Sedimentation equilibrium measurements of the intermediate-size tobacco mosaic virus protein polymers

Short-column sedimentation equilibrium methods have been applied for the first time to tobacco mosaic virus (TMV) protein (0.1 M ionic strength orthophosphate) at pH 6.5 and at pH 7.0 to estimate molecular weights. Previous sedimentation velocity experiments at pH 6.5, 20 degrees C have led to the conclusion that the major boundary with an S0(20),w value of 24.4 +/- 0.1 S consists of a distribution of polymers which are mainly three-turn, 48-51-subunit helical rod aggregates. The directly measured z-average molecular weights together with sedimentation velocity data are entirely consistent with this assignment of a three-turn aggregate. Molecular weights have also been determined under two conditions where a large mass fraction of the protein sediments with an S0(20),w value of 20.3 +/- 0.2 S. At pH 6.5, 6-8 degrees C, the aggregates in this boundary are metastable and correspond to 50-60% of the preparation. At pH 7.0, 20 degrees C at equilibrium, 65-75% of the protein sediments at 20.3 S. The 20.3S boundary is very similar under both conditions and is interpreted as being composed of a distribution of protein aggregates centered about 39 +/- 2 subunits. This result is important in the interpretation of previous kinetic measurements of TMV self-assembly. The current view is that the 34-subunit structure of TMV protein, in the form of a cylindrical disk which is made up of two 17-subunit layers and has been characterized in single-crystal X-ray diffraction studies, plays a central role in the initial binding steps with RNA. The present results are not consistent with the view that there is a significant concentration of the TMV protein disk structure in solution under the usual conditions of TMV self-assembly.     

The Donnan effect in tobacco mosaic virus and its components

Osmotic pressure studies were carried on tobacco mosaic virus (TMV) and its components, protein and RNA, as well as on bis(3,3′-aminopropyl)amine, reported to be present in TMV preparations. Solvents were phosphate and barbital buffers at different values of pH and ionic strength. Measurements were made at room temperature. The Donnan effect was exhibited by TMV protein in phosphate buffer of 0.01 ionic strength at pH values ranging between 5.8 and 7.5. The observed values of the Donnan effect at pH 5.8 and 5.97 were in reasonable agreement with theoretical values calculated from the charge obtained by hydrogen ion titration. TMV-RNA in phosphate buffer at pH 7.5 and ionic strength 0.01 did not exhibit more than 1% of the expected Donnan effect. This is explained tentatively as the result of firm binding of metal ions. Negative values of osmotic pressure were observed with bis(3,3′-aminopropyl)amine. Similar anomalous osmosis was sometimes observed with TMV protein and with TMV. In agreement with earlier observations, TMV did not exhibit the Donnan effect in phosphate buffer of 0.01 ionic strength at pH values ranging from 5.5 to 8.0. However, TMV dialysed extensively in the presence of EDTA at pH 8.5 and TMV produced by reconstitution of purified protein and RNA did exhibit the Donnan effect in both phosphate and barbital buffers. The magnitude was of the same order as that calculated from the net charge determined by hydrogen ion titration. When reconstituted TMV, which did exhibit Donnan effect, was treated with calcium ions, the effect was abolished.     

Coordinated two-disk nucleation, growth and properties, of virus-like particles assembled from tobacco-mosaic-virus capsid protein with poly(A) or oligo(A) of different length

Assembly of nucleoprotein rods from tobacco mosaic virus (TMV) coat protein and poly(A) depends on the presence of 20S disks in a manner very similar to nucleation and growth of virions in reconstitution with TMV RNA. Products assembled with (A) approximately equal to 5000 appear to have the same buoyant density in CsCl, the same nucleotide/protein ratio and the same nuclease stability, as reconstituted and native TMV. Their rate of formation is very similar to the rate of reconstitution with TMV RNA when high-molecular-mass (A) approximately equal to 5000 is used, but becomes a function of chain length particularly with (A) less than or equal to 185. The composition of assembly products can be described sufficiently with the relation between number of capsid polypeptide monomers/particle, np, to the number of nucleotide residues/chain, nnt, of np = 1/3 (nnt + 50) with two important restrictions: (1) particles of less than four turns of helically arranged capsid subunits are unstable, and (2) particles with about 150 or less nucleotides per chain deviate in structure from mature virus and virus-like (= longer) assembly products. This is indicated by changes in both buoyant density in CsCl and optical properties, while 'dislocation' of the disk to the helical arrangement of capsid subunits ('helicalization') and nuclease stability already become established with chains as short as (A) approximately equal to 58 +/- 20. Consequently, we suggest that assembly proceeds through three distinct phases: (1) nucleation (resulting in helicalization) by interaction of nucleic acid with the first disk; (2) stabilization of the primary (unstable!) nucleation complex by addition of a second disk and formation of a four-turn virus-like and stable nucleoprotein helix, which is then fit for (3) elongation by addition of further disks. The question of what makes the TMV protein disk select specifically TMV RNA during virion assembly is discussed in some detail.     

与TMV-RNA装配起始位点互补的cDNA片段对TMV体外装配的抑制

合成了与TMV-RNA病毒装配起始位点互补的、长度为二十个核苷酸的DNA片段。该片段用~(32)P标记后,代替反义RNA(antisense RNA)与TMV-RNA进行硝基纤维素膜点杂交和溶液杂交。结果表明,该cDNA片段在两种条件下均能与TMV-RNA进行杂交。将溶液杂交的RNA-cDNA复合体经酒精沉淀,再与TMV衣壳蛋白的20S聚合体制剂进行体外装配,用测定310nm吸收光谱变化和电子显微镜观察的方法鉴定装配结果。实验证明,该cDNA片段与TMV-RNA杂交后抑制了装配起始位点的活力,从而使TMV病毒颗粒的装配不能完成。这一结果提示,TMV基因装配起始位点顺宁的cDNA和反义RNA能够在体外抑制TMV病毒颗粒的装配。     

Hydrogen ion uptake upon tobacco mosaic virus protein polymerization.

To determine the stage at which H⁺ ions are bound during the entropy-driven polymerization of tobacco mosaic virus protein, acid-base titrations were carried out at a concentration of 5 mg/ml in 0.1 m-KCl from pH 8 to pH 5.2 and back to pH 8 at 4, 10, 15 and 20 °C. The titration was always completely reversible when the addition of acid or base was so slow that the experiment required seven hours in each direction. When the titration was started at pH 7 and performed down and up twice as rapidly, a hysteresis loop, indistinguishable from one previously published, was obtained at 20 °C.Ultracentrifugation experiments were carried out at selected pH values at the four temperatures. H⁺ ion uptake, as determined from the reversible titration curves, is correlated with the disappearance of the 4 S component and is independent of whether the polymerized species is in a 20 S or higher state of aggregation. At pH 7, approximately 1 mole of H⁺ ion is bound per mole of monomer. At pH values between 6.56 and 6.05, 1.5 moles of H⁺ ion are bound per mole of monomer upon polymerization. At pH 6.05, 0.5 mole of H⁺ ion is bound before any polymerization takes place.Tobacco mosaic virus protein at 20 °C in an unbuffered 0.1 m-KCl solution at pH 7.18 at a concentration of 41 mg/ml, largely in the 20 S state, was depolymerized entirely to the 4 S state by dilution with 0.1 m-KCl adjusted to the same pH. Under these conditions, there was no pH change, indicating that no H ⁺ ions are released.These seemingly contradictory findings can be explained by assuming that the 4 S component polymerizes to form either double discs without binding H⁺ ions, or, alternatively, two-turn helices accompanied by the binding of H⁺ ions. Both double discs and two-turn helices sediment at approximately 20 S. Whether polymerization in the neighborhood of pH 7 leads to helices or discs depends upon the availability of H⁺ ions.     

Efficiency of Nitrous Acid as An Inactivating and Mutagenic Agent of Intact Tobacco Mosaic Virus and Its Isolated Nucleic Acid

When tobacco mosaic virus (TMV) and its isolated nucleic acid (TMV-RNA) were treated with nitrous acid, the nucleic acid was inactivated six times faster than the intact virus. Inactivation of both the infectious entities was exponential with treatment time to 0.1% level of survival. Eight different mutant phenotypes were scored after inactivation of TMV and TMV-RNA to 50, 10, 1.0, and 0.1% survival levels. Significantly more mutants in relation to unaltered isolates were induced at all levels of survival upon nitrous acid treatment of TMV than of TMV-RNA. Furthermore, the proportion of two specific mutant phenotypes was significantly greater in treated TMV than in treated TMV-RNA. No qualitative differences, however, were observed between the mutational spectra of nitrous acid-treated TMV and TMV-RNA. These results indicate that, in the intact virus, the viral capsid protects some of the sites involved in lethality; thus, proportionately more mutants are induced on nitrous acid treatment of TMV versus TMV-RNA.     

用不同方法把烟草花叶病毒RNA基因导入植物原生质体的研究

应用电激法和聚乙二醇法以及脂质体协调的上述两种方法对烟草和青菜原生质体进行烟草花叶病毒ＴＭＶ－ＲＮＡ的导入试验,并应用酶标免疫技术、电镜观察、半叶接种和十二烷基磺酸钠－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）等方法对在原生质体中增殖的ＴＭＶ进行鉴定。实验证明,虽然电激法和聚乙二醇法均能有效地将外源病毒基因导入植物原生质体,但经阳离子脂质体处理后的ＴＭＶ－ＲＮＡ,其转染效率可提高１０倍以上。ＴＭＶ在原生质体转染４８小时后达到复制高峰。ＳＤＳ－ＰＡＧＥ显示,原生质体转染４８小时后,除出现ＴＭＶ外壳蛋白明显条带外,尚有１条分子量在５０～５５ｋｄ蛋白质条带也明显增强。这些研究结果对植物遗传工程和抗病毒基因有种研究提供重要的数据和基础。     

Entropy-driven polymerization of ribgrass virus protein

Holmes ribgrass virus (HRV), because of serological results, is regarded as a distantly related strain of tobacco mosaic virus (TMV). HRV protein differs substantially in amino acid sequence from TMV protein, especially in that it contains one histidine residue and three methionine residues, compared to none of either for TMV protein. Ultracentrifugation and hydrogen ion titration data on HRV protein, similar to those obtained previously for the early stage polymerization of TMV and E66 proteins, demonstrated some similarities and more distinct differences from those of the other two proteins. The major similarities are that the early polymerization of HRV protein is entropy driven and the first major polymerized product is a 20 S component, presumably a double disk or two-turn helix, as in the case of the other proteins. The major differences are that the unpolymerized HRV protein sediments at 3 S rather than at the 4 S for the others; it is presumably a dimer of the polypeptide chain. The enthalpy of polymerization per mole of A protein, delta H*, is 18,400 cal for HRV protein, compared to about 30,000 for TMV protein. One mol of H+ ion/mol HRV A protein, compared to 1.5 for TMV and E66 proteins, is bound during polymerization to the 20 S state. Contrasted with the other proteins, very little if any electrical work contribution was detected for the HRV protein. A major difference was found in hydrogen ion titration. Unpolymerized HRV protein binds hydrogen ions significantly in the unpolymerized A protein state, unlike the A proteins from the other two viruses.     

Preparation and properties of recombinant DNA derived tobacco mosaic virus coat protein

Recombinant DNA derived tobacco mosaic virus (vulgare strain) coat protein (r-TMVP) was obtained by cloning and expression in Escherichia coli and was purified by column chromatography, self-assembly polymerization, and precipitation. SDS-PAGE, amino terminal sequencing, and immunoblotting with polyclonal antibodies raised against TMVP confirmed the identify and purity of the recombinant protein. Isoelectric focusing in 8 M urea and fast atom bombardment mass spectrometry demonstrated that the r-TMVP is not acetylated at the amino terminus, unlike the wild-type protein isolated from the tobacco plant derived virus. The characterization of r-TMVP with regard to its self-assembly properties revealed reversible endothermic polymerization as studied by analytical ultracentrifugation, circular dichroism, and electron microscopy. However, the details of the assembly process differed from those of the wild-type protein. At neutral pH, low ionic strength, and 20 degrees C, TMVP forms a 20S two-turn helical rod that acts as a nucleus for further assembly with RNA and additional TMVP to form TMV. Under more acidic conditions, this 20S structure also acts as a nucleus for protein self-assembly to form viruslike RNA-free rods. The r-TMVP that is not acetylated carries an extra positive charge at the amino terminus and does not appear to form the 20S nucleus. Instead, it forms a 28S four-layer structure, which resembles in size and structure the dimer of the bilayer disk formed by the wild-type protein at pH 8.0, high ionic strength, and 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biophysical characterization of a designed TMV coat protein mutant, R46G, that elicits a moderate hypersensitivity response in Nicotiana sylvestris

The hypersensitivity resistance response directed by the N' gene in Nicotiana sylvestris is elicited by the tobacco mosaic virus (TMV) coat protein R46G, but not by the U1 wild-type TMV coat protein. In this study, the structural and hydrodynamic properties of R46G and wild-type coat proteins were compared for variations that may explain N' gene elicitation. Circular dichroism spectroscopy reveals no significant secondary or tertiary structural differences between the elicitor and nonelicitor coat proteins. Analytical ultracentrifugation studies, however, do show different concentration dependencies of the weight average sedimentation coefficients at 4 degrees C. Viral reconstitution kinetics at 20 degrees C were used to determine viral assembly rates and as an initial assay of the rate of 20S formation, the obligate species for viral reconstitution. These kinetic results reveal a decreased lag time for reconstitution performed with R46G that initially lack the 20S aggregate. However, experiments performed with 20S initially present reveal no detectable differences indicating that the mechanism of viral assembly is similar for the two coat protein species. Therefore, an increased rate of 20S formation from R46G subunits may explain the differences in the viral reconstitution lag times. The inferred increase in the rate of 20S formation is verified by direct measurement of the 20S boundary as a function of time at 20 degrees C using velocity sedimentation analysis. These results are consistent with the interpretation that there may be an altered size distribution and/or lifetime of the small coat protein aggregates in elicitors that allows N. sylvestris to recognize the invading virus.     

Tobacco mosaic virus protein aggregates in solution: structural comparison of 20S aggregates with those near conditions for disk crystallization

Previous X-ray studies (2.8-A resolution) on the crystals of tobacco mosaic virus protein (TMVP) grown from solutions containing high salt have characterized the structure of the protein aggregate as a bilayered cylindrical disk formed by 34 identical subunits [Bloomer, A.C., Champness, J.N., Bricogne, G., Staden, R., & Klug, A. (1978) Nature (London) 276, 362-368]. Under low-salt conditions, 20S aggregates are in equilibrium with 4S species and involved in the efficient nucleation of TMV assembly in vitro [Butler, P.J.G. (1984) J. Gen. Virol. 65, 253-279]. We have investigated by sedimentation velocity and near-UV circular dichroism (CD) measurements the structure of 20S aggregates in low salt (I = 0.1 potassium phosphate at pH 7.0 and 20 degrees C) and the aggregates in high salt [0.2 M (NH4)2SO4 in I = 0.1 tris(hydroxymethyl)aminomethane hydrochloride at pH 8.0 and 20 degrees C, close to the conditions under which TMVP crystallizes as disk aggregates]. At high salt, we observe structures (presumably stacks of disks) having s20,w values around 40, 45, and 50 S, but not the 20S species present in low-salt buffers. The near-UV CD spectrum of 20S aggregates has been obtained for the first time, using computer techniques, from the spectra of the 4S-20S equilibrium mixture and the 4S species. This spectrum of 20S aggregates differs dramatically from that of the stacks of disks examined at both high and low salt (into which the stacks can be returned by dialysis), indicating that the difference is not a solvent effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unusual NMR, EPR, and M?ssbauer properties of Chromatium vinosum 2[4Fe-4S] ferredoxin.

The ferredoxin from Chromatium vinosum (CvFd) exhibits sequence and structure peculiarities. Its two Fe4S4(SCys)4 clusters have unusually low potential transitions that have been unambiguously assigned here through NMR, EPR, and M?ssbauer spectroscopy in combination with site-directed mutagenesis. The [4Fe-4S]2+/1+ cluster (cluster II) whose coordination sphere includes a two-turn loop between cysteines 40 and 49 was reduced by dithionite with an E degrees ' of -460 mV. Its S = 1/2 EPR signal was fast relaxing and severely broadened by g-strain, and its M?ssbauer spectra were broad and unresolved. These spectroscopic features were sensitive to small perturbations of the coordination environment, and they were associated with the particular structural elements of CvFd, including the two-turn loop between two ligands and the C-terminal alpha-helix. Bulk reduction of cluster I (E degrees ' = -660 mV) was not possible for spectroscopic studies, but the full reduction of the protein was achieved by replacing valine 13 with glycine due to an approximately 60 mV positive shift of the potential. At low temperatures, the EPR spectrum of the fully reduced protein was typical of two interacting S = 1/2 [4Fe-4S]1+ centers, but because the electronic relaxation of cluster I is much slower than that of cluster II, the resolved signal of cluster I was observed at temperatures above 20 K. Contact-shifted NMR resonances of beta-CH2 protons were detected in all combinations of redox states. These results establish that electron transfer reactions involving CvFd are quantitatively different from similar reactions in isopotential 2[4Fe-4S] ferredoxins. However, the reduced clusters of CvFd have electronic distributions that are similar to those of clusters coordinated by the CysIxxCysIIxxCysIII.CysIVP sequence motif found in other ferredoxins with different biochemical properties. In all these cases, the electron added to the oxidized clusters is mainly accommodated in the pair of iron ions coordinated by CysII and CysIV.     

Effect of dipolar ions on the entropy-driven polymerization of tobacco mosaic virus protein

The effect of the dipolar ions, glycine, glycylglycine, and glycylglycylglycine on the polymerization of tobacco mosaic virus (TMV) protein has been studied by the methods of light scattering and ultracentrifugation. All three dipolar ions promote polymerization. The major reaction in the early stage is transition from the 4 S to the 20 S state. As in the absence of dipolar ions, the polymerization is enhanced by an increase in temperature; it is endothermic and therefore entropy-driven. The effect of the dipolar ions can be understood in terms of their action as salting-out agents; they increase the activity coefficient of TMV A protein, the 4 S material, and thus shift the equilibrium toward the 20 S state. The salting-out constants, K, for the reaction in 0.10 ionic strength phosphate buffer at pH 6.7 was found by the light scattering method to be 1.6 for glycine, 2.5 for glycylglycine, and 2.5 for glycylglycylglycine. A value of 2.7 was obtained by the ultracentrifugation method for glycylglycine in phosphate buffer at 0.1 ionic strength and pH 6.8 at 10 degrees C. For both glycine and glycylglycine, K increases when the ionic strength of the phosphate buffer is decreased. This result suggests that electrolytes decrease the activity coefficient of the dipolar ions, a salting-in phenomenon. However, the salting-in constants evaluated from these results are substantially higher than those previously determined by solubility measurements. The effect of glycine and glycylglycine on polymerization was studied at pH values between 6.2 and 6.8. The effectiveness of both dipolar ions is approximately 50% greater at pH 6.8 than at pH 6.2. The variation of the extent of polymerization with pH in the presence of the dipolar ions is consistent with the interpretation that approximately one hydrogen ion is bound for half of the polypeptide units in the polymerized A protein.     

Recognition of 16S rRNA by ribosomal protein S4 from Bacillus stearothermophilus

Protein S4 is essential for bacterial small ribosomal subunit assembly and recognizes the 5' domain (approximately 500 nt) of small subunit rRNA. This study characterizes the thermodynamics of forming the S4-5' domain rRNA complex from a thermophile, Bacillus stearothermophilus, and points out unexpected differences from the homologous Escherichia coli complex. Upon incubation of the protein and RNA at temperatures between 35 and 50 degrees C under ribosome reconstitution conditions [350 mM KCl, 8 mM MgCl2, and 30 mM Tris (pH 7.5)], a complex with an association constant of > or = 10(9) M(-1) was observed, more than an order of magnitude tighter than previously found for the homologous E. coli complex under similar conditions. This high-affinity complex was shown to be stoichiometric, in equilibrium, and formed at rates on the order of magnitude expected for diffusion-controlled reactions ( approximately 10(7) M(-1) x s(-1)), though at low temperatures the complex became kinetically trapped. Heterologous binding experiments with E. coli S4 and 5' domain RNA suggest that it is the B. stearothermophilus S4, not the rRNA, that is activated by higher temperatures; the E. coli S4 is able to bind 5' domain rRNA equally well at 0 and 37 degrees C. Tight complex formation requires a low Mg ion concentration (1-2 mM) and is very sensitive to KCl concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 9.3]. The protein has an unusually strong nonspecific binding affinity of 3-5 x 10(6) M(-1), detected as a binding of one or two additional proteins to the target 5' domain RNA or two to three proteins binding a noncognate 23S rRNA fragment of the approximately same size. This binding is not as sensitive to monovalent ion concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 6.3] as specific binding and does not require Mg ion. These findings are consistent with S4 stabilizing a compact form of the rRNA 5' domain.     

Solution behavior, circular dichroism and 22 HMz PMR studies of the bovine myelin basic protein.

Bovine myelin basic protein has been investigated with regard to its solution behavior, circular dichroism and 220 MHz PMR spectral properties. At pH 4.8 gamma/2=0.1 acetate buffer, light scattering yielded a Mr of 17 700 and a virial coefficient of 1.0-10(-4) mol-ml/g2. Above pH 7.0 the protein was found to aggregate to higher mol. wt species. Sedimentation experiments at pH 4.8 yielded s degrees 20,w of 1.27 S at gamma/2=0.1 and 1.46 S at gamma/2=0.35. The diffusion coefficient determined from ultracentrifugal experiments was 7.25-10(-7) cm2/s at gamma/2=0.1 and 0.35. The value of f/f0 from diffusion at pH 4.8 and gamma/2=0.35 was 1.64, corresponding to an axial ratio of 11 to 1. The radius of gyration was calculated as 4.28 nm and the root mean square end to end distance was 10.5 nm. At pH 9.0, gamma/2=0.1, s degrees 20,w was 1.71 S and D degrees 20,w was estimated at 7.4-10(-7) cm2/s. The behavior at pH 9.0 reverted to the behavior at pH 4.8 when the pH was readjusted. The E1%/1cm=5.64 at 276.4 nm and 225 at 196 nm. Titration of the protein with trifluoroethanol elicited three distinct regions of conformation stability having increasing helical content as the mol fraction of trifluoroethanol increased. The results of the present study have permitted some comparison of analogous properties and conformational behavior with the basic membrane protein cytochrome c.     

Comparison of glucocorticoid-binding proteins in normal and neoplastic mammary tissues of the rat.

Kinetic and molecular properties of components binding [3H]triamcinolone acetonide were studied using 105,000g supernatants of lactating mammary gland, R3230AC, and dimethylbenz[a]anthracene (DMBA) induced mammary tumors of the rat. Using a dextran-coated charcoal adsorption procedure, the relationship between specific glucocorticoid binding and protein concentration was linear in the range of 0.5-4.0 mg/reaction. These cytoplasmic macromolecules bound [3H]triamcinolone acetonide with limited capacity (50-400 fmol/mg of cytosol protein) and high affinity, Kd approximately 10(-8)-10(-9) M. Optimal binding was obtained when homogenizations were made in Tris buffers, at pH 7.4, containing monothioglycerol. Time course of association of [3H]triamcinolone acetonide and its binding sites showed maximal binding by 6-8 hr at 3 degrees which remained unchanged up to 24 hr. The rate constant of association at 3 degrees was in the range of 2-4 x 10(5) M-1 min-1. The rate constant of dissociation of bound [3H]triamcinolone acetonide could not be calculated accurately since the reaction was essentially irreversible for 5 hr at 3 degrees. Estimation of the half-life of the steroid-binding protein complexes from the Kd and the rate constant for association gave a value of 11-12 hr. From ligand specificity studies, the glucocorticoids, triamcinolone acetonide, corticosterone, cortisol, and dexamethasone competed well for [3H]triamcinolone acetonide binding sites. Progesterone, aldosterone, and the anti-glucocorticoid, cortexolone, were also good competitors while androgens and estrogens were weak inhibitors of binding. The binding compenents sedimented at 7-8 S in sucrose gradients of low ionic strength and dissociated into lower molecular weight components sedimenting at 4-5S in high ionic strength gradients. Studies in vivo using animals bearing the DMBA-induced tumor demonstrated that [3H]triamcinolone acetonide binding complexes were present in cytoplasmic and nuclear compartments. Sedimentation coefficients of the cytoplasmic and nuclear forms of these receptors labeled in vivo were 7-8S and 4-5S, respectively. These studies suggest that the molecular and kinetic binding properties of glucocorticoid receptors in neoplastic mammary tissues are similar to those of the normal mammary gland.     

The subunit structure of the hemocyanin from the crayfish Jasus edwardsii.

The hemocyanin from the crayfish Jasus edwardsii(=lalandii) has been studied using ultracentrifugation, viscosity, circular dichroism and oxygen binding techniques. Sedimentation velocity experiments at pH 7.0 indicated the presence of principal species with S 20w=16.4 S, and at higher pH the presence of a species with S20,w=5.2S. Sedimentation equilibrium experiments yielded molecular weights of 490 000 and 81 000 respectively, indicating that the larger unit is a hexamer of the monomer unit. However, preliminary experiments with gel filtration and electrophoresis under denaturing conditions indicate that more than one monomer species may be present with molecular weight in the range 76-100 000. Circular dichroism (CD) spectra are presented at pH 7.0,8.6,10.0 and 11.0 for oxy-, deoxy- and apo-hemocyanins. Slight differences were observed in the magnitude of the bands in the presence or absence of Mg++. Oxygen binding studies have been made at pH 6.1,7.0,8.8 and 10.6, in the presence of 0.01 M MgCl2. The extent of cooperative binding was indicated by a maximum value of n=3.7, and a pronounced bohr effect was observed.     

Isolation of Polyribosome from Tobacco Plants Infected with Tobacco Mosaic Virus

Polyribosomes have been isolated from tobacco leaves. Upon infection with TMV, a new polyribosome has appeared which is specific for infection, and TMV-antigenic protein is formed on this polyribosome. This new polyribosome has an S-value of 360–380. From these results, it is suggested that this TMV specific polyribosome is an aggregate of 60–80 ribosomes which is bound by TMV-RNA as messenger, and that TMV-RNA is translated polycistronically.