Histone Phosphatase Activity Research Articles

Tissue and subcellular distributions, and characterization of rat brain protein phosphatase 2A containing a 72-kDa delta/B" subunit.

A 74-kDa delta/B" subunit was isolated by heparin-Sepharose column chromatography from human erythrocyte protein phosphatase 2A (PP2A) consisting of a 34-kDa catalytic subunit (alpha/C) and 63- and 74-kDa regulatory subunits (beta/A and delta/B") in a ratio of 1:1:1. The purified delta/B" was used as an immunogen in mice, to prepare specific antisera against delta/B". Immunoblot analyses with the antisera detected an immunoreactive 72-kDa protein in the cytosol from various rat tissues including erythrocytes, brain, lung, testis, adrenal gland, heart, spleen, kidney, and liver. The 72-kDa protein was highly abundant in brain and was distributed evenly in cerebral cortex, cerebellum, and brain stem. The 72-kDa protein was also detected in mitochondria and microsome fractions. An immunoreactive 68-kDa protein was detected mainly in nuclear and microsome fractions. The 72-kDa protein from rat brain cytosol copurified with phosphorylated H2B histone phosphatase activity during successive chromatographies on DEAE-Toyopearl, AH-Sepharose, Sephadex G-150, H1 histone-Toyopearl, TSK DEAE-5PW, protamine-Toyopearl, and TSK G3000SW columns. The purified enzyme migrated as a single protein band on nondenaturing PAGE and as three protein bands of 34, 63, and 72 kDa in a ratio of 1:1:1 on SDS-PAGE. The molecular weight of the enzyme was estimated to be 170,000 from the s20,W value of 7.2 +/- 0.3 S and the Stokes radius of 5.5 +/- 0.1 nm. The rat brain enzyme was classified as PP2A, based on the following properties; (1) an IC50 for okadaic acid of 10(-9) M; (2) its preferential dephosphorylation of the a subunit of phosphorylase kinase; (3) its insensitivity to protein inhibitor 2; and (4) its heterotrimeric subunit structure. The Km value and the molecular activity of the enzyme for phosphorylated H2B histone were 72.3 +/- 0.3 microM and 192 +/- 2 mol Pi released/min/mol enzyme, respectively, and were comparable to those of human erythrocyte PP2A (alpha1 beta1 delta1/ CAB"). The 72-kDa subunit in the purified rat brain PP2A was phosphorylated in vitro by cAMP-dependent protein kinase.

Purification and Characterization of Protein Phosphatase 2C in Rat Parotid Acinar Cells: Two Forms of Mg2+-Activated Histone Phosphatase and Phosphorylation by cAMP-Dependent Protein Kinase

Two forms of Mg2+-activated histone phosphatase activities were partially purified from rat parotid acinar cells using Mono Q and gel filtration chromatography. Both enzymes activities were dependent on the presence of Mg2+, showing little activity in the presence of EDTA. The activities fractionated on the Mono Q column into two peaks: the first was a minor peak of histone phosphatase activity; the second was a major peak. These two peaks eluted at distinct positions on the gel filtration column. The molecular masses of the two peak fractions corresponded to 46 and 55 kDa, respectively on SDS–gels. The first 46-kDa peak immunoreacted with anti-PP2Cα phosphatase antibody and like PP2Cα phosphatase could be phosphorylated by cAMP-dependent protein kinase. The second 55-kDa peak showed neither reactivity with anti-PP2Cα phosphatase antibody nor phosphorylability by cAMP-dependent protein kinase, but retained a Mg2+or Mn2+dependence for its histone phosphatase activity. Ca2+showed a strong inhibition on this activity. On the basis of these observations, we have identified the first peak enzyme as PP2Cα phosphatase and the second peak as a novel PP2C-like phosphatase.

Three distinct forms of type 2A protein phosphatase in human erythrocyte cytosol.

Two type 2A protein phosphatases, phosphatases I (Mr = 180,000) and III (Mr = 177,000), were purified to near homogeneity from human erythrocyte cytosol. Phosphatase I was composed of alpha (34 kDa), beta (63 kDa), and delta (74 kDa) subunits in a ratio of 1:1:1. Phosphatase III comprised alpha, beta, and gamma (53 kDa) subunits in the same ratio. Heparin-Sepharose column chromatography converted most of phosphatase I and 20% of phosphatase III into alpha 1 beta 1 which were indistinguishable from phosphatase IV (Usui, H., Kinohara, N., Yoshikawa, K., Imazu, M., Imaoka, T., and Takeda, M. (1983) J. Biol. Chem. 258, 10455-10463). The catalytic subunit alpha and the beta subunit of phosphatases I, III, and IV displayed identical V8 and papain peptide maps, respectively, while the peptide maps of the alpha, beta, gamma, and delta subunits were clearly distinct. The molar ratio of phosphatases I, III, and IV in erythrocyte cytosol was estimated to be 6:1:14. Comparison of molecular activities of alpha, alpha 1 beta 1, alpha 1 beta 1 delta 1, and alpha 1 beta 1 gamma 1 revealed that beta suppressed phosphorylase and P-H2B histone phosphatase activities of alpha but stimulated the P-H1 histone phosphatase activity, and delta suppressed all the phosphatase activities of alpha 1 beta 1. The gamma subunit stimulated the P-histone phosphatase activity of alpha 1 beta 1 but inhibited the phosphorylase and P-spectrin phosphatase activities. The beta subunit increased the Mg2+ or Mn2+ requirement for P-H2B histone phosphatase activity of alpha, an effect which was counteracted by delta. The effects of heparin, H1 histone, protamine, and polylysine on the phosphorylase phosphatase activity of phosphatases I, III, IV, and alpha were described and discussed in connection with the functions of the subunits.

Chromatographic resolution of soluble and particulate protein phosphatases from Dictyostelium discoideum

We have examined protein phosphatase activities that are present during the cellular differentiation of Dictyostelium. Utilizing differential centrifugation, ion exchange, gel filtration, and concanavalin A affinity chromatography we found a number of distinct protein phosphatase activities. Three peaks of soluble Kemptide phosphatase activity and a very broad and heterogeneous soluble histone phosphatase activity were resolved by anion exchange chromatography. Histone phosphatase was associated with the particulate fraction, while Kemptide phosphatase was not. The protein phosphatase activities were able to dephosphorylate sites that had been phosphorylated by the cyclic AMP-dependent protein kinase. Therefore it is possible that their function in vivo may be to oppose the action of the cAMP-dependent protein kinase. In addition several paranitrophenyl phosphate phosphatase activities are shown to be largely separable from the protein phosphatases. An apparent heat-stable inhibitor of histone phosphatase is shown to be artifactual in that instead of interacting with the enzyme it acts by complexing With histone.

Protein phosphatases in developing Dictyostelium discoideum cells

Protein phosphatase activities in developing Dictyostelium discoideum cells were investigated. Substrates were prepared by phosphorylation of histone H2b and kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) using cAMP-dependent protein kinase. Two histone phosphatase activities ( M r 170 000 and 520 000) and one kemptide phosphatase activity ( M r 230 000) were found in the cytosolic cell fraction. Histone phosphatase was also present in the particulate fraction, kemptide phosphatase was not. All phosphatase activities were present throughout development. No differences in protein phosphatase activities were found in prespore and prestalk cells. A heat-stable factor which inhibits the particulate and both soluble histone phosphatase activities was isolated.

Demonstration of separate phosphotyrosyl- and phosphoseryl-histone phosphatase activities in the plasma membranes of a human astrocytoma

A plasma membrane preparation from a human astrocytoma contained p-nitrophenyl phosphate (pNPP), phosphotyrosyl histone, and phosphoseryl histone hydrolysis activities. The pNPPase and phosphotyrosyl histone phosphatase activities were inhibited by vanadate, whereas the phosphoseryl histone phosphatase activity was not; the latter activity was inhibited by pyrophosphate and nucleoside di- and triphosphates. When the membranes were solubilized by Triton X-100 and the solubilized proteins were subjected to column chromatography on DEAE-Sephadex, Sepharose 6B-C1, and wheat germ agglutinin-Sepharose 4B columns, the pNPPase activity coeluted with the phosphotyrosyl histone phosphatase activity, and was separable from the phosphoseryl histone phosphatase activity. The results from column chromatography also indicated that there may be multiple phosphotyrosyl and phosphoseryl protein phosphatases in the plasma membranes.

Phosphotyrosine and phosphoprotein phosphatase activity of alkaline phosphatase in mineralizing cartilage

We used embryonic skeletal cartilage known to have high levels of alkaline phosphatase activity to determine whether growing cartilage has phosphotyrosine phosphatase activity and phosphotyrosinyl histone phosphatase activity at physiologc pH. Embryonic chick pelvic cartilage and fetal pig scapular growth-plate cartilage were assayed using phosphotyrosine as substrate at pH 7.5 and the amount of tyrosine generated measured. Both cartilage models had K m for phosphotyrosine between 6 to 24 μmol/L. Phosphotyrosine phosphatase activity correlated with alkaline phosphatase activity as assessed by (1) distribution of histologic staining for alkaline phosphatase within the cartilages, (2) hormonal stimulation of cartilage alkaline phosphatase activity in vitro, (3) comparison of alkaline phosphatase and phosphotyrosine phosphatase activities in the presence of known inhibitors (vanadate, levamisole, homoarginine, and zinc), and (4) assaying chick epiphyseal cartilage alkaline phosphatase purified to homogeneity for phosphotyrosine phosphatase activity. Areas of cartilage with elevated alkaline phosphatase activity also had raised phosphotyrosine phosphatase activity. Triiodothyronine, a known stimulator of cartilage alkaline phosphatase, increased chick cartilage alkaline phosphatase activity 88% and phosphotyrosine phosphatase activity 106%, and stimulated porcine growth-plate cartilage alkaline phosphatase activity 91% and phosphotyrosine phosphatase activity 145% after 3 days of in vitro incubation. Each of the inhibitors block alkaline phosphatase and phosphotyrosine phosphatase activities. The purified alkaline phosphatase had a K m for phosphotyrosine of 18 μmol/L and V max of 5700 nmol tyrosine/mg protein/h, which is well over 1000-fold higher than the phosphotyrosine phosphatase activity found in the above preparations of pelvic and scapular cartilage. Furthermore, cartilage alkaline phosphatase dephosphorylated phosphotyrosinated histone and had little to no activity when histones phosphorylated at serine and threonine were used as substrate. Our studies support the hypothesis that alkaline phosphatase functions as a neutral phosphoprotein phosphatase, ie, a phosphotyrosine phosphatase, and is involved in the regulation of protein phosphorylation-dephosphorylation reactions.

The separation and properties of two phosphoprotein phosphatases from the dimorphic fungus Mucor rouxii

Soluble preparations from mycelium of the dimorphic fungus Mucor rouxii contained detectable amounts of phosphoprotein phosphatase activity. This cytosolic phosphatase activity exhibited a molecular weight below 80,000 and could be resolved into two different forms (enzymes I and II) by chromatography on DEAE-cellulose followed by gel filtration on Sephacryl S-300. Enzyme I ( M r 64,000) was mainly a histone phosphatase activity, absolutely dependent on divalent cations, with a K 0.5 for MnCl 2 of 2 m m. Enzyme II ( M r 40,000) was active with histone and phosphorylase. Its activity was independent or slightly inhibited by Mn 2+. This enzyme was strongly inhibited by 50 m m NaF or 1 m m ATP. When partially purified enzymes I and II were separately treated with ethanol, the catalytic properties of enzyme II were apparently not affected while those of enzyme I were drastically changed. The activity with histone, which was originally dependent on Mn 2+, became independent or slightly inhibited by the cation. The treatment was accompanied by a notable increase in phosphorylase phosphatase activity which was strongly inhibited by Mn 2+. Treated enzyme I eluted from DEAE-cellulose and Sephacryl S-300 columns at a position similar to that of enzyme II.

Chromatographic characteristics and subcellular localization of synthase phosphatase, phosphorylase phosphatase and histone phosphatase in human polymorphonuclear leukocytes.

Synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity in a leukocyte homogenate were found to have different sedimentation characteristics: both synthase phosphatase and phosphorylase phosphatase activity are associated with the microsomal fraction, while the majority of histone phosphatase activity (75-85%) was found in the cytosol. Synthase phosphatase, phosphorylase phosphatase and histone phosphatase activities accompanying the microsomal fraction are readily solubilized by 0.3% Triton X-100. When the solubilized microsomal enzymes were chromatographed on Sephadex G-200, the majority of synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity migrated in single peaks corresponding to apparent molecular weights of 380 000, 250 000 and 68 000, respectively. A minor peak of 30 000, which had phosphatase activity against all three substrates was also obtained. Ethanol treatment resulted in solubilization and dissociation of the three phosphatase activities. It was found that although ethanol treatment resulted in a 4-fold increase of phosphorylase phosphatase activity, histone phosphatase activity was decreased (by 60%), while synthase phosphatase activity remained stable. Similar results were obtained when ethanol treatment was performed on the 17 000 X g supernatant. Chromatography of the ethanol-treated microsomes (or homogenate) on Sephadex G-200 showed that the phosphatase activity towards synthase D, phosphorylase a and phosphohistone coincided a Mr 30 000 species. Heat treatment of the Mr 30 000 peak resulted in dissociation of synthase phosphatase and phosphorylase phosphatase activity. Synthase phosphatase was inhibited by phosphorylase a in a kinetically non-competitive manner while histone phosphatase activity was not inhibited by synthase D (8.5 unit/ml) or by phosphorylase a (12 unit/ml).

Resolution and reassociation of three distinct components from pig heart phosphoprotein phosphatase.

A partially purified pig heart phosphoprotein phosphatase was dissociated into three distinct components, namely alpha, beta, and gamma, by gel filtration on Sephacryl S-200 followed by chromatography on DEAE-Sephadex in the presence of 6 M urea. Although alpha itself had phosphatase activities toward P-H2B histone, P-H1 histone, phosphorylase a, and glycogen synthase b, beta and gamma had no activity toward these substrates even in the presence of 1 mM Mn2+. The beta component (Mr = 80,000) combined with alpha (Mr = 31,000) in the absence of urea to produce Form 2 (Mr = 123,000) with concomitant increase in P-H1 histone phosphatase activity and Mg2+ requirement for P-H2B histone phosphatase activity (Imazu, M., Imaoka, T., Usui, H., Kinohara, N., and Takeda, M. (1981) J. Biochem. 90, 851-862). The gamma component (Mr = 62,000) reassociated with Form 2 to produce Form 1 (Mr = 199,000) which was similar to the original phosphoprotein phosphatase in substrate specificity and Mg2+ requirement. Binding of gamma to Form 2 strongly suppressed the phosphatase activities toward phosphorylase a and glycogen synthase b with marginal effects on the other phosphatase activities and Mg2+ requirement. However, gamma alone could not associate with alpha. The gamma component was sensitive to treatment with heat (60 degrees C for 2 min) or trypsin and was resistant to treatment with DNase or RNase. The pig heart phosphoprotein phosphatase was further purified to near homogeneity, as judged by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis revealed that the purified enzyme (Mr = 171,000) was composed of three polypeptide components, namely alpha', beta', and gamma' with molecular weights of 34,000, 69,000, and 56,000, respectively. The component stoichiometry was determined to be alpha' 1 beta' 1 gamma' 1 by densitometric tracing of the Coomassie blue-stained bands on the acrylamide gel. After dissociation of alpha ' and other components by gel filtration of the purified enzyme on Sephacryl S-200 in the presence of 6 M urea, one alpha ' combined with one beta' to produce Form 2' of Mr = 106,000. Since Form 1 and the purified enzyme as well as Form 2 and Form 2' had similar catalytic properties and s20,w values, respectively, component compositions are suggested to be alpha 1 beta 1 gamma 1 for Form 1 and alpha 1 beta 1 for Form Form 2.

Histone variants and histone modifications in chromatin fractions from heterochromatin-rich Peromyscus cells

In order to investigate the relationship between condensed heterochromatin and histone modification by acetylation, phosphorylation and amino acid variation, chromatin from cultured Peromyscus eremicus cells, containing 35% constitutive heterochromatin, was fractionated into heterochromatin-enriched and heterochromatin-depleted fractions. The constitutive heterochromatin content of these fractions was determined from satellite DNA content. The distribution of phosphorylated and acetylated histones and amino acid variants of histone H2A in these chromatin fractions was examined by gel electrophoresis. Fractionation of histones demonstrated that endogenous histone phosphatase activity was high in chromatin fractions and could not be inhibited sufficiently to allow accurate histone phosphorylation measurements. However, sodium butyrate did inhibit deacetylation activity in the fractions, allowing histone acetylation measurements to be made. It was found that the constitutive heterochromatin content of these fractions was proportional to both their unacetylated H4 content and their more-hydrophobic H2A content. These observations support, by direct measurement, earlier experiments (Exp cell res 111 (1978) 373; 125 (1980) 377; 132 (1981) 201) suggesting that constitutive heterochromatin is enriched in unacetylated arginine-rich histones, and in the more hydrophobic variant of histone H2A.

Subcellular localisation and properties of histone phosphate phosphatase in human polymorphonuclear leukocytes: alterations in pregnancy and chronic granulocytic leukaemia and relationship to alkaline phosphatase

Using [ 32P]histone as substrate, an assay for histone phosphate phosphatase was optimised for human polymorphonuclear leukocytes. Kinetic studies showed that the activity was optimal at pH 6.8, was stimulated by Mn 2+ and Co 2+, and inhibited by sodium sulphite and zinc chloride. The apparent K m of the enzyme for histone phosphate was 0.89 μmol/l. Neutrophils were homogenized in isotonic sucrose and, after low speed centrifugation, the supernatant was subjected to analytical subcellular fractionation. Gradient fractions were assayed for principal marker enzymes and for histone phosphate phosphatase. Histone phosphate phosphatase activity was shown to be solely located to the cytosol. No activity was detected in the alkaline phosphatase-containing granules. Neutrophils were isolated from the blood of control subjects, patients with chronic granulocytic leukaemia and women in the third trimester of pregnancy. The specific activity (milliunits/mg protein) of histone phosphate phosphatase was significantly reduced in patients with chronic granulocytic leukaemia compared to control values but this decrease was considerably less than that found for alkaline phosphatase. The possible implication of the reduced histone phosphatase activity in leukaemia neutrophils is discussed. There was no significant change in histone phosphate phosphatase in leucocytes from pregnant women. These results, together with the subcellular fractionation experiments and inhibitor studies, strongly indicate that histone phosphate phosphatase is not attributable to neutrophil alkaline phosphatase.

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.

Purification and subunit structure of rat-liver phosphoprotein phosphatase, whose molecular weight is 260000 by gel filtration (phosphatase IB).

1. Phosphoprotein phosphatase IB is a form of rat liver phosphoprotein phosphatase, distinguished from the previously studied phosphoprotein phosphatase II [Tamura et al. (1980) Eur. J. Biochem. 104, 347-355] by earlier elution from DEAE-cellulose, by higher molecular weight on gel filtration (260000) and by lower activity toward phosphorylase alpha. This enzyme was purified to apparent homogeneity by chromatography on DEAE-cellulose, aminohexyl--Sepharose-4B, histone--Sepharose-4B, protamine--Sepharose-4B and Sephadex G-200. 2. The molecular weight of purified phosphatase IB was 260000 by gel filtration and 185000 from S20,W and Stokes' radius. Using histone phosphatase activity as the reference for comparison, the phosphorylase phosphatase activity of purified phosphatase IB was only one-fifth that of phosphatase II. 3. Sodium dodecyl sulfate gel electrophoresis revealed that phosphatase IB contains three types of subunit, namely alpha, beta and gamma, whose molecular weights are 35000, 69000 and 58000, respectively. The alpha subunit is identical to the alpha subunit of phosphatase II. While the beta subunit is also identical or similar to the beta subunit of phoshatase II, the gamma subunit appears to be unique to phosphatase IB. 4. When purified phosphatase IB was treated with 2-mercaptoethanol at -20 degrees C, the enzyme was dissociated to release the catalytically active alpha subunit. Along with this dissociation, there was a 7.4-fold increase in phosphorylase phosphatase activity; but histone phosphatase activity increased only 1.6-fold. The possible functions of the gamma subunit are discussed in relation to this activation of enzyme.

Isolation of an inactive component from pig heart phosphoprotein phosphatase and its reassociation with an active component

Treatment of a pig heart phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) of M r 224 000 with 40% ethanol followed by gelfiltration on Sephadex G-150, dissociated the enzyme into an active component of M m 31 000 and an inactive component of M r 80 000. The inactive component reassociated with the active component, resulting in the formation of an enzyme form of M r 123 000. A large excess of either component in the reassociation produced only this enzyme form. The ability of the inactive component to associate with the active component was lost by treatment of the inactive component with trypsin and heat (60°C, 2 min) but not with DNAase and RNAase. Effects of the inactive component on the activities of the active component by the association were as followings. The inactive component: (1) stimulated slightly the 32P-H2B histone phosphatase activity in the presence of either NaCl or Mg (CH 3COO) 2 but inhibited strongly in the absence of the salts; (2) stimulated the 32P-H1 histone phosphatase activity in the presence of the salts; (3) inhibited the phosphorylase α phosphatase activity in the presence and absence of the salts; (4) enhanced the response to the stimulatory effects of the salts on the dephosphorylation of 32P-histone; and (5) protected the phosphorylase α phosphatase activity from inhibition by the salts.

Enzymic properties and effects of androgen on nuclear histone phosphatase activity of rat ventral prostate.

Abstract Nuclei of rat ventral prostate have been demonstrated to possess a protein phosphatase activity utilizing 32 P-labelled, lysine-rich histone (calf thymus) as the phosphoprotein substrate. This phosphatase has a pH optimum of 7.1 and was stimulated by the sulfhydryl protective agents dithiothreitol and 2-mercaptoethanol. This nuclear protein phosphatase did not appear to require divalent cations; rather, small inhibitions of activity were found in the presence of 2.4 mM Mg 2+ , Mn 2+ , and Ca 2+ . Divalent cations such as Zn 2+ or Cu 2+ were found to be much stronger inhibitors, giving about 80% inhibition at 1 mM. Monovalent cations were also found to inhibit the histone phosphatase, e.g., 43% at 200 mM NaCl. Ammonium molybdate did not influence the enzyme activity whereas ADP and ATP reduced it by 72 and 82% respectively at 1 mM. There was no change in activity of the histone phosphatase up to 96 h post-orchiectomy when specific activity was based per unit of nuclear protein. However, a small decrease is noted if specific activity is expressed per unit of nuclear DNA (19% at 48 h and 36% at 96 h orchiectomy). This difference reflects the decreased nuclear protein content of the prostate observed following castration. Our data suggest that the decline in prostatic nuclear histone phosphorylation observed following orchiectomy is not due to increased phosphatase activity.

Evidence for the non-identity of proteins having synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity in rat liver

Synthase phosphatase, phosphorylase phosphatase and histone phosphatase in rat liver were measured using as substrate purified liver synthase D, phosphorylase a and 32P-labelled phosphorylated f1 histone, respectively. The three phosphatase enzymes had different sedimentation characteristics. Both synthase phosphatase and phosphorylase phosphatase were found to sediment with the microsomal fraction under our experimental conditions. Only 10% of histone phosphatase was in this fraction; the majority was in the cytosol. No change in histone phosphatase was observed in the adrenalectomized fasted rat whereas synthase phosphatase and phosphorylase phosphatase activities were decreased 5–10-fold. Fractionation of liver extract with ethanol produced a dissociation of the three phosphatase activities. When a partially purified fraction was put on a DEAE-cellulose column, synthase phosphatase and phosphorylase phosphatase both exhibited broad elution profiles but their activity peaks did not coincide. Histone phosphatase eluted as a single discrete peak. When the supernatant of CaCl 2-treated microsomal fraction was put on a Sepharose 4B column, the majority of synthase phosphatase was found to elute with the larger molecular weight proteins whereas the majority of phosphorylase phosphatase eluted with the smaller species. Histone phosphatase migrated as a single peak and was of intermediate size. Synthase phosphatase was inhibited by phosphorylase a ( K i < 1 unit/ml) and phosphorylase phosphatase by synthase D ( K 1 ≈ units/ml). The inhibition of synthase phosphatase by phosphorylase a was kinetically non-competitive with substrate. Histone phosphatase activity was not inhibited by synthase D or by phosphorylase a. The above results suggest that different proteins are involved in the dephosphorylation of synthase D, phosphorylase a and histone in the cell.

Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.

Phosphorylation of histone catalyzed by a bovine brain protein kinase.

The use of polyethyleneimine-cellulose thin layer sheets to follow the phosphorylation of histone and decomposition of ATP catalyzed by an adenosine 3':5'-monophosphate (cyclic AMP)-stimulated protein kinase, protein kinase I, has made possible a more detailed analysis of the time course of these reactions than has been achieved previously be observing only recovered phosphorylated protein. When [gamma-32P] ATP was employed, significant error was introduced by the presence of 32Pi at the solvent front on these sheets, and this limited the accuracy of the available information. However, the analysis of assays performed with [U-14C] ATP was straightforward and appeared to have an accuracy comparable to that of the present standard assay. This appears to be the first use of [U-14C] ATP to assay protein kinases. Our physical characterization of protein kinase I showed it to be a homogeneous protein species by polyacrylamide gel electrophoresis, sodium dodecyl sulfate gel electrophoresis and analytical ultracentrifugation. Kinetic studies with protein kinase I indicated the absence of histone phosphatase and cyclic AMP phosphodiesterase activity. Furthermore, the ATPase activity seen is believed to be intimately associated with the protein kinase action, particularly in view of the observed dependence of the rate of Pi production on the presence of cyclic AMP. The kinetic data for the phosphorylation of histone catalyzed by protein kinase I under full stimulation by cyclic AMP are consistent with a double displacement mechanism.

Relationship between chromosome condensation and metaphase lysine-rich histone phosphorylation.

Treatment of metaphase HTC cells with ZnCl2 inhibits histone phosphatase activity and leads to an increase in the hyperphosphorylated forms of the lysine-rich (F1) histone. Under normal conditions a massive phosphatase activity is triggered as the cells shift from M into G1 phase. In the presence of ZnCl2 this activity is abolished and thehyperphosphorylated form of F1 persists intact into G1. We have asked the simple question of whether the chromosome can still extend during the M-G1 transition even if the F1 histone is maintained in the hyperphosphorylated form. We observe an apparently normal extension os the chromosomal material under these conditions, though it is evident that high levels of ZnCl2 have rather substantial effects on other cell functions.