Preparation Of Phosphatase Research Articles

Prostatic acid phosphatase, purification and iodination using Iodogen

Prostatic acid phosphatase was purified from prostatic adenomas. The procedure involved chromatography on Concanavalin A-Sepharose, DEAE-cellulose, Bio-Gel P-150 and L-tartrate-Sepharose. The purified phosphatase hydrolyzed p-nitrophenyl phosphate at a rate of 270 μmol · mg −1 min −1 (25°C) and showed homogeneity upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The final prostatic acid phosphatase preparation was pure and the antisera were monospecific as judged by the highly sensitive technique of crossed immunoelectrophoresis. Of the procedures evaluated for the iodination of the purified enzyme, oxidation with lodogen was found to give the best iodinated product.

Purification, isolation and characterization of a phosphoglycolate phosphatase isoenzyme from human erythrocytes

1. 1. Preparation, purification and characterization of a phosphoglycolate phosphatase (PGP)‡ isoenzyme from human erythrocytes was achieved by DEAE-Sepharose CL.-6B chromatography and isoelectric focusing using carrier ampholytes. pH 4–6. 2. 2. The isoenzyme has an isoelectric point of 5.00 ± 0.05 and could be purified 33.000 fold to a specific activity of 32.7 U/mg of protein. It represents the PGP phenotype 1 consisting of a single isoenzyme. 3. 3. The enzyme is composed of two subunits (mol. wt 35,000) which are identical and not connected by SS-bridges. 4. 4. At 4°C the isoenzyme is more stable in the pH range of 7–9 than at acid pH values. 5. 5. Incubation at 30 and 40°C for 4 hr does not affect the activity of the isoenzyme. 6. 6. It has a K m -value of 0.28 mM for phosphoglycolate (PG) as substrate.

Purification of Gizzard Myosin Light-Chain Phosphatase, and Reversible Changes in the ATPase and Superprecipitation Activities of Actomyosin in the Presence of Purified Preparations of Myosin Light-Chain Phosphatase and Kinase1

Sepharose 4B conjugated with phosphorylated myosin light chains was used in affinity chromatography of a partially purified preparation of gizzard myosin light-chain phosphatase (MLCP) (Onishi et al. (1979) J. Biochem. 86, 1283-1290). The MLCP preparation thus purified contained, according to SDS gel electrophoresis, three components of 67,000 (67 K), 54,000 (54 K), 34,000 (34 K) daltons. In an accompanying report, Uchiwa et al. (J. Biochem. 91, 273-282 (1982)) described the purification of gizzard myosin light-chain kinase, which consisted of two subunits; 130 K and 17 K daltons. Using the purified preparations of MLCP and MLCK, it was demonstrated a) that reversible changes in the ATPase and superprecipitation activities occur as myosin light chains are enzymatically phosphorylated and dephosphorylated, and b) that addition of a very low concentration of Ca2+ and its removal cause reversible changes in the turbidity of actomyosin suspensions as well as in the state of phosphorylation of myosin light chains only when MLCK and MLCP are both present. These results provide strong support for the proposal (see Ikebe et al. (1977) J. Biochem. 80, 299-302) that MLCK and MLCP play a key role in the Ca2+ regulation in gizzard.

Preparation of homogeneous alkaline phosphatase from calf intestine by dye-ligand chromatography.

The paper deals with a simple and effective procedure for the isolation of calf intestinal alkaline phosphatase (EC 3.1.3.1) with a yield of 35 per cent by employing immobilized Procion Red HE-3B and Cibacron Blue F3G-A, respectively, as dye-ligands. The resulting enzyme is homogeneous and has a specific activity of about 2500 units per mg of protein. Because dye liganded gels are of low costs and can be used several times without loss of binding properties, the presented method is in particular suited for large scale application.

Selective dephosphorylation of proteins containing phosphotyrosine by alkaline phosphatases.

Histones specifically phosphorylated at either tyrosine (phospho-Tyr-histones) or serine and threonine (phospho-Ser-histones) were prepared and tested as substrates for so-called alkaline and protein phosphatases. Three different preparations of alkaline phosphatase (calf intestine, bovine liver, and Escherichia colg dephosphorylated phospho-Tyr-histones at 5-10 times the ratet hey dephosphorylated phospho-ser-histones. It was concluded that the p-nitrophenyl phosphatase and phospho-Tyr phosphatase activities reside in the same protein on the basis of high performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, inhibition of both activities by Pi and EDTA but not fluoride, and inhibition of phospho-Tyr phosphatase activity by p-nitrophenyl phosphate. Phosphoprotein phosphatase from rabbit muscle, which had little if any p-nitrophenyl phosphatase activity, dephosphorylated phospho-Ser-histones at 16 times the rate it dephosphorylated phospho-Tyrhistones. Thus, the ratio of the pseudo-first order rate constants for dephosphorylation of phospho-Tyr-histones/ phospho-Ser-histones was approximately 100 times higher for alkaline phosphatases compared to the rabbit muscle protein phosphatase. Membrane proteins from A-431 cells (phosphorylated at tyrosine) were more effective substrates than phospho-Tyr-histones as substrates for alkaline phosphatase; this was not the case for protein phosphatase. Phospho-Tyr phosphatase activity of the alkaline phosphatases was optimal in the pH range of 7-8. The so-called alkaline phosphatases, then, may be a group of membrane-bound glycoproteins that represent a class of phosphoprotein phosphatases that show selectivity for proteins phosphorylated at tyrosine residues.

Redox modulation of a phosphatase from Anacystis nidulans

Ascorbic acid (AA) increased the phosphatase activity (pH 6.8) in 10,000 g supernatants from Anacystis nidulans. The enzyme activated by AA was deactivated by dehydroascorbic acid (DHAA). The modulation by AA/DHAA of phosphatase activity in Anacystis appears to be specific; a number of other redox compounds, known to modulate other enzymes, had no effect on the Anacystis phosphatase. A purified phosphatase preparation from Anacystis was also deactivated by DHAA. In contrast, the purified enzyme was not activated by AA, suggesting that a factor mediating the effect of AA was lost during purification. Another factor was found to protect the purified phosphatase against deactivation by DHAA. The enzyme was characterized as a phosphatase with a broad substrate specificity, an apparent molecular weight of 19,000, and a pH optimum of 6.0-7.0. Dialysis of the enzyme preparation against EDTA abolished the phosphatase activity which could be restored by Zn(2+) ions and partially restored by Co(2+) ions. Crude extracts also contained a latent enzyme, the phosphatase activity of which could be detected in the presence of Co(2+) ions only. Zn(2+) ions did not activate this enzymatically inactive protein. The Co(2+)-dependent phosphatase had an apparent mol. wt. of 40,000, a broad substrate specificity, and an alkaline pH-optimum. Infection of Anacystis cultures by cyanophage AS-1 resulted in a decrease in phosphatase activity. The enzyme present in 10,000 g supernatants from infected cells could not be modulated by the AA/DHAA system.

Phosphoprotein phosphatase activity of bovine intestinal alkaline phosphatase.

The phosphoprotein phosphatase activity of a commercial preparation of bovine intestinal alkaline phosphatase (EC 3.1.3.1) was examined using phosvitin and dentine phosphoprotein as substrates. Over 90% and 70% of the phosphorus from dentine phosphoprotein and phosvitin were hydrolyzed in 2 h. The optimum pH of the enzyme for the dephosphorylation of phosvitin and dentine phosphoprotein was nearly 6. No protein phosphatase activity was observed when the alkaline phosphatases from bovine liver and pulp were investigated.

Dephosphorylation of pig heart pyruvate dehydrogenase phosphate complexes by pig heart pyruvate dehydrogenase phosphate phosphatase.

1. Pig heart pyruvate dehydrogenase phosphate complex in which all three sites of phosphorylation were completely phosphorylated was re-activated at a slower rate by phosphatase than complex predominantly phosphorylated in site 1. The ratio of initial rates of re-activation was approx. 1:5 with a comparatively crude preparation of phosphatase and with phosphatase purified by gel filtration and ion-exchange chromatography. 2. The ratio of apparent first-order rate constants during dephosphorylation of fully phosphorylated complex averaged 1/3.8/1.3 for site 1/site 2/site 3. Only site-1 dephosphorylation was linearly correlated with re-activation of the complex throughout dephosphorylation. Dephosphorylation of site 3 was linearly correlated with re-activation after an initial burst of dephosphorylation. 3. Because dephosphorylation of site 1 was always associated with dephosphorylation of site 2, it is concluded that dephosphorylation cannot be purely random. 4. The ratio of apparent first-order rate constants for dephosphorylation of site 1 (partially/fully phosphorylated complexes) averaged 1.72. This ratio is smaller than the ratio of approx. 5 for the initial rates of re-activation. Possible mechanisms involved in the diminished rate of re-activation of fully phosphorylated complex are discussed.

Interaction of calmodulin with histones. Alteration of histone dephosphorylation.

The Ca2+-dependent regulator protein (CDR), also frequently termed "calmodulin" was determined to influence the dephosphorylation of mixed calf thymus histones or purified histones 1, 2A, or 2B by a partially purified bovine brain phosphoprotein phosphatase. CDR increase the rate of dephosphorylation of mixed histones more than 20-fold. With increasing concentrations of mixed histones as substrate, a proportionate increase of CDR concentration was required to maintain maximal expression of histone phosphatase activity. Mixed histones suppressed the activation by CDR of a bovine brain cyclic nucleotide phosphodiesterase activity, with activation being restored by increased quantities of CDR. Dephosphorylation of casein and phosphorylase alpha by the phosphatase preparation was not affected by CDR. These observations support the interpretation that the effects of CDR on histone dephosphorylation are substrate-directed. The rates of dephosphorylation of histones 1, 2A, and 2B by the phosphatase were 4- to 12-fold more rapid at low (sub-micromolar) concentrations of free Ca2+ than at high (200 microM) Ca2+ in incubations containing CDR, but they were unaffected by Ca2+ in incubations without CDR. The addition of stoichiometric quantities of calmodulin increased the apparent Km of the phosphatase for the various histones 2- to 6-fold, while maximal velocities were 4- to 12-fold higher at low than at high added Ca2+. The inhibitory effect of Ca2+ on histone dephosphorylation was immediately reversible by chelation of Ca2+ with EDTA. Ca2+-dependent inhibition of histone 1 or 2B phosphatase activities was also produced by rabbit skeletal muscle troponin C, but not by rabbit skeletal muscle parvalbumin, by poly(L-aspartate) or poly(L-glutamate). The phosphorylated fragment from the NH2-terminal region of either H2A (generated by treatment with N-bromosuccinimide) or H2B (generated by treatment with cyanogen bromide) was dephosphorylated by the phosphatase, with the rates of dephosphorylation being reduced 3- to 6-fold by Ca2+ in incubations containing CDR.

Phosphopeptide substrates of a phosphoprotein phosphatase from rat liver.

The substrate specificity of a preparation of phosphoprotein phosphatase (Mr = 32 000) from rat liver was investigated. Phosphopeptides based on the structure Leu-Arg-Arg-Ala-Ser(P)-Val-Ala-Glx-Leu and Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-Val-Tyr-Glu-Pro-Leu-Lys were used. These phosphopeptides correspond to the phosphorylation sites of rat liver pyruvate kinase (type L) and the beta subunit of rabbit muscle phosphorylase b kinase, respectively. A decrease in the apparent Km values and a concomitant increase in Vmax values was observed when the number of amino acyl residues after the phosphoseryl residue in the respective phosphopeptides were increased from 2 to 4, 5, or 6. Most of the phosphopeptides investigated generally showed apparent Km values higher than the values obtained with phosphopyruvate kinase. Ala-Ser(P)-Val-Ala and Gly-Ser(P)-Val-Tyr appeared to be the shortest phosphopeptides that could be dephosphorylated rapidly. These findings support the hypothesis that a small part of the phosphoprotein may be sufficient to fulfill the minimal requirements for its dephosphorylation.

Short term reversible modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in isolated epithelial cells from rat ileum. Regulation of phosphorylation dephosphorylation by bicarbonate.

In ileal epithelial cells isolated through incubation of gut loops in phosphate-buffered saline containing EDTA, 3-hydroxy-3-methylglutaryl coenzyme A reductase was found to exist prdominantly in an active (dephosphorylated) form. However, in cells obtained by scraping the intestine, the enzyme was mostly inactive (phosphorylated) and could be activated several-fold on incubation with a crude phosphatase preparation. Reductase activity in microsomes from ileal epithelial cells could be inactivated extensively by incubation with Mg . ATP in the absence of any exogenous factors. When cells isolated in phosphate-buffered saline were incubated in bicarbonate-containing buffers, there was a time-dependent decrease in the apparent activity of 3-hydroxy-3-methylglutaryl CoA reductase with little change in the total activity of the enzyme. On transferring the cells to a buffer without bicarbonate, the decrease in the apparent activity of the enzyme was fully reversed. There was no change in both the apparent and the total activities of reductase when cells were incubated in buffers lacking bicarbonate. Sterol synthetic rates in isolated intestinal cells were reflective of the fraction of reductase in the active form. These results strongly suggest the operation of a phosphorylation-dephosphorylation mechanism for short term modulation of 3-hydroxy-3-methylglutaryl CoA reductase activity in the intact mucosal cell. The bicarbonate anion appears to function as an activator of this modulation system.

Recovery of sensitivity of J-41 cells to coxsackie B3 virus by treatment with exogenous alkaline phosphatase

It has been shown that injection of G-41 cell cultures, deficient as regards alkaline phosphatase and resistant to Coxsackie B3 virus, in conjunction with exposure to an alkaline phosphatase preparation from the calf intestine results in virus reproduction. Depending on the dose administered and multiplicity of infection there occur either complete destruction of the monolayer or death of some cells with the development of cytopathic changes specific for Coxackie virus.

Isolation and characterization of a tartrate-sensitive splenic acid phosphatase in Gaucher's disease

The acid phosphatase activity that is increased in the spleens of patients with Gaucher's disease can be separated into two principal isoenzymes by chromatography on sulphopropyl-Sephadex. The acid phosphatase species that is resistant to inhibition by l-(+)-tartrate is retained by the cation-exchange resin while the tartrate-sensitive species passes through. We have isolated and characterized the tartrate-sensitive acid phosphatase (designated SP I) from the spleen of a patient with the adult (type 1) form of Gaucher's disease. SP I acid phosphatase, representing approximately 30 to 50% of the total acid phosphatase activity in a detergent (Triton X-100) extract of spleen tissue, has been purified approximately 400-fold to a specific activity of 48 units/mg of protein (substrate, 4-methylumbelliferyl phosphate). The final preparation of acid phosphatase contains at least two protein components—each with phosphatase activity—when analyzed by polyacrylamide gel electrophoresis at pH 8.9 or isoelectric focusing. SP I acid phosphatase exhibits a broad substrate specificity and catalyzes the hydrolysis of a variety of artificial and natural phosphate-containing compounds including p-nitrophenyl phosphate, α-naphthyl phosphate, phosphoenolpyruvate, and CMP. The enzyme is inhibited by l-(+)-tartrate, sodium fluoride, and ammonium molybdate and has the following properties: pH optimum, 4.5; K m on 4-methylumbelliferyl phosphate, 44 μ m; p I, 3.8–4.1; M r, 177,400; s 20,w, 6.8.

Regulation of vertebrate liver HMG-CoA reductase via reversible modulation of its catalytic activity

We have investigated the comparative biochemistry of in vitro regulation of HMG-CoA reductase (EC 1.1.1.34) in microsomal preparations from the livers of nine vertebrates. In all instances, reductase activity was rapidly and profoundly decreased by addition of MgATP. Reductase activities were restored to near or above initial levels after removal of MgATP and incubation with a crude, low molecular weight phosphatase preparation from rat liver cytosol. Restoration of reductase activity was inhibited both by NaF and by pyrophosphate, known inhibitors of phosphoprotein phosphatase activity. Liver cytosol of species other than the rat exhibits reductase phosphatase activity. The converter enzymes that catalyze modulation of MG-CoA reductase activity (reductase kinase and reductase phosphatase) thus appear to be ubiquitous in vertebrate liver. Interconversion in vitro of active and inactive forms of reductase probably is general for vertebrate liver also. The majority of the reductase present in vertebrate liver may be present in a catalytically inactive or latent form in vivo. Under the experimental conditions used, the fraction present in the active form is, for a given species, quite constant. Species to species, from 20-45% of the reductase appears to be present in the active form.

Physiological function of periplasmic hexose phosphatase in Salmonella typhimurium

Hydrolysis of sugar phosphates by crude and purified preparations of periplasmic hexose phosphatase from Salmonella typhimurium followed Michaelis-Menten kinetics. The enzyme bound glucose 1-phosphate with high affinity (Km = 10 microM) but bound glucose 6-phosphate with low affinity (Km = 2,000 microM). The order of substrate affinities was glucose 1-phosphate greater than mannose 1-phosphate = galactose 1-phosphate greater than fructose 1-phosphate greater than glucose 6-phosphate. These results and others suggest that the physiological function of the enzyme is the periplasmic hydrolysis of hexose 1-phosphates.

A Comparison between Heat-Stable Phosphatase Inhibitors and Activators from Rabbit Skeletal Muscle and Liver and Their Effects upon Different Preparations of Phosphoprotein Phosphatase

The separation and properties of heat-stable phosphatase effectors from muscle and liver were studied using phosphoprotein phosphatases with molecular weights of 250000, 65000 and 30000. Muscle inhibitors that were only active when phosphorylated were eluted from DEAE-cellulose by 20 mM NaCl (inhibitor 1) and 65 mM NaCl (inhibitor 1 ′). Available evidence suggests that these are different forms of the same protein and that inhibitor 1′ more closely resembles the native inhibitor. Liver extracts contained only the form eluted by 20 mM NaCl. These inhibitors did not affect the activity of the smallest enzyme. Both tissues also contained a non-phosphorylatable inhibitor of phosphorylase phosphatase activity (inhibitor 2) and an activator of phosphohistone phosphatase activity that affected all three enzymes. There was no evidence for the presence of effectors that were specific for only one of the phosphatases employed. The ratio of phosphorylatable to non-phosphorylatable inhibitor was much lower in liver than in muscle. Corresponding effectors from muscle and liver had similar physical properties and similar effects upon the activities of the three enzymes. Phosphorylase phosphatase activity was not affected by the activator. The 250000-Mr enzyme was relatively less sensitive than the 65000-Mr enzyme to inhibitor 2, and more sensitive to inhibitor 1′. With phosphohistone as substrate, only the largest enzyme was inhibited by inhibitor 1 and no enzyme was inhibited by inhibitor 1′ or inhibitor 2. There was no apparent competition between effectors. The presence of one effector did not modify the action of any of the others.

A highly purified rat-liver phosphoprotein phosphatase preparation with activity towards phosphopyruvate kinase (Type L)

A highly purified rat-liver phosphoprotein phosphatase preparation with activity towards phosphopyruvate kinase (Type L)

Anion-stimulated phosphohydrolase activity of intestinal alkaline phosphatase.

Phosphohydrolase activity of a highly enriched commercial preparation of calf intestinal alkaline phosphatase was stimulated in the presence of HCO3. SO4, Cl, SCN, and acetate did not stimulate hydrolysis, whereas SO3 exhibited a bimodal effect, stimulating at low (25mM) concentration but inhibiting at high (100 mM) concentration. The pH optimum of this stimulation by HCO3 or SO3 was 8.5--9.0 and was maximal at a Mg concentration of 0.5 mM. HCO3 increased the Vmax of the reaction without changing the Km for ATP. ATP, GTP, UTP, and xanthosine triphosphate were equally effective as substrates, whereas AMP and p-nitrophenyl phosphate were much less effective. Alkaline phosphatase activity was inhibited by L-cysteine and L-phenylalanine, compounds that also inhibited the HCO3-ATPase activity of the preparation. Passage of the commercial preparation through an anion-exchange column yielded a fraction with enriched alkaline phosphatase and HCO3-ATPase activities; this fraction proved to be a single protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, on isoelectric focusing, and by immunologic techniques. These studies strongly suggest that alkaline phosphatase and anion-stimulated phosphohydrolase activities are properties of the same protein in small intestine. It is possible that alkaline phosphatase may function as a HCO3-ATPase involved in intestinal absorption and secretion.

Differential precipitation with concanavalin a as a method for the purification of glycoproteins: Human alkaline phosphatase

The purification of a glycoprotein from a mixture by first precipitating with a lectin and fractional solution of the precipitate could be applicable as an important preparative procedure. Another possibility could be fractional precipitation with the lectin by varying the concentration of Ca 2+ and Mn 2+ required for binding. These principles have been tested in relation to the preparation of human liver alkaline phosphatase. Both approaches have led to the purification of alkaline phosphatase from single human livers with a high recovery (28 and 34%) and purification factors of 16 × 10 3 and 13 × 10 3 over the initial aqueous butanl-ol extract. The specific activities for the preparations were 1610 and 1500 units/mg of protein, respectively; a unit is defined as the amount of enzyme catalyzing the hydrolysis of 1.0 μmol of p-nitrophenyl phosphate/min at 35°C in 0.1 m 2-amino-2-methylpropan-1-ol/HCl buffer, pH 10.5, containing 10 m m p-nitrophenyl phosphate, 0.1 m m MgCl 2, and 1 μ m ZnSO 4.

Complement fixation for study of placental-type alkaline phosphatase

Preparations of human placental alkaline phosphatase differing in specific enzyme activities were compared by microcomplement fixation assays using monospecific antisera. While both specific enzyme activity and complement fixation units increased 15,000-fold upon purification, the ratio between these units remained constant. Separation of an alkaline phosphatase preparation into ‘A’ and ‘B’ forms by ampholine isoelectric focusing indicated that these forms also possessed the same ratio of immunoreactive enzyme protein to enzyme activity. The correspondence of complement fixation units with specific enzyme activity indicates that complement fixation with monospecific antisera can be used to analyze structural differences among alkaline phosphatase isoenzymes.