Nuclear phosphoprotein phosphatase from calf liver.

Calf liver nuclear phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) has been purified approx. 850-fold. The enzyme has a mol. wt. of 34 000 as determined by SDS-polyacrylamide gel electrophoresis. The purified enzyme has a pH optimum between 7.0 and 7.5 with phosphophosphorylase, phosphohistones f1 and f2b, and phosphoprotamine as substrates. The enzyme activity towards these substrates follows the order, phosphophosphorylase greater than phosphohistone f1 greater than phosphohistone f2b greater than phosphoprotamine. The Km values toward phosphophospharylase and phosphohistone f1 are 17 and 28 micron phosphate, respectively. Dephosphorylated histone f1 and orthophosphate are competitive inhibitors of the enzyme with respective Ki values of 11 micron and 4.1 mM. NaCl and divalent metal ions inhibit the enzyme but CaCl2 is slightly stimulatory. It appears that metal ion inhibition occurs at two sites, one on the enzyme and the other on the substrate. The enzyme is also inhibited by NaF and EDTA. Nucleotides bearing the pyrophosphate structure are potent inhibitors of the enzyme while mononucleotides are slightly inhibitory. DNA and other polyions also inhibit the enzyme. The enzyme appears to require free sulfhydryl groups for activity since it is inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate; the latter inhibition can be reversed by mercaptoethanol and dithiothreitol.     

Properties of phosphoprotein phosphatase from the rat liver

Prosphoproteid phosphatase, an enzyme highly specific to lysyl-tRNA-synthetase and proteins of the high-molecular-multienzymic complex of aminoacyl-tRNA-synthetases, was isolated from the rat liver. The data of electrophoresis in 4-30% PAAG with the presence of DS-Na have shown that phosphoproteid phosphatase is homogeneous and its molecular mass is 56 kDa. The isolated phosphoproteid phosphatase is activated by 2.5 mM Mg2+, Mn2+ and is inhibited by ions of univalent metals ions--200 mM Na+, 5 mM K+ as well as by 1 mM ATP, ADP, AMP.     

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.     

Purification of a phosphoprotein from chromatin of rat liver.

A simple and effective method to purify a phosphoprotein (B2) (Mr 68,000, pI 6.2-8) from phenol-soluble non-histone chromatin proteins of rat liver is described. The purification involved only two steps, CM-cellulose chromatography and preparative SDS/polyacrylamide (10%)-gel electrophoresis. The purified phosphoprotein B2 was shown to be homogeneous by SDS/polyacrylamide-gel electrophoresis. The yield was 2% of total non-histone chromatin proteins. The acidic to basic amino acid ratio of phosphoprotein B2 was less than 1, with high contents of glutamic acid, aspartic acid, arginine, lysine, glycine and alanine. The phosphate content of this protein is 0.3%.     

Monoclonal antibodies to a phosphoprotein from chromatin of rat liver.

Three monoclonal antibody subclasses (IgG1, IgG2a, and IgM) were raised to the phosphoprotein B2 (Mr 68000, pI6.5-8.2) which has been shown previously to be associated with the nucleosomes of rat liver nuclei. These antibodies do not show any significant cross reactivity with CM-cellulose 'unbound' non-histone chromosomal proteins, bovine serum albumin or histones. Further verification of the specificity of these antibodies to this phosphoprotein was carried out using both 'dot' blot and immunological transfer analysis ('Western blot'). The monoclonal antibodies (IgG1 and IgG2a) could also be used to semi-quantify the phosphoprotein B2 in rat liver nuclei. The high specificity and unlimited availability of this type of probe provides a means to study the role(s) of this phosphoprotein in the overall scheme of actively transcribed chromatin.     

Purification and properties of a phosphoprotein phosphatase from rat liver.

A phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) has been partially purified from rat liver homogenates by (NH4)2SO4 and ethanol precipitations followed by DEAE-cellulose and Sepharose 6B chromatography. The phosphoprotein phosphatase is capable of cleaving [32P]phosphate from radiolabelled phosphopyruvate kinase (type L) (EC 2.7.1.40), phosphohistones, and phosphoprotamine. However, it did not detectably dephosphorylate ATP, ADP, DL-phosphorylserine or beta-glycerophosphate. Dephosphorylation of [32P]phosphopyruvate kinase was stimulated by divalent cations and inhibited by ATP, ADP, Fru-1,6-P2, and orthophosphate. Divalene cations could reverse inhibition induced by ADP or ATP. At least one function of the phosphoprotein phosphatase may be to remove phosphate groups from the phosphorylated form of pyruvate kinase in the liver.     

Calmodulin-dependent protein kinases purified from rat brain and rabbit liver

A calmodulin-dependent protein kinase was purified from rat brain by the same protocol used previously for a rabbit liver calmodulin-dependent glycogen synthase kinase. The rat brain kinase readily phosphorylated rabbit skeletal muscle glycogen synthase at sites 1b and 2, the same sites phosphorylated by rabbit liver calmodulin-dependent kinase. The two kinases have other similarities: substrate specificity, potent inhibition by sodium fluoride, and nearly equal Ka's (10-20 nM) for calmodulin. Also, both enzymes have similar Stokes radii, 70 A (rabbit liver) and 75 A (rat brain), but quite different sedimentation coefficients, 10.6 S and 17.4 S, respectively. Consequently, the calculated molecular weights are also different: 560,000 for the brain enzyme and 300,000 for the liver enzyme. The major subunit of the rat brain kinase appears to be a single 51-kDa peptide, not a doublet pattern of 51- and 53-kDa subunits that is characteristic of the rabbit liver enzyme. Our findings are consistent with the hypothesis that the rat brain and rabbit liver enzymes belong to a class of closely related calmodulin-dependent protein kinases, possibly isozymes. This class of enzymes may be responsible for regulating several of the known calcium-dependent physiological functions.     

Nuclear protein kinases in rat liver: evidence for increased histone H1 phosphorylating activity during liver regeneration.

Comparison of protein kinase activity in normal and regenerating rat liver nuclei indicates that exogenous histone H1 is hyperphosphorylated in 22-h regenerating nuclei. The protein kinase involved is not sensitive to protein kinase A inhibitor, is inhibited by staurosporine and by an anti-PKC polyclonal antibody, utilizes only ATP, and also phosphorylates the C-terminal fragment of histone H1. These data suggest that protein kinase C is responsible for the observed effects, in agreement with the presence of this enzyme in normal and regenerating nuclei demonstrated by immunoblotting.     

Nuclear precursors for ceruloplasmin-coding messenger RNA from rat liver

The distribution of the sequences coding for ceruloplasmin (CP) in rat liver heterogeneous nuclear RNA (hnRNA) was studied using highly specific CP cDNA as a hybridization probe. The content of CP-coding sequences in poly(A)-containing and poly(A)-free subfractions of hnRNA was shown to be respectively 1 and 27 equivalents of CP mRNA molecule per one hepatocyte. The gel electrophoresis of hnRNA under strongly denaturing conditions with the subsequent transfer of RNA to diazobenzyloxymethyl paper and hybridization with [32P]-cDNA probe showed that CP mRNA sequences were of multiple molecular weight distribution. In particular, 9.0, 6.6, 2.4 and 1.6 megadalton fractions of non-polyadenylate hnRNA carried CP-coding sequences while the only hand that hybridized to CP cDNA was detected in polyadenylated hnRNA. This band was of a molecular weight 1.1-1.2 megadaltons corresponding to that of cytoplasmic CP mRNA. The hybridization of high molecular weight hnRNA with full-length CP cDNA followed by the determination of the size of cDNA fragments protected against SI nuclease demonstrated that coding sequences of CP pre-mRNA are interrupted by intervening sequences.     

The protein kinases of rat liver nuclei

Two compounds with properties of Factor F-430 were purified from $\text{Methanobacterium}\text{bryantii}$ by column chromatography. Analysis of these compounds by neutron activation and atomic absorption spectroscopy revealed the presence of nickel and the absence of other metals commonly associated with molecules of biological origin. For the two compounds, the masses are 3300 daltons per mol Ni and 1500 daltons per mol Ni. The absorbance at 430 nm of both compounds is between 2.7?2.1 × 10⁴ cm^?1 L (mol Ni)^?1. Factor F-430 appears to be a unique, nickel-containing compound of low molecular weight.     

Partial purification and properties of a chromatin-associated phosphoprotein kinase from rat liver nuclei.

A phosphoprotein kinase (EC 2.7.1.37) KIVb, from rat liver nuclei, was purified 75-fold by phosphocellulose chromatography and gel filtration on Sephadex G-200. The enzyme, which has an apparent molecular weight of 55 000, phosphorylates casein and chromatin-bound nonhistone proteins more readily than histones or ribosomal proteins. It exhibits an absolute requirement for divalent cation with optimum activity at 15--20 mM Mg2+. Maximal kinase activity is achieved at 100 mM NaCl. The pH vs. activity curve is biphasic with optima at pH 6.5 and pH 8.0. The Km value for casein is 280 mug/ml and the Km for ATP is 6-10(-6) M. Kinase KIVb phosphorylates numerous nonhistone nuclear proteins as shown by electrophoretic analysis. The addition of kinase KIVb to reaction mixtures containing nonhistone proteins results in the phosphorylation of a spectrum of polypeptides similar to those that are phosphorylated by endogenous nuclear kinases. Nonhistone proteins bound to chromatin appear to be better substrates for KIVb than nonhistones dissociated from chromatin. A comparison of nuclear phosphoproteins phosphorylated either in the intact animal or in vitro (by the addition of kinase KIVb) indicates some differences and some similarities in the patterns of phosphorylation.     

Identification of three protein kinases which phosphorylate threonyl-tRNA synthetase from rat liver

Threonyl-tRNA synthetase is phosphorylated in Chinese hamster ovary cells labeled with 32Pi [(1984) J. Biol. Chem. 259, 11160-11161]. Phosphorylation of the purified synthetase from rat liver has been examined with five different protein kinases. Three of the enzymes phosphorylate the synthetase, protease activated kinase I, the cAMP-dependent protein kinase, and the Ca2+, phospholipid-dependent protein kinase. Phosphorylation occurs exclusively on seryl residues. Two-dimensional phosphopeptide maps of tryptic digests of the phosphorylated synthetase are distinct with each protein kinase. These data suggest that multiple phosphorylation of the synthetase may occur in vivo.     

Properties of rat liver nuclear protein kinases.

Two cyclic nucleotide-independent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) have been purified to homogeneity from rat liver nuclei. While these enzymes have many similar catalytic properties (preference for acid rather than basic proteins), they differ in molecular weight and subunit composition. Protein kinase NII will utilize ATP and GTP as phosphate donors while protein kinase NI will only effectively use ATP. Both enzymes reveal an unusual activation by Fe2+.     

Nucleolar phosphoprotein phosphatase from Novikoff hepatoma and rat liver: characterization and partial purification.

Phosphoprotein phosphatase which dephosphorylates 32P-labeled nucleolar protein substrates was found in nucleoli of Novikoff hepatoma ascites cells and normal rat liver. The activity was extracted in high yield from nucleoli with 0.01 M Bis/Tris (pH 7.0). Low ionic strength was also required for activity: the activity was only 50% of maximum in 0.075 M NaCl. Activity was affected differently by various divalent cations: MgCl2 had little effect: CaCl2, MnCl2 and CoCl2 above 4 mM inhibited the activity 30--60%; ZnCl2 above 2 mM completely destroyed the activity. EDTA had no effect, indicating that divalent cations are probably not required. The enzyme activity was enhanced 20% by 5--8 mM dithiothreitol and was inhibited 60% by 7--10 mM N-ethylmaleimide indicating a requirement for free sulfhydryl groups. The Km of the extracted enzyme for 32P-labeled nucleolar protein was 0.6 mg/ml. The phosphatase was capable of dephosporylating the major phosphorylated nucleolar proteins C23-24 and B23-24 and also histone H1. The enzyme was purified more than 200-fold on hydroxyapatite followed by DEAE-Sephadex, which resolved the activity into three major components. The activity of enzyme extracted from Novikoff hepatoma nucleoli was approximately 2.5 times greater than from normal liver nucleoli.