A simple theoretical model for the effects of cholesterol and polypeptides on lipid membranes

A simple theoretical model for the effects of impurities on biomembranes is proposed. The model accounts for the cholesterol-induced decrease of membrane phase transition temperature, membrane condensation above the gel to liquid crystalline phase transition, and increase in lateral compressibility. The model also predicts that addition of molecules such as cholesterol and polypeptides to membranes results in unmasking of a continuous phase transition. This results in a second broad peak in the calorimetric curves for melting of lipid-cholesterol mixtures, and the appearance of a second melting transition in membranes modified by the incorporation of polypeptides. The theory assumes that the membrane may be adequately described by a kink model, and that impurities are randomly distributed in the membrane. The difference in size and shape of impurity molecules, compared to membrane lipids, results in a spatial disordering in the membrane which in turn causes increased chain disorder and membrane condensation, as well as a decrease in the cooperativity of melting. The second transition results from a second expansion of the condensed, partially disordered membrane, which takes place over a several degree temperature range. This transition, although unmasked by boundary effects of non-lipid molecules, does not correspond to melting of a boundary annulus or phase separation.     

芹菜夜蛾核型多角体病毒D克隆株某些理化性质的研究

DNA of Syngrapha falcifera nuclear polyhedrosis virus D-clone(Sfa-D clone) was extracted and digested by three kinds of restriction endonuclease,We calculated its molecular weight and measure its melting temperature,G C%, virus particle and polyhedrin were purified.The structural polypeptides and polyhedrin are analysed by SDS-PAGE.     

Thermal melting and circular dichroism of DNA complexes with (Lys-Ala-Ala)10 and (Lys-Ala-Ala)n: effect of polypeptide chain length

DNA complexes with polypeptides (Lys-Ala-Ala)₁)] and (Lys-Ala-Ala)₃₄ have been studied using the methods of thermal melting and circular dichroism. Derivative melting curves of (Lys-Ala-Ala)₁₀ DNA differed substantially from those of (Lys-Ala-Ala)₃₄ prepared either by salt gradient dialysis or by direct mixing. Melting curves of the former complex were unimodal or bimodal with T_m increasing continuously withn input lysin-to-DNA phosphate ratio (r); those of the latter complex consisted of three separate transitions with T_m values almost independent of r. Complete reversibility of binding in the (Lys-Ala-Ala)₁₀-DNA system but a slow redistribution of (Lys-Ala-Ala)₃₄ on DNA at low temperature were found in the redistribution experiments Much faster redistribution from denatured to native DNA occurs at the temperature of melting, contributing to the unusual trimodal melting pattern. Circular dichroism curves are very similar for both complexes and indicate little change of DNA conformation upon polypeptide binding.     

Pair formation and promiscuity of cytokeratins: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides

Cytokeratins are expressed in different types of epithelial cells in certain combinations of polypeptides of the acidic (type I) and basic (type II) subfamilies, showing "expression pairs." We have examined in vitro the ability of purified and denatured cytokeratin polypeptides of human, bovine, and rat origin to form the characteristic heterotypic subunit complexes, as determined by various electrophoretic techniques and chemical cross-linking, and, subsequently, intermediate-sized filaments (IFs), as shown by electron microscopy. We have found that all of the diverse type I cytokeratin polypeptides examined can form complexes and IFs when allowed to react with equimolar amounts of any of the type II polypeptides. Examples of successful subunit complex and IF formation in vitro include combinations of polypeptides that have never been found to occur in the same cell type in vivo, such as between epidermal cytokeratins and those from simple epithelia, and also heterologous combinations between cytokeratins from different species. The reconstituted complexes and IFs show stability properties, as determined by gradual "melting" and reassociation, that are similar to those of comparable native combinations or characteristic for the specific new pair combination. The results show that cytokeratin complex and IF formation in vitro requires the pairing of one representative of each the type I and type II subfamilies into the heterotypic tetramer but that there is no structural incompatibility between any of the members of the two subfamilies. These findings suggest that the co-expression of specific pair combinations observed in vivo has other reasons than general structural requirements for IF formation and probably rather reflects the selection of certain regulatory programs of expression during cell differentiation. Moreover, the fact that certain cytokeratin polypeptide pairs that readily form complexes in vitro and coexist in the same cells in vivo nevertheless show preferential, if not exclusive, partner relationships in the living cell points to the importance of differences of stabilities among cytokeratin complexes and/or the existence of extracytokeratinous factors involved in the specific formation of certain cytokeratin pairs.     

Incorporation of synthetic peptide helices in membranes of tetraether lipids from Thermoplasma acidophilum. A calorimetric study

Four analogues of the membrane-modifying, alpha-helical polypeptide antibiotic alamethicin were synthesized. the alpha-helical deca-, undeca-, heptadeca-, and icosapeptides were mixed with the main tetraether lipid of the Archaebacterium Thermoplasma acidophilum (MPL), dipalmitoylphosphatidylcholine (DPPC) and dihexadecylmaltosylglycerol (DHMG) in various ratios and the modification of the lipid phase transition was determined by differential thermal analysis (DTA). The polypeptides form mixed phases with MPL and DPPC, however, not with DHMG. Heptadeca- and icosapeptide exert a much stronger reduction of enthalpy (delta H) than deca- and undecapeptide and bind about 0.5 molecule of MPL (or one molecule of DPPC) per peptide molecule. delta H of the DPPC pretransition is reduced by the deca- and the undecapeptides and completely disappears with heptadeca- and icosapeptides (at 0.2 mole of peptide/mole of lipid). The modulation of the melting point Tm by the incorporation of peptides is more pronounced with MPL than with DPPC, the heptadecapeptide exhibiting the strongest reduction (with MPL) and the strongest broadening of the transition peak (with DPPC). Helix length, amphiphilicity and charge of the polypeptides can be correlated with the observed modifications of the lipid phase transitions.     

General method for purification of α-amino acid-n-carboxyanhydrides using flash chromatography

We describe the application of flash column chromatography on silica gel as a rapid and general method to obtain pure α-amino acid-N-carboxyanhydride (NCA) monomers, the widely used precursors for the synthesis of polypeptides, without the need for recrystallization. This technique was effective at removing all common impurities from NCAs and was found to work for a variety of NCAs, including those synthesized using different routes, as well as those bearing either hydrophilic or hydrophobic side chains. All chromatographed NCAs required no further purification and could be used directly to form high molecular weight polypeptides. This procedure is especially useful for the preparation of highly functional and low melting NCAs that are difficult to crystallize and, consequently, to polymerize. This method solves many long-standing problems in NCA purification and provides rapid access to NCAs that were previously inaccessible in satisfactory quality for controlled polymerization. This method is also practical in that it requires less time than recrystallization and often gives NCAs in improved yields.     

Infrared linear dichroism investigations of deoxyribonucleic acid complexes with poly(L-arginine) and poly(L-lysine).

Complexes between DNAs from various sources and poly(L-lysine) and poly(L-arginine) were studied by means of infrared linear dichroism. The measurements of dichroic ratios allowed us to determine the orientation of the phosphate group of DNA in the complexes with basic polypeptides. At high relative humidities (higher than 90%, B form), the bisector of the less than OPO in the complexes forms an angle with respect to the helical axis which has a value lower by about 4 degrees than in the corresponding DNA sample. This change of orientation of the phosphate group of DNA indicates a modification of the B form upon binding of polylysine or polyarginine. The structural transitions B leads to A and B leads to C measured as a function of relative humidities were not affected by formation of complexes with both basic polypeptides. Similar results were obtained for complexes prepared by direct mixing or by salt gradient dialysis. The presence of A and C forms was observed in complexes of DNA with poly(L-lysine) and poly(L-arginine) at lower relative humidity. Thus, the conformational flexibility of DNA in complexes with polylysine and polyarginine is not changed despite a substantial increase in the Tm (melting temperature). These results are considered as a model for the understanding of interactions between DNA and histones particularly of the binding of the N-terminal fragment, lysine or arginine rich.     

Antibodies to the most tightly bound proteins in eukaryotic DNA : Formation of immuno-complexes with ‘nuclear matrix’ components

Chromosomal DNA is associated with polypeptides covalently bound to internal DNA ends. Since these polypeptides can only be released from chromosomal DNA by enzymes or other agents hydrolysing phosphodiester bonds they were termed 'the most tightly bound' (MTB) polypeptides in DNA. Antibodies developed against the MTB polypeptides are shown to form immunocomplexes with major 'nuclear matrix' polypeptides as well as with polypeptides which are still associated with 'nuclear matrix' DNA isolated by means of SDS/proteinase K and phenol. Immuno-complex formation is revealed by immunoblotting and by indirect immunofluorescence. Thus, since MTB polypeptides, major 'nuclear matrix' polypeptides and 'nuclear matrix' DNA-associated polypeptides share common antigenic sites they can be considered to be identical or at least closely related. This suggests that a fraction of distinct 'nuclear matrix' polypeptides is either transiently or permanently linked to DNA by covalent bonds. Consistently, isolated eukaryotic 'bulk' DNA is inevitably associated with residual 'nuclear matrix' polypeptides.     

Nonstructural proteins of herpes simplex virus. II. Major virus-specific DNa-binding protein

The major herpes simplex virus type 2 DNA-binding infected cell-specific polypeptides 11 and 12 have been purified to homogeneity from extracts of virus-infected cells. Monospecific antiserum to the purified protein has been made and used to examine virus temperature-sensitive mutants for defects in the synthesis of the protein and to probe virus DNA synthesis in isolated chromatin. The purified protein acted directly on a polydeoxyadenylic acid-polydeoxythymidylic acid helix, reducing its melting temperature. The results indicated that the protein functions in virus DNA synthesis.     

Immunoblotting of keratin polypeptides extracted from tissues preserved in standard histologic fixatives

Cytoskeletal polypeptides from fresh placental tissue, tissue stored at -30 degrees C, and tissue fixed in 10% buffered formalin, Bouin's solution, and Carnoy's solution were extracted, separated by electrophoresis, and immunoblotted using monoclonal antibodies immunoreactive with keratin polypeptides. Storage of the placental tissue at -30 degrees C, or fixation in Carnoy's solution did not alter the extractability, migration pattern, or immunoreactivity of the keratin polypeptides. Keratin polypeptides could not be adequately demonstrated in extracts prepared from formalin- or Bouin's solution-fixed tissues. Several unmasking procedures used on tissues before extraction and on nitrocellulose blots before application of primary antibodies failed to unmask keratin polypeptides, either in Coomassie blue-stained gels or in immunoblots reacted with anti-keratin antibodies. These data indicate that Carnoy's solution is the fixative of choice for tissues in which electrophoretic and immunoblotting analyses of keratin polypeptides might be required.     

Interaction of DNA with poly(L-Lys-L-Ala-Gly) and poly(L-Lys-L-Ala-L-Pro). Circular dichroism and thermal denaturation studies

Complexes of DNA with polypeptides composed of Lys, Ala, and Gly in both a sequential order, poly(L-lysine-L-alanine-glycine), and a statistical distribution, poly(L-lysine36-L-alanine28-glycine), were prepared using gradient dialysis. These polypeptide-DNA complexes were studied using ultraviolet absorption (UV) and circular dichroism (CD) to probe the conformation, binding, and melting behavior of DNA in the complex. Complexes with the sequential polypeptide showed no structural change in the DNA; however, the complexes with the random polypeptide yield CD spectra similar to phi DNA [Maniatis, T., Venable, Jr., J.S., and Lerman, L.S. (1974), J. Mol. Biol. 84, 37]. A second sequential polypeptide, poly(L-Lys-L-Ala-L-Pro)n, -DNA complex was also studied. It was found to exhibit pronounced structural changes as a function of ionic strength and poly-peptide-DNA ratio, more similar to the random sequence that the ordered sequence of the Lys, Ala, Gly polymer. Thus the importance of the composition and amino acid sequence in polypeptides which bind to DNA, even in such simple systems, is demonstrated. Evidence from thermal denaturation, employing simultaneous monitoring of CD and UV changes, supports a model in which specific polypeptides cause condensation of the DNA in the complex into an asymmetric tertiary structure. The relevance of these model systems to chromatin is discussed.     

Studies on the polypeptides of poxvirus. II. Comparison of virus-induced polypeptides in cells infected with vaccinia, cowpox and Shope fibroma viruses.

Virus-induced polypeptides in cells infected with vaccinia, cowpox and Shope fibroma viruses were examined by SDS-polyacrylamide gel electrophoresis followed by autoradiography. At least 42 vaccinia virus-induced polypeptides were identified among the polypeptides of cells pulse-labeled with [35S]-methionine and/or of fractionated cells labeled with [14C]-leucine for 24 hr. They consisted of 15 polypeptides (early polypeptides) which were synthesized even in the presence of cytosine-1-beta-D-arabinofuranosyl-HCl, and 27 polypeptides (late polypeptides) which were synthesized only in the absence of cytosine-1-beta-D-arabinofuranosyl-HCl. By the same procedure at least 40 cowpox virus-induced polypeptides (14 early polypeptides and 26 late polypeptides) and at least 31 Shope fibroma virus-induced polypeptides (13 early polypeptides and 18 late polypeptides) were identified. Comparative studies of virus-induced polypeptides on the basis of migration in SDS-polyacrylamide gel electrophoresis revealed that 11 polypeptides were early polypeptides common to both vaccinia and cowpox viruses; 21 were late polypeptides common to both vaccinia and cowpox viruses; 4 were early polypeptides common to both vaccinia and Shope fibroma viruses; 7 were late polypeptides common to both vaccinia and Shope fibroma viruses; 5 were early polypeptides common to both cowpox and Shope fibroma viruses; 9 were late polypeptides common to both cowpox and Shope fibroma viruses; 4 were early polypeptides common to all three viruses; and 7 were late polypeptides common to all three viruses.     

An evaluation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the identification of microsporidia

Microsporidian spore polypeptides separated with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) can be used to identify isolates of microsporidia. The spore polypeptides separated with SDS/PAGE provided unique, reproducible electrophoretic profiles which were not influenced by host species or the temperature at which the host larvae were maintained for development. Furthermore, host proteins were not detected in electrophoretic profiles of the spore polypeptides. Spore mixtures of two microsporidian species can be detected when the spore polypeptides of either or both species have been previously separated with SDS/PAGE.     

Interactions between mucopolysaccharides and cationic polypeptides in aqueous solution: Chondroitin 4-sulfate and dermatan sulfate

Circular dichroism spectroscopy has been used to study the interactions of both dermatan sulfate and chondroitin 4-sulfate with the cationic polypeptides; poly(L -arginine), poly(L -lysine), and poly(L -ornithine). The results indicate that the mucopolysaccharides have a conformation directing effect on both poly(L -arginine) and poly-(L -lysine) such that these polypeptides adopt the α-helical conformation. The extent of interaction in each polypeptide-polysaccharide system can be judged by the degree of induced helicity and the “melting temperature” at which the interaction is disrupted On comparison of these results with those previously obtained for chondroitin 6-sulfate-polypeptide mixtures, the extent of interaction can be seen to depend on the length of the amino acid side chain and the positions of the anionic groups on the mucopolysaccharide chain. Such considerations place the three mucopolysaccharides in order of increasing interaction: chondroitin 4-sulfate < chondroitin 6-sulfate < dermatan sulfate. These results are correlated with observations that dermatan sulfate is bound more tightly to collagen in connective tissues than are the other two polysaccharides.     

In vitro membrane association of the F0 polypeptides of the Escherichia coli proton translocating ATPase.

The F0 polypeptides a, b, and c of the H+-translocating ATPase associated with membranes when synthesized in vitro. This association occurred when the membranes were present either cotranslationally or post-translationally. In addition, the F0 polypeptides associated with liposomes. The membrane association seemed to be an insertion process since there was protection of polypeptides a and c from proteolysis. The in vitro insertion of the F0 polypeptides a, b, and c was independent of the synthesis of each polypeptide and of the F1 polypeptides.     

Regulation of Herpesvirus Macromolecular Synthesis I. Cascade Regulation of the Synthesis of Three Groups of Viral Proteins

Based on evidence that 50% of herpes simplex 1 DNA is transcribed in HEp-2 cells in the absence of protein synthesis we examined the order and rates of synthesis of viral polypeptides in infected cells after reversal of cycloheximide- or puromycin-mediated inhibition of protein synthesis. These experiments showed that viral polypeptides formed three sequentially synthesized, coordinately regulated groups designated alpha, beta, and gamma. Specifically: (i) The alpha group, containing one minor structural and several nonstructural polypeptides, was synthesized at highest rates from 3 to 4 h postinfection in untreated cells and at diminishing rates thereafter. The beta group, also containing minor structural and nonstructural polypeptides, was synthesized at highest rates from 5 to 7 h and at decreasing rates thereafter. The gamma group containing major structural polypeptides was synthesized at increasing rates until at least 12 h postinfection. (ii) The synthesis of alpha polypeptides did not require prior infected cell protein synthesis. In contrast, the synthesis of beta polypeptides required both prior alpha polypeptide synthesis as well as new RNA synthesis, since the addition of actinomycin D immediately after removal of cycloheximide precluded beta polypeptide synthesis. The function supplied by the alpha polypeptides was stable since interruption of protein synthesis after alpha polypeptide synthesis began and before beta polypeptides were made did not prevent the immediate synthesis of beta polypeptides once the drug was withdrawn. The requirement of gamma polypeptide synthesis for prior synthesis of beta polypeptides seemed to be similar to that of beta polypeptides for prior synthesis of the alpha group. (iii) The rates of synthesis of alpha polypeptides were highest immediately after removal of cycloheximide and declined thereafter concomitant with the initiation of beta polypeptide synthesis; this decline in alpha polypeptide synthesis was less rapid in the presence of actinomycin D which prevented the appearance of beta and gamma polypeptides. The decrease in rates of synthesis of beta polypeptides normally occurring after 7 h postinfection was also less rapid in the presence of actinomycin D than in its absence, whereas ongoing synthesis of gamma polypeptides at this time was rapidly reduced by actinomycin D. (iv) Inhibitors of DNA synthesis (cytosine arabinoside or hydroxyurea) did not prevent the synthesis of alpha, beta, or gamma polypeptides, but did reduce the amounts of gamma polypeptides made.     

The polymorphism and structural homology of storage polypeptides (hordein) coded by the Hor-2 locus in barley (Hordeum vulgare L.)

Two-dimensional mapping (isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of the polypeptide components of “B” hordein fractions from eight barley varieties of widely different ancestry has been carried out. The relative positions of 47 different polypeptides were mapped, there being between 8 and 16 present in any one variety. The individual polypeptides differed in their distribution patterns; some were present in a number of varieties, while others were restricted to one or two. They also differed in their relative contributions to the total hordein fraction, both within and between varieties. The structural homology of the major polypeptides was compared by cleavage at methionine residues with cyanogen bromide and separation of the peptides on gradient gels. The polypeptides were classified into three groups which gave cleavage patterns with either two (class I), four (class II), or five (class III) low molecular weight bands. Class III polypeptides were found in all eight varieties, but in seven of the varieties class I or class II polypeptides were also present. With one exception, polypeptides migrating in the same position in different varieties gave identical or almost identical patterns. The three classes of polypeptides showed different distributions on the two-dimensional gels. Classes II and III polypeptides had a similar range of isoelectric points (pH 6.5–8.0), but all of the class II polypeptides were of slightly lower molecular weight. Class I polypeptides had a wider range of pI and molecular weight; the most alkaline and the lowest molecular weight polypeptides were in this group. The hordein fractions from a number of other barley varieties were compared with that of Julia. All had major polypeptides which migrated with ones present in Julia, but they differed in the relative amounts of these and in the absence of some polypeptides and the presence of others. B hordein is coded for by a single locus which has been suggested to be a complex multigenic family derived by duplication and divergence of a single gene. The data reported here provide support for this hypothesis and suggest that both mutations in the duplicated genes and recombination within the locus may have contributed to the polymorphism of the polypeptides.