Detection of covalent DNA-protein complexes: the adenovirus DNA-terminal protein complex and HeLa DNA-protein complexes.
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Adenoviruses, Human↗
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Immunological identification of complex proteins resolved by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis.
Immunological identification of an antigen resolved from a protein complex by sodium dodecyl sulfate polyacrylamide gel electrophoresis has been attained. The identification is based on the formation of immunoprecipitin lines after the antigen diffuses laterally from acrylamide gel transverse slices into a surrounding agarose gel. This technique was designed for study of contractile and regulatory protein complexes of non-muscle cells where the scarcity of tissue precludes easy purification or high yield of muscle-like proteins. It complements double-gel immunodiffusion or immunoelectrophoresis and its use may be extended to other protein complexes.
Actinin↗
Studies on the binding of the ribosomal protein complex L7/12-L10 and protein L11 to the 5'-one third of 23S RNA: a functional centre of the 50S subunit.
The RNA binding sites of the protein complex of L7/12 dimers and L10, and of protein L11, occur within the 5'-one third of 23S RNA. Binding of the L7/12-L10 protein complex to the 23S RNA is stimulated by protein L11 and vice-versa. This is the second example to be established of mutual stimulation of RNA binding by two ribosomal proteins or protein complexes, and suggests that this may be an important principle governing ribosomal protein-RNA assembly. When the L7/12-L10 complex is bound to the RNA, L10 becomes strongly resistant to trypsin. Since the L7/12 dimer does not bind specifically to the 23S RNA, this suggests that L10 constitutes a major RNA binding site of the protein complex. Only one of the L7/12 dimers is bound strongly in the (L7/12-L10)-23S RNA complex; the other can dissociate with no concurrent loss of L10.
Escherichia coli↗
Filter-binding assay for covalent DNA-protein complexes: adenovirus DNA-terminal protein complex.
A rapid, simple, and quantitative filter-binding assay using glass fiber filters has been developed to detect the convalent adenovirus DNA-terminal protein complex. The assay is unusually sensitive because binding of protein-free DNA generally is less than 0.1%. Binding of the adenovirus complex to filters is mediated by terminal protein. We have found that: (i) the adenovirus complex binds maximally to filters in NaCl at concentrations higher than 0.2 M; (ii) noncovalent complexes between protein-free DNA and adenovirus proteins bind to filters in salt at concentrations lower than 0.4 M but not in concentrations higher than 0.7 M; and (iii) protein-free DNA alone binds to filters in guanidine.hydrochloride at concentrations higher than 0.8 M. By varying the ionic conditions, "all or none" modulation of these interactions can be achieved.
Adenoviridae↗
Purification of an adenosine deaminase complexing protein from human plasma.
A protein which specifically complexes with adenosine deaminase (complexing protein) has been purified to homogeneity from human plasma. This protein was compared with complexing protein isolated from human kidney. The two proteins produce electrophoretically different forms of high molecular weight adenosine deaminase when combined with the Mr = 36,000 enzyme monomer from erythrocytes. This difference may, at least in part, be due to the greater sialic acid content of complexing protein from plasma. By other criteria, including amino acid composition, total carbohydrate content, and subunit structure, the two proteins are quite similar. In addition, plasma complexing protein shows complete cross-reactivity with anti-kidney complexing protein serum. These results suggest that plasma and kidney complexing proteins are products of the same gene.
Adenosine Deaminase↗
Enhancement of encephalitogenic activity by the formation of myelin basic protein-brain acidic protein complex.
Encephalitogenic protein fraction (BEC) was isolated from bovine brain tissue by extraction with salt-ethanol mixture at neutral pH, instead of employing dilute mineral acid. The fraction BEC was separated into two fractions. An acid-soluble protein was encephalitogenic and the major component was very alike to the basic protein of myelin (Al). The other was acid-insoluble acidic protein that was not encephalitogenic even at a dose of 100 mug. The acidic protein formed an insoluble complex with Al rotein which was purified by Eylar's method. Encephalitogenic activity of the complex was higher than Al protein in young guinea pigs when injected with complete Freund's adjuvant.However, this enhancement of encephalitogenic activity was not observed in aged guinea pigs. The complex showed higher blastogenic activity than Al protein alone with peripheral blood lymphocytes from guinea pigs immunized with Al protein and complete Freund's adjuvant. These results show that an adjuvant-like acidic protein is present in brain tissue and the complex with Al protein enhances the induction of experimental allergic encephalomyelitis (EAE).
Age Factors↗
Influence of light on activity of adenylate kinase and pterin-protein complexes from pea chloroplasts.
The activity of adenylate kinase (AK) and of pterin-protein complexes (PPC), whose proteins have adenylate kinase activity comparable to that of the enzyme was studied. It was established that light inhibits adenylate kinase activity and that this effect is partially eliminated by phosphate ions. The forward and reverse reactions catalyzed by AK and PPC were studied and it was found that the activity of native protein complexes is different in the forward and reverse reactions. The thermostable protein both of adenylate kinase and of the pterin-protein complexes had identical activity in the ADP dismutation and the reverse reaction.
Adenosine Diphosphate↗
Enhanced infectivity of adenovirus type 2 DNA and a DNA-protein complex.
The infectivity of adenovirus type 2 DNA and a DNA-protein complex was studied in 293 cells, a human embryonic kidney cell line transformed by sheared adenovirus type 5 DNA, and in human KB cells. Adenovirus type 2 DNA was more infectious (up to about 40-fold) in 293 cells than in KB cells, whereas a DNA-protein complex (prepared by a rapid procedure) had about the same infectivity in both cell lines. These data may mean that a factor present in 293 cells (perhaps a viral-coded protein) enhances the infectivity of free viral DNA. The infectivity of DNA and the DNA-protein complex was increased up to fivefold by brief treatment of cell monolayers with 25% dimethyl sulfoxide after transfection. Under these conditions, (i) the infectivity of native adenovirus type 2 DNA ranged from 400 to 1,300 PFU/microgram of DNA in 293 cells and from about 9 to 14 PFU/microgram of DNA in KB cells, and (ii) the infectivity of the DNA-protein complex was 6 X 10(3)to 2 X 10(4) PFU/microgram in 293 cells and 1.4 X 10(4) to 1.6 X 10(4) PFU/microgram in KB cells.
Adenoviruses, Human↗
Characteristics of lipid A-protein complex from endotoxin of Shigella dysenteriae type 1 (S and R strains).
Mild acetic acid hydrolysis of endotoxin (lipopolysaccharide-protein complex) of Shigella dysenteriae type 1 (S and R forms) yielded a lipid A-protein complex that consisted of amino acids, fatty acids, and sugar and, in terms of chemical composition, displayed no marked differences between the S and R forms. Its protein portion (53 to 56%) consisted of at least 16 amino acids. In the fatty acid portion (14 to 18%), myristic, 3-hydroxymyristic, palmitic, and stearic acids accounted for 50%. The sugar portion (10 to 12%) consisted solely of glucosamine. The remainder was unidentified substances, most of which contained phosphorus. Lipid A-protein complexes derived from both S and R forms were not toxic for mice in doses up to 1,000 microgram/mouse, but their Linulus test activity had increased considerably as compared with the starting lipopolysaccharide-protein complex material: from 10(-6) to 10(-10--10(-12) mg/ml. The lipid A-protein complexes were readily soluble in a water solution of triethylamine, in dimethyl sulfoxide, and in pyridine.
Amino Acids↗
[Histone-nonhistone protein complex reconstruction].
A complex of chromatin proteins composed of five histon fractions and nine nonhiston fractions and stable in 2M NaCl solution was destructed by treating with urea, changing pH and decreasing the NaCl concentration, No reconstruction of the complex was observed after the said factors has been removed and the initial solvent composition restored. Nevertheless the reconstruction was detected after dissociation of the complex by desalting in the presence of DNA. The reconstruction was successful when stimulated both dimers and tetramers, the chromatin protein complex under study cannot be reconstructed by "self-organization", the only way being through the stage of nucleoproteid formation, and is similar in this respect to some other complex protein structures of the cell.
Animals↗
Characterization of a DNA-protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis-N,N'-tetraacetic acid and dithiothreitol.
Treatment of polyoma virions with ethyleneglycol-bil-N,N'-tetraacetic acid (EGTA) and dithiothreitol (DTT) at pH 8.5 resulted in the dissociation of the virions into a DNA-protein complex and individual structural capsomere subunits. The sedimentation value of the DNA-protein complex in sucrose gradients was approximately 48S, and it had a density of 1.45 g/cm3 in equilibrium CsCl gradients. Alkaline sucrose analysis of the DNA within this DNA-protein complex demonstrated that approximately 75% of the DNA is component 1. The proteins associated with the DNA were dissociated by treatment with either NaCl or the anionic detergent Sarkosyl. VP1 and the histone proteins VP 4--7 were the major proteins associated with the DNA. Treatment of the DNA-protein complex with alkaline pH resulted in the specific removal of FP1. Electron microscopy of the 48S DNA-protein complex demonstrated that it is a very tightly coiled structure that is slightly larger than the intact virion. Treatment of the complex with either NaCl or with pH 10.5 buffer resulted in the loss of protein and subsequent loosening of the DNA-protein complex such that the DNA could be visualized. The capsomere subunits released as a result of the EGTA-DTT treatment sedimented as 18S, 12S, and 5S subunits in sucrose gradients. Electrophoretic analysis of the isolated capsomeres demonstrated that VP1, VP2, and VP3 were present in each species, although the ratios of the proteins varied. In addition to the structural proteins, histones VP 4--7 were found to be predominantly associated with the 5S capsomere subunit.
Capsid↗
Phagocytic activity of reticuloendothelial system of mice in shock-like state following administration of polysaccharide-protein complex isolated from Candida albicans.
Fever and shock are symptoms of acute disseminated candidiasis. Phagocytic activity of the reticuloendothelial system (RES), following a single administration of polysaccharide-protein complex, was studied with respect to the role of the RES in the pathophysiology of circulatory shock. The index of phagocytic activity was determined 15 or 120 minutes following IV administration of polysaccharide-protein complex (50 mg/kg) to mice, from the rates of clearance of carbon particles and of heterologous erythrocytes labeled with 51Cr. The mice were pretreated with hydrocortisone 300 mg/kg, methylprednisolone, 30 mg/kg, or saline 120 minutes before administration of the polysaccharide-protein complex. Depression of the phagocytic activity was observed following administration of polysaccharide-protein complex in both time intervals investigated. This depression could be prevented successfully by administration of both glucocorticoids studied. The fractional distribution of 86Rb was determined in other groups of identically treated mice. An increase of 86Rb uptake was found in the heart, lungs, and adrenals 15 minutes after administration of polysaccharide-protein complex. A decrease of 86Rb uptake was found in the intestines and spleen. These changes were also found in mice pretreated with glucocorticoids. A return to the normal picture of 86Rb fractional distribution was found 120 minutes after polysaccharide-protein administration. It was suggested that changes in phagocytic activity were not a result of altered hemodynamics.
Animals↗
The 5-S RNA . protein complex from an extreme halophile, Halobacterium cutirubrum. Purification and characterization.
A 5-S RNA . protein complex has been isolated from the 50-S ribosomal subunit of an extreme halophile, Halobacterium cutirubrum. The 50-S ribosomal subunit from the extreme halophile requires 3.4 M K+ and 100 mM Mg2+ for stability. However, if the high K+ concentration is maintained but the Mg2+ concentration lowered to 0.3 mM, the 5-S RNA . protein complex is selectively extracted from the subunit. After being purified on an Agarose 0.5-m column the complex had a molecular weight of about 80000 and contained 5-S RNA and two proteins, HL13 and HL19, with molecular weights (by sedimentation equilibrium) of 18700 and 18000, respectively. No ATPase or GTPase activity could be detected in the 5-S RNA . protein complex. The amino acid composition and electrophoretic mobility on polyacrylamide gels indicated both proteins were much more acidic than the equivalent from Escherichia coli or Bacillus stearothermophilus. Partial amino acid sequence data suggest HL13 is homologous to EL18 and HL19 to EL5.
Adenosine Triphosphatases↗
Resonance Raman spectroscopy of chlorophyll-protein complexes.
Resonance Raman spectra of chlorophyll a (Chl a) and of Chl b were selectively obtained, at low temperature, from chlorophyll-protein complexes prepared from green and blue-green algae and from higher plants. Antenna Chl a in the Chl a-P700-protein complexes (CP I) and in the light-harvesting Chl a/b-protein complexes (CP ii gives resonance Raman spectra extremely close in all their features to those previously obtained from intact cells and chloroplasts. In particular, the same multiplicity of binding sites for the ketone carbonyl groups of Chl a is observed in both CP I and CP II as in intact membranes. These bindings sites are probably the same types as those observed in the intact membranes and are not the magnesium atoms of other chlorophylls. The magnesium atoms of most Chl a molecules in both CP I and CP II bind a single external ligand. Resonance Raman spectra of Chl b in CP II preparations, although very similar to those from intact membranes, show partial rearrangement of one of the two environmental subspecies of Chl b previously found in intact membranes. These results provide evidence that chlorophyll-protein complexes closely represent the state of the bulk of antenna chlorophyll in vivo.
Binding Sites↗
Uptake of polynucleotides by mammalian cells. XV. Properties and function of a DNA-protein complex situated in the outer membrane of Ehrlich ascites tumor cells.
1) DNA-protein complexes are supposed to be original constituents of the membrane of Ehrlich ascites tumor cells. These complexes can be attacked at the surface of viable cells by DNase or protease. The DNA is partially embedded in protein structures. 2) The net charge of this complex is of major importance for the RNA uptake capacity of the cells. Negatively charged DNA which is situated at the surface hinders RNA uptake. This is the explanation for the stimulation of RNA uptake by DNase or the decrease in RNA uptake after protease treatment. 3) Upon treatment of DNA-deficient complexes with homologous or heterologous DNA the original RNA uptake capacity of the cells is restored but the original conformation of the complex cannot be regained. 4) The DNase action on the complex is temperature dependent in a sigmoidal fashion. It is markedly slowed down at temperatures below 12 degrees C. This implies that structural changes in the complex occur at this transition temperature which make surface DNA susceptible to DNase. This effect can only be observed in original structures but not in reconstituted ones. 5) Polyanion treatment of the cells [poly(L-lysine)] which increases their RNA uptake capacity, most probably does not interact with the DNA-protein complex. Poly(L-lysine) appears to act at other membrane sites. 6) The DNA-protein complex has been investigated entirely in situ, i.e. situated in the membrane of viable cells.
Animals↗
ATPase and GTPase activities associated with the 5-S RNA-protein complex of Escherichia coli ribosomes.
Specific 5-S RNA-protein complexes were reconstituted from Escherichia coli 5-S RNA and 50-S ribosomal proteins. These complexes consist of 5-S RNA and two major proteins, namely E-L18 and E-L25. Analysis for enzymatic activities shows that ATP and GTP are hydrolyzed and that this hydrolysis is independent of elongation factors.
Adenosine Triphosphatases↗
Structure of a nicked DNA-protein complex isolated from simian virus 40: covalent attachment of the protein to DNA and nick specificity.
A portion of the nicked circular DNA isolated from purified simian virus 40 contains a protein-DNA complex in which protein(s) is covalently attached to the end of a DNA single strand. (Nicked DNA is double-stranded DNA that contains at least one single-strand scission.) The protein was visualized by electron microscopy and labeled in vitro with 125I. The bond between the protein and the DNA is stable in alkali, 4 M guanidine-hydrochloride, 3.86 M hydroxylamine (pH 4,23), and in 98% formamide. Most of the molecules in the nicked circular DNA fraction contained one nick. The nick occurs on either of the two complementary strands; the specific nick sites on the two strands are staggered, but lie within a few hundred nucleotides of each other.
DNA, Circular↗
Characterization of DNA-protein complexes from simian adenovirus SA7.
DNA-protein complexes prepared from purified simian adenovirus SA7 virions and from lytically infected monkey kidney cells exhibited similar properties when compared with respect to size by sucrose gradient centrifugation, to configuration by electron microscopy, and to susceptibility to a variety of treatments by electron microscopy and electrophoresis in agarose gels.
Adenoviridae↗