Determination of the number and relative position of tryptophan residues in various albumins.

Trace metals were measured by neutron-activation analyses in purified nucleic acids and histone(s) of lymphocytes from patients with acute lymphocytic leukaemia or infectious mononucleosis and from normal donor DNA isolated from lymphocytes of a patient with infectious mononucleosis and a normal donor showed a high a high content of Cr2+, Sb2+, Fe2+, and Zn2+, whereas DNA of lymphoblasts from a patient with acute lymphocytic leukaemia had a lower content of these trace metals, but the Co2+ content was 20-fold higher than in DNA or normal donor lymphocytic cells. Total histones from leukaemic cells had higher contents of most of the trace metals except for Zn2+, which was present in lesser concentration than in histones from normal donor lymphocytic cells. Lysine-rich (F1) histones showed lower contents of Cr2+, Sb2+ and Co2+, whereas arginine-rich (F3) histones had significantly higher contents of these trace metals. These observations may be of interest in that F3 histones more effectively inhibit RNA synthesis in human lymphocytic cells than do other species of histones.     

Manganese-phospholipid-stimulated protein kinase activity of human leukemic cells

A protein kinase system with unusual characteristics was noted in extracts of HL-60 cells using endogenous proteins as substrates. This system exhibited a cation preference for manganese at an optimal concentration of 0.5 mM. Moderate activity was detectable with magnesium at an optimal concentration of 5.0 mM, but calcium was inactive. Activity was markedly stimulated by phospholipid, with phosphatidylglycerol and phosphatidylinositol exhibiting greater activity than phosphatidylserine. In isolated plasma membranes, the major substrate of this system was a 73-kDa protein, while cytoplasmic extracts exhibited larger amounts of a 42-kDa substrate. 73-kDa phosphorylating activity of membranes was comparably active at 0 and 31 degrees C, although in cytosol activity was greater at 31 degrees C. No 73-kDa protein phosphorylation was observed in the presence of Ca2+, Mg2+, and phosphatidylserine. Phosphoamino acid analysis of the 73-kD band revealed phosphothreonine and phosphoserine. The 42-kDa substrate was distinguishable from actin by two-dimensional gel electrophoresis, which disclosed that both major substrates were highly basic in the isoelectric focusing dimension. Protamine and histones (H2B greater than H1 greater than H3) exhibited phospholipid-stimulated phosphorylation in the presence of Mn2+, but phosvitin, casein, and vinculin were not substrates. High levels of phosphorylative activity involving the 73-kDa substrate were noted in nuclear extracts. Complex patterns of increase of this activity were noted in both cytosol and nuclear extracts following induction of differentiation with dimethyl sulfoxide, retinoic acid, or phorbol 12-myristate 13-acetate. This study thus demonstrated the presence of a previously undescribed type of protein kinase activity which exhibited alterations during leukemic cellular differentiation.     

Studies on DNA bound to histones in chromatin]

Regions of DNA protected by histones against the action of DNAse 1 in the chromatin were isolated. Such DNA fragments ("subhistones" DNA) have 80% double helix structure, their nucleotide composition is close to that of total DNA, and their sedimentation constant is within the range of 2-2.7S for completely denatured molecules. Kinetics of renaturation of "subhistone" DNA was studied: within a wide range of Cot values, renaturation curves of total and "subhistone" DNA are almost identical. According to the data on hybridization with nuclear d-RNA, "subhistone" DNA is transcribed in the cell. The data obtained witness for uniform character of distribution of histones along the DNA chain in the chromatin. DNA sites which are active in RNA synthesis seem to be bound to histones as well as the non-active ones. No significant difference was found in the hybridization of "subhistone" DNA from rat liver and thymus with ibver nuclear RNA.     

Helix-coil transition in nucleoprotein: renaturation of polylysine-DNA and polylysine-nucleohistone complexes

Thermal denaturation and renaturation of directly mixed and reconstituted polylysine–DNA, directly mixed polylysine–nucleohistone complexes, and NaCl-treated nucleohistones in 2.5 × 10^?4 M EDTA, pH 8.0 have been studied. At the same input ratio of polylysine to DNA, the percent of renaturation of free base pairs in a directly mixed polylysine–DNA complex is higher than that in a reconstituted complex. For a directly mixed complex, the renaturation of free base pairs is proportional to the fraction of DNA bound by polylysine or inversely proportional to the sizes of free DNA loops. A of large amount of renaturation of free base pairs has also been observed for 0.6 M and 1.6 M NaCl-treated nucleohistones. The binding of polylysine to nucleohistone enhances the renaturation of histone-bound base pairs. The percent of renaturation of polylysine–bound base pairs is high and is approximately independent of the extent of binding on DNA by polylysine. This is true in polylysine–DNA complexes prepared either by reconstitution or by directly mixing. It also applies for polylysine–nucleohistone complexes. The model where polylysine-bound base pairs collapse at T_m′ with two complementary strands still bound by polylysine is favored over the model where polylysine is dissociated from DNA during melting. The low renaturation of histone-bound base pairs in nucleo-histone indicates that either histones do not hold two complementary strands of DNA tightly or that histones are fully or partially dissociated from DNA when the nucleo-histone is fully denatured.     

Interaction of renatured histones f3 and f2a1

Ultracentrifuge measurements show that the individual histones f2a1 and f3 interact to form tetramers in solution. The conditions which facilitate renaturation are discussed.     

Nucleosome assembly of simian virus 40 DNA in a mammalian cell extract.

We report here a mammalian cell-free system that can support chromatin assembly. Effective nucleosome assembly in HeLa cell extracts occurred at 125 to 200 mM KCl or potassium glutamate. At this physiological K+ ion concentration, two types of chromatin assembly were observed. The first was interfered with by Mg2+. Other cations such as Mn2+, Ca2+, Fe3+, and spermidine also inhibited this type of nucleosome assembly. The second type of assembly occurred in the presence of Mg2+ and at least equimolar ATP. However, even in the presence of ATP, excess Mg2+ inhibited assembly and promoted catenation of DNA; these effects could be circumvented by excess ATP, GTP, EDTA, or polyglutamic acid. The critical DNA concentration for optimum assembly in both pathways suggested a stoichiometric association of histones with DNA. The spacing of nucleosomes formed by both types of assembly on linear and circular DNA was reasonably regular, but chromatin assembled in the presence of ATP and Mg2+ was more stable.     

The histone H3/H4.N1 complex supplemented with histone H2A-H2B dimers and DNA topoisomerase I forms nucleosomes on circular DNA under physiological conditions

We have fractionated the whole cell extract of Xenopus oocytes (oocyte S-150) and isolated the endogenous components required for DNA supercoiling and nucleosome formation. Histone H2B and the three oocyte-specific H2A proteins were purified as free histones. Histones H3 and H4 were purified 100-fold in a complex with the acidic protein N1. In the presence of DNA topoisomerase I or II, histone H3/H4.N1 complexes supercoil DNA in a reaction that is inhibited by Mg2+, and this inhibition is relieved by NTPs. The supercoiling reaction induced by H3/H4.N1 complexes is enhanced by free histone H2A-H2B dimers, which by themselves do not supercoil DNA. Nuclease digestions and protein analyses indicate that H3/H4.N1 complexes form subnucleosomal particles containing histones H3 and H4. Nucleosomes containing 146-base pair DNA and the four histones are formed when histones H2A and H2B complement the reaction.     

Identification of a novel casein kinase activity in HeLa cell nuclei

Three casein kinase activities have been resolved by column chromatography of HeLa cell nuclear extracts. In addition to casein kinases NI and NII, which have been described in other cell types, HeLa nuclei contain a third casein kinase activity which we have named NIII. NIII is a cyclic nucleotide-independent casein kinase which uses either Mg2+ or Mn2+ as a divalent cation, but is inhibited by increasing NaCl concentrations in the presence of Mg2+ and has optimal activity at 50 mM NaCl in the presence of Mn2+. In Mg2+, NIII uses only ATP as a phosphate donor, but in Mn2+ NIII transfers phosphate from either ATP or GTP. NIII phosphorylates the serine and threonine residues of casein, but does not phosphorylate phosvitin or calf thymus histones.     

Enzymic characteristics of ecto-phosphoprotein phosphatase in goat epididymal intact spermatozoa

Intact washed spermatozoa from goat cauda epididymis possess an ecto-phosphoprotein phosphatase that causes dephosphorylation of phosphoserine and phosphothreonine residues of exogenous 32P-labelled histones. The cell-bound ecto-enzyme has high affinity for proteins (histones, casein, phosvitin, and protamine) rather than phosphate esters, such as p-nitrophenyl phosphate, beta-glycerophosphate, AMP, and ATP. The activity of the enzyme is inhibited by 4 mM Mg2+, Ca2+, Mn2+, or Co2+. Pi (10 mM), NaF (10 mM), and Zn2+ (1 mM) inhibit the enzyme by approximately 50, 35, and 100%, respectively. Polyamines such as spermine and spermidine at 10 mM each caused significant inhibition (60 and 30%, respectively) of the cell-bound phosphoprotein phosphatase activity, whereas cAMP, orthovanadate, and calmodulin (with or without Ca2+) had no appreciable effect. Under the standard assay conditions, spermatozoa remain intact as evidenced by assay of cytosolic enzyme markers. Both the washed and "native" intact spermatozoa showed nearly the same specific activity of the ectoenzyme. The product of the reaction (Pi) was found in the extracellular medium. Sonication doubled the enzymic activity of the intact cells. The specific activity of the enzyme was nearly fourfold higher in the intact forwardly motile cells than the "composite" spermatozoa. These data provide further support for the localization of a phosphoprotein phosphatase on the external surface of spermatozoa and that the ectoenzyme may have a role in the regulation of flagellar motility.     

Histones and DNA methylation in mammalian chromatin. II. Presence of non-inhibitory tightly-bound histones.

After removal, by high-salt extraction, of the loosely-bound components present in human placenta chromatin, tightly-bound cationic proteins could be solubilized, by acid extraction, from the 'stripped' chromatin, as well as from the 'stripped' loops or from the 'digested matrix'. These acid-soluble tightly-bound proteins are, in terms of apparent molecular mass and immunoreactivity, quite similar to the 'typical', loosely-bound histones, and, similarly to their 'loosely-bound' counterparts, they can be subdivided in distinct H1-, H2A-, H2B-, H3- and H4-like components, the 'digested matrix' being however characterized by the absence of tightly-bound H1. These tightly-bound histones, at variance from the 'typical' ones, readily find a right-handed helical conformation upon renaturation by progressive dialyses. The H1 components strongly differ also in their effects on enzymic DNA methylation: while 'typical' H1 has a strong inhibitory effect, its tightly-bound counterpart exerts a slight but definite stimulation.     

Interaction of lysine-rich histone and DNA in chromatin structure.

The heat denaturation and renaturation curves of rat liver and ascites hepatoma (AH 108A) chromatins were measured. In these renaturation curves, there are small sigmoidal regions. These sigmoidal regions remained in redenaturation curves and were largely stable to DNAase I digestion. When the chromatins were treated stepwise with NaClO4 and lysine-rich histones were removed, the sigmoidal regions in the renaturation curves disappeared. These results suggested that the sigmoidal regions reflected the interaction of DNA and lysine-rich histones.     

Glycosylation, ADP-ribosylation, and methylation of Tetrahymena histones

We have examined some of the postsynthetic modifications that occur in macronuclear histones from Tetrahymena thermophila. When purified macronuclei are incubated with [32P]NAD+, histones H1, H2A, H2B, and H3 are ADP-ribosylated. Furthermore, histones H1, H2A, H2B, and H3 contain fucose and mannose residues as evidenced by the incorporation of [3H]fucose and by the specific binding to these proteins of gorse seed lectin and concanavalin A. Finally, our studies on incorporation of methyl groups into histones show that histone H2A, together with the related nonhistone protein A24, is methylated in Tetrahymena.     

A human RNA viral cysteine proteinase that depends upon a unique Zn2+-binding finger connecting the two domains of a papain-like fold .

A cysteine proteinase, papain-like proteinase (PL1pro), of the human coronavirus 229E (HCoV) regulates the expression of the replicase polyproteins, pp1a and ppa1ab, by cleavage between Gly111 and Asn112, far upstream of its own catalytic residue Cys1054. In this report, using bioinformatics tools, we predict that, unlike its distant cellular homologues, HCoV PL1pro and its coronaviral relatives have a poorly conserved Zn2+ finger connecting the left and right hand domains of a papain-like fold. Optical emission spectrometry has been used to confirm the presence of Zn2+ in a purified and proteolytically active form of the HCoV PL1pro fused with the Escherichia coli maltose-binding protein. In denaturation/renaturation experiments using the recombinant protein, its activity was shown to be strongly dependent upon Zn2+, which could be partly substituted by Co2+ during renaturation. The reconstituted, Zn2+-containing PL1pro was not sensitive to 1,10-phenanthroline, and the Zn2+-depleted protein was not reactivated by adding Zn2+ after renaturation. Consistent with the proposed essential structural role of Zn2+, PL1pro was selectively inactivated by mutations in the Zn2+ finger, including replacements of any of four conserved Cys residues predicted to co-ordinate Zn2+. The unique domain organization of HCoV PL1pro provides a potential framework for regulatory processes and may be indicative of a nonproteolytic activity of this enzyme.     

Nuclear proteins. V. Studies of histone-binding proteins from mouse liver by affinity chromatography

We examined four extracts of mouse liver for histone-binding proteins using histone affinity chromatography and positively charged resins. The extracts used were cytoplasm and washes from isolated nuclei with buffers containing 0.05 M Tris, 0.15 M NaCl or 0.35 M NaCl. Proteins from the nuclear washes showed greater binding to the columns than proteins from the cytoplasm. The binding fractions were heterogeneous in gel electrophoresis systems. Proteins bound to affinity columns of individual histones were similar to those bound to columns of whole histone, polylysine and DEAE. A 25,000 dalton polypeptide (J2), found only in nuclear washes was a prominent histone-binding protein. It could be competitively eluted from DEAE with histones, suggesting polypeptide J2 may show a specific affinity for histones. Polypeptide J2 has an acidic to basic amino acid ratio of 1.58, and its amino acid composition is not similar to that of the high mobility group 1 protein. Polypeptide J2 binds to hydrophobic columns and may play a role in modifying histone-histone and histone-DNA interactions.     

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.     

Evidence that the peripheral cyclic AMP phosphodiesterase of rat liver plasma membranes is a metalloenzyme.

Cyclic nucleotide phosphodiesterase activity in salt extracts of rat liver plasma membranes was progressively inactivated by treatment with the metal chelators 8-hydroxyquinoline and o-phenanthroline, but not the non-chelating m-phenanthroline isomer. Activity at 20 microM-cyclic AMP was lost more slowly than activity at 0.4 microM-cyclic AMP. The activity of treated preparations was partially restored by incubation with Zn2+ or Mn2+ ions (in the presence of 1 mM-MgCl2) but not with Ca2+, Cd2+, Co2+, Cu2+ or Fe2+ ions, nor by MgCl2 alone. The results suggest the presence in the membrane extracts of a cyclic AMP phosphodiesterase containing tightly bound metal, possibly Zn or Mn, that affects the enzyme's affinity for cyclic AMP.     

Partial characterization of a histone acetyltransferase from trout testis.

Histone acetyltransferase activity of trout testis was studied both in intact nuclei, and in high salt nuclear extracts. With intact nuclei, the distribution of incorporated [14C]acetate in the various histones was similar to that observed in vivo; the arginine-rich histones H3 and H4 showed the highest specific activities, and lower amounts of label were detected in histones H2a and H2b. Histone H1 incorporated little or no label. Acetyltransferase activity could be detected in purified, sheared chromatin after the addition of MgCl2 or KCl, suggesting that the enzyme is bound to chromatin. Treatment of nuclei with 0.4 M NaCl caused the dissociation of acetyltransferase activity. Most of this solubilized activity failed to bind to DEAE Sephadex and behaved as a high molecular weight heterogeneous complex on Sephadex G-100, suggesting that the enzyme is present as an aggregate with other proteins in the extract. The pH optimum of this preparation was approximately 8.5, and the enzyme showed a preference for histones H3 and H4 as substrates.     

pp54 microtubule-associated protein 2 kinase. A novel serine/threonine protein kinase regulated by phosphorylation and stimulated by poly-L-lysine

An hepatic protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) on Ser/Thr residues is markedly activated after intraperitoneal injection of cycloheximide in the rat. The enzyme has been purified greater than 10,000-fold to near homogeneity and corresponds to a 54-kDa polypeptide, based on auto-phosphorylation, renaturation of activity from sodium dodecyl sulfate gels, and gel filtration. The protein kinase activity is unaffected by prior autophosphorylation, Ca2+, diacylglycerol and phospholipids, cyclic nucleotides, staurosporine, and protein kinase inhibitor, but can be totally and specifically deactivated by the Ser/Thr protein phosphatase 2A. The enzyme is inhibited completely but reversible by transition metals and p-chloromercuribenzoate, and is strongly stimulated by poly-L-lysine toward most, but not all protein substrates. The activity of the cycloheximide-stimulated MAP-2 kinase (pp54 MAP-2 kinase) toward potential polypeptide substrates was compared to that of an insulin-stimulated MAP-2 kinase (pp42 MAP-2 kinase). Although both MAP-2 kinases exhibited little or no ability to phosphorylate histones and casein, the two kinases had a distinguishable substrate specificity. At comparable MAP-2 phosphorylating activities, pp42 MAP-2 kinase, but not pp54 MAP-2 kinase, phosphorylated and activated the Xenopus S6 protein kinase II. Moreover, pp42 MAP-2 kinase phosphorylated myelin basic protein at 10-12-fold higher rates than did pp54 MAP-2 kinase. Cycloheximide-activated pp54 MAP-2 protein kinase appears to be a previously uncharacterized protein kinase that is itself regulated through Ser/Thr phosphorylation and, perhaps, polypeptide regulators with basic domains. The identity of the upstream regulatory elements and the native substrates remain to be established.     

The histone chaperone Asf1 is dispensable for direct de novo histone deposition in <Emphasis Type="Italic">Xenopus</Emphasis> egg extracts

Histone chaperones that escort histones during their overall lifetime from synthesis to sites of usage can participate in various tasks. Their requirement culminates in the dynamic processes of nucleosome assembly and disassembly. In this context, it is important to define the exact role of the histone chaperone Asf1. In mammals, Asf1 interacts with two other chaperones, CAF-1 and HIRA, which are critical in DNA synthesis-coupled and synthesis-uncoupled nucleosome assembly pathways, respectively. A key issue is whether Asf1 is able or not to deposit histones onto DNA by itself in both pathways. Here, to delineate the precise role of Asf1 in chromatin assembly, we used Xenopus egg extracts as a powerful system to assay de novo chromatin assembly pathways in vitro. Following characterization of both Xenopus Asf1 and p60 (CAF-1), we used immunodepletion strategies targeting Asf1, HIRA, or CAF-1. Strikingly, the depletion of Asf1 led to the simultaneous depletion of HIRA and consequently impaired the DNA synthesis-independent nucleosome assembly pathway. The rescue of nucleosome assembly capacity in such extracts was effective when adding HIRA along with H3/H4 histones, yet addition of Asf1 along with H3/H4 histones did not work. Moreover, nucleosome assembly coupled to DNA repair was not affected in these Asf1/HIRA-depleted extracts, a pathway impaired by CAF-1 depletion. Thus, these data show that Asf1 is not directly involved in de novo histone deposition during DNA synthesis-independent and synthesis-dependent pathways in egg extracts. Based on our results, it becomes important to consider the implications for Asf1 function during early development in Xenopus.     

Specific thymic peptides-DNA interaction. Correlation with the possible stereochemical kinking scheme of DNA

Low molecular weight peptides from calf thymus cause a strong dose-dependent stabilization of the DNA. The strength of DNA-peptide interaction is pH-dependent and decreases rapidly above pH 6.5. Moreover the complete kinetics of DNA denaturation and renaturation demonstrates that the peptide fraction increases significantly the DNA renaturation mostly at low temperature, showing that the interaction DNA-thymic effector helps the recombination of complementary DNA segments. The DNA stabilization rate by the peptide fraction is comparable to that obtained by means of high concentration of histones or synthetic polycationic peptides. However, the lack of basic amino acids in the peptide structure is not in favor of strong electrostatic interactions and implies a specific binding of peptide to DNA. The possible correlation of the specific thymic peptides-DNA interaction with the stereochemical kinking scheme of DNA is discussed.