The four structural proteins of bacteriophage PM2 have been related to the morphological structures in the virion. Protein I can be selectively removed from the virus by degradation with bromelain, a proteolytic enzyme. From electron microscopic studies and from experiments showing that the virus cannot attach to its host cell after bromelain treatment, we conclude that protein I forms the outer spikes at the vertices of the icosahedron. Protein I1 can be labeled with [35S]sulfanilic acid diazonium salt. As shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, proteins I and I1 can be removed if the virus is treated with 1 M urea. From the above studies and from packing considerations, we conclude that protein I1 forms the outer shell of the virus.The dissociation of the virus in 1 M urea leads to: (a) a nucleocapsid core containing viral lipid, DNA, protein 111 and IV, (b) a second fraction which consists of the same elements obtained by treatment of the virus with bromelain. Both particles have been identified by electron microscopy.The dissociation of the virus in 1 M urea plus 0.5 Triton X-100, a nonionic detergent, leads to a third fraction representing an empty shell which contains all of the lipid, proteins I1 and 111, and about 5076 of protein IV, but no DNA. We have also been able to isolate a second type of nucleocapsid core by dissociation of the virus in the presence of 4.5 M urea. This core, which has an approximate sz0,.+ = 133 S and which has been identified in the electron microscope as a core with icosahedral shape, contains proteins 111 and IV, as well as the DNA, but not phospholipid.In the presence of 4.5 M urea this core is resistant to Triton X-100 concentrations up to 0.1 %.Higher concentrations of the nonionic detergent dissociate the core into DNA and the solubilized proteins I11 and IV. In the absence or in the presence of low concentrations of urea (1 M) the core is resistant to TritonX-100 concentrations up to 0.5%. The DNA in the core is digestible by pancreatic DNAase I and the core structure is broken down after DNAase treatment. Protein IV interacts with the DNA as shown by transformation of non-covalent interactions between DNA and protein into covalent bonds induced by a nucleophilic reagent, In conclusion, protein I forms the spikes, protein I1 the outer icosahedral shell and protein I11 the inner icosahedral shell around the DNA, which is closely associated with protein IV. The phospholipid bilayer is sandwiched between these two shells. Protein I11 may also form the protein bridges to protein I which connect the inner and outer shells through the bilayer.Extensive studies on the molecular biology of the lipid-containing bacteriophage PM2 were initiated by Espejo and Canelo [1,2]. This bacteriophage was isolated from the Pacific Ocean and the host organism is a marine pseudomonad, Pseudomonas BAL-31 [3]. Since PM2 is readily dissociated by ethyl ether or ionic detergents [l], it is reasonable to propose that the integrity of the virus depends o...
The lipid-containing bacteriophage PM2 has four distinct structural proteins, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate or electrophoresis on cellulose acetate strips in the presence of 6-8 M urea. The molecular weights of these four polypeptides determined in several polyacrylamide gel systems are: I = 43000, I1 = 26000-27000, 111 = 12500, IV = 4700. The isoelectric points of the four viral proteins were determined on disrupted virus particles by electrophoresis on cellulose acetate strips in the presence of 6 M urea at various pH values. The mobilities were extrapolated to zero mobility, a method preferable to isoelectric focusing where the denatured viral proteins aggregate and precipitate near the isoelectric point. The method is well suited for the determination of the isoelectric point of proteins in the native or only partially denatured state. Whereas proteins I, 111 and IV have isoelectric points slightly under neutrality (pH 6.2, 5.8 and 5.5, respectively), the isoelectric point of protein I1 is around 12.3. Calcium ions are tightly bound to protein 11. After removal of the calcium ions in the presence of 6 M guanidine hydrochloride and EDTA, the p l of protein I1 is 9. The isoelectric point of the intact virus, determined on cellulose strips, is around 7.3. Protein I, the spike protein, is the only structural polypeptide which is soluble in aqueous solutions around pH 7. All of the proteins are soluble in 6-8 M urea or 6 M guanidine hydrochloride at pH 7-8.5. Proteins 111 and IV, the nucleocapsid core proteins, have the solubility properties of proteolipids or highly lipophilic polypeptides as shown by their solubility in nonionic detergents and by their distribution in a Bligh and Dyer fractionation. Neither glycolipids nor glycoproteins are present in the virus. The distribution of phospholipids has been determined by chemical modification of the outer lamella or outer and inner lamellae of the bacteriophage lipid bilayer. The viral lipid bilayer is asymmetric ; most of the phosphatidylglycerol residues form the outer lipid layer, most of the phosphatidylethdnolamine residues the inner lipid layer.A complete model of the virus, including the assembly process, is discussed at the end of this report.In the previous report [ l ] we described the correlation of the individual proteins of the lipid-containing bacteriophage PM2 with its structure and substructure. In this paper we estimate the number and physical properties of the viral polypeptides, look at the distribution of the lipids in the bilayer and discuss a model of the virus and possible assembly processes.As described by Datta et af.[2], bacteriophage PM2 has four structural proteins with molecular weights of I = 43000, I1 = 34000, 111 = 12500 and IV = 7700-4650 (the higher value was obtained This is paper number XV in the series. Abbreviations. PI, isoelectric point; p.f.u., plaque-forming unit; ESR. electron spin resonance. from gel electrophoresis and the lower from gel filtration in the presenc...
Ca2+ uptake into Ehrlich ascites tumor cells was studied at 0 degrees C in the presence of mitochondrial inhibitors, conditions that minimized complications caused by sequestration of Ca2+ into organelles or by excretion. Under these conditions Ruthenium Red inhibited Ca2+ uptake, but other previously implicated ions, such as Pi or Mg2+, had no effect. Valinomycin either inhibited or slightly stimulated Ca2+ uptake depending on the presence of excess K+ on the outside or inside of the cell, respectively. Nigericin inhibited Ca2+ transport. Based on these data we propose an electrogenic uptake of Ca2+, possibly via a Ca2+/H+ antiport mechanism. The observation that glucose inhibited Ca2+ uptake suggested that in Ehrlich ascites tumor cells an energy-driven Ca2+ expulsion mechanism is operative, similar to that in erythrocytes. Plasma membrane preparations of ascites tumor cells were found to contain a Ca2+-dependent ATPase. These preparations, when incorporated into liposomes in an inside-out orientation, catalyzed an ATP-dependent uptake of Ca2+.
The four structural proteins of the lipid-containing bacteriophage PM2 have been purified by dissociation of the virus in the presence of acetic acid followed by a combination of gel filtration and ion-exchange chromatography in the presence of sodium dodecylsulfate and guanidine hydrochloride. Amino acid analyses of each of the proteins were performed and correlated with the properties and functions of the proteins. Protein I has the highest polarity and is the only watersoluble protein. Protein I1 has a rather high polarity and hydrophobicity index and probably interacts electrostatically and hydrophobically with the bilayer. Proteins I11 and IV have low polarities and possess the solubility properties of proteolipids. At least protein 111 and perhaps also protein IV may interact with the bilayer. No fatty acids are covalently linked to these proteins. Tryptic fingerprints showed that proteins I and I1 contain a high proportion of hydrophobic peptides, but especially protein I also contains a large number of hydrophilic peptides. Proteins 111 and IV have relatively few hydrophobic peptides despite their relatively high hydrophobicity. Protein IV has two distinct regions, as shown by partial sequence studies. Basic amino acids at the N-terminus would serve for interaction with the viral DNA, the following hydrophobic sequence might interact with protein 111 or with the bilayer.
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