-100. However, when he precipitated the acid phosphatase with acetone, washed the precipitate twice in acetone, then extracted the resulting dry powder with 0.14 Jlf NaCl, he obtained an active preparation of acid phosphatase. Application of aliquots of this extract to Sephadex G-75 and elution with 0.14 M NaCl resulted in two peaks. The first peak had two pH optima, one at pH 5.0 and another at pH 6.0. It was activated by Mg optimally at a concentration of 6.6 mJlf. The second peak had a pH optimum at 5.0 and was not affected by Mg. Conversely, fluoride at a concentration of 10 mM inhibited the enzyme activity in the first peak to the extent of 47% but did not affect that in the second. As Georgatsos (Gl) has pointed out, the conflicting results obtained by different investigators may be due to the change in proportion of these two components as purification proceeds from the crude hemolysate. 

The lysosomal and the soluble fractions of acid phosphatase were compared in several other ways. The values (milf) for the lysosomal fractions on various substrates were -glycerophosphate, 1.6 fructose 1,6-diphosphate, 2.0 p-nitrophenyl phosphate, 1.6 AMP, 0.43 they were not significantly different from those obtained with the soluble fraction. The pH-activity curves with these substrates were similar for the two fractions. Inhibition of phosphatase activity of the lysosomal and soluble fractions occurred at approximately the same concentration of fluoride or n- (+) -tartrate when j8-glycerophosphate, AMP, or fructose 1,6-diphosphate were used as substrates. However, with p-nitrophenyl phosphate... 

Mice overexpressing acid phosphatase (Acp5) develop osteoporosis whereas mice lacking this enzyme have decreased bone resorption and develop mild osteopetrosis (overly dense, brittle bones). Fluoride at 0.12 mM (2.3 ppm) inhibits Acp5b and stimulates osteoblast bone deposition in vitro, but fluoride therapy does not inhibit human osteoporosis (Sect. 16.2.2). 

Since disturbed acid phosphatase activity has been associated with pathological conditions, the research has focused on the development of diagnostic methods for detection of activity as a marker for the onset of the disease, and in some extent to the development of inhibitors rather than activators to treat those conditions in which the increase in enzyme activity has a direct effect on the evolution of the disease. In particular, only the development of bisphospho-nate derivatives as inhibitors for tartrate-resistant acid phosphatase found their way to the market for treatment of osteoporosis [41], Typical inhibition of phosphatase activity includes anionic species such as L-(+)-tartrate, phosphate, vanadate, molybdate, and fluoride and neutral molecules such as formaldehyde. Vanadate ion,, is a competitive unspecific inhibitor for acid phosphatases by forming transition state analogs. Other oxoanions such as molybdate and tungstate also show inhibitory effects on... 

Most acid phosphatases and phytases are inhibited by fluoride, which is a strong... 

The phosphate group of a purine nucleotide can be lost in at least three different types of reactions catalyzed by phosphatases with broad specificity, nucleotidases, and transferases. Intestinal and liver phosphatase, prostatic acid phosphatase, and bone phos-phomonoesterase hydrolyze purine 5 - and 3 -mononucleotides. A 5 -nucleotidase acting on 5 -AMP, 5 -UMP nicotinamide 5-nucleotide and R5P prepared from bull has been studied more extensively. It acts optimally at pH 8.5 and is activated by magnesium and inhibited by fluorides and borates. 

Viewing the fact that only a portion (but not all of the acid phosphatase) of the lysosomal fraction is readily released upon physical disruption of the lysosomal membrane by freezing and thawing or by hypoos-motic pressure, Baccino et al. (1971) suggested that at least two varieties of acid phosphatase were associated with the lysosomal fraction, the first being readily, and the other not readily, dissociable from lysosomal structures. This interpretation coincides with the earlier observation of Sloat and Allen (1969) who showed two varieties of acid phosphatase associated with lysosomal fractions of rat liver. One form is readily released after physical disruption of lysosomal fractions, the other form is associated with the lysosomal membrane and became soluble only with 5% Triton X—100 treatment. This membrane-associated enzyme accounted for 40% of the total lysosomal acid phosphatase, is heat-stable, and can be separated from the soluble form by electrophoresis. But these two enzymes have similar pH optima and a common response to inhibitions by L-tartrate, fluoride, alloxan, and formaldehyde. 

Acid phosphatase associated with the hamster kidney plasma membrane also has been characterized (Gahmberg and Simons, 1970). The plasma membrane acid phosphatase differs from the lysosomal enzyme in pH optimum, electrophoretic patterns, and inhibitions by fluoride, tartrate, and sulfate ions. The plasma membranes in this study are isolated from microsomal fractions but the possibility of contamination by offier cytomembranes cannot be ruled out. 

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. 

Many reagents are capable of inhibiting the enzymic action of the purple acid phosphatases. They include oxidants such as hydrogen peroxide and ferricyanide tetrahedral oxyanions including phosphate, molybdate, vanadate, sulfate, and arsenate sulfhydryl reagents such as heavy metal ions and p-chloromercuribenzoate and fluoride ions. 

Extracellular, acidic, and alkaline phosphatases differ not only in their pH optima, but also in their requirements for Mg2+ and Zn2+, in their inhibition by chelators such as EDTA, and in their sensitivity to fluoride (Cembella et al., 1984). These enzymes, known to occur simultaneously in certain algal species, appear to be common among algae and bacteria, although in some, such as Dunaliella tertiolecta, they are lacking. Some phosphatases, particularly the intracellular ones are always present and active in cells, but the activity of their extracellular counterparts is dependent on phosphorus availability. Like the extracellular deaminases, the synthesis and activity of phosphatases is regulated by the nutritional state of the organisms. 

The activity of the membrane-bound protein phosphatase, which dephosphorylates the phosphorylated p42/44 proteins, is fiilly inhibited by fluoride ions, but is insensitive to Na3V04 and okadaic acid (Liu et al., 1999), implying that this is a member of the protein phosphatase 2C family (PP2C). A full-length complementary 4119-bp DNA containing an open reading ftame of 1146-bp that encodes a protein of 382 amino acids... 

---