Disulfide bond engineering to monitor conformational opening of apolipophorin III during lipid binding.

Apolipophorin III (apoLp-III) from the Sphinx moth, Manduca sexta, is an exchangeable, amphipathic apolipoprotein that alternately exists in water-soluble and lipid-bound forms. It is organized as a five-helix bundle in solution, which has been postulated to open at putative hinge domains to expose the hydrophobic interior, thereby facilitating interaction with the lipoprotein surface (Breiter, D. R. , Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603-608). To test this hypothesis, we engineered two cysteine residues in apoLp-III, which otherwise lacks cysteine, by site-directed mutagenesis at Asn-40 and Leu-90. Under oxidizing conditions the two cysteines spontaneously form a disulfide bond, which should tether the helix bundle and thereby prevent opening and concomitant lipid interaction. N40C/L90C apoLp-III was overexpressed in Escherichia coli and characterized for disulfide bond formation, secondary structure content, and stability, under both oxidizing and reducing conditions. Functional characterization was carried out by comparing the abilities of the oxidized and reduced protein to associate with modified lipoproteins in vitro. While the reduced form behaved like wild type apoLp-III, the oxidized form was unable to associate with lipoproteins. These results suggest that opening of the helix bundle is required for interaction with lipoproteins and provide a molecular basis for the dual existence of water-soluble and lipid-bound forms of apoLp-III. However, in phospholipid bilayer association assays, wild type, reduced, and oxidized N40C/L90C apoLp-III exhibited similar abilities to transform dimyristoylphosphatidylcholine multilamellar vesicles to disc-like complexes, as judged by electron microscopy. These data emphasize that underlying differences exist in initiating or maintaining a stable interaction of apoLp-III with phospholipid disc complexes versus spherical lipoprotein surfaces.

Branched structures in the intracellular DNA of herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) replication produces large intracellular DNA molecules that appear to be in a head-to-tail concatemeric arrangement. We have previously suggested (A. Severini, A.R. Morgan, D.R. Tovell, and D.L.J. Tyrrell, Virology 200:428-435, 1994) that these DNA species may have a complex branched structure. We now provide direct evidence for the presence of branches in the high-molecular-weight DNA produced during HSV-1 replication. On neutral agarose two-dimensional gel electrophoresis, a technique that allows separation of branched restriction fragments from linear fragments, intracellular HSV-1 DNA produces arches characteristic of Y junctions (such as replication forks) and X junctions (such as merging replication forks or recombination intermediates). Branched structures were resolved by T7 phage endonuclease I (gene 3 endonuclease), an enzyme that specifically linearizes Y and X structures. Resolution was detected by the disappearance of the arches on two-dimensional gel electrophoresis. Branched structures were also visualized by electron microscopy. Molecules with a single Y junction were observed, as well as large tangles containing two or more consecutive Y junctions. We had previously shown that a restriction enzyme which cuts the HSV-1 genome once does not resolve the large structure of HSV-1 intracellular DNA on pulsed-field gel electrophoresis. We have confirmed that result by using sucrose gradient sedimentation, in which both undigested and digested replicative intermediates sediment to the bottom of the gradient. Taken together, our experiments show that the intracellular HSV-1 DNA is held together in a large complex by frequent branches that create a network of replicating molecules. The fact that most of these branches are Y structures suggests that the network is held together by frequent replication forks and that it resembles the replicative intermediates of bacteriophage T4. Our findings add complexity to the simple model of rolling-circle DNA replication, and they pose interesting questions as to how the network is formed and how it is resolved for packaging into progeny virions.

Induction of ferritin synthesis in cells infected with Mengo virus.

We have recently identified ferritin as a cellular protein particle whose synthesis is stimulated in mouse or human cells infected by the picornavirus Mengo. Immunoprecipitation of the particle from infected murine L929 cells showed a 4- and 6-fold increase in the intracellular concentrations of H and L apoferritin subunits, respectively. This differential expression altered the H/L subunit ratio from 3.0 in uninfected cells to 2.2 in Mengo virus-infected cells. The induction is not due to an increase in transcription of the apoferritin L and H genes, nor is it due to an increase in stability of the apoferritin mRNAs. At the level of translation, the iron regulatory protein (IRP) remained intact, with similar amounts being detected in uninfected and infected cells. The Mengo virus RNA genome does not compete with the iron regulatory element (IRE) for the binding of IRP, and sequence analysis confirmed that there are no IREs in the virus RNA. The IRE binding activity of IRP in infected cells decreased approximately 30% compared with uninfected cells. The decrease in binding activity could be overcome by the addition of Desferal (deferoxamine mesylate; CIBA) an intracellular iron chelator, which suggests that virus infection causes an increase in intracellular free iron. Electron paramagnetic resonance (EPR) studies have confirmed the increase in free iron in Mengo virus infected cells. The permeability of cells for iron does not change in virus infected cells, suggesting that the induction of ferritin by Mengo virus is due to a change in the form of intracellular iron from a bound to a free state.

Characterization of Mengo virus neutralization epitopes. II. Infection of mice with an attenuated virus.

A panel of five neutralizing monoclonal antibodies was generated from mice immunized with an attenuated strain of Mengo virus. Four of the antibodies were used to select mutants of Mengo virus which were able to escape neutralization by the selecting antibody, but it was not possible to select mutants which could escape neutralization by the fifth antibody. The capsid coding region of the RNA genome of each mutant was directly sequenced to identify the mutation(s) responsible for the neutralization escape phenotype. These results are compared to those of a previous study in which immunogenic determinants recognized by neutralizing antibodies generated against pentameric capsid subunits were located on the external surface of the Mengo virion. We have confirmed the existence of the previously identified immunogenic determinant in VP3 (site 2) as well as an immunodominant determinant in VP2 (site 1). Two previously uncharacterized determinants, located in surface loops of VP1 (sites 3 and 4A), were also identified. None of the mutations conferring the neutralization escape phenotype was found near the surface depressions on the virion which are believed to be the receptor binding sites.

Spectroscopic and lipid binding studies on the amino and carboxyl terminal fragments of Locusta migratoria apolipophorin III.

The structural basis for the lipid binding capability of Locusta migratoria apolipophorin III (apoLp-III) was assessed by characterizing the amino and carboxyl terminal halves of the protein. The native molecule (approximately 20 kDa) was deglycosylated with endoglycosidase F (molecular mass of deglycosylated species approximately 18 kDa) and cleaved with endoproteinase Arg-C to yield two fragments with molecular masses of approximately 9 kDa each. The two fragments were purified by reversed-phase HPLC and identified by mass spectrometry, amino acid analysis and N-terminal sequencing as the amino terminal (N9) and carboxyl terminal (C9) halves. Due to the apparent discrepancy of the protease digestion pattern obtained compared to that expected from the deduced amino sequence of apoLp-III cDNA, we carried out partial amino acid sequencing of the fragments and cDNA sequencing for the entire protein. Circular dichroism spectroscopy of the N9 and C9 peptides revealed that both exist in buffer in a random coil state. However, addition of trifluoroethanol, a helix-inducing agent, resulted in the formation of an alpha-helix, reflecting an innate propensity of the peptides to adopt a helical conformation. When cosonicated with dimyristoylphosphatidylcholine (DMPC) both peptides assumed an alpha-helical conformation, indicative of interaction with the phospholipid. In the presence of phospholipids, a 22 nm blue shift in Trp fluorescence emission was observed in the case of the C9 peptide, suggesting that the Trp residues are located in a more hydrophobic environment. Electron microscopy revealed that, compared to native apoLp-III, both peptides possessed a reduced ability to transform DMPC vesicles to disklike complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

A stable interaction between separated pyrimidine.purine tracts in circular DNA.

Pyrimidine.purine tracts are widespread in eukaryotic genomes and have the potential to form a number of unusual structures including triplexes. Two such tracts, which could form triplexes with each other but not with themselves, were cloned into a plasmid at separate sites. Upon lowering the pH, linear, open circular and relaxed plasmid molecules formed a number of novel structures that were observed on agarose gels and directly by electron microscopy. In open circles a stable join was formed between the two Pyr.Pur tracts giving rise to molecules resembling dumbells, trefoils and tetrafoils, which collectively are termed T-loops. The structure was stable at pH 8 and contains a single-stranded region that was sensitive to P1 nuclease. Thus, there is no apparent topological impediment to the formation of triplex-mediated loops in circular molecules. These structures may be important for gene regulation and chromosome condensation.

Studies on the binding of integration host factor (IHF) and TraM to the origin of transfer of the IncFV plasmid pED208.

The origin of transfer (oriT) of the IncFV plasmid pED208 contains a region with three binding sites for both the plasmid-encoded TraM protein and the integration host factor (IHF) of Escherichia coli, a sequence-specific DNA-binding protein. One region, containing overlapping TraM and IHF binding sites, could be interpreted as containing two binding sites for each protein. Using gel retardation assays, an affinity constant for IHF binding to the three main sites was estimated in the presence and absence of 0.1 M potassium glutamate, which increased the avidity of IHF binding to the weaker sites by two orders of magnitude. DNase I protection analyses and electron microscopy were used to determine the affinity of IHF for oriT-containing DNA in the presence and absence of TraM. The binding of IHF and TraM was found to be non-cooperative by the two techniques employed. Electron microscopy also demonstrated that IHF bent the oriT region in a manner consistent with its previously determined mode of action, while TraM had no discernible effect on the appearance of the DNA. This suggested that IHF and TraM interact with a 295 bp sequence in the oriT region and organize it into a higher order structure that may have a role in the initiation of DNA transfer and control of traM expression.

Identification of Mengo virus T helper cell epitopes.

To identify Mengo virus-specific T cell epitopes in mice (the natural host for the virus), lymph node cells were obtained from BALB/c (H-2d) mice, previously immunized with u.v.-inactivated virus, and stimulated in vitro with each of 116 overlapping peptides (10 to 18 residues long) covering the entire capsid coding region (834 amino acids). T cell epitopes were defined on the basis of specific peptide-induced lymphocyte proliferation. Where proliferation occurred, immunological characterization showed that it was the CD4+ T helper (Th) cell subpopulation that was responsible for the Mengo virus-specific response. Surprisingly, no Mengo virus Th cell epitopes were found in capsid protein VP1 or VP4. Six peptides in VP2 (residues 1 to 15, 99 to 108, 118 to 132, 133 to 147, 227 to 236 and 247 to 256) identified the positions of separate Th cell epitopes, and two overlapping peptides (residues 173 to 182 and 178 to 192) defined an additional Th cell immunogenic sequence. Three individual peptides in VP3 (residues 46 to 58, 136 to 150 and 198 to 212) and two overlapping peptides (residues 1 to 15 and 11 to 20) also represent Th cell epitopes. Similar assays with C57BL/6 (H-2b) and SJL/J (H-2s) mice showed that the pattern of recognition of these peptides was H-2 restricted. Each of the previously identified sites of B cell antigenicity in VP2 and VP3 are associated with one Th epitope. Comparison of the experimentally determined Th epitopes with potential T cell epitopes identified by several predictive strategies revealed only a low correlation between authentic and predicted epitopes.

Effect of phospholipase C and apolipophorin III on the structure and stability of lipophorin subspecies.

Four distinct subspecies of the insect hemolymph lipoprotein, lipophorin, that range in diacylglycerol (DAG) content from approximately 100 to 1000 molecules per particle, were treated with phospholipase C. Lipid analysis demonstrated that both phosphatidylcholine and phosphatidylethanolamine were hydrolyzed to DAG. Phospholipase C was used to remove 74-82% of the phospholipid of different lipophorins and these were analyzed for aggregation. Low density lipophorin (LDLp), the largest subspecies, with a diameter of approximately 23 nm, developed turbidity (monitored by sample absorbance at 340 nm) suggesting the formation of lipoprotein aggregates. High density lipophorin-adult (HDLp-A) and high density lipophorin-wanderer 1 (HDLp-W1) also displayed an increase in A340 when incubated with phospholipase C, although the maximal increase observed was considerably less than that for LDLp on a per particle basis. Phospholipase C caused only a minimal increase in A340 in a fourth subspecies, high density lipophorin-wanderer 2 (HDLp-W2), which contains an even lower amount of DAG. Electron microscopy was used to evaluate changes in particle morphology as a result of phospholipid depletion. HDLp-W2 and HDLp-W1 showed signs of progressive aggregation and particle fusion. A similar aggregation/fusion was seen in the case of high density lipophorin adult (HDLp-A) while LDLp samples contained multiple aggregation/fusion foci and resultant very large particles. In the presence of exogenous apolipophorin III (apoLp-III), phospholipase C-induced lipophorin aggregation/fusion was prevented. Electron microscopy of LDLp and HDLp-A samples revealed that apoLp-III-stabilized, phospholipase C-treated particles had a morphology similar to that of control particles.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of the U-particle, a cellular constituent whose synthesis is stimulated by Mengovirus infection.

We have isolated a cellular protein particle whose synthesis is induced by infection with Mengovirus or TMEV. The U-particle inhibits translation in vitro and binds to both capped and uncapped mRNA's. It is spherical, 12 nm in diameter, and is composed of multiple copies of two polypeptide subunits having molecular weights of 23,000 and 25,000 which do not appear to be glycosylated or phosphorylated. U-particles are capable of inhibiting mRNA translation in vitro.

The cellular U-particle, whose synthesis is induced by mengovirus infection, is homologous to apoferritin.

Mengo virus infection of mouse L-cells results in induction of the synthesis of a cellular protein-containing particle, 12 nm in diameter, which was designated U (Boege et al. (1987) Virology 159, 358-367). We have purified the U-particle from virus-infected cells by a series of chromatographic steps and found it to be composed of two polypeptide species (MW 23,000 and 25,000), present in a ratio of approximately 7:3. Neither of these polypeptides is measurably glycosylated or phosphorylated and the U-particle contains no detectable nucleic acid. Several amino acid sequences obtained from CNBr fragments of the U-polypeptides identified them as the H- and L-chains of mouse apoferritin. This finding was supported by immunoblotting and electron microscopy. In terms of function, the U-particle/apoferritin effectively inhibits the translation of mRNAs in reticulocyte lysates. These experiments indicate that apoferritin may perform important functions in eukaryotic cells in addition to iron storage. Finally, we propose mechanisms to explain how Mengo virus infection could specifically induce the synthesis of apoferritin and how increasing amounts of cytoplasmic apoferritin could facilitate virus replication.

Impaired biosynthesis of phosphatidylcholine causes a decrease in the number of very low density lipoprotein particles in the Golgi but not in the endoplasmic reticulum of rat liver.

We have investigated the mechanism by which inhibition of phosphatidylcholine biosynthesis in rat hepatocytes by choline deprivation causes a reduction in the secretion of very low density lipoprotein (VLDL) (Yao, Z., and Vance, D. E. (1988) J. Biol. Chem. 263, 2998-3004). Rats ingested a choline-deficient or control diet for 3 days, and subcellular fractions of liver were prepared. No change in the amount of apolipoprotein B in the lumina of the endoplasmic reticulum was observed, but there was a 40-50% decrease of apolipoprotein B in the lumina of the Golgi from choline-deficient compared with control rats. Incubation of microsomes, derived from choline-deficient and -supplemented hepatocytes, with trypsin showed similar degradation of apolipoprotein B, indicating similar quantities of this protein are present on the surface and within the lumina. The VLDL particles in the Golgi of liver cells and in plasma, on average, were larger in samples derived from choline-deficient compared with choline-supplemented animals. Incubation of plasma VLDL with proteases demonstrated that the apolipoprotein B of plasma VLDL particles from choline-deficient animals had a different susceptibility to digestion than did VLDL from choline-supplemented animals. These data indicate that the number of VLDL particles assembled in the endoplasmic reticulum of liver is similar in choline-deficient and -supplemented rats, but the number of particles is decreased in the Golgi from choline-deficient animals.

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 alpha-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 +/- 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.

Conversion of human low density lipoprotein into a very low density lipoprotein-like particle in vitro.

The lipid substrate specificity of Manduca sexta lipid transfer particle (LTP) was examined in in vitro lipid transfer assays employing high density lipophorin and human low density lipoprotein (LDL) as donor/acceptor substrates. Unesterified cholesterol was found to exchange spontaneously between these substrate lipoproteins, and the extent of transfer/exchange was not affected by LTP. By contrast, transfer of labeled phosphatidylcholine and cholesteryl ester was dependent on LTP in a concentration-dependent manner. Facilitated phosphatidylcholine transfer occurred at a faster rate than facilitated cholesteryl ester transfer; this observation suggests that either LTP may have an inherent preference for polar lipids or the accessibility of specific lipids in the donor substrate particle influences their rate of transfer. The capacity of LDL to accept exogenous lipid from lipophorin was investigated by increasing the high density lipophorin:LDL ratio in transfer assays. At a 3:1 (protein) ratio in the presence of LTP, LDL became turbid (and aggregated LDL were observed by electron microscopy) indicating LDL has a finite capacity to accept exogenous lipid while maintaining an overall stable structure. When either isolated human non B very low density lipoprotein (VLDL) apoproteins or insect apolipophorin III (apoLp-III) were included in transfer experiments, the sample did not become turbid although lipid transfer proceeded to the same extent as in the absence of added apolipoprotein. The reduction in sample turbidity caused by exogenous apolipoprotein occurred in a concentration-dependent manner, suggesting that these proteins associate with the surface of LDL and stabilize the increment of lipid/water interface created by LTP-mediated net lipid transfer. The association of apolipoprotein with the surface of modified LDL was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and scanning densitometry revealed that apoLp-III bound to the surface of LDL in a 1:14 apoB:apoLp-III molar ratio. Electron microscopy showed that apoLp-III-stabilized modified LDL particles have a larger diameter (29.2 +/- 2.6 nm) than that of control LDL (22.7 +/- 1.9 nm), consistent with the observed changes in particle density, lipid, and apolipoprotein content. Thus LTP-catalyzed vectorial lipid transfer can be used to introduce significant modifications into isolated LDL particles and provides a novel mechanism whereby VLDL-LDL interrelationships can be studied.

Effect of particle lipid content on the structure of insect lipophorins.

Four distinct subspecies of the major insect lipoprotein, lipophorin, that range in overall lipid content from 20 to 51% of the particle mass, were isolated from the hemolymph or oocytes of the tobacco hornworm, Manduca sexta. Examination of these subspecies by electron microscopy revealed distinctive morphologies. Adult high density lipophorin (HDLp-A) was found to be an approximately spherical particle with a diameter of 15 +/- 1 nm, while HDLp-Wanderer 1 (W1), was more rectangular in shape and had a distinct cleft extending into the particle at one end. In the case of HDLp-Wanderer 2 (W2) the cleft was deeper and wider than that in HDLp-W1. In egg very high density lipophorin (VHDLp-E) the cleft was increased in size to the extent that the particle had an overall crescent-like conformation. Circular dichroism spectroscopy of the three lipophorin subspecies that contain only apolipophorin I and II revealed that only minor differences in the global protein secondary structure occur as the particle lipid content is decreased. The VHDLp-E apolipoproteins are an exception in that, while having the same alpha-helix content as HDLp-W1 and HDLp-W2, they contain less beta-structure and correspondingly more random coil. Limited digestion of the apolipoprotein components of the lipophorin subspecies with trypsin revealed that as the lipid content of the particles decreases the susceptibility of the apolipoprotein to proteolytic degradation increases. Likewise, tryptophan fluorescence quenching experiments demonstrated that the relative exposure of lipophorin apolipoprotein tryptophan residues also increases as the particle lipid content decreases.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of Mengo virus neutralization epitopes.

A set of four monoclonal antibodies which neutralized the infectivity of Mengo virus was used to select 20 non-neutralizable (escape) mutants. Altered amino acids were identified by sequence analyses of the capsid-coding regions of the mutant virus genomes. Mutations were found predominantly in proteins VP2 and VP3, while mutations in VP1 were detected only as second mutations. The Mengo virus VP2 mutations at amino acid residues 2144, 2145, 2147, and 2148 align with site Nlm II in human rhinovirus-14 and site 2 in polioviruses 1 and 3. The mutation at 2075 as well as those at 3057, 3061, and 3068 in VP3 correspond to site 3 in poliovirus. These alignments notwithstanding, the results of cross-neutralization experiments indicate the existence of a single composite neutralization site on the Mengo virion. Considering the three-dimensional structure of the Mengo capsid, the amino acids which are altered in the escape mutants are all exposed on the outer surface and none are found in the "pit," the probable site for binding of a cellular receptor. The VP3 mutations are located in the VP3 "knob" and the VP2 mutations on a nearby ridge. Together these mutations define a set of epitopes within a single composite antigenic determinant which forms a crescent-shaped area around the three-fold icosahedral axes of the Mengo virion.

Lipid transfer particle-induced transformation of human high density lipoprotein into apolipoprotein A-I-deficient low density particles.

Incubation of human high density lipoprotein (HDL) particles (density = 1.063-1.21 g/ml) with catalytic amounts of Manduca sexta lipid transfer particle (LTP) resulted in alteration of the density distribution of HDL protein such that the original HDL particles were transformed into new particles with an equilibrium density = 1.05 g/ml. Concomitantly, substantial amounts of protein were recovered in the bottom fraction of the density gradient. The LTP-induced alteration in HDL protein density distribution was dependent on the LTP concentration and incubation time. Electrophoretic analysis revealed that the lower density fraction contained apolipoprotein A-II (apoA-II) as the major apoprotein component while nearly all of the apoA-I was recovered in the bottom fraction. Lipid analysis of the HDL substrate and product fractions revealed that the apoA-I-rich fraction was nearly devoid of lipid (less than 1%, w/w). The lipid originally associated with HDL was recovered in the low density, apoA-II-rich, lipoprotein fraction, and the ratios of individual lipid classes were the same as in control HDL. Electron microscopy and gel permeation chromatography experiments revealed that the LTP-induced product lipoprotein population comprised particles of larger size (19.7 +/- 1.4-nm diameter) than control HDL (10.6 +/- 1.4-nm diameter). The results suggest that facilitated net lipid transfer between HDL particles altered the distribution of lipid such that apoprotein migration occurred and donor particles disintegrated. Similar results were obtained when human HDL3 or HDL2 density subclasses were employed as substrates for LTP. The lower surface area to core volume ratio of the larger, product lipoprotein particles compared with the substrate HDL requires that there be a decrease in the total exposed lipid/water interface which requires stabilization by apolipoprotein. Selective displacement of apoA-I by apoA-II or apoC, due to their greater surface binding affinity, dictates that apoA-I is preferentially lost from the lipoprotein surface and is therefore recovered as lipid-free apoprotein. Thus, it is conceivable that the structural arrangement of HDL particle lipid and apoprotein components isolated from human plasma may not represent the most thermodynamically stable arrangement of lipid and protein.

Organization of dimethyl sulfoxide reductase in the plasma membrane of Escherichia coli.

Dimethyl sulfoxide reductase is a trimeric, membrane-bound, iron-sulfur molybdoenzyme induced in Escherichia coli under anaerobic growth conditions. The enzyme catalyzes the reduction of dimethyl sulfoxide, trimethylamine N-oxide, and a variety of S- and N-oxide compounds. The topology of dimethyl sulfoxide reductase subunits was probed by a combination of techniques. Immunoblot analysis of the periplasmic proteins from the osmotic shock and chloroform wash fluids indicated that the subunits were not free in the periplasm. The reductase was susceptible to proteases in everted membrane vesicles, but the enzyme in outer membrane-permeabilized cells became protease sensitive only after detergent solubilization of the E. coli plasma membrane. Lactoperoxidase catalyzed the iodination of each of the three subunits in an everted membrane vesicle preparation. Antibodies to dimethyl sulfoxide reductase and fumarate reductase specifically agglutinated the everted membrane vesicles. No TnphoA fusions could be found in the dmsA or -B genes, indicating that these subunits were not translocated to the periplasm. Immunogold electron microscopy of everted membrane vesicles and thin sections by using antibodies to the DmsABC, DmsA, DmsB subunits resulted in specific labeling of the cytoplasmic surface of the inner membrane. These results show that the DmsA (catalytic subunit) and DmsB (electron transfer subunit) are membrane-extrinsic subunits facing the cytoplasmic side of the plasma membrane.

Functional aspects of the capsid structure of Mengo virus.

The three-dimensional structure of the Mengo virus capsid has been determined at a resolution of 3.0 A. This achievement is discussed in an historical context, and the general features of picornavirus capsid design are presented. The dynamic functional aspects of the Mengo virus capsid--namely its ability to interact with specific receptors on host cells, to dissociate and release the viral genomic RNA into the cellular cytoplasm, to assemble with progeny RNA molecules and form new virions, and to alter its external surface in order to evade neutralization by circulating antibodies--are discussed. Comparisons with other picornaviruses whose capsid structures have also been elucidated (poliovirus serotype 1 and 3, human rhinovirus types 14 and 1A, and foot-and-mouth disease virus type O) illustrate both the similarities and the distinctive features of capsid design found within this family of mammalian viruses.

Studies of the morphology and structure of the plasma lipid transfer particle from the tobacco hornworm, Manduca sexta.

Morphological features of Manduca sexta plasma lipid transfer particle (LTP) have been investigated by electron microscopy. LTP was found to be an asymmetric particle with two major structural features: a roughly spherical head and an elongated, hinged tail. The hinge occurs approximately at the midpoint of the tail section with the two halves forming angles ranging from 30 degrees to 180 degrees. A molecular mass estimate of 1.4 x 10(6) daltons based on the dimensions of LTP suggests that multiple copies (two or three) of each of the three LTP apoproteins exist in the native complex. Limited digestion studies of LTP suggest that apoLTP-III is less susceptible to trypsin cleavage than apoLTP-I or -II, and therefore may be less exposed to the aqueous environment. Digestion for 1 h at a 1:50 trypsin-LTP protein ratio did not alter the flotation properties of LTP or its morphological features; thus, although significant proteolysis occurred, the particle retained its overall structure. Transfer activity, on the other hand, was affected by trypsin digestion with 30 +/- 14% inhibition of LTP activity occurring upon proteolysis at a 1:50 trypsin-LTP protein ratio. Treatment of LTP with phospholipase A2 resulted in the conversion of LTP-associated phosphatidylcholine and phosphatidylethanolamine to their corresponding lyso forms. Phospholipase A2 treatment did not, however, alter the SDS-PAGE profile, transfer activity, flotation pattern, or the microscopic features of LTP. These results suggest that the products of the phospholipase reaction remain associated with the particle.(ABSTRACT TRUNCATED AT 250 WORDS)