Histone Type Research Articles

Phenol-Extracted Plant Proteins Can Be Renatured and Assayedin Gelfor Protein Kinase Activity

Extraction of proteins by phenol has been shown to be a rapid and reproducible method for the preparation of samples for two-dimensional gel electrophoresis (1–3). An advantage of this method, which has been commercially exploited (4), is that it yields an aqueous phase containing the extracted nucleic acids and thus allows for the simultaneous analysis of gene expression at RNA and protein levels on the same experimental sample. For protein analysis, a further advantage of phenol extraction is the rapid inactivation of proteases and protein-modifying enzymes. Thus, this method has been successfully used for analyzing the in vivo modulation of protein synthesis (2) and protein phosphorylation (3) in response to environmental stimuli. However, phenol extraction may result in reversible or irreversible inactivation of proteins of interest. In the case of reversible inactivation, one should be able to renature the phenol-extracted enzyme protein and determine its activity. Here, we have tested this possibility by analyzing protein kinase activity of proteins renatured after phenol extraction. We show that phenol extraction is an efficient and cost-effective method for analyzing protein kinase activity in polyacrylamide gels. To demonstrate the relative efficiency of phenol in sample preparation, we compared the in gel renaturable activities of protein kinases extracted by phenol to those extracted by utilizing Triton or sodium dodecyl sulfate (SDS). Three-week-old alfalfa seedlings were used as experimental material. Since cold acclimation (exposure to low but nonfreezing temperatures) is known to alter levels of protein kinase transcripts and activities (5, 6), one set of seedlings was cold acclimated for 6 h by placing them at 4°C. Preparation of protein samples was carried out at 4°C as follows. Extraction with phenol. Extraction was essentially as described elsewhere (1), except for the modifications indicated. Seedlings were ground (3 ml/g fresh wt) in Tris– HCl-buffered phenol, pH 8 (7). Then an equal volume of buffer A (0.05 M Tris, pH 8.0, 0.002 M EDTA, 0.7 M sucrose, 0.5 M NaCl, 0.07 M 2-mercaptoethanol) was added and vigorously mixed with the phenol-containing homogenate. Phases were separated by centrifugation at 10,000g for 5 min. Proteins in the phenol phase (upper) were precipitated with 4 volumes of 0.1 M ammonium acetate in methanol. The protein precipitate was washed three times with 0.1 M ammonium acetate in methanol and once with acetone, dried under vacuum, and resuspended in Laemmli’s sample buffer (8). Extraction with Triton. Seedlings were ground (2 ml/g fresh wt) in buffer B [0.05 M Hepes, pH 7.2, 0.04 M EDTA, 0.001 M EGTA, 0.001 M 4-(2-aminoethyl)benzenesulfonyl fluoride (ICN, Costa Mesa, CA), 0.005 M cysteine, 0.001 M benzamidine, 0.01 M NaF, 0.25 M sucrose, and 0.024 M 2-mercaptoethanol]. Triton X-100 was added to the homogenate to a final concentration of 2% (v/v), and the mixture was stirred for 10 min. The stirred homogenate was cleared by centrifugation at 12,000g for 15 min and the supernatant filtered through glass wool. For SDS–polyacrylamide gel electrophoresis (SDS–PAGE), aliquots of the filtrate were mixed with an equal volume of 2X Laemmli sample buffer (8). Extraction with SDS. Seedlings were ground (2 ml/g fresh weight) in buffer C (0.125 M Tris, pH 6.8, 4% SDS, 1.7 M 2-mercaptoethanol). The homogenate was incubated at 95°C for 5 min (2 min per each milliliter of homogenate) and cleared by centrifugation at 12,000g for 5 min. For SDS– PAGE, aliquots of the supernatant were mixed with an equal volume of 2X Laemmli sample buffer (8). To assess renaturable proteins, protein extracts were fractionated by SDS–PAGE, renatured, and assayed for protein kinase activity in situ. Aliquots of each sample, 25 mg of protein, were separated on minigels containing 10% polyacrylamide and 0.5 mg/ml casein or histone type III-S. Casein or histone was added at the same concentration as above in the electrode buffers to prevent changes in their concentration in the gel during electrophoresis. Protein concentrations were determined by the Coomassie protein assay reagent kit of Pierce (Rockford, IL) and further verified for equal loading by SDS–PAGE (Fig. 1, top). After electrophoresis, washing, denaturation, and renaturation of proteins in the gel were as described (9). Protein kinases were 1 To whom correspondence should be addressed. Fax: (514) 3985069. E-mail: RDHINDSA@BIO1.LAN.MCGILL.CA.

Increased protein kinase C activity in myotrophin-induced myocyte growth.

Myotrophin, a novel protein that has been shown to stimulate myocyte growth, has been isolated, purified, and sequenced from the hearts of spontaneously hypertensive rats and dilated cardiomyopathic human tissue. Recently, the cDNA clones encoding myotrophin have been isolated and expressed in Escherichia coli, and the recombinant myotrophin was found to be as biologically and immunologically active as natural myotrophin. The mechanism by which myotrophin stimulates protein synthesis and initiates myocardial hypertrophy is not known. To evaluate the involvement of protein kinase C (PKC) in myotrophin-induced hypertrophy, PKC activity and its distribution in the subcellular fraction were determined in cultured neonatal and adult myocytes. PKC activity was determined by measuring the incorporation of 32P into histone type III-S and PKCepsilon substrate peptide (epsilon(pep)) from [gamma-32P]ATP in neonatal myocytes. Myotrophin significantly stimulated PKC activity in neonatal myocytes and was associated with a significant increase in protein synthesis. The effect of myotrophin on the stimulation of PKC activity and [3H]leucine incorporation was abolished by pretreatment with either staurosporine or H-7, two selective, pharmacological PKC inhibitors. Pretreatment of myocytes with staurosporine also reduced the myotrophin-induced mRNA levels of c-fos and beta-myosin heavy chain. To evaluate the subcellular events whose occurrence was due to myotrophin and translocation of PKC, we studied the effect of genistein, a tyrosine kinase inhibitor, on myotrophin-induced neonatal myocyte growth. Genistein attenuated the [3H]leucine incorporation induced by myotrophin. To define the specificity of the PKC isoform(s) involved in myotrophin-stimulated myocyte growth, both neonatal and adult myocytes were treated with myotrophin, and Western blot analyses were performed by using the antibodies of different PKC isoforms. Results showed that both PKCalpha and PKCepsilon isoforms participated in the myotrophin-induced neonatal myocyte growth, whereas only the PKCepsilon isoform was involved in myotrophin-induced adult myocyte hypertrophy. PKCdelta and PKCzeta do not seem to participate in either neonatal or adult myocyte growth induced by myotrophin. Treatment with antisense oligonucleotides specific for PKCalpha and PKCepsilon isoforms further supported this result. PKCalpha is the major PKC isoform in neonatal myocytes and needs Ca2+ and phospholipids for its activation, and PKCepsilon (the Ca2+-independent PKC isoform) is present in both neonatal and adult myocytes; the 15-mer antisense oligodeoxynucleotides of each were used for this study. Treatment of neonatal myocytes with the PKCalpha and PKCepsilon antisense oligodeoxynucleotides for 5 days significantly reduced Ca2+-dependent and Ca2+-independent PKC activity, respectively, as well as the [3H]leucine incorporation induced by myotrophin. Furthermore, myotrophin-induced PKC activity was primarily located in the particulate fraction and did not result in a concomitant decrease in the cytosolic fraction. Myotrophin does not change PKC isoform expression (both Ca2+ dependent and independent PKC isoforms used in this study) in rat neonatal cardiac fibroblasts. Our data suggest that myotrophin exerts its action on protein synthesis, possibly through a tyrosine kinase-coupled pathway and translocation of PKC from the cytosol to the cell membrane.

Reconstitution of native-like nucleosome core particles from reversed-phase-HPLC-fractionated histones.

We have reconstituted nucleosome core particles from reversed-phase-HPLC-purified chicken erythrocyte core histones and 145 bp random-sequence DNA fragments. Characterization of the resulting nucleoprotein complexes by sedimentation velocity, CD and DNase I footprinting showed that they are structurally indistinguishable from native nucleosome core particles. Furthermore, we have shown that the ability to reproduce these native-like structural features in these reconstituted nucleosome core particles is basically independent of the biological source or the method used (i.e. salt versus acid) for the extraction of histones before their HPLC fractionation. The usefulness and relevance of this approach for the reconstitution of native-like chromatin structures from histone types (histone variants/post-translationally modified histones), which are usually available only in relatively small amounts, is discussed.

Modulation of cAMP-dependent protein kinase by derivatives of chondroitin sulfate.

Here, we report investigations about the direct effect of glycosaminoglycans, such as dermatan sulfate, chondroitin 4- and 6-sulfate upon cAMP-dependent protein kinase activity. The results indicate that glycosaminoglycans strongly influence the phosphorylation activity of this enzyme against histone type IIa and [Val6, Ala7]-kemptide. While chondroitin 4-sulfate and dermatan sulfate exhibit inhibitory effects, chondroitin 6-sulfate shows a stimulating effect. In addition, the chondroitin 6-sulfate is also able to reduce the chondroitin 4-sulfate and dermatan sulfate specific inhibition.

An Abscisic Acid-Activated and Calcium-Independent Protein Kinase from Guard Cells of Fava Bean.

Abscisic acid (ABA) regulation of stomatal aperture is known to involve both Ca2+-dependent and Ca2+-independent signal transduction pathways. Electrophysiological studies suggest that protein phosphorylation is involved in ABA action in guard cells. Using biochemical approaches, we identified an ABA-activated and Ca2+- independent protein kinase (AAPK) from guard cell protoplasts of fava bean. Autophosphorylation of AAPK was rapidly (~1 min) activated by ABA in a Ca2+- independent manner. ABA-activated autophosphorylation of AAPK occurred on serine but not on tyrosine residues and appeared to be guard cell specific. AAPK phosphorylated histone type III-S on serine and threonine residues, and its activity toward histone type III-S was markedly stimulated in ABA-treated guard cell protoplasts. Our results suggest that AAPK may play an important role in the Ca2+-independent ABA signaling pathways of guard cells.

The use of histone as a facilitator to improve the efficiency of retroviral gene transfer.

Vectors based on murine C-type retroviruses are commonly used in biology. The efficiency of viral infection is normally increased by a facilitator, for example polybrene, DEAE-dextran or a liposome. The receptor for ecotropic viruses is a transporter for basic amino acids; we therefore explored the use of a highly basic protein, histone type IIA, as a facilitator. We show in several cell types that histone is as efficient as the other agents tested, and in some cases more so. This readily available reagent is thus likely to be useful in the wide range of studies that employ retroviral vectors.

Steady-state two-dimensional maps of very alkaline proteins in an immobilized pH 10–12 gradient, as exemplified by histone types

Two-dimensional steady-state patterns of histones are here reported for the first time. The first dimension run consists in nonlinear immobilized pH gradients, spanning the pH 10–12 range. The second dimension run is a standard SDS-PAGE in a constant concentration (15% T) gel slab, in presence of a 6% T stacking gel. All the histone fractions analysed (II-AS, VI-S, VII-S and VIII-S) exhibit p I values between pH 11 and 12. The M r values range from 13 to 32 kDa, with the heaviest distribution around 18 kDa. When running all different histone fractions in a single mixture, and analysing the 2-D gel by computerized image data acquisition, a total of 35 individual spots is detected.

Cytochemistry and Polarization Microscopy of Amphibian Sperm Cell Nuclei Presumed to Differ in Basic Protein Composition.

The pattern of availability of free DNA phosphates, and the kind of DNA-protein complex arrangement, both induced by nuclear basic proteins, and the richness in arginine residues in these proteins were investigated cytochemically and cytophysically in spermatozoa of the South-American Hylidae species, Hyla fuscovaria and Hyla biobeba. The aim was to demonstrate differences at the level of sperm histones in two species of Hyla until recently considered to be congeneric. The results indicated differences in the spermatozoal nuclear basic proteins and DNA-protein complexes when the two species were compared. The spermatozoa of Hyla biobeba were assumed to be likely to contain a Bloch's “type 3” protein type (intermediate sperm basic protein), similarly to Hyla species of North and Central America. On the other hand, the data obtained for the spermatozoa of Hyla fuscovaria indicated that they contain a protamine or protamine-like protein, differing from Hyla biobeba and Hyla species of North and Central America. It is suggested that the differences reported here may be genusspecific, since Hyla fuscovaria has recently been reclassified as Scinax fuscovaria based on parameters other than sperm histone types. These findings are in agreement with the general view of a wide variability in sperm nuclear proteins in the Anura group.

Benzo[a]pyrene inhibits protein kinase C activity in subcultured rat aortic smooth muscle cells

Recent studies in this laboratory have shown that benzo[ a]pyrene (BaP) interferes with protein kinase C (PKC)-mediated phosphorylation of aortic smooth muscle cell (SMC) proteins in vivo. To evaluate the biochemical basis of this response, the present studies have been conducted to examine the time- and concentration-dependent effects of BaP on PKC activity in vitro. Growth-arrested subcultures of rat aortic SMCs were exposed to 0.3, 3, or 30 μM BaP in the presence of fetal bovine serum for various times and then processed for measurements of exogenous histone Type III-S phosphorylation under PKC-activating conditions. Challenge of SMCs with BaP for 8 h was associated with a concentration-dependent inhibition of PKC activity in both cytosolic and particulate fractions. While no changes of enzymatic activity were observed in either fraction following exposure of SMCs to 0.3 μM BaP, higher concentrations of BaP inhibited PKC in both cytosolic and particulate fractions. A 49% and 68% reduction of cytosolic PKC activity was observed in SMCs treated with 3 and 30 μM BaP, respectively. The inhibitory response elicited by BaP was more pronounced in the particulate fraction where 61% and 89% decreases in PKC activity were observed in cultures treated with 3 and 30 μM BaP, respectively. Time course studies revealed that inhibition of PKC activity by 30 μM BaP occurred as early as 30 min and was sustained for up to 24 h in both fractions. Benzo[ a]pyrene (30 μM) did not interfere with the ability of phorbol-12-myristate-13-acetate to induce PKC translocation from the cytosolic to particulate compartment since maximal translocation occurred by 5 min and lasted for up to 60 min in both control and BaP-treated cultures. The inhibitory effects of BaP were independent of new protein or RNA synthesis, but appear to involve oxidative metabolism of the parent compound since 3-hydroxy-BaP, the major P450-derived BaP metabolite in SMCs, also inhibited cytosolic and particulate PKC activity. Collectively, these data demonstrate that BaP and its 3-hydroxy metabolite inhibit PKC activity in rat aortic SMCs and raise the possibility that interference with PKC-mediated protein phosphorylation participates in the deregulation of SMC growth and differentiation induced by BaP.

Protein kinase C (PKC)-mediated protein phosphorylation in rat aortic smooth muscle cells is enhanced by allylamine

The profile of endogenous protein phosphorylation mediated by protein kinase C (PKC) was examined in cell fractions prepared from subcultured aortic smooth muscle cells (SMCs) isolated from rats treated with 70 mg/kg allylamine (AAM) or tap water for 20 days. Increased phosphorylation of endogenous proteins was observed under unstimulated conditions in the particulate, but not cytosolic, fraction of cells from AAM-treated animals (i.e. AAM cells) relative to control cells. Although the same phosphorylation bands were observed in the particulate or cytosolic fraction of control and AAM cells following phorbol ester stimulation of the enzyme, enhanced PKC-mediated phosphorylation was observed in both fractions of AAM cells relative to control cells. Measurements of exogenous histone Type III-S phosphorylation by PKC following in vitro exposure of naive SMCs to 100 μM AAM for up to 60 min revealed that AAM selectively increased histone phosphorylation in the cytosolic fraction of SMCs. These results demonstrate that AAM treatment enhances PKC-mediated protein phosphorylation in rat aortic SMCs and raise the possibility that such alterations participate in the angiotoxic response to AAM.

Chromatin Transitions during Early Xenopus Embryogenesis: Changes in Histone H4 Acetylation and in Linker Histone Type

We describe major transitions in the type and modification of chromatin-associated proteins during the early development of Xenopus laevis. Histone H4 is stored in the diacetylated form in the egg and is progressively deacetylated during normal development. If histone deacetylases are inhibited with sodium butyrate, hyperacetylated histone H4 only accumulates after the mid-blastula transition. The type of linker histone in chromatin also changes during embryogenesis, from predominantly the B4 protein at the mid-blastula transition to predominantly histone H1 at the end of gastrulation. These transitions in chromatin composition correlate with major changes in the replicative and transcriptional activity of embryonic nuclei.

Decreased levels of protein kinase C enzyme activity and protein kinase C mRNA in primary colon tumors.

We have previously reported decreased protein kinase C (PKC) enzyme activity in primary human colorectal carcinomas. The purpose of this study was to extend these findings to a larger number of cases and to also examine the levels of expression of mRNAs that encode specific isoforms of PKC in these tumors. Colorectal carcinomas and paired grossly normal adjacent mucosal samples were collected from 39 patients. Complete histopathologic analyses were performed on all samples. PKC enzyme activity in both the cytosolic and particulate fractions was quantitated by measuring the amount of 32P incorporated into histone Type III-S. Northern blot nucleic acid hybridization was performed using polyA+ RNA extracted from both the tumor and normal tissue samples and 32P-labeled probes for specific isoforms of PKC. The paired sample t-test was used to determine the statistical significance of tumor to normal ratios of both enzyme activity and mRNA levels. The mean value for cellular PKC enzyme activity in the colon tumors from 39 patients was about 60 percent of that found in the paired adjacent grossly normal mucosa samples (P < 0.001). The subcellular distribution of PKC activity was similar in normal and tumor samples (about 70 percent in the particulate fraction). The abundance of PKC alpha mRNAs varied considerably among 28 tumor/normal pairs, with a mean tumor to normal (T:N) ratio of 1.0 +/- 0.6 for the 9.9-kb mRNA band and 1.4 +/- 0.7 for the 3.5-kb band. The abundance of PKC beta mRNAs was decreased in 30 of 39 tumors, with a mean T:N ratio of 0.6 +/- 0.4 for both the 9.4- and 3.5-kb bands for all 39 samples (P < 0.001). None of the parameters measured correlated with Dukes stage or the grade of the tumor. These studies extend previous evidence that total PKC enzyme activity is frequently decreased in primary human colon tumors. Our finding that this is often associated with decreased levels of PKC beta mRNA suggest that this is not simply due to post-translational down-regulation of this enzyme system. Further studies are required to determine whether these changes in PKC alpha and PKC beta mRNAs are due to altered de novo transcription or mRNA stability. It will also be of interest to examine the expression of other isoforms of PKC in colon tumors.

Structural analysis of Trypanosoma brucei brucei chromatin by limited proteolysis

The sensitive electric-birefringence method was used to reveal structural differences between the soluble chromatin of procyclic Trypanosoma brucei brucei and the chromatin of the higher eukaryotes. The orientation of the nucleosomal chains and the presence of extended DNA were analysed from the sign and amplitude of the steady-state birefringence, and the conformational properties (overall dimensions and flexibility) were studied in relation to the orientational relaxation times. In contrast to the higher eukaryotes, the birefringence of T. brucei brucei is negative and of low amplitude, corresponding to that of H1-depleted rat liver nucleosomes. Furthermore, the relaxation times are very small, about 10 microseconds. If salt is added, the birefringence as well as the relaxation time decreases dramatically, indicating that condensation affects T. brucei brucei chromatin although it behaves like nucleosome filaments, with less stable DNA-protein interaction than for the higher eukaryotes. However, this condensation does not induce the formation of regular higher-order structure. This complies with the hypothesis that typical histone H1 is absent from T. brucei brucei chromatin and that a protein or protein domain fulfils the role of histone H1. The accessibility and structural role of histone-like proteins in T. brucei brucei chromatin were also investigated using limited proteolysis with enzymes covalently bound to nylon spheres. The analysis of protein products obtained after digestion with immobilized trypsin and subtilisin shows that proteins a and d, which are classified as H3 and H4 histones, respectively, are the first to be attacked. The changes in chromatin conformation indicate that chromatin undergoes a structural transition, leading to decondensation, as indicated by increases in negative birefringence and relaxation time, and to a change in its orientation mechanism, indicated by the appearance of a permanent moment. This result is very interesting since, in rat liver, H4 was very resistant and was the last histone to be attacked, suggesting internal location and its involvement in nucleosome stabilization rather than higher-order condensation. Therefore, in T. brucei brucei chromatin, the characteristic properties of proteins a and d (their composition and interaction with DNA), as well as their external location on the nucleosome surface, suggest that if these proteins play a role similar to that played by H3 and H4 in higher eukaryotes, probably through their N-terminal regions and interaction either with DNA or protein domains, the mechanisms involved in chromatin compaction are quite different.(ABSTRACT TRUNCATED AT 400 WORDS)

Can a protein influence the fate of its own coding sequence?: the amino- and carboxyl-terminal regions of H1 histone.

The amino- and carboxyl-terminal regions of H1 histone are afforded an opportunity to bind to their own coding segments. While such binding is of no consequence to the type of H1 histone that is expressed only in terminally differentiated cells (here referred to as H5), the same binding occurring in H1 of proliferating cells might affect the course of evolution of its amino- and carboxyl-terminal regions. With the above in mind, amino acid sequences of H5 from three species were compared with those of H1 from five species. The spherical core was more conserved in H1 than in H5, whereas the amino- and carboxyl-terminal regions of both were hypervariable. A distinct evolutionary pattern, however, was observed only in these regions of H1 but not of H5. Tandem repeats of short palindromes often generated nearly identical long palindromes in distantly related species. Thus, as far as the 37-residue-long amino-terminal region was concerned, the greatest homology was between the rat and human rather than between the rat and the mouse. With regard to the last 25 residues in the carboxyl-terminal region, the greatest homology was between the chicken and the rainbow trout; the next was between the mouse and the trout. Successions of long palindromes populating the amino- and carboxyl-terminal regions of H1 might be the direct consequence of the autologous binding between peptides and their coding sequence.

Characteristics of arachidonic-acid-mediated brain protein kinase C activation: evidence for concentration-dependent heterogeneity

Arachidonic acid (AA) activates brain protein kinase C (PKC) in a specific manner, and which differs from that of diacylglycerol (DG)-mediated PKC activation in cofactor Ca 2+ and phosphatidylserine (PtdSer) requirements. We presently report that characteristics of AA-mediated activation are heterogenous, and are dependent upon the concentrations of AA. Highly sensitive PKC activation (HS) occurring at concentrations of 20 μM AA can be distinguished from less sensitive PKC activation (LS) requiring concentrations of at least 160 μM AA, on the basis of the effects of phorbol ester TPA or DG, phosphatidylcholine (PtdCho) and sodium deoxycholate (DOC). TPA, like DG suppressed the HS reaction whereas it enhanced the LS reaction. PtdCho, a phospholipid which does not affect DG-mediated activation, also prevented the HS reaction without affecting the LS reaction. This latter was inhibited at 100 μM DOC, a concentration which slightly stimulated the HS reaction. The substrate specificity was also different in the two reactions: the preferential substrate for PKC in HS was histone type VII-S, while it was histone type V-S in LS. Both reactions were similarly affected by PtdSer. In 0.1 mM CaCl 2, PtdSer stimulated AA-mediated activation without evoking additive responses while this phospholipid prevented this activation in 0.5 mM EGTA, suggesting that AA and PtdSer bind PKC on the same or related sites. Together these results provide evidence for the existence of different modes of AA-mediated PKC activation with unique characteristics which presumably involve two different binding sites for AA on the same molecule and/or different PKC isoforms.

Direct assay of membrane-associated protein kinase C activity in B lymphocytes in the presence of Brij 58

This paper describes a simple and direct procedure for assaying Ca 2+-dependent protein kinase C (PKC) activity in membrane fractions isolated from purified murine B lymphocytes (B cells) treated with phorbol 12-myristate 13-acetate (PMA). The results indicate that membrane-bound PKC in B cells, treated with PMA, can be measured directly in the presence of 0.5% Brij 58 by assaying the transfer of 32P from [γ- 32P]ATP to histone type III-S. This method obviates the need for partial purification of the protein kinase by ion-exchange chromatography prior to assaying PKC activity. The properties of membrane-associated PKC activity in B cells have been characterized, and the kinetics of PMA-induced translocation of PKC in cultured murine B cells, the rat glial tumor clone C 6, and primary neonatal osteoblastic cells have been defined by this direct assay. The results obtained with B cells and the other cell lines indicate that this direct assay procedure could be useful for studies on the factors controlling PKC translocation in a variety of cultured mammalian cells.

Ca 2+-dependent protein kinase from the halotolerant green alga Dunaliella tertiolecta: Partial purification and Ca 2+-dependent association of the enzyme to the microsomes

Ca 2+-dependent protein kinase (CDPK) was purified 900-fold from the soluble fraction of Dunaliella tertiolecta cells by ammonium sulfate precipitation, DEAE—Toyopearl, phenyl-Sepharose, and hydroxylapatite column chromatography. The CDPK was activated by micromolar concentration of Ca 2+ and required neither calmodulin nor phospholipids for its activation. The enzyme phosphorylated casein, myosin light chain, and histone type III-S (histone H-1), but did not phosphorylate protamine and phosvitin. The K m values for ATP and casein were 11 μ m and 300 μg/ml, respectively. Phosphorylation of casein was inhibited by calmodulin antagonists, calmidazolium, trifluoperazine, and compound 48 80 , but not affected by calmodulin. CDPK bound to phenyl-Sepharose in the presence of Ca 2+ and was eluted by ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA). This suggests that hydrophobicity of the enzyme was increased by Ca 2+. CDPK was also bound to the microsomes isolated from Dunaliella cells in the presence of micromolar concentration of Ca 2+ and released in the presence of EGTA, suggesting the possibility of in vivo Ca 2+-dependent association of the enzyme. The enzyme phosphorylated many proteins in the microsomes but few in the cytosol, if at all.

Change in chromatin organization related to in vivo transcriptional activity and histone synthesis independent of DNA replication during differentiation (germination) of Physarum spherules.

During the germination of Physarum spherules, increases have been observed, at the same moment, in the level of in vivo transcriptional activity as measured by [3H] uridine incorporation, and the accessibility of DNA for ethidium bromide staining as shown by flow cytometric measurements. We suppose that the changes observed in these two processes are due to a difference in chromatin organization between the first and the second period of the premitotic germination stage. In the second period, the four nucleosome core histories are synthesized in the absence of DNA replication and may correspond to a replacement of spherulation histone variants by plasmodial histone types in nucleosomes. The synthesis of historic H4 clearly distinguishes the second period of the premitotic germination stage from a growing plasmodium G2 phase, though nuclei exhibit a G2 phase DNA content. The same pattern of histone synthesis has been found during the cell cycle following the first mitosis after germination and the growing plasmodium cell cycle, with a synthesis of two histories H2B and H2A and the high mobility group (HMG)-like protein AS3 during the G2 phase, i.e. in the absence of DNA synthesis.

Identification of basic nuclear proteins by their boronate complex.

A new histochemical reaction for the identification of histone type basic proteins has been developed. Carbonyldiimidazol is used to activate the basic proteins of TCA-extracted nuclei, their m-aminophenylboronic acid complex is prepared, and the DNA-free, histone-containing nuclei are stained with toluidine blue at pH 5.5.

Chromatin organization in Entamoeba histolytica

The chromatin structure of Entamoeba histolytica was investigated. It was found that this protozoan organizes its chromatin in nucleosome-like particles 10 nm in diameter, but digestion of the chromatin with micrococcal nuclease did not render a regularly spaced DNA ladder in agarose gels. Southern blot analysis of the products of Entamoeba chromatin digestion using total amebic DNA and a non-transcribed repetitive sequence produced a banding pattern characteristic of eukaryotic chromatin with a repetitive size of approximately 130 bp. Conversely, hybridization with two active gene probes, actin and ribosomal RNA, showed that these sequences are not part of the chromatin organized in nucleosomes. It was also found that the basic nuclear proteins differ from histones of higher eukaryotes in electrophoretic mobility. Screening of an E. histolytica HM1-IMSS genomic library with Saccharomyces cerevisiae H3 and H4 genes and attempts to amplify E. histolytica sequences, homologous to these yeast histone genes, gave negative results suggesting that the Entamoeba proteins involved in chromatin organization are not typical histones.