Region Of Sucrose Gradient Research Articles

Biochemical characterisation of purified human wild‐type p53 overexpressed in insect cells

Conditions for the overexpression of human wild-type p53 using a baculovirus construct were optimised in insect cells which produced up to 20 mg p53/1 culture. Milligram amounts of p53 were purified to apparent homogeneity using chromatography on double-stranded DNA-cellulose (approximately 58% yield) followed by immunoaffinity chromatography with an epitope elution step (up to 48% yields) at 4 degrees C. The M(r) of extracted p53 both from insect cell lysates and after purification was 54,000 by SDS/PAGE. Isoelectric focusing showed recombinant p53 to be an acidic protein, focusing at pI 6.0 under non-denaturing conditions. Expressed p53 at all stages of purification reacted by immunoblotting with specific p53 monoclonal antibodies, indicating the presence of intact epitopes at the C-terminus, N-terminus and central region of the protein. From ultracentrifugation studies, pure p53 exhibited significant oligomerisation, and sedimented broadly within the 7-12-S region of sucrose gradients. Pure p53 slowly precipitated out of solution at concentrations between 1-6 mg/ml even in the presence of 1% detergent. Using metal affinity chromatography, we have established that pure p53 binds the immobilised divalent ions Zn2+, Ni2+ and Co2+ with high affinity.

Purification and properties of dyneins from Paramecium cilia

Dynein ATPases purified from Paramecium cilia by salt extraction followed by sucrose density gradient centrifugation and anion exchange chromatography. The two major dyneins sedimented in sucrose gradients as species of 22 S and 12 S. After purification by anion exchange chromatography, their specific activities were about 0.4 and 0.5 μmol/min per mg, respectively. The dyneins could be distinguished by subunit composition and immunological crossactivity. Sucrose density gradient centrifugation revealed additional ATPase activity in the region between the 22 S and 12 S dyneins, including a 19 S activity. Mg 2+-ATPase activities of the dyneins and the 19 S activity were inhibited by vanadate and Zn 2+, and were activated by Triton X-100. Antibodies against the 22 S dynein from Paramecium reacted on immunoblots with most of the polypeptides of 22 S dynein, and showed that the heavy chains of 22 S dynein are not indentical to those that sediment at 19 S and 12 S. Several minor ATPase activities were revealed by anion exchange chromatography of fractions from the 22 S, 19 S and 12 S regions of sucrose gradients. These minor activities were stimulated by Mg 2+, inhibited by vanadate, and could be distinguished from each other by their elution positions and polypeptide compositions.

Polycyclic aromatic hydrocarbon binding macromolecules. Identification, characterization and temperature activation of a 4.5 S binding nucleoprotein.

A macromolecule binding 3H-methylcholanthrene (3H-MCA) and 3H-benzo(a)pyrene (3H-BaP) and sedimenting in the 4-5 S region of sucrose gradient (4.5 S) was identified in rat liver cytosol. The binding was displaced by 100-fold molar excess unlabeled ligands whereas 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was ineffective. The dissociation constant for both polycyclic aromatic hydrocarbons (PAHs) was of the order of 10(-8) M or lower. Both 3H-MCA and 3H-BaP bound to 4.5 S in a non covalent manner, since 92% of the bound radioactivity was extractable with ethyl ether. Furthermore the binding was strongly reduced by urea 8 M and by guanidine. HCl 4 M (99 and 70% respectively). Thin layer chromatography of the ethyl ether-solubilized radioactivity showed a peak comigrating with PAHs used as standards. When chromatographed on Sephadex G-200, 4.5 S was eluted as a sharp peak with an apparent molecular weight of 50-60,000 daltons. Enzyme treatment of liver cytosol showed that the 4.5 S binding sites were destroyed by micrococcal nuclease (92% of inhibition). Papain and phosphodiesterase I and II reduced the binding to 50%, whereas DNase I, DNase II, RNase, phospholipase A2 and C and trypsin were ineffective. These data suggest that the PAHs binding macromolecule of rat liver cytosol is a protein associated with a polynucleotide. The binding of both PAHs was enhanced by increasing the incubation temperature, the maximum being reached after 20-30 min at 37 degrees C. After 2.5 min at 65 degrees C, binding sites were completely destroyed. The same temperature-induced "activation" was obtained also by prewarming the cytosol at 37 degrees C in the absence of ligands.

Identification of 1,25-dihydroxyvitamin D3 receptors and activities in muscle.

Cytosols from cultured myoblast cells (G-8 and H9c2) prepared in high salt (0.3 M KCl) possesses receptor like proteins for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) that sediment in the 3.2 S region of sucrose gradients. These receptors were characterized as having high affinity (Kd less than 0.1 nM) for 1,25-(OH)2D3 and are in low capacity (less than 80 fmol/mg of cytosol protein). Analog competition for receptor binding revealed that 1,25-(OH)2D3 was more potent than 24,25-(OH)2D3, or 25-(OH)2D3 for displacement of 1,25-(OH)2[3H]D3 from these 3.2 S region sedimenting receptors. Furthermore, the receptor proteins had affinity for DNA and eluted from Sephacryl S-200 as a macromolecule with Stokes radius (Rs) of 32 A. High salt cytosol from collagenase-dispersed skeletal muscle cells was also found to possess a 3.2 S 1,25-(OH)2D3 receptor-like protein. The 1,25-(OH)2D3 receptor concentration in both G-8 and H9c2 myoblast lines was found to down-regulate by 50-70% when cells were stimulated to differentiate to myotubes by lowering fetal calf serum to 5% of the medium. Moreover, we demonstrated that 1,25-(OH)2D3 can inhibit DNA synthesis and cell proliferation of the G-8 myoblast cells in a dose-dependent manner. 1,25-(OH)2D3 was more potent at inhibiting cell proliferation in cells grown in 5% serum than in 20% serum. The data suggest that 1,25-(OH)2D3 can act directly on muscle myoblast via a 1,25-(OH)2D3 receptor that is similar to those found in intestine and bone. The data support the possibility that muscle is a target tissue for 1,25-(OH)2D3 and the hormone may act to initiate terminal differentiation of myoblast cells.

Na +, K +, H + and Cl − permeability properties of rabbit skeletal muscle sarcolemmal vesicles

The ion permeability properties of rabbit skeletal muscle sarcolemmal vesicles were investigated by means of radioisotope flux, membrane potential, and light-scattering measurements. An enriched sarcolemmal fraction was obtained from the 22–27% region of sucrose gradients after isopycnic centrifugation. The presence of contaminating sarcoplasmic reticulum was assessed with the use of a purified sarcoplasmic reticulum vesicle fraction. 22Na +, 86Rb +, 36Cl −, and [ 3H]sucrose flux measurements indicated that the sarcolemmal fraction possessed isotope spaces ranging between 1.5 and 4 μl/mg protein. Membrane potential measurements using the voltage-sensitive fluorescent probe 3,3′-dipentyl-2,2′-oxadicarbocyanine iodide (diO-C 5-(3)) indicated that sarcolemmal vesicles were impermeable to H + and Na + but that 10–15% of the vesicles were permeable to K +. Light-scattering measurements indicated a small fraction of sarcolemmal vesicles were permeable to both K + and Cl −. Whether the low permeability of sarcolemmal vesicles to Na +, K +, and Cl − is the result of a low concentration of ion channels or the inactivation of these channels during isolation is at present uncertain.

Similarities and differences of the binding of estradiol and 4-hydroxytamoxifen (an antiestrogen) in the chick oviduct cytosol

The binding characteristics of [ 3H]estradiol and 4-[ 3H]hydroxytamoxifen (a powerful estradiol antagonist) in the chick oviduct cytosol was analyzed by sucrose gradient centrifugation and dissociation kinetics experiments at 28°C. Heating the cytoplasmic estradiol-estrogen receptor complexes led to the ‘transformation’ of the receptor; as with the estrogen receptor in other target tissues and species, the transformed receptor sedimented in the 5 S region of sucrose gradients containing 0.4 M KCI and had a slower rate of dissociation of bound estradiol. Upon heating, the cytoplasmic 4-hydroxytamoxifen complexes also appeared to undergo similar changes in their physical states as analyzed by sedimentation rates and dissociation kinetics, and we conclude that antiestrogen can transform the receptor. Sodium molybdate inhibited the temperature mediated changes with both estrogen and antiestrogen complexes. Slight but consistent differences in the sedimentation coefficient and rate of ligand dissociation were observed between the complexes formed by estradiol and 4-hydroxytamoxifen but the relevance to opposite biological activities remains unknown.

Chromosomal organization of the herpes simplex virus type 2 genome

Nucleoprotein complexes containing herpes simplex virus type 2 DNA were extracted from CV-1 cells with NP-40 and (NH 4) 2SO 4. The viral DNA-protein complex sedimented in neutral sucrose as a relatively homogeneous peak at around 120 S. Pooled material from the 120 S region of sucrose gradients contained DNA which, when deproteinized, sedimented at 55 S and had a density of 1.728 g/cm 3 as expected for herpesvirus DNA. Buoyant density studies with glutaraldehyde-fixed complexes indicate that protein is bound to viral DNA at a ratio of approximately 1.1 to 1. Micrococcal nuclease digests of isolated viral nucleoprotein complexes indicate that these complexes do not have a nucleosomal structure. Chromatin prepared from nuclei following isolation of viral nucleoprotein complexes contained viral DNA. When infected nuclei were treated with micrococcal nuclease, nucleosome-sized fragments were obtained which contained both virus and cell DNA.

Sodium-calcium ion exchange in skeletal muscle sarcolemmal vesicles.

The Ca2+ permeability of rabbit skeletal muscle sarcolemmal vesicles was investigated by means of radioisotope flux measurements. A membrane vesicle fraction highly enriched in sarcolemma, as revealed by enzymatic markers, was obtained from the 22-27% region of sucrose gradients after isopycnic centrifugation. The ability of sarcolemmal vesicles to exchange Na+ for Ca2+ was investigated by measuring Ca2+ influx into and efflux from sarcolemmal vesicles in the presence and absence of a Na+ gradient. It was found that Ca2+ movements were enhanced in the direction of the higher Na+ concentration. When intra-and extravesicular Na+ concentrations were high, Na+-Na+ exchanged predominated and Na+-Ca2+ exchange was low or absent. The presence of the Ca2+ ionophore A23187 in the dilution medium resulted in the rapid release of Ca2+ and the elimination of the Na+-enhanced efflux of Ca2+, suggesting that internal rather than bound external Ca2+ was exchanged with Na+ . La3+ abolished Na+-Ca4+ exchanged was not due to sarcoplasmic reticulum or mitochondrial contaminants. This investigation suggests that skeletal muscle, like cardiac muscle and neurons, is capable of a transmembranous Na/-Ca2+ exchange.

Separation of satellite DNA chromatin and main band DNA chromatin from mouse brain.

Using restriction endonucleases which preferentially digest mouse main band DNA and leave satellite DNA intact, we have isolated highly purified chromatin fractions containing only mouse satellite or main band DNA. Following the digestion of mouse brain nuclei with EndoR Alu I, main band DNA chromatin is selectively extracted with 10mM Tris, 10mM EDTA. Satellite DNA chromatin is subsequently extracted from the nuclear pellet with Tris-3M urea and further purified on sucrose gradients. Chromatin extracted from digested nuclei with Tris-EDTA contains only main band DNA and has a molecular weight lower than 2 x 10(6). Chromatin fractions obtained from the lower regions of sucrose gradients of the Tris-Urea extracts contain 40--95% satellite DNA and have a molecular weight of 6 to 8 x 10(6). Both the satellite DNA and main band DNA chromatins contain all five histones and have a protein to DNA ratio of 1.3 to 1.

Cellular retinol-binding protein

1. 1. A protein which binds retinol in vitro with high affinity and specificity was detected by sucrose gradient centrifugation or by gel filtration after preincubating rat tissue cytosols with all- trans-[ 3H]retinol. This protein sediments in the 2 S region of sucrose gradients. Molecular size determination by gel filtration indicates a molecular weight of 16 000. 2. 2. Competition studies revealed that only all- trans-retinol, not retinal or retinoic acid, competes for binding. The binding of radioactive retinol is reversible. 3. 3. This protein was detected in cytosols of rat liver, lung, spleen, brain, testis, ovaries, uterus and intestinal mucosa whereas heart or gastrocnemius muscle seem to lack this protein. 4. 4. The cellular retinol binding protein was found in fetuses as early as day 12 of the gestation period and possessed the same specifity for the ligand as the one in adult tissues. 5. 5. This binding component was not detected in cytosols prepared from Novikoff hepatoma, ascites hepatoma AS-30D, mouse Ehrlich ascites tumor and mouse pituitary tumor cell line AtT 20. 6. 6. The cellular retinol binding protein seems to be different from that described to be present in the serum as suggested by difference in size and by the inability of the antisera against the serum retinol binding protein to remove the cellular binding protein from the cytosol preparations.

The subunit character of soluble estrogen receptors from rat uteri and their modification in vitro

The soluble receptor-estrogen complexes from mature rat uteri, which sediment in the 9-S region of sucrose gradients, are reversibly dissociated into two dissimilat units (A and B) when exposed to 0.3 M KCl; only the A unit binds estradiol. Partial separation of the units is obtained by sedimentation through sucrose gradients and by gel filtration on 6% agarose, columns. Aging the receptors in the presence of 0.3 M KCl at 0° prevents the reassociation of the subunits when they are sedimented into gradients lacking KCl. A small subunit (C) appears to be released from its original association within the 9-S complex during the aging process. This unit, which can be extracted with 0.3 M KCl from receptor complexes adsorbed on PVM-estradiol resin, modifies the A units and prevents them from reassociating with intact B units. A possible role of the modified receptors in the mechanism of estrogen action is discussed.

Unstable nuclear RNA synthesis following estrogen stimulation

The administration of estradiol-17β in vivo stimulates the rate of synthesis into all classes of uterine and liver RNA. The earliest observable effects on macromolecule synthesis involve transcriptional changes in the spectrum of RNA molecules synthesized in target tissues. RNA/DNA hybridization experiments indicate differences in the populations of DNA-like RNA in both the liver and uterus after hormone treatment. No change was observed in lung cells. The results of RNA/DNA hybridization on total cell RNA indicate that the hormone-stimulated RNA species of the liver and uterus are not homologous. Therefore, the estrogen-induced RNA synthesis is partly organ specific. Both normal and hormone-treated tissue contain some RNA molecules which are restricted to the nucleus and do not occur in the cytoplasm. Some RNA molecules restricted to the nucleus in normal liver cells appear in the cytoplasm of hormone-treated liver cells. However, the spectrum of RNA molecules restricted to uterine nuclei before hormone treatment appears much more dramatic. The size pattern of rapidly labeled RNA molecules restricted to the nucleus is similar in normal and hormone-treated uterine cells except in 20–40-S and 4-S regions of sucrose gradients. It is suggested that selective transport of RNA to the cytoplasm may be an important device in the regulation of translation of potential messengers. The organ specificity of hormone action therefore appears to be mediated through three steps: (1) the receptor recognition of the target tissue, (2) transcriptional RNA changes which appear partially organ specific, and (3) selective regulation of nuclear RNA transport to the cytoplasm in tissues with primary response.