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Methods are desceibed which permit rapid isolation of chromatographically purified histone and non-histone chromatin proteins under relatively mild chemical conditions. Chromatin is isolated from purified nuclei, dissociated in guanidine - HCl-urea and the nucleic acids removed by ultracentrigugation. This can be accomplished in 10 h by employing maximum-force rotors (500 000 x g). The proteins are then fractionated by a batch ion-exchange method, which leads to a rapid and complete separation of the histones and non-histone components, in apparently undegraded form. With these methods it is possible to obtain mg quantities of chromatographically pure histone and non-histone proteins in less than a single working day. 相似文献
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Pi-Kwang Tsung S.W. Kegeles E.L. Becker 《Biochimica et Biophysica Acta (BBA)/General Subjects》1977,499(2):212-227
The intracellular localization and properties of the chymotrypsin-like esterase activity ( esterase acitivity) of the rabbit peritoneal neutrophil has been studied and shown to differ from that of the human neutrophil.The major portion of the esterase activity in the rabbit neutrophil is in the 100 000 × g supernatant fraction with distinctly less activity in the lysosomal fraction. The 100 000 × g supernatant contained the highest relative specific activity of any of the subcellular fractions. Rabbit peripheral blood neutrophils gave the same distribution.The 100 000 × g supernatant esterase is 95% esterase 1 and 5% esterase 3, whereas, the lysosomal esterase is 78% esterase 1, 10–16% esterase 2 and 9% esterase 3 as defined by their ability to be inhibited by p-nitrophenyllethyl-5-chloropentylphosphonate. The 100 000 × g supernatant The 100 000 × g supernatant and lysosomal esterase activities further differ in their susceptibility to other inhibitors, their pH optima, ease of elution from DEAE and isoelectric points. Two molecular weight species of 174 000 and 70 000 were found in the 100 000 × g supernatant fraction and extracts of the lysosomal fraction but usually in differing proportions.In confirmation of others, essentially all of the chymotrypsin-like esterase activity ( esterase activity) of the human neutrophil is in the lysosomal fraction, unlike the rabbit cell. The human neutrophil esterase was less susceptible to inhibition by p-nitrophenylethyl-5-chloropentylphosphonate and diisopropylphosphofluoridate but more susceptible to soybean trypsin inhibitor than rabbit esterase activity. The pH optimum of the human neutrophil esterase differed from either the rabbit lysosomal or 100 000 × g supernatant esterase, as did the isoelectric point and molecular weights. 相似文献
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Release and activation of phosphorylase phosphatase upon rupture of organelles from rat liver 总被引:6,自引:0,他引:6
Liver extracts (8000 × for 10 min) from fasted rats contain about 4 times more phosphorylase phosphatase activity when the liver was homogenized in a hypotonic medium or frozen before homogenization. This increase is caused by: (i) release of partially latent phosphatases (r=60 000 and 45 000 in sucrose gradient centrifugation) from ruptured organelles; (ii) rapid activation of phosphatase in the ruptured pellet by endogenous protease(s) which can be blocked by p-tosyl-L-lysine chloromethyl ketone. Only the r=60 000 enzyme, associated with the nuclei, can be activated proteolytically, with conversion to an r=45 000. 相似文献
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Polyamine induced changes in the ADP-ribosylation of nuclear proteins from rat liver 总被引:1,自引:0,他引:1
Purified rat liver nuclei were incubated with [3H]NAD. Altered patterns of ADP-ribosylation of nuclear proteins occurred with 1 mM spermidine or spermine with the latter polyamine causing the greater change. Spermine treated nuclei showed a two-fold increase in ADP-ribose incorporation into H1 histones and a decrease in the other histones. Likewise, the incorporation into the more acidic non-histone nuclear proteins was greater with spermine than spermidine. These results suggest that polyamines may exert a regulatory function by altering the pattern of ADP-ribosylation of both histone and non-histone nuclear proteins. 相似文献
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An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at followed by sedimentation of a microsomal fraction at . The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591–1.1083 were characterized by ( activity and [3H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 μm. These fractions contained which appeared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca2+ concentration for enzyme activity was 5–10 μM. Both Na+ and K+ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca2+ from the cardiac cell. 相似文献
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A study was made of the phosphorylation of chromatographically purified histone H1 subfractions from the liver of premetamorphic tadpoles (). Two H1 subfractions were obtained which differed in terms of net incorporation of [32P]phosphate . Analysis of N-bromosuccinimide cleavage products further revealed that the two subfractions also differed in the relative distribution of [32P]phosphate in N- and C-terminal regions of the molecule. Incorporation of [32P]phosphate into both regions of the molecule occurred virtually exclusively in serine residues. 相似文献
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S H Quarfordt R S Jain L Jakoi S Robinson F Shelburne 《Biochemical and biophysical research communications》1978,83(3):786-793
Ubiquitin was first found in nuclei in protein A24 where its carboxyl terminal is covalently bound to histone 2A by an isopeptide linkage (Goldknopf, I. L. and Busch, H. (1977) Proc. Natl. Acad. Sci. USA , 864–868). Two-dimensional polyacrylamide gel electrophoresis of the 0.4 N H2SO4 soluble proteins from fractionated rat liver chromatin showed that protein A24 and histones H1, H2A, H2B, H3 and H4 were present in fractions P1 and P2 and markedly diminished in relative amounts in fraction S2. Conversely, a spot designated Ub was found in fraction S2 along with an increased amount and number of non-histone proteins. The Ub spot was not found in chromatin fractions P1 and P2. Ub was identified as ubiquitin by migration on two-dimensional gels and after purification by preparative polyacrylamide gel electrophoresis by its methionine NH2-terminal amino acid and its amino acid composition. 相似文献
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Deoxyribonucleoprotein (DNP)1 prepared by shearing chromatin of mouse cells may be fractionated in 2-phase aqueous Dextran-polyethyleneglycol mixtures. A partial separation of DNPs with different non-histone protein/DNA ratios may be obtained in a single-step partition. Separation of a spectrum of fractions of DNP has been obtained by countercurrent distribution using the same 2-phase polymer system. DNP fractions which bear nascent RNA (representing approximately of the total DNA) may be separated from the major fraction of DNP; they are found in the same region of the distribution pattern as DNP fractions with the highest non-histone protein/DNA ratio. 相似文献
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Dorothy A. Schafer Donald E. Hultquist 《Biochemical and biophysical research communications》1980,95(1):381-387
Microsomal NADH-cytochrome reductase has been purified from bovine liver by an improved procedure which employs affinity chromatography on ADP-agarose in combination with anion exchange chromatography. The reductase was extracted from a 105,000 × microsomal pellet with Triton X-100. The overall purification from isolated microsomes was 98-fold and the yield was 10%. The preparation was nearly homogeneous on SDS-PAGE. This procedure requires less time and effort than previously described procedures. Partially purified cytochrome is also obtained. 相似文献
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Hiroto Okayama Osamu Hayaishi 《Biochemical and biophysical research communications》1978,84(3):755-762
Nuclear protein A24, which is composed of histone H2A and ubiquitin, a nonhistone protein, joined by an isopeptide linkage [Goldknopf and Busch (1977) . . . . , 864–868], is found to be ADP-ribosylated in isolated rat liver nuclei. 相似文献
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P Thomopoulos B Dastugue N Defer 《Biochemical and biophysical research communications》1974,58(2):499-506
incubations of non-histone proteins from rat liver nuclei with labelled L-3, 5, 3′ triiodothyronine demonstrate the existence of high affinity, limited capacity binding sites for the hormone in this protein group; the affinity was found identical for triiodothyroacetic acid and lower for L-thyroxine. Binding ability was highly temperature dependent. At 4°C, the rate constant of association was 0.9 × 107 M?1 h?1 and the rate constant of dissociation was 0.015 h?1. The dissociation constant Kd was calculated from these data or measured by Scatchard analysis and found to be between 1.6 and 5 × 10?9 M. The maximum binding capacity was 10?13 moles of L-3, 5, 3′ triiodothyronine per 100 μg non-histone proteins or 6000 hormone molecules per nucleus. Protein binding had a half-life of 20 hours at 4°C, in the absence of hormone, but was found to be very stable in the presence of hormone. 相似文献
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