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A Zlotnick

Publications and source records attributed to A Zlotnick.

At least 37 records · Page 2Linked to original sources
PubMed1993

A monoclinic crystal with R32 pseudo-symmetry: a preliminary report of Nodamura virus structure determination.

We have crystallized Nodamura virus, a T = 3 icosahedral virus that can infect both mammalian and insect hosts. Crystals are monoclinic, with two crystallographically independent virus molecules per asymmetric unit. Packing analysis reveals a pseudo-rhombohedral (pseudo-C2 in the monoclinic setting) arrangement of virus particles in the crystal lattice. Crystals differ from the R32 symmetry by rotational and translational deviations. The rhombohedral packing arrangement and its failure to describe the exact virus packing is analyzed in detail. The icosahedral threefold axis is rotated from the body diagonal of the pseudo-rhombohedral cell, breaking the rhombohedral symmetry. The C2 pseudo-symmetry breaks down rotationally and/or translationally.

Journal Article↗
PubMed1993

Analysis of two distinct single-stranded DNA binding sites on the recA nucleoprotein filament.

The binding stoichiometry of Escherichia coli recA protein to single-stranded DNA (ssDNA) determined by two separate assays differs by a factor of 2.2-2.4. Using the fluorescence of etheno-DNA (epsilon DNA), a chemically modified ssDNA, the stoichiometry was found to be 7.0 +/- 0.6 bases/recA protein monomer in a nucleo-protein filament. Using a competition assay, a similar stoichiometry, 7.5 bases/recA, is found for unmodified poly(dT). Using the DNA-dependent ATPase of recA, which monitors bound protein rather than bound DNA, we find that each recA monomer needs to bind only 3.1 +/- 0.5 bases to fully activate the ATPase. The difference in site size determined by the two assays indicates that there are two DNA binding sites with differential effects on ATPase activation. When recA protein is mixed with ssDNA at a ratio of 7 bases/recA or greater, the complex that forms contains 7 bases/recA and acts as a kinetic trap for the ssDNA. Upon further addition of recA protein, no additional ATPase activity is observed. If, on the other hand, the ssDNA is initially mixed with excess recA (at a ratio of 3-3.5 bases/recA or less) the ATPase activity is twice as high. Analysis of the binding curves suggests that the first DNA strand binds recA to form a filament with a stoichiometry of 3-3.5 bases/protein monomer. The ATPase activity of recA is completely active in this complex. A second strand of DNA can then be bound to this filament yielding a final stoichiometry of approximately 7 bases/protein monomer. The presence of this second strand neither enhances nor inhibits ATP hydrolysis. This ternary complex may mimic the structures formed by recA in searching for homologous DNA sequences and/or in the strand exchange reaction.

Adenosine Triphosphatases↗
PubMed1991

Analysis of the nucleotide sequence of bean pod mottle virus middle component RNA.

The complete nucleotide sequence of the middle component RNA (M RNA) of the comovirus bean pod mottle virus (BPMV) has been determined. The sequence consists of 3662 nucleotides and contains a single long open reading frame sufficient to code for a protein of 113,353 Da. The proteolytic processing sites within this protein have been identified by comparison with the known three-dimensional structure of the virion and cleavage at these sites would lead to a range of products consistent with those observed during processing of the M RNA-encoded polyproteins in vitro. We have performed computer-aided searches for reiterated sequences within BPMV M RNA which might explain why ordered RNA is visible in the electron density map of BPMV middle component particles (Chen, Z., Stauffacher, C. V., Li, Y., Schmidt, T., Bomu, W., Kamer, G., Shanks, M., Lomonossoff, G., and Johnson, J. E., 1989, Science 245, 154-159). These searches revealed both the presence of overrepresented pentameric sequences and a consensus sequence which was repeated 15 times within the RNA sequence.

Amino Acid Sequence↗
PubMed1990

RecA protein self-assembly. II. Analytical equilibrium ultracentrifugation studies of the entropy-driven self-association of RecA.

We have investigated the self-association of RecA protein from Escherichia coli by equilibrium ultracentrifugation. Monomeric RecA (Mr = 37,842) was observed in reversible equilibrium with trimers, hexamers and dodecamers in the presence of 1.5 M-KCl, 5 mM-Hepes, 1 mM-EDTA, 2 mM-ATP (pH 7.0) at 1 degrees C. The equilibrium was strongly temperature-dependent, with polymerization being favored as the temperature was raised from 1 degrees C 21 degrees C, and was reversible with respect to temperature. The values of both the standard enthalpy and entropy of self-association were positive, indicating that it is an entropy-driven process under these conditions. In the absence of KCl, in 50 mM-citrate, 5 mM-ATP, 5% (v/v) glycerol (pH 6.0) at 4 degrees C, only small amounts of RecA monomer could be detected, while in 10 mM-Tris-acetate, 10% glycerol (pH 7.5) at 4 degrees C, the smallest species present in significant concentration appeared to be the trimer. The majority of the species observed had molecular weights between 228,000 and 456,000, suggesting dominant stoichiometries of six to 12 monomers per oligomer. At pH 6.0, in the absence of ATP, much larger oligomers containing at least 24 monomers also appeared to be present. The data are consistent with an equilibrium mixture of monomers, trimers, hexamers, dodecamers, 24-mers and higher oligomers, with the distribution of oligomers being dependent on solution conditions. Thermodynamic analysis indicates that these oligomeric species are in reversible equilibrium with each other. It is not certain whether trimers assemble directly into hexamers, or whether disassembly into monomers is a prerequisite for the formation of higher oligomers. The possible role of higher-order RecA oligomers in the formation of RecA nucleoprotein filaments is discussed.

Adenosine Triphosphate↗
PubMed1990

recA protein filaments bind two molecules of single-stranded DNA with off rates regulated by nucleotide cofactor.

To probe the role of nucleotide cofactor in the binding of single-stranded DNA to recA protein, we have developed a sedimentation assay using 5'-labeled 32P-poly(dT).recA.poly(dT) complexes sediment quantitatively when centrifuged at 100,000 x g for 45 min, whereas free poly(dT) remains in the supernatant. In the presence of ATP, between 6 and 7 bases cosediment per recA monomer; but when ADP is present or in the absence of added nucleotide cofactor, only 3-3.5 bases/recA monomer cosediment. In competition experiments in which recA.32P-poly(dT) complexes are incubated with unlabeled poly(dT), we again find 3-3.5 bases of labeled poly(dT) cosedimenting per recA monomer when no nucleotide cofactor is present. However, when the same experiment is performed with ATP, only half of the expected 6-7 bases of labeled poly(dT) remain bound to the DNA, demonstrating that half of the poly(dT) in the complex exchanges rapidly with free poly(dT), whereas the other half equilibrates slowly, like poly(dT) in the absence of nucleotide. The rate of exchange of the second more tightly bound poly(dT) is accelerated when ADP is present. Our observations are rationalized by a model in which each recA protein helical filament binds two strands of poly(dT) with a stoichiometry of 3-3.5 bases/recA monomer/strand.

Adenosine Diphosphate↗
PubMed1989

An alpha-helical peptide model for electrostatic interactions of proteins with DNA. The N terminus of RecA.

A series of synthetic peptides have been studied as models for non-specific protein-DNA interactions. In an alpha-helical conformation, the charged amino acid residues of the N-terminal 24 residues of RecA protein are asymmetrically distributed; at neutral pH there is a +4 charge on one face of the helix and a -3 charge on the other face. Modeling suggests that the positive face of the helix can bind five DNA phosphate groups by electrostatic interactions. Circular dichroism (c.d.) spectra indicate that the analogous peptide, Rec24 (AIDENKQKALAAALGQIEKQFGKG-amide), is largely unstructured in water but becomes highly helical in the presence of DNA. Peptide titrations of fluorescent etheno-DNA confirm that the changes in the c.d. spectrum of the peptide are associated with binding, although a dependence of the c.d. signal on the degree of DNA saturation is observed, indicating that peptide can be bound in more than one conformation. At saturation the peptide binds to 5.0(+/- 0.5) DNA phosphate groups as predicted and the electrostatic nature of the binding is confirmed by a strong dependence on salt concentration. A "mutant" peptide where an acidic glutamate residue replaces an alanine on the basic face of the Rec24 helix exhibits weaker binding to single-stranded DNA, also consistent with the electrostatic nature of the proposed peptide-DNA interaction. Extending Rec24 by ten amino acid residues, where the additional residues do not participate in the helical motif, does not noticeably affect binding. Thus, we show experimentally that an asymmetric charge distribution on an alpha-helix can represent an important element for binding nucleic acids.

Alanine↗
PubMed1988

RecA protein self-assembly. Multiple discrete aggregation states.

Light scattering, sedimentation and electron microscopy have been used to investigate the aggregation states of highly purified RecA protein in solution. We show that RecA protein will self-assemble into a discrete series of quaternary structures depending upon protein concentration, ionic environment, and nucleotide cofactors. In a stock solution at moderate concentration (10 to 50 microM) RecA protein exists as small particles approximately 4 nm in diameter, larger particles approximately 12 nm in diameter (most probably rings of RecA protein), 10 nm diameter rods varying from 50 to 200 nm in length, and finally as much larger bundles of rods. The addition of monovalent salt shifts the distribution of RecA protein between its various oligomeric states. Increasing protein concentration favors more highly aggregated structures. At a given protein concentration, addition of mM levels of MgCl2 promotes the rapid formation of rods and slow formation of bundles. Under conditions typical of in vitro strand exchange reactions, RecA protein was found to exist as a mixture of rods and 12 nm particles with relatively few monomers.

Adenosine Triphosphate↗
PubMed1986

Fluorescence quenching of cytochrome b5 in vesicles with an asymmetric transbilayer distribution of brominated phosphatidylcholine.

Several fluorescence techniques have been used to estimate the depth, in the membrane, of the endogenous tryptophans of membrane-bound proteins. We reported recently the use of phosphatidylcholines specifically brominated at different positions of the sn-2 acyl chain for this purpose (Markello, T., Zlotnick, A., Everett, J., Tennyson, J., and Holloway, P. W. (1985) Biochemistry 24, 2895-2901). The membranes made from these brominated lipids will have the brominated lipid in both monolayers, and so the estimated depth of the fluorophore will be relative to either the inner or outer surface of the membrane, but will not distinguish between these two extremes. To differentiate between these two models vesicles have now been made with an asymmetric distribution of brominated lipid, by use of phosphatidylcholine exchange protein. The asymmetric vesicles were isolated by virtue of their density, and their asymmetry was established by addition of an amphipathic fluorescent carbazole compound. With these vesicles it was shown that the tryptophan in the membrane-binding domain of cytochrome b5 which is quenched by bromolipid is located 0.7 nm below the outer surface of the membrane vesicles, rather than 0.7 nm from the inner surface.

Animals↗
PubMed1985

Determination of the topography of cytochrome b5 in lipid vesicles by fluorescence quenching.

Cytochrome b5, a protein isolated from the endoplasmic reticulum by detergent extraction, interacts spontaneously with small unilamellar phosphatidylcholine vesicles. When the vesicles are made from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), the tryptophan fluorescence of the cytochrome is enhanced, and when they are made from 1-palmitoyl-2-(dibromostearoyl) phosphatidylcholine (BRPC), the fluorescence is quenched. A series of BRPC were synthesized with bromine atoms at the 6,7, 9,10, 11,12 or 15,16 positions. The vesicles synthesized from each of these lipids were similar in size to those made from POPC. The relative fluorescence intensities of the cytochrome b5 in POPC and 6,7-, 9,10-, 11,12- and 15,16- BRPC were 100, 19.4, 29.4, 37.1, and 54.0, respectively. These data suggest that the exposed tryptophan(s) is (are) at a depth of 0.7 nm below the surface of the vesicle. Bromine is a collisional quencher; hence, these data may indicate the relative position of the lipid annulus around the protein rather than the depth of the protein below the average vesicle surface. Cytochrome b5 contains three potentially fluorescent tryptophans, and determinations of fluorescent quantum yield indicate all three potentially fluorescent tryptophans, and determinations of fluorescent quantum yield indicate all three are fluorescent with an average quantum yield, when in POPC vesicles, of 0.21. Fluorescence lifetime measurements by the demodulation technique indicated heterogeneity of fluorescence lifetimes in all vesicles. The lifetimes in the BRPC vesicles ranged from 2.0 to 2.4 ns compared to a value of 3.3 ns in POPC.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗
PubMed1985

In vivo function and membrane binding properties are correlated for Escherichia coli lamB signal peptides.

Wild-type and pseudorevertant signal peptides of the lamB gene product of Escherichia coli interact with lipid systems whereas a nonfunctional deletion mutant signal peptide does not. This conclusion is based on interaction of synthetic signal peptides with a lipid monolayer-water surface, conformational changes induced by presence of lipid vesicles in an aqueous solution of signal peptide, and capacities of the peptides to promote vesicle aggregation. Analysis of the signal sequences and previous conformational studies suggest that these lipid interaction properties may be attributable to the tendency of the functional signal peptides to adopt alpha-helical conformations. Although the possibility of direct interaction between the signal peptide and membrane lipids during protein secretion is controversial, the results suggest that conformationally related amphiphilicity and consequent membrane affinity of signal sequences are important for function in vivo.

Bacterial Outer Membrane Proteins↗
PubMed1985

Prognostic factors in polymyositis/dermatomyositis. A computer-assisted analysis of ninety-two cases.

An effort was made to identify all patients with polymyositis/dermatomyositis (PM/DM) admitted to hospitals in Israel from 1956-1976. The diagnosis of PM/DM was retrospectively reviewed in 92 (46 definite, 26 probable, and 20 possible) cases. The most common complaints and physical findings in the course of the disease were muscle weakness (86 patients), rash (53 patients), arthritis or arthralgia (39 patients), and dysphagia (35 patients). Elevated serum aldolase levels were found in 64% of the patients for whom data were available; 92% had abnormal electromyogram results, and 60.9% had muscle histopathology consistent with PM/DM. Malignancy was diagnosed in 13 patients. Malignancy, ischemic heart disease, and pulmonary complications were the most common causes of death. The actuarial survival curve was heterogeneous, with an accelerated mortality during the first year after diagnosis and a slower mortality during the following 7 years. Independent unfavorable prognostic signs were: failure to induce remission, leukocytosis, fever, older age, a shorter disease history, and dysphagia.

Adolescent↗
PubMed1985

A central anti-DNA idiotype in human and murine systemic lupus erythematosus.

The monoclonal anti-DNA autoantibody A52 (IgG2b) was obtained from a (NZB X NZW)F1 (B/W) hybridoma. Two rabbits were immunized with the pure monoclonal antibody and produced anti-idiotypic (Id) antibodies. The purified anti-Id reacted with three different B/W monoclonal anti-DNA antibodies at or close to their DNA binding sites. Moreover, the rabbit antibodies had a profound inhibitory effect on the polyclonal anti-DNA activity in the majority of sera derived from B/W mice and human systemic lupus erythematosus (SLE) patients. The A52 IgG must, therefore, represent a major cross-reactive Id of anti-DNA immunoglobulins. In addition, the rabbit anti-Id antibodies may act as the "internal image" of antigen and should prove useful in modulation of the autoimmune response to DNA in SLE.

Animals↗
PubMed1985

Autoantibodies to anti-DNA with anti-allotypic and anti-idiotypic specificities in (NZB X NZW)F1 mice.

The monoclonal A52 (IgG2b, kappa) anti-DNA autoantibody represents a major cross-reactive idiotype in the murine and human autoimmune response to DNA. Examination of sera and purified IgG derived from (NZB X NZW)F1 mice showed that these mice develop an age-dependent binding reactivity with the pure anti-DNA IgG. Three monoclonal antibodies possessing this reactivity were prepared from unprimed female (NZB X NZW)F1 mice. One of these monoclonal antibodies appeared to be directed against allotypic determinants present in the NZB IgG2b; the other two antibodies exhibited a marked preference for idiotypic determinants of the A52 IgG. The IgG anti-allotype and anti-idiotype activities in (NZB X NZW)F1 mice may, therefore, represent the products of a deregulated immune system and/or constitute the normal elements of a functional immune regulation system.

Animals↗
PubMed1985

Coexistence of Kaposi's sarcoma and chronic lymphocytic leukemia in the same lymph node.

A patient with chronic lymphocytic leukemia in whom a carcinoma of the gall bladder was previously found is described. The patient was treated with prednisone and chlorambucil and developed a significant lymphadenopathy. A biopsy of one of the lymph nodes revealed the coexistence of Kaposi's sarcoma and chronic lymphocytic leukemia in the same node. This and the presence of three malignancies in the same patient are discussed.

Aged↗
PubMed1981

Albumin-immunoglobulin complexes in human serum: classification and immunochemical analysis.

A complex between serum albumin and immunoglobulins was observed on immunoelectrophoresis in six patients. Two patients with multiple myeloma had monoclonal IgA-albumin complexes; one of these complexes was formed by covalent bonds and the other by non-covalent bonds. Four patients displayed non-covalent IgG-albumin complexes: of these, one had multiple myeloma, two had been treated with nitrofurantoin for prolonged periods, and one had diabetes mellitus. The IgG-albumin complex of the last patient was subjected to a detailed immunochemical analysis. The albumin-specific antibodies were isolated by affinity chromatography and analysed, using a sensitive tritium labelling technique. The antibodies were polyclonal, complexed with serum albumin through their Fab portion, and showed a high specificity for the human albumin as compared with bovine and rabbit albumins. The serum albumin of two patients displayed an abnormal behaviour in reduced SDS-polyacrylamide gel electrophoresis (PAGE). The abnormal albumins had an apparent molecular weight of 52,000 and could react with rabbit anti-human serum albumin like the normal protein. No abnormal albumin could be detected in 20 other patients' sera, including nitrofurantoin-treated patients and normal controls. These findings suggest a possible role for an altered self-component in the triggering of a specific autoimmune reaction.

Animals↗