Staphylococcal acid phosphatase: preliminary physical and chemical characterization of the loosely bound enzyme

At temperatures between 45 and 50 C, staphylococcal acid phosphatase purified 44-fold had maximal activity at pH 5.2 to 5.3. However, the enzyme was most stable in the alkaline range (pH 8.5 to 9.5) at temperatures below 50 C. Iodoacetate and ethylenediamine-tetraacetic acid were effective inhibitors, whereas mercaptoethanol and Cu(2+) acted as stimulators. The energy of activation for hydrolytic cleavage of the synthetic substrate, p-nitrophenyl phosphate, was 19.5 Kcal/mole. K(m) for the same substrate was 4.5 x 10(-4)m. The purified enzyme was most active against the substrates p-nitrophenyl phosphate and glyceraldehyde 3-phosphate.     

On the reliability of the lead salt precipitation method of acid phosphatase localization in plant cells

Summary Fixation of cells with glutaraldehyde (5.0%, pH 6.7) was found to facilitate both the penetration of substrate (p-nitrophenyl phosphate) into cells and the leaking out of intracellular phosphate ions. 64% of the original activity survived the fixation for at least 24 hours. Lead ions added to the incubation medium at 6 mM neither accelerated nonenzymatic hydrolysis of the substrate, nor completely inactivated the enzyme activity. Lead ions at concentrations above 6 mM formed an insoluble compound with p-nitrophenyl phosphate, resulting in a decrease in the concentration of free substrate and lead ions. Phosphate ions liberated from substrate could not be completely trapped by lead ions even at above 6 mM, suggesting the possibility of intracellular migration of phosphate ions.In the presence of 4 mM p-nitrophenyl phosphate, 6 mM lead nitrate, and 0.2 M sucrose at pH 6.5, lead salt precipitates were deposited on the outer surface of cell walls, within cell walls, at tonoplast membranes, in nuclei, and occasionally in proplastids. No deposition of lead salt was formed in the control test from which the substrate was omitted. When cells were treated at first with lead nitrate and then with potassium phosphate, lead salt deposits were formed in the same sites as those of cells incubated in a complete reaction medium.It is concluded that although the result of the lead salt precipitation procedure reflects the presence of enzyme activity, it cannot directly show the site of the enzyme.     

Role of ATP and enzyme-bound nascent peptides in the control of elongation for mycobacillin synthesis.

The presence of a Zn2+-dependent acid p-nitrophenyl phosphatase (EC 3.1.3.2) in bovine liver was described. The enzyme was purified to apparent homogeneity and migrates as a single band during electrophoresis on polyacrylamide gel. The enzyme requires Zn2+ ions for catalytic activity, other bivalent cations have little or no effect. The enzyme, of Mr 118,000, optimum pH 6-6.2 and pI 7.4-7.5, was inhibited by EDTA, tartrate, adenine and ATP, but not by fluoride. The common phosphate esters are poor substrates for the enzyme, which hydrolyses preferentially p-nitrophenyl phosphate and o-carboxyphenyl phosphate. The Zn2+-dependent acid p-nitrophenyl phosphatase of bovine liver was different from the high-Mr acid phosphatases previously detected in mammalian tissues.     

Bis-(4-methylumbelliferyl) phosphate as a substrate for the surface membrane-associated phosphodiesterase activity of pig platelets.

It was found that the newly-available compound, bis-(4-methylumbelliferyl) phosphate, could be used as a substrate for the pig platelet surface membrane-associated phosphodiesterase activity, usually assayed with bis-(p-nitrophenyl) phosphate. This enzyme activity is distinct from the phosphodiesterase activity towards 5'-dTMP-P-nitrophenyl ester, which is probably associated with intracellular membrane structures in platelets. Consequently, the use of the 4-methylumbelliferyl derivative as substrate for the phosphodiesterase activity provides a sensitive, fluorimetric assay for this marker enzyme of the platelet surface membrane.     

P-nitrophenyl phosphate as substrate for rabbit liver fructose diphosphatase

Purified rabbit liver fructose diphosphatase has been found to catalyze the hydrolysis of p-nitrophenyl phosphate, PNPP. It has been established that the hydrolysis of p-nitrophenyl phosphate is due to fructose diphosphatase through studies of the chromatographic properties of the enzyme, its temperature sensitivity, dependence on divalent cations and its inhibition by fructose diphosphate. The K_m for PNPP is 6 × 10⁻³M at pH 9.2, 5 × 10⁻⁴M at pH 7.5. This substrate should facilitate studies of the kinetics and mechanism of action of fructose diphosphatase and the comparison of this enzyme with other alkaline phosphatases.     

Purification and characterization of an esterase isozyme involved in hydrolysis of organophosphorus compounds from an insecticide resistant pest, Helicoverpa armigera (Lepidoptera: Noctüidae)

An esterase isozyme was purified from the insecticide resistant pest, Helicoverpa armigera collected from field crops. Purification involved ammonium sulfate precipitation, hydrophobic interaction and ion exchange chromatography followed by gel filtration chromatography. The purification was 212-fold with 1% yield of the enzyme. The optimum pH of the isozyme was found to be 10.5 and 8.5 for p-nitrophenyl phosphate and paraoxon, respectively. The enzyme was unstable at temperature >50 degrees C. The molecular mass determined by SDS-PAGE was 66 kDa. Cations such as Hg(+2), Ag(+2), Cd(+2) inhibited the activity while Zn(+2) stimulated it. Kinetic studies indicated that the enzyme had low K(m) values of 0.238 and 0.348 mM for p-nitrophenyl phosphate and paraoxon, respectively. The enzyme had broad substrate specificity with high K(m) values for ATP, ADP and beta-glycerophosphate. This enzyme was partially sequenced and identified as an alkaline phosphatase.     

Enzymatic characterization of the chondrocytic alkaline phosphatase isolated from bovine fetal epiphyseal cartilage.

Purified chondrocytic alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and inorganic pyrophosphate. Optimal activities for p-nitrophenyl phosphate, ATP and inorganic pyrophosphate are found at pH 10.5, 10.0 and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations. p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasng substrate concentration. Heat inactivation studies indicate that both phosphorolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg2+ (0.1-10.0 mM) and Mn2+ (0.01-0.1 mM) show a small stimulation of p-nitrophenyl phosphate-splitting activity at pH 10.5. Levamisole, Pi, CN-, Zn2+ and L-phenylalanine are all reversible inhibitors of the phosphomonoesterase activity. Pi is a competitive inhibitor with a Ki of 10.0 mM. Levamisole and Zn2+ are potent non-competitive inhibitors with inhibition constants of 0.05 and 0.04 mM, respectively. The chondrocytic alkaline phosphatase is inhibited irreversibly by Be2+, EDTA, EGTA, ethane-1-hydroxydiphosphonate, dichloromethane diphosphonate, L-cysteine, phenyl-methylsulfonyl fluoride, N-ethylmaleimide and iodoacetamide. NaCL, KCL and Na2SO4 at 0.5-1.0 M inhibit the enzyme. At pH 8.5, the cleavage of inorganic pyrophosphate (pyrophosphate phosphohydrolase, EC 3.6.1.1) by the chondrocytic enzyme is slightly enhanced by low levels of Mg2+ and depressed by concentrations higher than 1mM. Ca2+ show only inhibition. Similar effects of Mg2+ and Ca2+ on the associated ATPase (ATP phosphohydrolase, EC 3.1.6.3) activity were observed. Arrhenius studies using p-nitrophenyl phosphate and AMP as substrates have accounted for the ten-fold difference in V in terms of small differences in both the enthalpies and entropies of activation which are 700 cal/mol and 2.3 cal/degree per mol, respectively.     

Human liver acid phosphatases.

Human liver contains three chromatographically distinct forms of non-specific acid phosphatase (EC 3.1.3.2). Acid phosphatases I, II and III have molecular weights of greater than 200 000, of 107 000, and of 13 400, respectively. Following partial purification, isoenzyme II was obtained as a single activity band, as assessed by activity staining with p-nitrophenyl phosphate and alpha-naphthyl phosphate on polyacrylamide gels run at several pH values. With 50mM p-nitrophenyl phosphate as a substrate, enzymes II and III exhibit plateaus of activity over the pH range 3 - 5 and 3.5 - 6, respectively.Acid phosphatase II is not significantly inhibited by 0.5% formaldehyde. The activity of human liver acid phosphatase II and of human prostatic acid phosphatase towards several substrates is compared. The liver enzyme, is marked contrast to the prostatic enzyme, does not hydrolyze O-phosphoryl choline.     

Purification and characterization of acid phosphatase from cotyledons of germinating soybean seeds

Soybean acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) was completely separated from phytase (EC 3.1.3.8) isolated from cotyledons of germinating seeds and purified to homogeneity. A four-step purification regimen consisting of ammonium sulfate fractionation, and ion-exchange, affinity, and chromatofocusing gel chromatographies was employed to achieve a homogeneous preparation. Acid phosphatase activity appeared as a major band of the three forms of acid phosphatase identified on native gels. The purified enzyme had a molecular weight of 53,000 when electrophoresed on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a molecular weight of 53,000 from its mobility in a Fracto-gel TSK HW-50F gel permeation column. The molar extinction coefficient of the enzyme at 278 nm was estimated to be 4.2 X 10(4) M-1 cm-1. The isoelectric point of the protein, as revealed by chromatofocusing, was about 6.7. The optimal pH for activity, like other plant acid phosphatases, was 5.0. While the enzyme failed to accommodate phytate as a substrate, the enzyme did exhibit a broad substrate selectivity. The affinity of the enzyme for p-nitrophenyl phosphate was high (Km = 70 microM), and activity was competitively inhibited by orthophosphate (Ki = 280 microM). The estimated catalytic turnover number (Kcat) of the enzyme for p-nitrophenyl phosphate was about 430 per second. Although the purified enzyme was stable at 0 degrees C and exhibited maximum catalytic activity at 60 degrees C, thermal inactivation studies indicated that the enzyme lost 100% activity after treatment at 68 degrees C for 10 min.     

Resistance to inactivation by EGTA of the enzyme-substrate and enzyme-phosphate complexes of alkaline phosphatase.

Bovine intestinal mucosal alkaline phosphatase is inactivated by the chelating agent EGTA. Several concentrations of the enzyme were incubated with EGTA and a range of concentrations of the substrate p-nitrophenyl phosphate to determine the substrate concentration as a function of time. As predicted by a recently developed theory [Duggleby (1986) J. Theor. Biol. 123, 67-80], catalysis ceases before all substrate is exhausted. An analysis of these final substrate concentrations according to the theory revealed that, whereas the free enzyme is unstable, the effect of EGTA is counteracted when either the substrate or product (phosphate) is bound. Comparison of the results with those obtained by direct stability measurements and steady-state kinetic experiments gave a qualitatively and quantitatively consistent body of evidence in support of this interpretation.     

Modification of human placental alkaline phosphatase by periodate-oxidized 1,N6-ethenoadenosine monophosphate.

Oxidation of 1,N6-ethenoadenosine monophosphate (epsilon AMP) with periodate cleaved the cis-diol of the ribose ring and resulted in the formation of a dialdehyde derivative (epsilon AMP-dial). At room temperature epsilon AMP-dial was unstable and underwent beta-elimination to give 4',5'-anhydro-1,N6-ethenoadenosine dialdehyde acetal (A epsilon Ado-dial). These nucleotide analogues were found to inactivate human placental alkaline phosphatase in a time- and concentration-dependent manner. epsilon AMP-dial was shown to be an affinity label for the enzyme on the basis of the following criteria. (a) Kinetics of the enzyme activity loss over a wide range of epsilon AMP-dial concentration showed a saturating phenomenon. Removal of the phosphate group made the reagent (A epsilon Ado-dial) become a general chemical modifying reagent. (b) The artificial substrate p-nitrophenyl phosphate gave substantial protection of the enzyme against inactivation. (c) epsilon AMP-dial was a substrate and a partial mixed-type inhibitor for the enzyme. Results of the inhibition and protection studies indicated that the reagent and substrate could combine with the enzyme simultaneously. Besides the phosphate-binding domain, an induced hydrophobic region is proposed for the substrate-binding site for human placental alkaline phosphatase.     

Alkaline phosphatase from rat osseous plates: purification and biochemical characterization of a soluble form

A soluble form of an alkaline phosphatase obtained from rat osseous plates was purified 204-fold with a yield of 24.3%. The purified enzyme showed a single protein band of Mr 80,000 on SDS-PAGE and an apparent molecular weight of 163,000 by gel filtration on Sephacryl S-300 suggesting a dimeric structure for the soluble enzyme. The specific activity of the enzyme at pH 9.4 in the presence of 2 mM MgCl2 was 19,027 U/mg and the hydrolysis of p-nitrophenyl phosphate (K0.5 = 92 microM) showed positive cooperativity (n = 1.5). The purified enzyme showed a broad substrate specificity, however, ATP, bis(p-nitrophenyl) phosphate and pyrophosphate were among the less hydrolyzed substrates assayed. Surprisingly the enzyme was not stimulated by cobalt and manganese ions, in contrast with a 20-25% stimulation observed for magnesium and calcium ions. Zinc ions exerted a strong inhibition on p-nitrophenylphosphatase activity of the enzyme. This paper provides a simple experimental procedure for the isolation of a soluble form of alkaline phosphatase which is induced by demineralized bone matrix during endochondral ossification.     

Vanadate inhibition of ATP and p-nitrophenyl phosphate hydrolysis in the fragmented sarcoplasmic reticulum.

The vanadate inhibition of the Ca(2+)-ATPase activity was analysed both in intact sarcoplasmic reticulum vesicles and in the presence of low concentrations of Tween 20, using ATP and p-nitrophenyl phosphate as substrates. The saturation of the internal low-affinity calcium-binding sites protects the enzyme against vanadate inhibition, because: (1) p-nitrophenyl phosphate hydrolysis is not inhibited by vanadate in intact vesicles, but inhibition developed after solubilization with detergents; (2) the vanadate inhibition of the p-nitrophenyl phosphate hydrolysis in solubilized preparations is prevented by free Ca2+ concentrations higher than 10(-3) M and vanadate competes with calcium (10(-5)-10(-3) M); and (3) the vanadate inhibition of ATP hydrolysis is decreased with an increase in vesicular Ca2+ concentration. The presence of magnesium ions is indispensable for the vanadate effect. The vanadate inhibition is non-competitive with respect to Mg-p-nitrophenyl phosphate and uncompetitive with respect to Mg-ATP. However, in the presence of dimethyl sulfoxide, which facilitates phosphorylation of the enzyme, the inhibition is converted to a competitive one with respect to a substrate. The results suggest, that in the process of enzyme operation vanadate interacts with the unliganded E form of Ca(2+)-ATPase, occupying probably an intermediate position between the E2 and E1 forms, with the formation of an E2 Van complex, that imposes the inhibition on the Ca(2+)-ATPase activity.     

The catalytic-centre activity and kinetic properties of bovine milk alkaline phosphatase

1. The phosphorylation of milk alkaline phosphatase was studied under various conditions: maximum incorporation occurred at pH5.0 and 50% incorporation at pH6.6-7.0. 2. The phosphorylation was shown to be specific and the results suggest that the active centre of the enzyme is involved in the process. 3. Phosphoryl-enzyme is rapidly hydrolysed at alkaline pH. at pH7.0 the results suggest that a phosphoryl-enzyme could occur as a transient intermediate in the hydrolysis of phosphate esters by the phosphatase. 4. The catalytic-centre activity of the enzyme was found to be 2700sec.(-1) at pH10.0 and 25 degrees with p-nitrophenyl phosphate as substrate.     

Studies on kinetic properties of acid phosphatase from nuclei-free rat liver homogenate using different substrates

Kinetic properties of rat liver acid phosphatase were evaluated using the conventional synthetic substrates sodium beta glycerophosphate (betaGP) and p-nitrophenyl phosphate (PNPP) and physiologically occurring phosphate esters of carbohydrates, vitamins and nucleotides. The extent of hydrolysis varied depending on the substrates; phosphate esters of vitamins and carbohydrates were in general poor substrates. Kinetic analysis revealed the presence of two components of the enzyme for all the substrates. Component I had low Km and low Vmas. Opposite was true for component II. The Km values were generally high for betaGP, PNPP and adenosine diphosphate (ADP). Amongst the nucleotides substrates AMP showed high affinity i.e. low Km. The increase in enzyme activity in general at high substrate concentration seems to be due to substrate binding and positive cooperativity. AMP which showed highest affinity was inhibitory at high concentration beyond 1 mM. The results suggest that in situ the nucleotides may be the preferred substrates for acid phosphatase.     

Kinetic mechanism of the Zn-dependent aryl-phosphatase activity of myo-inositol-1-phosphatase

Myo-inositol-1-phosphatase (EC 3.1.3.25) is able to hydrolyze myo-inositol-1-phosphate in the presence of Mg(2+) ions at neutral pH, and also p-nitrophenyl phosphate in the presence of Zn(2+)-ions at acidic pH. This enzyme plays a role in phosphatidylinositol cell signalling and is a putative target of lithium therapy in manic depression. We elucidate here the kinetic mechanism of the Zn-dependent activity of myo-inositol-1-phosphatase. As part of this analysis it was necessary to determine the basicity constants of p-nitrophenyl phosphate and the stability constant of its metal-complex in the presence of zinc chloride. We find that the Zn-dependent reaction may be described either by a rapid-equilibrium random mechanism or an ordered steady-state mechanism in which the substrate binds to the free enzyme prior to the metal ion. In both models the Zn-substrate complex acts as a high affinity inhibitor, yielding a dead-end species through its binding to the enzyme-Zn-substrate in rapid-equilibrium or to the enzyme-phosphate complexes in a steady-state model. Phosphate is a competitive inhibitor of the enzyme with respect to the substrate and an uncompetitive inhibitor with respect to zinc ions.     

Rat intestinal phosphodiesterase II. Properties of the highly purified enzyme and its inactivation by iodoacetic acid.

A highly purifed preparation of rat intestinal phosphodiesterase II (oligonucleate 3'-nucleotidohydrolase, EC 3.1.4.18) has been studied using a synthetic substrate, thymidine 3'(2,4-dinitrophenyl) phosphate. The enzyme was most active between pH 6.1 and pH 6.7 and was inhibited by Cu2+ and Zn2+ but unaffected by EDTA, Mg2+, Co2+, and Ni2+. The reaction rate decreased at high levels of enzyme because of competitive inhibition by deoxythymidine 3'-phosphate, a reaction product, which showed a Ki of 2-10(-5) M. The molecular weight of the enzyme by gel-filtration was 150 000-170 000. In electrofocusing experiments multiple peaks of activity were found at pH 3.4, 4.2-4.5and 7.2. Polyacrylamide gel electrophoresis of freshly purified phosphodiesterase II showed up to 10 protein bands in the gels. If the preparations were stored at 4 degrees C for some time only one or two bands appeared. Investigation of the reaction of rat intestinal phosphodiesterase II with a number of possible phosphodiesterase substrates indicated that the enzyme required a nucleoside 3'-phosphoryl residue for the initiation of hydrolysis. Thus compounds such as NAD, ATP, bis-(p-nitrophenyl)phosphate, thymidine 5'-(p-nitrophenyl)phosphate, glycerylphosphorylcholine, guanylyl-(2' leads to 5')-adenosine and 3',5'-cyclic AMP which contain phosphodiester bonds, nevertheless were not substrates for the enzyme. The enzyme was inhibited reverisbly by p-chloromercuribenzoate and p-chloromercuriphenylsulfonate and inactivated irreversibly by iodoacetic acid. Activity of the phosphodiesterase II was reduced to 50% by incubation with 2.0-10(-3)--5.0-10(-3) M iodoacetate for 20--30 min at 24 degrees C at pH 5.0--6.1. Iodoacetamide had no effect. The degree of inactivation by iodoacetate was reduced by the presence of a substrate for the enzyme or, more effectively by deoxythymidine 3'-phosphate, a competitive inhibitor. It is concluded that iodoacetic acid alkylates an essential residue at the active centre of the enzyme.     

Purification and characterization of phosphodiesterase from Crotalus venom.

A procedure for the purification of phosphodiesterase from Crotalus venom on DEAE-cellulose at alkaline pH is described. The enzyme gives a single band in polyacrylamide gels and is free of contaminating nucleolytic enzymes. The molecular weight is about 115000. Concentration in an Amicon ultrafiltrator gave a highly concentrated active enzyme. Phosphodiesterase is relatively stable and can be stored at 4 degrees C in the presence of Mg2 and serum albumin for years. For the detection of contaminating endonuclease, an assay was used in which tRNA was the substrate and possible internal breaks were detected in polyacrylamide gel after denaturation. With bis(p-nitrophenyl) phosphate as substrate, 15mM Mg2 was necessary for optimal activity. The reaction remained linear for at least 15 min at 22 degrees C. At 45 degrees C, the liberation of p-nitrophenol was highest within 25 min of incubation. At 75 degrees C, inactivation of the enzyme occurred after 4 min.     

Inactivation of gastric and pancreatic lipases by diethyl p-nitrophenyl phosphate

Reacting gastric and pancreatic lipases with mixed diethyl p-nitrophenyl phosphate/bile salt micelles resulted in a stoichiometric inactivation of these enzymes as tested on emulsified tributyroylglycerol and trioleoylglycerol as substrates. Diethyl p-nitrophenyl phosphate treated gastric lipases were also inactive on water-soluble p-nitrophenyl acetate, whereas the modified pancreatic lipase was still able to hydrolyze this water-soluble substrate. The binding of diethyl p-nitrophenyl phosphate modified pancreatic and gastric lipases to tributyroylglycerol/water interface was comparable to that of native lipases. The essential free sulfhydryl group of gastric lipases underwent no chemical changes due to the reaction with micellar diethyl p-nitrophenyl phosphate. All in all, these results indicate that, in both gastric and pancreatic lipases, the essential serine residue which was stoichiometrically labeled by this organophosphorus reagent is involved in catalysis and not in lipid binding.     

Inhibition of glycosyltransferases by bis-(p-nitrophenyl)phosphate: general effect and relation to their membrane integration

The effect of bis-(p-nitrophenyl)phosphate on various glycosyltransferases involved in protein glycosylation (sialyl-, fucosyl-, galactosyl-, mannosyl- and glucosyltransferases) have been studied using crude enzyme preparations solubilized from rat spleen lymphocytes. Bis-(p-nitrophenyl)phosphate appears as a common inhibitor for every glycosyltransferase reaction utilizing sugar nucleotides as direct donors. In most cases 10 mM inhibitor is sufficient to obtain a 90 per cent inhibition. Kinetic studies achieved with a purified galactosyltransferase preparation reveal that bis-(p-nitrophenyl)phosphate exerts a competitive inhibition towards UDP-galactose binding. Concerning membrane-bound enzymes, the interaction of bis-(p-nitrophenyl)phosphate depends on its accessibility to the enzyme active site. This is shown by the different effect obtained with two UDP-Glc utilizing membrane-bound enzymes : UDP-Glc : phospho-dolichyl glucosyltransferase and UDP-Glc : ceramide glucosyltransferase : the first one not being affected but the second one being markedly inhibited under the same condition, although both are inhibited when the membrane environment is disturbed by detergent. Bis-(p-nitrophenyl)phosphate appears to be a tool to study membrane topology of glycosyltransferases.