Bovine liver phosphoamidase as a protein histidine/lysine phosphatase.

A 13-kDa phosphoamidase was isolated as a single band on SDS-PAGE from bovine liver. Its Stokes' radius, sedimentation coefficient, molecular mass, and optimal pH were estimated to be 1.6 nm, 1.8 s, 13 kDa, and 6.5, respectively. The enzyme released P(i) from 3-phosphohistidine, 6-phospholysine, and amidophosphate at rates of 0.9, 0.6, and 2.6 micromol/min/mg protein, respectively. However, it did not dephosphorylate phosphocreatine, N(omega)-phosphoarginine, imidodiphosphate, or O-phosphorylated compounds including inorganic pyrophosphate. It also dephosphorylated succinic thiokinase and nucleoside diphosphate kinase autophosphorylated at His residues, indicating that it works as a protein histidine phosphatase. A thiol reagent, 30 microM N-ethylmaleimide, depressed the activity by half, while a thiol compound, 2-mercaptoethanol, protected the enzyme from heat-inactivation. Five millimolar divalent cations, such as Mg2+ and Mn2+, and 5 mM EDTA, had no effect on the activity.

3-phosphohistidine and 6-phospholysine are substrates of a 56-kDa inorganic pyrophosphatase from bovine liver.

A 56-kDa inorganic pyrophosphatase isolated from bovine liver hydrolyzed PPi, imidodiphosphate, 3-phosphohistidine, and 6-phospholysine at rates of 0.11, 0.44, 1.09, and 1.22 mumol/min/mg protein, respectively, in a reaction mixture containing 1 mM MgCl2 at pH 8.2. The hydrolysis of imidodiphosphate was influenced by various treatments in a different manner from that of N-phosphorylated amino acids, indicating that the pyrophosphatase has two different catalytic sites for imidodiphosphate and N-phosphorylated amino acids, respectively. Evidence for separate catalytic sites consists of the following findings: the activity on hydrolysis of imidodiphosphate gave a bell-shaped pH curve with a peak at pH 6.5, while the activity on hydrolysis of N-phosphorylated amino acids maintained a high level in the pH range between 6.0 and 9.5. One hundred micromolar p-chloromercuriphenyl sulfonate inhibited the hydrolysis of imidodiphosphate by 35% and did not inhibit that of N-phosphorylated amino acids. Two millimolar magnesium chloride repressed the hydrolysis of imidodiphosphate and had no inhibitory effect on the hydrolysis of N-phosphorylated amino acids. Moreover, methylenediphosphonic acid, an analog of imidodiphosphate, stimulated the hydrolysis of imidodiphosphate in the presence of MgCl2, while it potentiated the substrate inhibition on hydrolysis of N-phosphorylated amino acids.

Purification and characterization of hepatic inorganic pyrophosphatase hydrolyzing imidodiphosphate.

A 56-kDa inorganic pyrophosphatase consisting of 33-kDa subunits was purified from bovine liver almost to homogeneity. This hydrolase required divalent cations such as MgCl2, CoCl2, and MnCl2 to hydrolyze PPi and was insensitive to 2 mM sodium fluoride. The purified hydrolase released 2.1 mumol Pi from PPi per minute per milligram of protein in the presence of 1 mM MgCl2, and the Km for PPi was 0.14 mM. It also hydrolyzed imidodiphosphate to yield Pi and ammonia even in the absence of a divalent cation. The purified hydrolase liberated 2.2 mumol Pi from imidodiphosphate per minute per milligram of protein and the Km for imidodiphosphate was 0.12 mM. Addition of 80 microM MgCl2, CoCl2, or MnCl2 to the reaction mixture increased the hydrolysis rate of imidodiphosphate by 1.5-, 2.0-, and 3.4-fold, respectively. In the absence of divalent cations, the enzymatic hydrolysis of imidodiphosphate was inhibited competitively by PPi (Ki = 0.13 mM). Moreover, one-half of the maximal hydrolysis of imidodiphosphate was inhibited by 10 microM trans-1,2-diaminocyclohexane N,N,N',N'-tetraacetic acid or 45 microM p-chloromercuriphenyl sulfonate. When the hydrolase was treated with heat or SDS, two activities capable of hydrolyzing PPi and imidodiphosphate gave similar inactivation profiles, indicating that one hydrolase participated in the hydrolysis of both substrates.

N(omega)-phosphoarginine phosphatase (17 kDa) and alkaline phosphatase as protein arginine phosphatases.

Seven synthetic polymers, (Glu4, Tyr)n, (Arg)n, (Arg, Pro, Thr)n, (Arg-Gly-Glu)6, (Arg-Gly-Phe)6, (Glu-Arg-Gly-Phe)5, and (Ala-Leu-Arg-Arg-Ile-Arg-Gly-Glu-Arg)2, were treated with phosphoryl chloride to phosphorylate their Tyr, Thr, and Arg residues. Protamines and histones were phosphorylated similarly. These phosphorylated peptides were examined as to whether or not they serve as substrates for intestinal alkaline phosphatase [EC 3.1.3.1] and liver N(omega)-phosphoarginine phosphatase [Kuba, M., Ohmori, H., and Kumon, A. (1992) Eur. J. Biochem. 208, 747-752]. Phosphorylated polyarginine was hydrolyzed with a lower Km with alkaline phosphatase than with N(omega)-phosphoarginine phosphatase, while the phosphorylated forms of (Arg-Gly-Phe)6 and culpeine were better substrates for N(omega)-phosphoarginine phosphatase. When (Arg, Pro, Thr)n and culpeine were phosphorylated chemically after treatment with phenylglyoxal, these phosphorylated peptides were worse substrates for N(omega)-phosphoarginine phosphatase than for alkaline phosphatase. Moreover, the results of proton-decoupled 31P NMR analysis indicated that N(omega)-phosphoarginine phosphatase released Pi from N(omega)-phosphoarginine residues of phosphopeptides. These results indicate that both phosphatases function as protein arginine phosphatases in different manners, and that N(omega)-phosphoarginine phosphatase is useful for selectively detecting N(omega)-phosphoarginine residue in peptides containing various kinds of phosphorylated amino acids.

3-Phosphohistidine/6-phospholysine phosphatase from rat brain as acid phosphatase.

A phosphatase hydrolyzing 3-phosphohistidine and 6-phospholysine was purified from rat brain cytosol to 90% homogeneity on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. One milligram of protein of the purified phosphatase released inorganic phosphate from 3-phosphohistidine, 6-phospholysine, AMP, GMP, and p-nitrophenyl phosphate at velocities of 6.5, 15.6, 15.0, 6.9, and 8.3 mumol/min, respectively. However, the purified enzyme could not hydrolyze N omega-phosphoarginine and phosphocreatine, which are substrates for phosphoamidase [EC 3.9.1.1]. The molecular masses of the holoenzyme and the subunit were 94 and 50 kDa, respectively, and the sedimentation coefficient of the native enzyme was 6.3 s, indicating that it was a dimeric enzyme of identical subunits. The enzyme functioned well under acidic conditions, and 50% of the activity was inhibited by 30 microM tartrate, 4 microM vanadate, 20 microM molybdate, 4 microM VCl3, or 13 microM MoCl5. These results indicate that the present hydrolase belongs to the acid phosphatase group [EC 3.1.3.2].

N omega-phosphoarginine phosphatase from rat renal microsome was alkaline phosphatase.

Activity hydrolyzing both N omega-phosphoarginine and glucose-6-phosphate was detected in rat renal microsome but not in hepatic microsome. Renal microsome was solubilized with 1% n-octyl-beta-D-thioglucoside and purified with DEAE-Sepharose column chromatography. Fractions hydrolyzing both N omega-phosphoarginine or glucose-6-phosphate were subjected to 7.5%-polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate. Phosphatase activity in the gels was detected by a lead nitrate stain using N omega-phosphoarginine or glucose-6-phosphate as substrates. Both substrates produced a stain in the region of the gel corresponding to a protein with a mass of 150 kDa. Extracts of slices from this region of the gel also hydrolyzed phosphocreatine, inorganic pyrophosphate, and O-phosphotyrosine. Moreover, the phosphatase had its optimal pH in the alkaline range and was inhibited completely by 20 microM sodium vanadate, 1 mM cysteine, and 1 mM tetramisole. All these properties indicate that the microsomal phosphoamidase (EC 3.9.1.1) of rat kidney was identical with alkaline phosphatase (EC 3.1.3.1).

Nucleoside monophosphoramidate hydrolase from rat liver: purification and characterization.

1. Adenosine 5'-phosphoramidate hydrolase of 29 kDa was isolated from rat liver cytosol. 2. It consisted of two subunits of 14 kDa. 3. It hydrolyzed nucleoside 5'-monophosphoramidates into nucleoside 5'-monophosphates and ammonia, while it did not hydrolyze adenylyl phosphoramidate, adenylyl imidodiphosphate and N-phosphorylated compounds like phosphocreatine, N omega-phosphoarginine, 6-phospholysine and 3-phosphohistidine. 4. Divalent cations and cyclic AMP had no effect on the hydrolytic activity.

Two phosphatases for 6-phospholysine and 3-phosphohistidine from rat brain.

6-Phospholysine phosphatase (30 kDa) and 3-phosphohistidine/6-phospholysine phosphatase (150 kDa) were purified partially from rat brain cytosol. The former hydrolyzed 6-phospholysine to inorganic phosphate and lysine, and the Km value was 0.83 mM. The latter hydrolyzed 6-phospholysine or 3-phosphohistidine to inorganic phosphate and lysine or histidine, and the Km values were 0.78 and 0.37 mM for 6-phospholysine and 3-phosphohistidine, respectively. Moreover, the latter enzyme preparation dephosphorylated AMP, GMP, and p-nitrophenyl phosphate. Substrate specificities of both enzymes were distinct from those of N omega-phosphoarginine phosphatase (Kuba, M., Ohmori, H., and Kumon, A. (1992) Eur. J. Biochem. 208, 747-752) and phosphoamidase (EC 3.9.1.1), indicating that there are phosphatases corresponding to N-phosphorylated basic amino acids. The possibility that these enzymes work in vivo as protein phosphatases to regulate the level of N-phosphorylated proteins is discussed.

Isolation of N omega-phosphoarginine hydrolase from rat liver and its physical properties.

N omega-Phosphoarginine hydrolase from rat liver cytosol was purified to apparent homogeneity on SDS-PAGE, by employing column chromatographies on Sephadex G-75, DEAE-cellulose, QAE-Toyopearl, and glutathione-2-pyridyl-disulfide-Superose. One milligram protein of the final preparation released 4 mumol/min of inorganic phosphate from N omega-phosphoarginine. The molecular mass on SDS-PAGE, the Stokes' radius and the sedimentation coefficient were estimated to be 17.3 kDa, 1.63 nm, and 2.0 s, respectively, indicating that this enzyme consists of a single peptide. The stability of the enzyme to heat depended on the buffers employed and treatment of the enzyme preparation in 50 mM Tris-HCl, pH 7.0 at 50 degrees C, reduced the hydrolytic activity with a decay constant of 0.099 per min.

Characterization of N omega-phosphoarginine hydrolase from rat liver.

N omega-Phosphoarginine hydrolase from rat liver hydrolyzed N omega-phosphoarginine into arginine and inorganic phosphate, whereas it did not release inorganic phosphate from 19 other phosphorylated compounds containing a N-P bond, an O-P bond or a C-P bond. In addition, it was not able to transfer the phosphoryl moiety from N omega-phosphoarginine to ADP. These results indicated that this enzyme was distinct from both phosphoamidase and arginine kinase. Its properties were as follows: thiol compounds were essential for its activity; it was stimulated by 1.5-2-fold in the presence of 0.001% Lubrol, Tween 20, poly(oxyethylene) 9-lauryl ether and Nonidet P-40, while 0.004% sodium lauryl sulfate inhibited the activity completely; concentrations of sodium molybdate and sodium vanadate necessary for 50% inhibition were 7 microM and 12 microM, respectively; some proteins stimulated the activity, while lysophosphatidic acid, lysophosphatidylinositol, and phosphatidic acid suppressed the activity even in the presence of poly(oxyethylene) 9-lauryl ether.

Myosin and actin from aortae of spontaneously hypertensive and normotensive rats.

Contractile proteins were extracted from thoracic aortae of 9-month-old spontaneously hypertensive stroke-prone (SHRSP) and normotensive Wistar Kyoto rats (WKY). The average wet weight of the aortae from SHRSP was approximately 1.6-fold heavier than that of WKY. On polyacrylamide gel electrophoresis of the myofibrillar extracts prepared from normalized, equal weights of aortae of SHRSP and WKY, both the compositions and concentrations of major polypeptides including myosin heavy chains and actin were nearly identical in these extracts. The myosins purified from these extracts contained identical light chains of about 20- and 17 kDa. Proteolytic peptide maps of the heavy chains of myosin from SHRSP were also indistinguishable from those of WKY, suggesting that the same isoforms of myosin are expressed in both aortae. From these results it is suggested that the qualitative differences may be small, if any, between the major contractile proteins in the aortae of SHRSP and those of WKY.

Physical, enzymatic, and contractile properties of brain myosin with anti-brain myosin Fab fragment bound on its tail.

An antibody obtained by immunizing a rabbit with purified bovine brain myosin was found to react with the tail portion of the myosin heavy chain. An Fab fragment obtained by limited papain digestion of the antibody was allowed to bind to brain myosin, and the complex of the Fab fragment and brain myosin (Fab-myosin) was isolated. On examination of the rotary-shadowed Fab-myosin by electron microscopy, most of the Fab fragment was located on the middle to C-terminal regions of the tails of the myosin molecules. The solubility of Fab-myosin in low salt solutions was higher than that of control brain myosin. Fab-myosin was found to form small irregular aggregates in low salt solutions instead of regular bipolar filaments, and the relative population of the monomeric form of myosin molecules observed for the Fab-myosin was much larger than that observed for the control myosin. The actin-activated Mg2+-ATPase activity of Fab-myosin was stimulated two- to threefold by phosphorylation of the light chains with myosin light chain kinase, as observed for the control brain myosin. Furthermore, the levels of the ATPase activity of the phosphorylated and dephosphorylated Fab-myosins were similar to those of the phosphorylated and dephosphorylated control myosins, respectively. The superprecipitation activity of Fab-myosin was also highly dependent on phosphorylation of the light chains. Although control brain myosin formed a large superprecipitate network which contracted to a dense particle, Fab-myosin generated only numerous tiny superprecipitates under the same conditions. From these results it was deduced that a regular filamentous state of brain myosin was not prerequisite for its actin-activated Mg2+-ATPase and superprecipitation activities but was indispensable for the formation of a large and well contractible superprecipitate.

A basic protein from bovine brain that co-precipitates with tubulin in vitro.

A 193 kDa protein consisting of 58 kDa subunits, which has pI values of 8.50 and 8.65, was purified from bovine brain cytosol. It formed heavy precipitates with tubulin, and the molar ratio of tubulin dimer to this protein in the precipitate was 3.2. In contrast to microtubules containing ordinary microtubule-associated proteins, these complexes remained stable against cold and 1 mM CaCl2.

Studies on the physical states of human platelet myosin in crude extracts.

The physical properties of human platelet myosin in crude extracts were studied by means of Sepharose 4B gel filtration and sucrose density gradient centrifugation in the presence or absence of Mg-ATP. Platelet myosin extracted with a buffer containing 0-0.15 M KCl gave a Stokes radius of about 12.0-12.5 nm irrespective of the presence or absence of Mg-ATP. The sedimentation coefficients obtained in the presence of Mg-ATP were about 10-11 and 8.5S at 0.05-0.10 and 0.15 M KCl, respectively, whereas the values obtained in the absence of Mg-ATP were about 16, 9-12, and 8.5S at 0.05, 0.10, and 0.15 M KCl, respectively. The apparent molecular weight in the presence of Mg-ATP, therefore, was about 500,000 and 420,000 at 0.05-0.10 and 0.15 M KCl, respectively, while the molecular weight in the absence of Mg-ATP was about 790,000, 460,000-620,000, and 440,000 at 0.05, 0.10, and 0.15 M KCl, respectively. The purified monomeric platelet myosin that had been solubilized with Mg-ATP at 0.10 M KCl had a Stokes radius of about 12.5 nm, a sedimentation coefficient of about 9S, and an apparent molecular weight of 460,000. On the other hand, while crude platelet myosin extracted at 0.6 M KCl with Mg-ATP gave a Stokes radius of about 20 nm, a sedimentation coefficient of about of 6S, and an apparent molecular weight of about 490,000, each of these physical parameters obtained in the absence of Mg-ATP was much larger than that obtained in the presence of Mg-ATP because the myosin was associated with F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of the heavy chain of skeletal muscle myosin by casein kinase II: localization of the phosphorylation site to the amino terminus.

While the heavy chain of rabbit skeletal muscle myosin is not phosphorylatable by casein kinase II, it turned out to be phosphorylatable after removal of all of the light chains. The phosphorylation site for the kinase was determined to be Ser-1 and/or Ser-2 at the amino terminus.

The effects of phosphorylation and dephosphorylation of brain myosin on its actin-activated Mg2+-ATPase and contractile activities.

Purified bovine brain myosin contained approximately 1 and 3 mol of protein-bound phosphate/mol myosin in the light chains and heavy chains, respectively. Large portions of this light chain- and heavy chain-bound phosphate (about 0.8 and 2.4 mol, respectively) were removed by incubation with a brain phosphoprotein phosphatase and potato acid phosphatase, respectively. Upon phosphorylation of the dephosphorylated brain myosin with myosin light chain kinase and casein kinase II, about 1.6 and 3.0 mol of phosphate was incorporated into the light chains and heavy chains, respectively, while much lower levels of phosphate were incorporated into the non-dephosphorylated brain myosin under the same conditions. The actin-activated Mg2+-ATPase activity of brain myosin rephosphorylated with myosin light chain kinase was about twice as high as that of dephosphorylated brain myosin (about 30 and 15 nmol phosphate/mg/min, respectively). On the other hand, whereas the rephosphorylated brain myosin superprecipitated rapidly with F-actin, the rate of superprecipitation of the dephosphorylated brain myosin was extremely low. Under appropriate conditions, a loose network of tiny superprecipitates, which formed initially throughout the solution, contracted to form eventually a large and dense particle. These results indicate that phosphorylation of the light chains of brain myosin is a prerequisite for the contraction of brain actomyosin. The role of phosphorylation of the heavy chains by casein kinase II remains to be elucidated.

Regions necessary for pH-dependent assembly of gizzard myosin rod.

Aggregated and disaggregated forms of gizzard myosin rod and its fragments in various concentrations of NaCl (0-0.30 M) at various pH (7.4-8.6) were distinguished from each other by their permeability through a Sepharose 4B column. The rod existed in three forms, namely: large aggregates impermeable to the column, small aggregates eluted at the void volume of the column and a disaggregated monomer which penetrated the column. The relative proportions of the three forms varied depending on the salt concentration and pH. The monomeric rod was detected in NaCl solutions above 0.20 M and its relative proportion at 0.25 M NaCl was larger than those of the small and large aggregates. The small aggregates of the rod were predominant at below 0.05 M NaCl and, upon decrease in pH from 8.6 to 7.4, these small aggregates in NaCl solutions between 0.10 M and 0.15 M were replaced by the large aggregates. Light meromyosin, which corresponded to the C-terminal two-thirds of the rod, existed exclusively as large aggregates in NaCl solutions below 0.15 M; increase of NaCl concentration to above 0.20 M resulted in the formation of its monomer, instead of the large aggregates. In contrast to the rod, no small aggregated form of the light meromyosin was detected. Truncated light meromyosin which had lost a small segment from either the C-terminal or N-terminal of light meromyosin was eluted only as a monomer in any NaCl concentration at any pH. It may be deduced from the above results that a small segment in the light meromyosin is requisite for the assembly of both rod and light meromyosin in the NaCl solutions below 0.15 M and that the relative proportion of small and large aggregates of the rod is determined in a pH-dependent manner by the subfragment 2 segment, the N-terminal third of the rod.

Two chymotrypsin-susceptible sites of myosin rod from chicken gizzard.

The rod prepared from chicken gizzard myosin has been found to have two sites sensitive to limited digestion with chymotrypsin; these sites were located at a subfragment 2/light meromyosin junction (site 1), and at a site 10 kDa remote from either C-terminal or N-terminal of light meromyosin (site 2). The site 1 was more sensitive to the digestion than the site 2. The cleavage at site 2 of the light meromyosin yielded a 74-kDa fragment that was soluble in a low ionic strength solution, contrary to the insolubility of the parent light meromyosin in the same solution. Studies on the effects of MgCl2, ATP and pH on the susceptibilities of these sites to chymotrypsin have given following results. (a) Millimolar concentrations of MgCl2 protected site 1 and site 2 from the chymotryptic cleavage. (b) The cleavage at site 1 of myosin rod in the low salt solution free of Mg2+ at pH 7.0 and pH 8.5, was not affected by the presence of 5 mM ATP. However, MgCl2-induced protection of site 1 was relieved by addition of ATP. On the other hand, the cleavage at site 2 was stimulated by addition of ATP, irrespective of the presence or absence of MgCl2. (c) The alkaline condition of pH 8.5 was more favorable for the chymotryptic cleavages at both site 1 and site 2 than the neutral condition of pH 7.0. These results suggest that myosin rod contains two flexible regions, the structures of which are influenced by such an ambient factor as MgCl2, ATP or pH.

Proteolytic substructure of brain myosin.

Individual bovine brain myosin molecules visualized by electron microscopy consist of two globular heads and a fibrous tail, like myosin molecules from other sources. Brain myosin, however, showed much lower solubility at moderate to high ionic strength (0.2 to 0.4 M KCl) than gizzard myosin, and the filaments formed at low ionic strength in the presence of Mg2+ were fairly resistant to low concentrations of ATP, by which gizzard myosin filaments were completely solubilized. Brain myosin was digested with low concentrations of papain, alpha-chymotrypsin, or trypsin, and the fragmentation patterns were analyzed by means of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, sedimentation at low ionic strength, and electron microscopy of the fragments produced. The results indicate that all of the proteases cleave the myosin molecule primarily at sites located in the neck or in the head close to the neck, suggesting that the brain myosin molecule contains a hinge region or an open peptide stretch around these sites. The differences as well as the similarities between the proteolytic fragmentation patterns of brain myosin and other myosins are discussed.

Requirement of the 20-kDa light chain for the papain-resistant conformation of gizzard myosin.

The limited chymotryptic digestion of unphosphorylated gizzard myosin in 0.15 M NaCl converted a papain-insensitive myosin in ATP to a papain-sensitive one. This conversion without phosphorylation of its 20-kDa light chain was accompanied with truncation of a 200-kDa heavy chain to a 195-kDa fragment and with the degradation of a 20-kDa light chain. Papain also yielded the 195-kDa fragment from the heavy chain, irrespective of the presence or absence of ATP. However, the ATP-induced protection of unphosphorylated myosin from the papain-digestion disappeared concurrently with degradation of the 20-kDa light chain by papain rather than the truncation of heavy chain. Papers from two laboratories [Onishi, H. & Watanabe, S. (1984) J. Biochem. (Tokyo) 95, 903-905; Kumon, A., Yasuda, S., Murakami, N., and Matsumura, S. (1984) Eur. J. Biochem. 140, 265-271] have reported that the ATP-protection of unphosphorylated myosin against papain is not observed after the 20-kDa light chain has been phosphorylated. The present results might indicate that the ATP-induced protection is also abolished through the chymotryptic degradation of the 20-kDa light chain.