Douglas G. Scraba

Prevention of phospholipase‐C induced aggregation of low density lipoprotein by amphipathic apolipoproteins

Phospholipase C (PL‐C) digestion of human low density lipoprotein (LDL) results in hydrolytic cleavage of the phosphocholine head group of phosphatidylcholine, thereby generating diacylglycerol. Loss of amphiphillic surface lipids and/or accumulation of diacylglycerol causes LDL samples to develop turbidity. Examination of PL‐C treated LDL by electron microscopy revealed a progressive aggregation of LDL as a function of phosphatidylcholine hydrolysis: fused particles, clusters, and multiple stacked aggregates were observed. Lipid analysis of untreated and aggregated LDL confirmed that the phosphatidylcholine content of the latter had decreased with a corresponding increase in diacylglycerol. It is likely that phospholipolysis created hydrophobic gaps within the surface monolayer of LDL, thereby inducing LDL fusion and aggregation. When amphipathic α‐helix‐containing apolipoproteins, such as human apoA‐I or Manduca sexta apolipophorin III (apoLp‐III) were present, PL‐C treated LDL did not aggregate. Compositional analysis of apolipoprotein‐containing PL‐C LDL showed that phospholipolysis was not affected by the presence of apolipoproteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of lipoproteins re‐isolated following incubation with PL‐C revealed an association of apoA‐I or apoLp‐III with PL‐C digested LDL. Electron microscopy showed no major morphological differences between native LDL and apoprotein stabilized PL‐C treated LDL and the average particle diameter of apoA‐I stabilized PL‐C LDL was 22.5 ± 0 2.2 nm versus 22.8 ± 1.6 nm for control LDL. Incubation of tritium‐labeled apoLp‐III with LDL and PL‐C demonstrated that association of apoLp‐III with PL‐C LDL correlated with the extent of phospholipid hydrolysis, the apolipoproteins apparently being recruited to compensate for the increased hydrophobic surface created by conversion of phosphatidylcholine into diacylglycerol. The results suggest that transient association of amphipathic apolipoproteins with damaged or unstable LDL may provide a mechanism to obviate formation of atherogenic LDL aggregates in vivo.

Structure of the Mengo virion: I. Polypeptide and ribonucleate components of the virus particle

Abstract The genome of Mengo virus, “35 S” RNA, is a single polynucleotide chain having a molecular weight of 2.44 ± 0.08 × 10 6 daltons. This value has been determined from studies using the methods of polyacrylamide gel electrophoresis, sedimentation in dimethyl sulfoxide, sedimentation after reaction with formaldehyde and electron microscope measurements of the viral replicative form. SDS-polyacrylamide gel electrophoresis of solubilized purified virus revealed the presence of four major polypeptides (α, β, γ, and δ) having estimated molecular weights of 32,500, 29,600, 23,700, and 10,600 daltons, respectively. In addition, two minor components were detected. The first (D2) has a molecular weight of 57,700 daltons and the second (ϵ), a molecular weight of 39,000 daltons.

Recovery of SDS-proteins from polyacrylamide gels by electrophoresis into hydroxylapatite.

Abstract A method is described which permits the purification of proteins in quantities necessary for physicochemical characterization. The protein to be purified is separated from contaminants by electrophoresis in a number of sodium dodecyl sulfate (SDS)-containing polyacrylamide disc gels. The region of each gel which contains the desired protein band is then cut out and the slices are stacked one above another in a tube. After the protein is electrophoresed from the gel slices into a bed of hydroxylapatite, it is eluted from the hydroxylapatite with 0.5 m sodium phosphate (pH 6.4) containing 0.1% SDS and 1 m m dithiothreitol. Protein recovery is greater than 90%.

Structure of the mengo virion V. Distribution of the capsid polypeptides with respect to the surface of the virus particle

Abstract The disposition of the Mengo capsid polypeptides (α, β, γ, and δ) with respect to the external surface of the virion has been investigated by measuring their relative susceptibilities to lactoperoxidase-catalyzed iodination and their reactivities in immunological tests with specific antisera. When intact virions were subjected to iodination for a brief period of time (1 min), radioactive iodine was incorporated predominantly into the a polypeptides and to a lesser extent into β polypeptides. Only with longer incubation times (15 min or more) did label appear in the γ and δ polypeptides; and this coincided with a progressive loosening and ultimate collapse of the viral capsid. Antisera specific for each of the capsid polypeptide species were produced in rabbits using isolated proteins as antigens. Reaction of virions with these antisera in plaque-neutralization and hemagglutination-inhibition tests showed that only the anti-α serum was capable of blocking virus-cell interactions. Complement-fixation and immunodiffusion tests confirmed the observations that the α polypeptides occupy most of the external surface of the virus particle and that the β polypeptides are partially exposed. The γ and δ polypeptides apparently occupy internal locations in the capsid of the Mengo virion.

Physico-chemical studies of three variants of Mengo virus and their constituent ribonućleates☆

Methods are described for the isolation in pure form of three variants of Mengo virus and their constituent ribonucleates. The resulting preparations have been subjected to a detailed physico-chemical investigation using the techniques of sedimentation velocity, diffusion at low centrifugal fields (method of Moller, 1964), optical rotatory dispersion and electron microscopy. It was established that all three variants are identical with respect to the measured hydro dynamic parameters (S20,w0 and D20,w0), and a similar conclusion was drawn for their RNA constituents. The particle weight of the virus, as estimated from the Svedberg equation, is 8.32 ± 0.7 × 106, while that of the RNA is 1.74 ± 0.2 × 106. In the electron microscope the three Mengo variants are indistinguishable from each other and appear as spherical particles with diameters of 26.2 to 27.2 mμ. The hydrated diameter calculated from the experimental diffusion coefficient is 29.3 mμ, which is consistent with a hydrated sphere having 0.23 g water per gram dry virus. The frictional ratio of the virus particle, estimated from S20,w0 and D20,w0, is 1.10, which also suggests that it is essentially spherical in shape. A comparison of the optical rotatory dispersion of the three variants of Mengo virus and their constituent ribonucleates, shows that the curves are essentially identical to one another, with similar cross-over points, positions of troughs and peaks, and amplitudes. This would suggest that the manner in which the proteins are arranged around the RNA chain in the virus is precise and similar in all three cases. By subtracting the contribution of the RNA from that of the virus, the optical rotatory dispersion curve of the protein in situ was obtained. The small amplitude of [m′]234 suggests that the protein in its native environment possesses negligible α-helical content.

The role of bacteriophage T7 exonuclease (gene 6) in genetic recombination and production of concatemers

Abstract Genetic analysis reported here shows that bacteriophage T7 exonuclease (gene 6) is necessary for intragenic and intergenic recombination in several areas of the T7 genetic map. This supports our previous conclusion ( Lee & Miller, 1974 ) that the enzyme is necessary for T7 molecular recombination. Results of sucrose gradient analysis show that DNA concatemers are formed when both the T7 exonuclease (gene 6) and the T7 endonuclease (gene 3) are absent. Further results show that concatemers cannot be maintained in the absence of the exonuclease unless the endonuclease is also eliminated. Therefore, concatemers are formed by a process other than normal phage recombination. Selective defects in the recombination system do interfere with the stability of concatemers, however.

Structure of the mengo virion: VI. Spatial relationships of the capsid polypeptides as determined by chemica cross-linking analyses

Abstract The asymmetric structure unit of the Mengo virus capsid comprises one molecule each of the polypeptides α, β, and γ. Structure units are clustered in pentamers which are centered at each of the 12 vertices of a T = 1 icosahedral lattice (Mak et al., 1974). The location of the ∼-60 δ polypeptides in the capsid has not been established. In order to obtain information about the arrangement of α, β, and γ in the structure unit and to determine which polypeptides participate in the noncovalent interactions responsible for pentamer formation and capsid stabilization, virions were reacted with bifunctional crosslinking reagents and the polypeptide complexes produced were identified by gel electrophoresis. Using the reversible crosslinkers dimethylsuberimidate (DMS) and dithiobis(succinimidyl propionate) (DSP), positive identification of βγ, αγ, αβγ, α2, α3, and α4 complexes was made. Complexes involving S were not detected, nor were βn or γn. The latter observation indicated that the hydrophobic interactions among αβψ structure units in a pentamer involve α-α contacts. When virions crosslinked with DMS, DSP, or dimethyladipimate (DMA) were subsequently dissociated by 0.1 M NaCl at pH 6 and examined in the electron microscope, it was shown that only treatment with DSP prevented complete capsid dissociation. Since DSP crosslinking alone produced αβ complexes, it was concluded that the interactions between adjacent pentamers probably result from α-β contacts. Treatment of Mengo virions with formaldehyde produced crosslinks between β and γ polypeptides and the RNA. Based upon these data, a model for the organization of individual polypeptide species within the Mengo virus capsid is presented.

Structure of the Mengo virion. IV. Amino- and carboxyl-terminal analyses of the major capsid polypeptides.

Abstract Improvements have been made in the procedure for isolating each of the major capsid polypeptides of Mengo virus by chromatography on hydroxylapatite in the presence of sodium dodecyl sulfate (SDS). These polypeptides have been subjected to N- and C-terminal analyses. The δ polypeptide appears to have a blocked amino terminal, while the N-terminal residues of α, β, γ are glycine, aspartate, and serine, respectively. In addition, the first 10 amino acid residues in the α, β, and γ polypeptides have been sequenced by an automated Edman procedure ( Edman and Begg, 1967 ). The carboxyl-terminal amino acids in the δ, α, and β polypeptides are alanine, leucine, and glutamine, respectively, while the C-terminal of γ is probably glutamine. Based upon this information, and information from other laboratories regarding the N- and C-terminal amino acids of the polio and foot-and-mouth disease virus capsid proteins, a model for the post-translational processing of picornaviral structural polypeptides is presented. The model envisions the participation of a virus-coded protease with a specificity for peptide bonds involving the carbonyl function of glutamine residues.

A comparative study of BK and polyoma viruses

Abstract A comparative study of BK and polyma (Py) viruses has shown that the molecular anatomy of the two virions is strikingly similar. The capsids of both are composed of 72 capsomeres arranged in a T = 7d icosahedral lattice. Analysis of the DNAs of the two viruses suggest that the full-length DNA of BK virus may be slightly smaller than that of Py virus. The two virions contain the same number of structural polypeptides, but the molecular mass of BK-VP1 is about 4000 daltons less, and those of BK-VP2 and 3 about 4000 daltons more than those of the corresponding Py polypeptides. The observation that the major capsid polypeptide, BK-VP1, is smaller than Py-VP1, is compatible with the observed difference in the diameters of the two virions (BK = 405 ± 10A; Py = 430 ± 15A). The two viruses differ sharply in the relative efficiencies with which they hemagglutinate guinea pig and human erythrocytes. BK virus, unlike Py virus, replicates only in certain human or monkey cells; and of the cells examined in this study, human fetal kidney cells are the most satisfactory for the propagation of this agent.

Isolation and characterization of the tubular organelles induced by fumarate reductase overproduction in Escherichia coli.

Strains of Escherichia coli amplifying the intrinsic membrane enzyme fumarate reductase accommodate the overproduced enzyme by increasing the amount of membrane material, in the form of intracellular tubular structures. These tubules have been observed in strains harbouring multicopy frd plasmids and in ampicillin hyper-resistant strains. A procedure has been developed for isolation of tubules nearly free of cytoplasmic membrane. Using protein A-gold labelling and optical diffraction of electron micrographs, a model for tubule structure is proposed. The tubules have a lower lipid/protein ratio than the cytoplasmic membrane, with the enzyme accounting for greater than 90% of the protein in the tubules. Both cytoplasmic membranes and tubules from amplified strains are enriched in cardiolipin and have a more fluid fatty acid composition than wild-type strains. Mutants defective in cardiolipin synthesis produce tubules in response to excess fumarate reductase, but these tubules have an altered appearance, indicating that lipid-protein interactions may be important for tubule assembly.