Phospholipids differently modulate the activity of cytosolic protein-tyrosine kinase from porcine spleen

Effect of membrane phospholipids on the activity of cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) has been studied. Using poly(Glu Na, Tyr)4:1 as a substrate, phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine had stimulatory effects on that phosphorylation activity, however phosphatidic acid had inhibitory and phosphatidylinositol had no effects. Similar results were obtained using[Val5]angiotensin II as a substrate. On the other hand using basic protein (H2B histone and myelin basic protein) as substrates, phosphatidic acid stimulated the activity of CPTK-40, while phosphatidylinositol inhibited the activity. Phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine caused different effect on the activity of CPTK-40 depending on the substrate employed. However using acidic protein (tubulin and casein) as substrates, the activity of CPTK-40 was neither stimulated nor inhibited by any phospholipids. These results suggest that phospholipids may modulate the activity of CPTK-40.     

Regulation of cytosolic protein-tyrosine kinase from porcine spleen by polyamines and negative-charged polysaccharides

In vitro regulation of cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) by various positive or negative charged compounds was studied. Spermine and spermidine stimulated the activity of CPTK-40 about two-fold using (Val5)angiotensin II as a substrate. This stimulation was not specific for the peptide but was also observed in the case of tubulin phosphorylation indicating a direct effect of these compounds on the enzyme itself. On the contrary, negative-charged polysaccharides were shown to be strong inhibitors of CPTK-40. The possibility of the physiological regulation of CPTK-40 by these compounds is briefly discussed.     

Characterization of partially purified cytosolic protein-tyrosine kinase from porcine spleen

Biochemical properties of a cytosolic protein-tyrosine kinase (CPTK-40) partially purified from porcine spleen were characterized and compared with p40 kinase, a cytosolic protein-tyrosine kinase from bovine thymus. When CPTK-40 was incubated with MnCl2 and (gamma-32P)ATP, only a phosphoprotein with a molecular weight of 40 kilodalton was observed. CPTK-40 efficiently phosphorylated tubulin with the rate of 0.5 nmol/min/mg. Unlike p40 kinase, casein was also a substrate for CPTK-40. Among various divalent cations tested, Co2+, Mn2+ and Mg2+ were effective metal ions for the enzyme activity. Ca2+ could also serve as a divalent cation for the activity although the rate was low. These results suggest that CPTK-40 is similar but not identical to p40 kinase.     

Diverse effects of poly-basic amino acids, heparin and ionic strength on the phosphorylation of various substrates by cytosolic protein-tyrosine kinase from porcine spleen

1. Effects of poly-basic amino acids, heparin and ionic strength on the activity of cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) have been studied. 2. Both polylysine and polyarginine stimulated the phosphorylation of [Val5]angiotensin II and E11 G1 (synthetic peptide of EDAEYAARRRG), but could neither stimulate nor inhibit the phosphorylation of random copolymers; poly(EY)4:1 and poly(EAY)6:3:1. 3. Heparin stimulated the phosphorylation of poly(EY)4:1 by 2.5-fold, however, it inhibited those of E11G1, poly(EAY)6:3:1, casein and H2B histone. 4. Elevation of ionic strength of either NaCl, KCl or (NH4)2SO4 stimulated the phosphorylation of poly(EY)4:1 by greater than 5-fold, but inhibited those of casein, tubulin, H2B histone, E11G1 and poly(EAY)6:3:1. 5. These effectors did not change the Km for substrates but increased the Vmax. 6. These results suggest that the effects of poly-basic amino acids, heparin and ionic strength on the activity of CPTK-40 are mainly on the substrates employed rather than on the enzyme itself.     

Histone H1 inhibits eukaryotic DNA topoisomerase I.

Histone H1 inhibits the catalytic activity of topoisomerase I in vitro. The relaxation activity of the enzyme is partially inhibited at a molar ratio of one histone H1 molecule per 40 base pairs (bp) of DNA and completely inhibited at a molar ratio of one histone H1 molecule per 10 base pairs of DNA. Increasing the amount of enzyme at a constant histone H1 to DNA ratio antagonizes the inhibition. This indicates that topoisomerase I and histone H1 compete for binding sites on the substrate DNA molecules. Consistent with this we show on the sequence level that histone H1 inhibits the cleavage reaction of topoisomerase I on linear DNA fragments.     

S-adenosylmethionine: protein-arginine methyltransferase. Purification and mechanism of the enzyme.

Protein methylase I (S-adenosylmethionine: protein-arginine methyltransferase, EC 2.1.1.23) has been purified from calf brain approximately 120-fold with a 14% yield. The final preparation is completely free of any other protein-specific methyltransferases and endogenous substrate protein. The enzyme has an optimum pH of 7.2 and pI value of 5.1. The Km values for S-adenosyl-L-methionine, histone H4, and an ancephalitogenic basic protein are 7.6 X 10(-6), 2.5 X 10(-5), and 7.1 X 10(-5) M, respectively, and the Ki value for S-adenosyl-L-homocysteine is 2.62 X 10(-6) M. The enzyme is highly specific for the arginine residues of protein, and the end products after hydrolysis of the methylated protein are NG,NG-di(asymmetric), NG,N'G-di(symmetric), and NG-monomethylarginine. The ratio of [14C]methyl incorporation into these derivatives by enzyme preparation at varying stages of purification remains unchanged at 40:5:55, strongly indicating that a single enzyme is involved in the synthesis of the three arginine derivatives. The kinetic mechanism of the protein methylase I reaction was studied with the purified enzyme. Initial velocity patterns converging at a point on the extended axis of abscissas were obtained with either histone H4 or S-adenosyl-L-methionine as the varied substrate. Product inhibition by S-adenosyl-L-homocysteine with S-adenosyl-L-methionine as the varied substrate was competitive regardless of whether or not the enzyme was saturated with histone H4. On the other hand, when histone H4 is the variable substrate, noncompetitive inhibition was obtained with S-adenosyl-L-homocysteine under conditions where the enzyme is not saturated with the other substrate, S-adenosyl-L-methionine. These results suggest that the mechanism of the protein methylase I reaction is a Sequential Ordered Bi Bi mechanism with S-adenosyl-L-methionine as the first substrate, histone H4 as the second substrate, methylated histone H4 as the first product, and S-adenosyl-L-homocysteine as the second product released.     

Possible endogenous substrate proteins for cytosolic protein-tyrosine kinase from porcine spleen.

1. Endogenous phosphate acceptor proteins by cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) were studied using subcellular fractions of porcine spleen and supernatant fraction of rat various tissues. 2. At least 13 phosphate acceptor proteins ranging from 94 to 26 kDa were observed in all but mitochondrial subcellular fractions. 3. Among the supernatant fraction of rat tissues, brain, testis and spleen contained many phosphate acceptor proteins. 4. The most heavily phosphorylated band of around 55 kDa which was commonly recognized among various tissues was confirmed as tubulin by the immunoreactivity with anti-tubulin antibody. 5. The results obtained in this paper indicate that CPTK-40 has the potential to catalyze the phosphorylation of numerous endogenous proteins including tubulin.     

Histone acetylation in Zea mays.I. Activities of histone acetyltransferases and histone deacetylases

DEAE-Sepharose chromatography of extracts from Zea mays meristematic cells revealed multiple histone acetyltransferase and histone deacetylase enzyme forms. An improved method for nuclear isolation allowed us to discriminate nuclear and cytoplasmic enzymes. Two nuclear histone acetyltransferases, A1 and A2, a cytoplasmic B-enzyme and two nuclear histone deacetylases, HD1 and HD2, have been identified. The histone specificity of the different enzyme forms has been studied in an in vitro system, using chicken erythrocyte histones as substrate. The cytoplasmic histone acetyltransferase B is the predominant enzyme, which acetylates mainly histone H4 and to a lesser extent H2A. The nuclear histone acetyltransferase A1 preferentially acetylates H3 and also H4, whereas enzyme A2 is specific for H3. This substrate specificity was confirmed with homologous Z. mays histones. The two histone deacetylases differ from each other with respect to ionic strength dependence, inhibition by acetate and butyrate, and substrate specificity. The strong inhibitory effect of acetate on histone deacetylases was exploited to distinguish different histone acetyltransferase forms.     

Purification of a protein histidine kinase from the yeast Saccharomyces cerevisiae. The first member of this class of protein kinases

An enzyme of molecular weight 32,000 comprising a single subunit has been isolated from whole cell extracts of the yeast Saccharomyces cerevisiae. In vitro, the enzyme transfers the gamma phosphate of ATP to a protein substrate, histone H4, to produce an alkali-stable phosphorylation. Modification of the substrate histidine with diethylpyrocarbonate prevented phosphorylation. Phosphoamino acid analysis of the phosphorylated substrate showed the presence of 1-phosphohistidine. Hence, the isolated enzyme is a protein histidine kinase. A novel assay for acid-labile alkali-stable protein phosphorylation was used in the purification of the kinase activity to a final specific activity of 2,700 nmol/15 min/mg. The purified enzyme phosphorylates specifically histidine 75 in histone H4 and does not phosphorylate histidine 18 nor histidine residues in any other core histone. Steady state kinetic data are consistent with an ordered sequential reaction with Km values for Mg-ATP and histone H4 of 60 and 17 microM, respectively. The protein histidine kinase requires a divalent cation such as Mg2+, Co2+, or Mn2+ but will not use Ca2+, Zn2+, Cu2+, Fe2+, spermine, or spermidine. This is the first purification of an enzyme that catalyzes N-linked phosphorylation in proteins.     

Specific substrate for histone kinase II: a synthetic nonapeptide

Based on the previously determined intrinsic substrate specificity of histone kinase II, a nonapeptide was synthesized which was a specific substrate for this enzyme. The Vmax value of phosphorylation of the peptide (Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide) was about the same as that for H1 histone and the apparent Km for the phosphorylation of the peptide was 0.2 mM, an order of magnitude higher than that for H1 histone. H1 histone inhibited the phosphorylation of the peptide, while the peptide did not inhibit the phosphorylation of H1 histone. In the crude extracts of calf thymus, spleen and liver, histone kinase II was the only enzyme which phosphorylated the synthetic peptide. The rate of phosphorylation of this peptide was used to determine the activity of histone kinase II in the crude extracts of several tissues obtained from different species.     

Effects of polyamine hydrochlorides and salts on phosphoprotein phosphatase.

Polyamine hydrochlorides, NaCl and magnesium acetate stimulated the enzymatic dephosphorylation of phosphorylated H2B histone by two forms (large form, mol. wt. 250 000; small form, mol. wt. 30 000) of a pig heart phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16). These ionic compounds stimulated the large form of the enzyme 5--9-fold but stimulated the small form of theenzyme only 2-fold. With phosphorylated H2B histone as substrate, these effectors caused an increase in both Km and V values of the two forms of the enzyme. On the other hand, when a tryptic phosphodecapeptide derived from phosphorylated H2B histone was used as substrate, these effectors were always inhibitory apparently non-competitively with respect to the substrate. Using phosphorylated H1 histone as substrate, these effectors stimulated the large form of the enzyme 2-fold but inhibited the small form. With phosphorylase a as substrate, the reactions were also inhibited by these effectors irrespective of the enzyme employed. With respect to phosphorylase a, this inhibition was apparently of a competitive type for the large form and a non-competitive type for the small form of the enzyme.     

Purification and properties of a histone acetyltransferase from Artemia salina, highly efficient with H1 histone.

An histone acetyltransferase has been purified from nuclei of 40-h-old Artemia salina larvae. The enzyme is very unstable at 0 degrees C, requires free -SH groups for activity and is rapidly inactivated at 40 degrees C. The optimal pH for activity is 8.5 and the activity is half inhibited by millimolar concentrations of Mn2+, Ca2+ or Mg2+ or decimolar concentrations of Na+ and K+. The molecular weight of the enzyme, determined by gel filtration chromatography, changed with the ionic strength of the medium (280,000 in 10 mM Tris . HCl, 170,000 in 0.2 M KCl). The very-lysine-rich histone H1 is a better substrate acceptor than the arginine-rich histones H3 or H4. Under proper conditions, the enzyme can modify all the internal lysyl residues in histones H1 and H4. The acetylation of H1 is inhibited when all the other histone fractions are present in the assay mixture.     

Histone acetyltransferases and histone deacetylases of Physarum polycephalum

DEAE-Sepharose chromatography of plasmodial extracts of the myxomycete Physarum polycephalum reveals the presence of multiple histone acetyltransferases and histone deacetylases. Five putative histone acetyltransferases and histone deacetylases. Five putative histone acetyltransferase forms with different substrate specificity can be discriminated: one enzyme which acetylates all core histones and four enzymes with a preference for each of H3, H2A, H2B or H4. Two histone deacetylases, HD1 and HD2, can be discriminated. They differ with respect to substrate specificity and pH-dependence. The substrate specificity of histone deacetylases is determined using HPLC-purified individual core histone species. The order of acetylated substrate preference is H2A>>H3≥H4> H2B for HD1, H3>H2A>H4 for HD2, respectively; HD2 is inactive with H2B as substrate.     

Molecular basis for Gcn5/PCAF histone acetyltransferase selectivity for histone and nonhistone substrates

Histone acetyltransferase (HAT) proteins often exhibit a high degree of specificity for lysine-bearing protein substrates. We have previously reported on the structure of the Tetrahymena Gcn5 HAT protein (tGcn5) bound to its preferred histone H3 substrate, revealing the mode of substrate binding by the Gcn5/PCAF family of HAT proteins. Interestingly, the Gcn5/PCAF HAT family has a remarkable ability to acetylate lysine residues within diverse cognate sites such as those found around lysines 14, 8, and 320 of histones H3, H4, and p53, respectively. To investigate the molecular basis for this, we now report on the crystal structures of tGcn5 bound to 19-residue histone H4 and p53 peptides. A comparison of these structures with tGcn5 bound to histone H3 reveals that the Gcn5/PCAF HATs can accommodate divergent substrates by utilizing analogous interactions with the lysine target and two C-terminal residues with a related chemical nature, suggesting that these interactions play a general role in Gcn5/PCAF substrate binding selectivity. In contrast, while the histone H3 complex shows extensive interactions with tGcn5 and peptide residues N-terminal to the target lysine, the corresponding residues in histone H4 and p53 are disordered, suggesting that the N-terminal substrate region plays an important role in the enhanced affinity of the Gcn5/PCAF HAT proteins for histone H3. Together, these studies provide a framework for understanding the substrate selectivity of HAT proteins.     

Effects of cell cycle dependent histone H1 phosphorylation on chromatin structure and chromatin replication.

We have reconstituted salt-treated SV40 minichromosomes with differentially phosphorylated forms of histone H1 extracted from either G0-, S- or M-phase cells. Sedimentation studies revealed a clear difference between minichromosomes reconstituted with S-phase histone H1 compared with histone H1 from G0- or M-phase cells, indicating that the phosphorylation state of histone H1 has a direct effect on chromatin structure. Using reconstituted minichromosomes as substrate in the SV40 in vitro replication system, we measured a higher replication efficiency for SV40 minichromosomes reconstituted with S-phase histone H1 compared with G0- or M-phase histone H1. These data indicate that the chromatin structure induced by the phosphorylation of histone H1 influences the replication efficiency of SV40 minichromosomes in vitro.     

Phosphorylation of calf thymus H1 histone by muscle glycogen phosphorylase kinase

Muscle glycogen phosphorylase kinase [EC 2.7.1.38] has the ability to phosphorylate five fractions of calf thymus histone. H1 histone is the most preferable substrate, and maximally about 1.3 mol of phosphate is incorporated into every mole of this histone. This reaction absolutely depends on CA2+, and the molecular activity is about one third of that of cyclic AMP-dependent protein kinase (protein kinase A). The affinity of phosphorylase kinase for H1 histone is higher than that of protein kinase A. Calmodulin stimulates this histone phosphorylation. Analysis of the N-bromosuccinimide-bisected fragments of fully phosphorylated H1 histone has revealed that the enzyme phosphorylates mostly seryl residues in both amino- and carboxyl-terminal portions, although phosphorylation of the carboxyl-terminal portion is twice as much as that of the amino-terminal portion. Fingerprint analysis indicates that the phosphorylation sites in H1 histone for this enzyme are different from the sites phosphorylated by protein kinase A. This catalytic activity also differs from that of a newly found multifunctional protein kinase which may be activated by the simultaneous presence of Ca2+ and phospholipid.     

Study by the spin-label method of relaxation properties of protein kinase, its subunits and the catalytic subunit--histone H1 complex

Using the spin label method, the rotational relaxation in solution of adenosine 3',5'-monophosphate-dependent protein kinase and its subunits as well as the complexes of the enzyme with the substrate, histone H1, was studied. The rotational correlation time of the spin labeled macromolecules was measured on the basis of the quantitative estimation of the label mobility in relation to the protein globule. The holoenzyme molecule was found to be a rigid sphere. Whereas the complex of the globular catalytic subunit of the enzyme with a specific protein substrate, the spin labeled histone H1, appeared a flexible formation. The relaxation properties of the histone H1 molecule selectively labeled by the spin label in its globular part were investigated.     

植物PHD结构域蛋白的结构与功能特性

植物同源结构域(plant homeodomain,PHD)是锌指结构域家族的一类转录调控因子,其最主要的功能是可以识别各种组蛋白修饰密码,包括组蛋白甲基化和乙酰化等;此外PHD结构域还可以与DNA结合.含有PHD结构域的蛋白,或者本身具有组蛋白修饰酶活性,或者可以与各类组蛋白修饰酶相互作用,还有部分与DNA甲基化相关...