Selective interaction of 5'-bromodeoxyuridine substituted DNA with different chromosomal proteins.

Chromosomal proteins selectively interact with 5'-bromodeoxyuridine (BrdUrd) substituted DNA relative to unsubstituted DNA. The relative affinities of chromosomal proteins for BrdUrd-DNA and unsubstituted DNA were measured by both thermal chromatography on hydroxylapatite and selective retention on nitrocellulose filters. Certain chromosomal proteins have a high affinity for hydroxylapatite; thus, during thermal chromatography of chromatin, the single-stranded DNA component percolates across a bed of adsorbed proteins as it elutes. We have measured the relative affinities of Brd-Urd-DNA and normal DNA for chromosomal proteins by chromatographing appropriate mixtures on hydroxylapatite. The results show that, under these conditions, the histone components, rather than the nonhistone chromatin proteins, retard the BrdUrd-substituted DNA. In addition, the individual histones vary in the degree of their affinity for BrdUrd-DNA in the order H3 greater than H4 greater than H2A greater than H2B greater than H1. We have used the property that protein-DNA complexes have a preferential affinity for nitrocellulose filters over naked DNA to measure the selective binding of BrdUrd-DNA and unsubstituted DNA's to both histone and nonhistone chromosomal proteins at low temperatures. The histones selectively retained BrdUrd-DNA on filters in the order H4 greater than H2A greater than H3 greater than H2B greater than H1. Using this assay, the nonhistones displayed greater selectivity toward BrdUrd-DNA than the histone fraction. We interpret these results to mean BrdUrd-containing DNA has a specific affinity for certain chromosomal proteins with BrdUrd-DNA may be the basis for selective inhibition of cytodifferentiation by the thymidine analogue, BrdUrd.     

Attempted separation of transcriptively active and inactive chromatin by hydroxylapatite thermal chromatography

Chromatin and purified DNA were fractionated by hydroxylapatite thermal chromatography. Fractions of varying thermal stability were tested for the proportions of transcribed sequences and repetitive sequences relative to the unfractionated genome. The first 80--85% of either total chromatin or purified DNA eluted from hydroxylapatite contained the same proportion of hybridizable sequences as total DNA. The remaining 15--20% of chromatin eluting at the highest temperatures was depleted of transcribed sequences. Analysis of the 20% highest melting fraction of purified DNA showed that, while the first two-thirds of this fraction contained the same proportion of transcribed sequences as unfractionated DNA, the last third, comprising about 6% of total DNA, was depleted of active sequences. Although no major differences were detected in nonrepetitive sequence complexity of chromatin fractions, there was a correlation between relative thermal stability and repetitive sequence content in fractions of both chromatin and DNA separated by thermal chromatography. Fragments eluting at higher temperatures contained a greater proportion of repetitive sequences, as indicated by a rapidly renaturing component. Most likely, the latest eluting fractions from both chromatin and purified DNA were enriched for a nontranscribed, highly reiterated, G+C rich satellite component of the chicken genome.     

Alpha-aminoadipate and kynurenine aminotransferase activities from rat kidney. Evidence for separate identity

alpha-Aminoadipate aminotransferase and kynurenine aminotransferase activities from rat kidney are reportedly associated with the same protein. We observed that when the supernatant fraction was maintained at pH 4.5 for 75 min, 100% of kynurenine aminotransferase activity was lost, whereas only 40% of aminoadipate aminotransferase activity was lost. We purified alpha-aminoadipate aminotransferase and kynurenine aminotransferase from rat kidney supernatant fraction to electrophoretic homogeneity by ammonium sulfate fractionation, DEAE-Sephacel, and hydroxylapatite chromatography. Kynurenine aminotransferase activity was precipitated by pH treatment. The remaining aminoadipate aminotransferase activity was concentrated and injected into rabbits to raise antibodies that were used to prepare an affinity column. A mixture of aminoadipate aminotransferase and kynurenine aminotransferase activities obtained after hydroxylapatite chromatography was subjected to affinity chromatography. Aminoadipate aminotransferase and kynurenine aminotransferase activities resolved as separate peaks, providing evidence that the two activities are associated with two different proteins.     

Isolation of antiserum against rat transcortin and characteristics of specific antibodies

Repeated chromatography of rat plasma protein on DEAE-cellulose, hydroxylapatite and subsequent gel-filtration through Sephadex G-200 were used to obtain a pure rat transcortin homogeneous upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of transcortin was about 66 kDa as determined by SDS-polyacrylamide gel electrophoresis. Immunization of a rabbit with the homogeneous preparation of rat plasma transcortin caused development of antibodies to transcortin. It was shown that the antibodies of rabbit antisera in the experiments made in vitro and in vivo neutralized 60 and 65% of 3H-corticosterone-transcortin complexes, respectively. Specific antibodies to the transcortin were isolated from the homogeneous fraction of IgG by affinity chromatography on transcortin-sepharose 4B. 125J-labelled antibodies were adsorbed by protein A-sepharose; IgG can be eluted by IM acetic acid as a sharp peak. The SDS-polyacrylamide gel electrophoresis demonstrated that affinity-eluted material contains 25 and 50 kDa polypeptides.     

Specificity of Kirkman-Robbins hepatoma non-histone chromatin proteins: electrophoretic and immunological analyses

The specificity of Kirkman-Robbins hepatoma and hamster liver non-histone chromatin proteins has been studied by comparing polypeptide patterns in polyacrylamide gel electrophoresis and by their immunological activity in the complement fixation test. Non-histone proteins were separated from DNA with a polyethylene glycol-dextran mixture and fractionated by hydroxylapatite chromatography into three classes named NHCP1, NHCP2, and NHCP3. Electrophoretic analysis indicated that among the non-histone proteins of Kirkman-Robbins hepatoma and hamster liver differences mainly of a quantitative nature can be observed. However, the polypeptides with molecular weight 25 000, 31 000, 36 000, 73 000 in NHCP1; 20 000, 40 000 in NHCP2 and 20 000, 23 000, 32 000, 38 000, 44 000, 75 000, 80 000 in NHCP3 were found to be specific for hepatoma chromatin. Application of antibodies against NHCP1, NHCP2 and dehistonized chromatin of Kirkman-Robbins hepatoma revealed that the highest specificity of NHCP2 eluted from hydroxylapatite with 100 mM phosphate buffer at pH 6.8. The NHCP1 of hepatoma shares some common antigenic determinants with analogous proteins of liver. On the other hand non-histone proteins specific for hepatoma dehistonized chromatin can be localized in the NHCP3 and partially in the NHCP1 fractions.     

Hydroxylapatite thermal fractionation of chromatin and DNA

Chromatin and DNA from developing muscle cultures were fractionated by hydroxylapatite thermal chromatography on the basis of differential thermal stability. A thermal chromatography system was developed in which protein mediated thermal stability of chromatin DNA was maximally expressed. The resulting chromatin and DNA elution profiles were similar to thermal denaturation profiles in low ionic strength solution. Additional studies showed this system was able to detect protein stabilization of DNA in in vitro nucleohistone preparations. Although some protein remained bound to hydroxylapatite during chromatin thermal elution, it did not affect the denaturation or elution behavior of free DNA on the same column. These studies show that fragments of chromatin or DNA can be segregated on the basis of differential thermal stability by hydroxylapatite chromatography.     

Properties of androphilic proteins in cytosols of human benign prostatic hypertrophy.

Androphilic proteins in the cytosol from the human benign prostatic hypertrophy are separated into two fractions by Sephadex chromatography; void volume fraction and IgG fraction which was eluted near the site of hIgG. In the present study, properties of these two androphilic proteins were compared. Association constants of these proteins were in the order of 10(9) M-1. However, the binding capacity of the former was smaller than that of the latter. These two androphilic proteins well bound to nuclei, and the high-affinity and saturable binding to nuclei was observed in the 3H-dihydrotestosterone-IgG fraction complex, while binding of 3H-dihydrotestosterone-void volume fraction complex to nuclei was low affinity and unsaturable. The binding of the complexes to chromatin seems to be of low affinity and nonsaturable. These androphilic proteins did not bind to calf thymus DNA. Salt extractability of the bound void volume fraction after incubation with nuclei was not different from that of the bound IgG fraction. It was observed that the chromatographic behavior of the androphilic protein in IgG fraction was changed after incubation with nuclei.     

Preferential association of acidic actin with nuclei and Nuclear matrix from mouse leukemia L5178Y cells

Nuclear matrix prepared from mouse leukemia L5178Y cells contained not only the two common actin isomers, beta and gamma actins, but also two additional acidic species of actin (pI 5.1 and 5.3). An anti-actin antibody recognized these acidic species as well as beta and gamma actins on a nitrocellulose filter following western blotting of two-dimensional electrophoresis. These acidic species were co-purified with beta and gamma actins using DNase I-Sepharose affinity chromatography on the nuclear matrix. Limited digestion of the acidic actin with protease V8 or trypsin gave very similar peptide fragments as did digestion of beta and gamma actins. These acidic actins were found to be distributed in the nuclear fraction, but were scarcely detectable in the cytoplasmic fraction. One of the acidic actins (pI 5.3) was found in all subnuclear fractions (DNase extract, high-salt extract and nuclear matrix), while the other species, the most acidic actin (pI 5.1), was localized predominantly in the nuclear matrix.     

Purification of chondroitinase B and chondroitinase C using glycosaminoglycan-bound AH-Sepharose 4B

Chondroitinase B and chondroitinase C were separated from an extract of Flavobacterium heparinum induced with chondroitin 6-sulfate by using column chromatography on hydroxylapatite. Chondroitinase C was eluted together with the activities of hyaluronidase, delta4,5glycosiduronase, and sulfatase. The latter two activities were eliminated exclusively by passing the crude chondroitinase C fraction through a phosphono-cellulose column pre-equilibrated with 0.07M sodium phosphate buffer (pH 6.8). Chondroitinase C was then purified by affinity chromatography using dermatan sulfate-bound AH-Sepharose 4B coated with the same glycosaminoglycan. Purification of the enzyme was achieved 18-fold and in 73% yield. On the other hand, the activities of delta4,5glycosiduronase and sulfatase were decreased to 50 and 60%, respectively, as compared with those in the crude chondroitinase B fraction, after passing the fraction through a column of phosphono-cellulose pre-equilibrated with 0.1M sodium phosphate buffer (pH 6.8). The remaining activities of these two enzymes were then eliminated from chondroitinase B by affinity chromatography with heparin-bound AH-Sepharose 4B coated with dermatan sulfate. In the affinity chromatography used in the present study, non-covalent coating of the glycosaminoglycan-bound (covalently) AH-Sepharose 4B with the same or another glycosaminoglycan was found to be important.     

The use of poly(L-proline)-Sepharose in the isolation of profilin and profilactin complexes

In the purification of proline hydroxylase by affinity chromatography on poly(L-proline)-Sepharose it was found earlier that two other components, profilin and the complex profilin-actin, also bind with high affinity to this matrix. We have exploited this observation to develop a rapid procedure for the isolation of profilin and profilin-actin complexes in high yields directly from high-speed supernatants of crude tissue-extracts. Through an extensive search for elution conditions, avoiding poly(L-proline) as the desorbant, we have found that active proteins can be recovered from the affinity column with a buffer containing 30% dimethyl sulphoxide. Subsequent chromatography on hydroxylapatite separates free profilin and the two isoforms of profilactin, profilin-actin_β and profilin-actin_γ. The profilin-actin complexes produced this way have high specific activities in the DNAase-inhibition assay, give rise to filaments on addition of Mg²⁺, and can be crystallized. From the isolated profilin-actin complexes the β- and γ-actin isoforms of non-muscle cells can easily be prepared in a polymerization competent form. Pure profilin is either obtained from an excess pool present in some extracts or by dissociation of profilin-actin complexes and removal of the actin.     

Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks

The repair of DNA double-strand breaks (DSBs) is facilitated by the phosphorylation of H2AX, which organizes DNA damage signaling and chromatin remodeling complexes in the vicinity of the lesion. The disruption of DNA integrity induces an alteration of chromatin architecture that has been proposed to activate the DNA damage transducing kinase ataxia telangiectasia mutated. However, little is known about the physical properties of damaged chromatin. In this study, we use a photoactivatable version of GFP-tagged histone H2B to examine the mobility and structure of chromatin containing DSBs in living cells. We find that chromatin containing DSBs exhibits limited mobility but undergoes an energy-dependent local expansion immediately after DNA damage. The localized expansion observed in real time corresponds to a 30-40% reduction in the density of chromatin fibers in the vicinity of DSBs, as measured by energy-filtering transmission electron microscopy. The observed opening of chromatin occurs independently of H2AX and ATM. We propose that localized adenosine triphosphate-dependent decondensation of chromatin at DSBs establishes an accessible subnuclear environment that facilitates DNA damage signaling and repair.     

A nucleolus-specific phosphoprotein in mouse ascites tumor cells

Analysis of proteins in nucleoli and chromatin of mouse ascites tumor cells labeled with [³²P]orthophosphate by SDS-polyacrylamide gel electrophoresis showed that a highly radioactive protein was localized in the nucleoli. This protein was purified and the final preparation appeared as a single component on hydroxylapatite column chromatography with or without SDS. This protein was found to be a nucleolus-specific phosphoprotein with a molecular weight of 120 000. The phosphate moiety in this protein turned over very rapidly whereas the protein itself was stable. When the nucleoli were disrupted by EDTA treatment, this unique protein was found as a major protein constituent of the ultracentrifugal supernatant.     

Reconstitution of nativelike nuclear acceptor sites of the avian oviduct progesterone receptor: evidence for involvement of specific chromatin proteins and specific DNA sequences

A specific fraction of avian oviduct chromosomal proteins can be reannealed to pure avian DNA to reconstitute nativelike specific nuclear binding sites (acceptor sites) for the oviduct progesterone receptor (PR). These specific nuclear binding sites represent the difference between the binding to the reconstituted NAP and that to pure DNA. The specific fraction of chromatin protein which contains the acceptor activity, fraction CP-3, is very tightly bound to hen DNA in a complex termed nucleoacidic protein (NAP). Removal of the CP-3 fraction from NAP results in a loss of specific PR binding sites. Resins containing chromatin adsorbed to hydroxylapatite are used as a rapid method to isolate the CP-3 fraction. Reconstitution of the CP-3 fraction to DNA by the described method involving a regressing gradient of 6-0 M guanidine hydrochloride (Gdn-HCl) results in a reconstituted NAP which displays specific PR binding sites identical with those in native (undissociated) NAP and whole chromatin. Optimal conditions and potential problems for reconstituting these nucleoproteins are described. Only partially purified receptor preparations were used in these cell-free binding analyses since they have been shown to bind with similar properties and patterns as the nuclear binding in vivo. Therefore, the binding of PR to the reconstituted NAPs was demonstrated to be receptor dependent, saturable, and of high affinity. Further, the pattern of binding to the reconstituted sites mimics those which are observed in vivo. Thus, nonfunctional receptors that cannot translocate and bind to the nuclear acceptor sites in vivo also failed to bind to the acceptor sites on the reconstituted NAPs generated by the acceptor proteins. In contrast, the binding to pure DNA does not reflect these receptor differences in receptor bindings. Specific binding of PR to reconstituted NAP can be reversed by again removing the protein fraction. Moreover, the specific binding can be destroyed by proteases and protected by protease inhibitors, indicating that acceptor activity is proteinaceous in nature. The reconstitution of the activity is both a concentration-dependent and time-dependent process. During the reconstitution, acceptor activity appears to reconstitute on the DNA when the Gdn-HCl concentration reaches 2.0 M. By use of the reconstitution method as an assay for acceptor activity, the activity in the CP-3 fraction was shown by molecular sieve chromatography to elute in a relatively broad molecular weight range between 13 000 and 25 000. The activity also focuses in isoelectric focusing resins with apparent pI's of 5.2 and 6.4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of ribonucleic acid polymerase activity in vitro by prostatic steroid–protein receptor complexes

A system has been developed which allows the stimulation in vitro of prostatic RNA polymerase by prostatic 5alpha-dihydrotestosterone-protein receptor complexes prepared from the tissues of castrated rats. The reconstitution in vitro of such a system necessitates the purification of several subcellular components. Two 5alpha-dihydrotestosterone-receptor complexes are located in the prostatic soluble supernatant fraction, separable by selective ammonium sulphate fractionation, and one complex can be isolated from the nuclear fraction. In the presence of all these complexes, stimulation of RNA polymerase in intact nuclei and nucleoli was observed. The complexes also increased the activity of the enzyme solubilized from whole nuclei. Greater stimulation of this system was noted in the presence of prostatic chromatin as template, as compared with that observed with calf thymus DNA or liver chromatin as template. The effects of the complexes on subnuclear forms of RNA polymerase, of nucleolar and extranucleolar origin, are also described. RNA polymerase solubilized from nucleoli is more susceptible to stimulation by the 5alpha-dihydrotestosterone-receptor complexes than is the ;nucleoplasmic' enzyme. Stimulation occurs less readily in the presence of Mn(2+) and at high ionic strength than in the presence of Mg(2+) and at low ionic strength. Preliminary experiments show that prostatic nucleolar RNA polymerase transcribes prostatic chromatin poorly as compared with the nucleoplasmic enzyme. The observations reported indicate an involvement of non-histone proteins associated with DNA in the process by which stimulation of enzyme activity by the 5alpha-dihydrotestosterone-receptor complexes is achieved. The implications of these findings in the mechanism of steroid hormone action is considered.     

Purification, characterization, and molecular cloning of a 60-kDa phosphoprotein in rabbit skeletal sarcoplasmic reticulum which is an isoform of phosphoglucomutase.

A 60-kDa substrate of calmodulin-dependent protein kinase in rabbit "heavy" skeletal sarcoplasmic reticulum (SR) was characterized by purification and cDNA cloning. Purification was achieved by column chromatography using DEAE-Sephacel, heparin-agarose, and hydroxylapatite in 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS). Analyses of amino acid sequence and composition indicated that the CHAPS-soluble 60-kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding two isoforms of PGM were isolated from rabbit skeletal muscles. The translated amino acid sequences show that the isoforms, PGM1 and PGM2, differ in the N-terminal 77 amino acids and that PGM2 is identical to the 60-kDa protein in the SR. Northern blot analysis showed that the size of the mRNA encoding PGM2 is 2.4 kilobases. The PGM enzyme activity was markedly inhibited in SR membranes, while perturbation of the membranes with CHAPS or guanidine-HCl recovered the enzyme activity. KCl (0.15-1 M) led to a partial recovery of the enzyme activity suggesting that the charge interaction is not the primary force for PGM-SR interaction. PGM is localized in the heavy fraction of SR, where calsequestrin and Ca2+ release channel are enriched. Our results demonstrate that an isoform of PGM localized in junctional skeletal SR is the 60-kDa substrate of calmodulin-dependent protein kinase.     

Reconstitution of a partially purified Na+-dependent D-glucose transport system from rat jejunal brush border membranes

The Na+-dependent D-glucose transport system of rat jejunal brush border membranes was partially purified and reconstituted into functional proteoliposomes. Brush border membrane vesciles isolated from villous cells were first extracted with 0.3% cholate to remove extrinsic proteins and the insoluble residual pellet was reextracted with 1.2% cholate. The 1.2% cholate-extracted soluble fraction was then further purified by hydroxylapatite and Concanavalin A affinity chromatography in tandem. When the HLP-unadsorbed-ConA-unadsorbed fraction was reconstituted into proteoliposomes, it showed a characteristic Na+-coupled, phlorizin inhibitable, D-glucose transport activity that was 3 fold higher than that of the reconstituted proteoliposomes of the 1.2% cholate-extracted fraction. This partially purified fraction also displayed the simplest polypeptide composition pattern among all the membrane fractions analysed in SDS-polyacrylamide gels.     

The use of poly(L-proline)-Sepharose in the isolation of profilin and profilactin complexes

In the purification of proline hydroxylase by affinity chromatography on poly(L-proline)-Sepharose it was found earlier that two other components, profilin and the complex profilin-actin, also bind with high affinity to this matrix. We have exploited this observation to develop a rapid procedure for the isolation of profilin and profilin-actin complexes in high yields directly from high-speed supernatants of crude tissue-extracts. Through an extensive search for elution conditions, avoiding poly(L-proline) as the desorbant, we have found that active proteins can be recovered from the affinity column with a buffer containing 30% dimethyl sulphoxide. Subsequent chromatography on hydroxylapatite separates free profilin and the two isoforms of profilactin, profilin-actin beta and profilin-actin gamma. The profilin-actin complexes produced this way have high specific activities in the DNAase-inhibition assay, give rise to filaments on addition of Mg2+, and can be crystallized. From the isolated profilin-actin complexes the beta- and gamma-actin isoforms of non-muscle cells can easily be prepared in a polymerization competent form. Pure profilin is either obtained from an excess pool present in some extracts or by dissociation of profilin-actin complexes and removal of the actin.     

A possible role of rabbit heart cytosol tocopherol binding in the transfer of tocopherol into nuclei.

An alpha-tocopherol-binding macromolecule was isolated from the heart cytosol of rabbits fed for 1 month with an alpha-tocopherol-deficient diet. The amount of [3H]-tocopherol bound to nuclear chromatin was increased when the alpha-tocopherol-deficient heart nuclei were incubated in the presence of [3H]tocopherol-cytosol complex. In this condition, large amounts of [3H]tocopherol were associated with a subnuclear fraction that contained non-histone acidic proteins.     

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [³H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [³H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.