Steady state kinetics and effect of SH inhibitors on acid phosphatase from bovine brain.

1. Product inhibition studies and transphosphorylation to methanol using two different substrates indicate that acid phosphatase from bovine brain forms a phosphoryl enzyme and that the phosphorylation step can not be rate limiting. 2. Acid phosphatase from bovine brain is inhibited by 5,5'-dithiobis-(2-nitrobenzoic acid); this inhibition can be counteracted by inorganic phosphate. Incubation of the enzyme with p-nitrophenyl phosphate in the presence of p-chloromercuribenzoate leads, initially, to a higher degree of inhibition than that found with the same concentration of inhibitor in the absence of substrate. Both the titration by 5,5'-dithiobis-(2-nitrobenzoic acid) and inhibition by p-chloromercuribenzoate are consistant with the presence of 2 SH groups per mol of enzyme.     

Activation of low molecular weight acid phosphatase from bovine brain by purines and glycerol.

Low molecular weight acid phosphatase (orthophosphoric monoester phosphophydrolase (acid optimum), EC 3.1.3.2) from bovine brain is activated up to 4-fold by guanosine, guanine, adenine, adenosine, and 6-ethylmercapto-purine. Several pyrimidines and other purines were tested and did not show any activation effect. The rate enhancement induced by purines is uncompetitive and not caused by transphosphorylation to the activator. Using transphosphorylation to glycerol as a probe, it is proposed that the activator binds to one of the phosphorylated intermediates in the reaction pathway. These findings are discussed in terms of the catalytic mechanism of low molecular weight acid phosphatase.     

Enhanced catalysis by active-site mutagenesis at aspartic acid 153 in Escherichia coli alkaline phosphatase.

Bacterial alkaline phosphatase catalyzes the hydrolysis and transphosphorylation of phosphate monoesters. Site-directed mutagenesis was used to change the active-site residue Asp-153 to Ala and Asn. In the wild-type enzyme Asp-153 forms a second-sphere complex with Mg2+. The activity of mutant enzymes D153N and D153A is dependent on the inclusion of Mg2+ in the assay buffer. The steady-state kinetic parameters of the D153N mutant display small enhancements, relative to wild type, in buffers containing 10 mM Mg2+. In contrast, the D153A mutation gives rise to a 6.3-fold increase in kcat, a 13.7-fold increase in kcat/Km (50 mM Tris, pH 8), and a 159-fold increase in Ki for Pi (1 M Tris, pH 8). In addition, the activity of D153A increases 25-fold as the pH is increased from 7 to 9. D153A hydrolyzes substrates with widely differing pKa's of their phenolic leaving groups (PNPP and DNPP), at similar rates. As with wild type, the rate-determining step takes place after the initial nucleophilic displacement (k2). The increase in kcat for the D153A mutant indicates that the rate of release of phosphate from the enzyme product complex (k4) has been enhanced.     

Substrate positioning by His92 is important in catalysis by purple acid phosphatase

Proteolysis of single polypeptide mammalian purple acid phosphatases (PAPs) results in the loss of an interaction between the loop residue Asp146 and the active site residues Asn91 and/or His92. While Asn91 is a ligand to the divalent metal of the mixed-valent di-iron center, the role of His92 in the catalytic mechanism is unknown. Site-directed mutagenesis of His92 was performed to examine the role of this residue in single polypeptide PAP. Conversion of His92 into Ala, which eliminates polar interactions of this residue with the active site, resulted in a 10-fold decrease in catalytic activity at the optimal pH. Conversely, conversion of this residue into Asn, which cannot function as either a proton donor or acceptor, but can provide hydrogen-bonding interactions, resulted in a three-fold increase in activity at the optimal pH. Both mutant enzymes had more acidic pH optima, with pK(es,1) values consistent with the involvement of an iron(III) hydroxide unit or a hydroxide in the second coordination sphere in catalysis. These results, together with EPR data, support a role of His92 in positioning either the nucleophile or the substrate, rather than directly in acid or base catalysis. The existence of an extensive hydrogen-bonding network that could fine-tune the position of His92 is consistent with this proposal.     

Activation of bovine brain calmodulin-dependent protein phosphatase by limited trypsinization

A calmodulin-dependent protein phosphatase isolated from bovine brain [Tallant, E.A., & Cheung, W.Y. (1983) Biochemistry 22, 3630-3635] is stimulated by limited trypsinization to the same activity level as that by calmodulin. Prolonged trypsinization caused gradual loss of phosphatase activity, a process retarded in the presence of Ca2+, and even more in the presence of calmodulin. Trypsinized phosphatase, when fully activated, had a molecular weight of 60 000 and was composed of two protein species of 43 000 and 16 000 daltons. Trypsinization decreased the Km of phosphatase for casein from 10.8 to 1.2 microM and increased the Vmax from 4.9 to 30.9 nmol (mg of protein)-1 min-1. The proteolyzed enzyme was insensitive to calmodulin and did not bind to a calmodulin-Sepharose affinity column. It was, however, stimulated by Ca2+, requiring 0.4 microM Ca2+ for half-maximal activation. Both native and trypsinized phosphatase were stimulated by Mn2+ to a level considerably higher than that by Ca2+.     

Activation of acid ribonuclease from bovine brain by aluminum.

An acid ribonuclease (optimum pH 6.0) has been purified from bovine brain in a five-step procedure. The preparation appeared homogeneous on SDS-polyacrylamide gel electrophoresis. The molecular size of the acid ribonuclease is 70 kDa and it is a dimeric protein with a subunit molecular size of 35 kDa. The acid RNase was activated by aluminum at low concentration. Preincubation of the acid RNase with 10 microM increased the specific activity of the enzyme 2.3-fold at acid pH, while the effect of aluminum was much weaker at alkaline pH under otherwise the same conditions. A stoichiometry of 1: 1 for the binding aluminum to brain acid RNase was estimated. None of the enzyme-bound aluminum was dissociated by dialysis against 50 mM HEPES, pH 7.0 at 4 degrees C for 24 h. Citrate, EDTA, NaF, and apotransferrin abolished the effects of aluminum on the enzyme. Ribonucleic acid also protected the enzyme against the activation caused by aluminum. These results suggest that accumulation of aluminum in brain may change the regulation of ribonucleic acid metabolism.     

Characterization of bovine brain calmodulin-dependent protein phosphatase

Calmodulin-dependent protein phosphatase of bovine brain exhibited a pH optimum of 7 and appeared to require sulfhydryl groups for activity. Phosphatase activity was inhibited by both NaF and ZnCl2, but was stimulated approximately 2-fold by MnCl2. The enzyme exhibited broad substrate specificity, dephosphorylating casein, troponin I, protamine, histone, and phosvitin, and was not phosphorylated by cAMP-dependent protein kinase. With 32P-labeled casein as a substrate, phosphatase was activated 15-fold by calmodulin; the dissociation constant of phosphatase for calmodulin was 30 nM. Activation of the enzyme by calmodulin as a function of Ca2+ was highly cooperative; the Hill coefficient was 4.9. At a saturating concentration of calmodulin, half-maximal activation of phosphatase was obtained at 0.3 microM Ca2+. Calmodulin increased the Vmax from 1.7 to 41 nmol mg protein-1 min-1 with no significant change in its Km. Formation of a Ca2+-dependent complex between calmodulin and the phosphatase was demonstrated by a calmodulin-Sepharose affinity column, gel-filtration chromatography, and sedimentation on a sucrose density gradient. The rate of formation and dissociation of the calmodulin X phosphatase complex was rapid and readily reversible in response to changes in Ca2+ concentration. The calmodulin X phosphatase complex consists of 1 mol of calmodulin and 1 mol of phosphatase.     

Demonstration and purification of multiple bovine brain and bovine lung calmodulin-stimulated phosphatase isozymes.

Calmodulin (CaM)-stimulated phosphatase in bovine brain or bovine lung CaM-binding protein fractions were fractionated on a heparin-Sepharose column into three activity peaks, designated in order of column three activity peaks, designated in order of column elution as the brain peak I (BPI), peak II (BPII), and peak III (BPIII) or the lung peak I (LPI), peak II (LPII), and peak III (LPIII) phosphatases, respectively. The pooled individual peak fractions were further purified on a fast protein liquid chromatography Superose 12 column. Analysis of the purified samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that they all contained major peptides corresponding to alpha and beta subunits of the brain CaM-stimulated phosphatase. The phosphatases had similar specific activities and were similarly stimulated by Ni2+, Mn2+, Mg2+ + Ca2+, and CaM. They showed differential reactivity on immunotransblots with an alpha subunit-specific monoclonal antibody VJ6, which reacted strongly toward BPI and weakly toward BPIII and LPI, but showed no reactivity toward BPII, LPII, and LPIII. Each of the alpha subunits of the purified phosphatases had a distinct V8 protease and chymotrypsin peptide map. The results suggest that both bovine brain and bovine lung contain multiple CaM-stimulated phosphatase isozymes. The suggestion of three mammalian brain CaM-stimulated phosphatase isozymes is in agreement with the results of recent molecular cloning studies (Kuno, T., Takeda, T., Hirai, M., Ito, A., Mukai, H., and Tanaka, C. (1989) Biochem. Biophys. Res. Commun. 165, 1352-1358; Guerini, D., and Klee, C.B. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9183-9187; da Cruz e Silva, E. F., and Cohen, P. T. W. (1989) Biochim. Biophys. Acta 1009, 293-296). The successful purification of the individual isozymes may facilitate the elucidation of molecular basis and physiological significance of the isozymes.     

Reactive thiol groups in rat liver acid phosphatase

Dimeric rat liver acid phosphatase P1 of Mr 92,000 is inactivated by p-chloromercuribenzoate and fluorescein mercuriacetate (FMA). The enzyme is protected against the mercurials by the substrate analogue Pi. The reaction with FMA is accompanied by changes in absorbance at 495 nm and in fluorescence emission at 520 nm that are characteristic of reaction of this compound with thiol groups. Titration of P1 with FMA monitored by spectrophotometry or by fluorimetry indicated that equivalence is reached at an FMA/P1 ratio of 3. Since FMA can act as a bifunctional reagent, it is likely that P1 contains either 3 or 6 reactive thiol groups per molecule. Analysis of FMA inactivation/modification data by a statistical method suggests that of 6 reactive thiol groups, 2 are essential so that there are probably 3 thiol groups per subunit, one of which is located at the active site. If the total thiol number is 3, analysis suggests 1 essential thiol per subunit.     

Heterogeneity of SH groups in sarcoplasmic reticulum.

The incorporation of [¹⁴C] N-ethylmaleimide reveals fast and slow-reacting sulfhydryl groups in sarcoplasmic reticulum. Two proteins react with the label: a fast-reacting glycoprotein recently isolated (Ikemoto, Cucchiaro and Garcia (1976) J. Cell Biol.$\text{70}$, 290a), and the Ca²⁺-ATPase. Labeling sarcoplasmic reticulum with a maleimide spin label gives a similar pattern. The spectra of maleimide-spin-labeled sarcoplasmic reticulum have both ‘strongly’ and ‘weakly’ immobilized components. Maleimide-spin-labeled purified Ca²⁺-ATPase, or sarcoplasmic reticulum labeled first with N-ethylmaleimide, and then with maleimide spin label, show spectra devoid of the ‘weakly’ immobilized component; the latter is enhanced in partially purified glycoprotein obtained from spin-labeled sarcoplasmic reticulum. This indicates that spectra from maleimide-spin-labeled sarcoplasmic reticulum do not reflect exclusively the state of the Ca²⁺-ATPase enzyme.     

Removal of phosphate groups from casein with potato acid phosphatase.

Potato acid phosphatase (EC 3.1.3.2) was used to remove the eight phosphate groups from alphas1-casein. Unlike most acid phosphatases, which are active at pH 6.0 or below, potato acid phosphatase can catalyze the dephosphorylation of alphas1-casein at pH 7.0. Although phosphate inhibition is considerable (K1=0.42 mM phosphate), the phosphate ions produced by the dephosphorylation of casein can be removed by dialysis, allowing the reaction to go to completion. The dephosphorylated alphas1-casein is homogeneous on gel electrophoresis with a slower mobility than native alphas1-casein and has an amino acid composition which is identical to native alphas1-casein. Thus the removal of phosphate groups from casein does not alter its primary structure. Potato acid phosphatase also removed the phosphate groups from other phosphoproteins, such as beta-casein, riboflavin binding protein, pepsinogen, ovalbumin, and phosvitin.     

Characterization of SH groups in porin of bovine heart mitochondria. Porin cysteines are localized in the channel walls.

Porin from bovine heart mitochondria contains probably two cysteines (Cys126 and Cys230 in human porin, Kayser, H., Kratzin, H. D., Thinnes, F. P., G?tz, H., Schmidt, W. E., Eckart, K. & Hilschmann, N. (1989) Biol. Chem. Hoppe-Seyler 370, 1265-1278). Reduced and oxidized forms of these cysteines were investigated in purified protein and in intact mitochondria using the agents dithioerythritol, cuprous(II) phenantroline, diamide and performic acid. Furthermore, intact mitochondria were labelled with the sulfhydryl-alkylating agents N-[14C]ethylmaleimide, eosin-5-maleimide and N-(1-pyrenyl)-maleimide. Affinity chromatography of bovine heart porin was performed with cysteine-specific material. The results can be summarized as follows: (1) Porin has one reduced and two oxidized forms of apparent molecular masses between 30 and 35 kDa. The native form of porin is the reduced 33 kDa form. The oxidized forms only appear after denaturation with SDS. (2) The 35-kDa reduced and the 33.5-kDa oxidized forms of porin show the same pore-forming properties after reconstitution of the protein into lipid bilayer membranes. (3) Labelling of cysteines by eosin-5-maleimide and N-(1-pyrenyl)-maleimide suggested their location at a boundary between the water-phase and the lipid-phase. Incubation of intact mitochondria with N-ethylmaleimide prior to eosin-5-maleimide and N-(1-pyrenyl)maleimide treatment resulted in the inhibition of the fluorescent labelling. Among the cysteines present in the primary structure, Cys126 is the most sensitive to N-ethylmaleimide binding. (4) Bovine heart mitochondrial porin covalently bound to Affi-Gel 501 (with a 1.75 nm long spacer), but not to Thiopropyl-Sepharose 6B (with a 0.51 nm spacer). This suggests that at least one of the cysteines is localized between 0.51 nm and 1.75 nm deep in the protein micelle.     

The divalent cation dependence of bovine brain calmodulin-dependent phosphatase

The divalent cation dependence of a calmodulin-stimulated phosphatase from bovine brain has been characterized kinetically using phosphorylated myelin basic protein and casein as substrates. At saturating concentrations of calmodulin, dephosphorylation of both myelin basic protein and casein was catalyzed 8- to 10-fold more rapidly at saturating concentrations of Mn2+ than at saturating concentrations of Ca2+. Half-maximal rates of dephosphorylation of both substrates occurred at either 15 microM Mn2+ or 1 microM Ca2+, and the Kact for each ion was not influenced appreciably by the presence of calmodulin. Half-maximal rates of dephosphorylation were observed at concentrations of calmodulin ranging from 3 X 10(-8) to 10(-6) M at saturating concentrations of divalent cations depending on the substrate used and the particular cation chosen. Trypsin treatment of the phosphatase activated the enzyme several-fold, eliminated its calmodulin dependence, but did not alter the Mn2+ concentration dependence of the activity. Ca2+ (10 microM) increased dephosphorylation rates without altering the Mn2+ concentration dependence of the phosphatase activity regardless of the presence of calmodulin. Mg2+ at millimolar concentrations did not alter the Ca2+ or Mn2+ concentration dependence of the activity. As measured without calmodulin, Ca2+ (90 microM) or Mn2+ (200 microM) produced nearly identical alterations of the far ultraviolet circular dichroic spectrum of the phosphatase.     

Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme. Comparison with the phosphotyrosyl protein phosphatase activity of skeletal alkaline phosphatase

A partially purified bovine cortical bone acid phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phosphotyrosyl histones. The apparent Km values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein since these activities could not be separated by Sephacryl S-200, CM-Sepharose, or cellulose phosphate chromatographies, he ratio of these activities remained relatively constant throughout the purification procedure, each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and phosphotyrosine and p-nitrophenyl phosphate appeared to be alternative substrates for the acid phosphatase. Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotyrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phosphotyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions.     

Characterization of intermediate-molecular-weight acid phosphatase from bovine kidney cortex

The distribution of acid phosphatases of intermediate molecular weight was determined in various mammalian tissues. The intermediate-molecular-weight acid phosphatases (designated P-II-1 and 2) comprised about 25% of the p-nitrophenyl phosphatase activity in the supernatant of bovine kidney cortex homogenate. The P-II-1 and 2 purified 2,000 fold showed the pI values of 5.9 and 5.7, respectively, on isoelectric focusing. Apparent molecular weights of both P-II-1 and 2 were estimated to be 42,000 by Sephadex G-100 gel filtration and 44,000 by SDS-polyacrylamide disc gel electrophoresis. Both the enzymes catalyzed the hydrolysis of a wide variety of natural phosphomonoesters, except for the phosphoproteins phosphoserine and o-phosphocholine. The enzymes showed a high activity on pyridoxal phosphate, beta-glycerophosphate, and 2'-AMP. The optimum activity pH was near 5 with p-nitrophenyl phosphate, but was shifted to the neutral range when pyridoxal phosphate was the substrate. The cations Hg2+ and Ag+ had a marked inhibitory effect. Neither enzyme was inhibited significantly by L-(+)-tartrate or pCMB. The two other types of acid phosphatases, the high-molecular-weight (designated P-I) and low-molecular-weight (designated P-III), were also purified to homogeneity from bovine kidney cortex, and were compared with P-II from several aspects including substrate specificity and susceptibility to various compounds.