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PubMed1979

Macromolecular complexes of aminoacyl-tRNA synthetases from eukaryotes. 1. Extensive purification and characterization of the high-molecular-weight complex(es) of seven aminoacyl-tRNA synthetases from sheep liver.

Starting from homogenates of sheep liver, extensive co-purification of seven aminoacyl-tRNA synthetases to high specific activities was achieved by a three-step procedure involving fractional precipitation by poly(ethylene glycol) 6000, gel filtration on 6% agarose and chromatography on Sepharose-bound tRNA. The purified material is composed of nine major protein components as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has an apparent molecular weight of about 10(6) estimated by gel filtration on 6% agarose. It contains aminoacyl-tRNA synthetase activities specific for methionine, lysine, arginine, leucine, isoleucine, glutamine and glutamic acid. The rigorous co-elution of these seven enzymes at each chromatographic step suggests, but does not conclusively prove, that they are physically associated within the same complex. The enzyme composition of the high-molecular-weight complex purified from sheep liver is identical to that of the complex previously isolated from human placenta by Denney in 1977 (Arch. Biochem. Biophys. 183, 156--167).

Amino Acyl-tRNA Synthetases↗
PubMed1977

In vitro binding of tritiated hormone-cytostatic complexes in the cytosol of various rat tissues and the incorporation of these complexes into nuclei.

Cytosols from rat ventral prostate, liver and muscle were incubated with tritiated estrogen-cytostatic complexes or tritiated estrogen alone. Considerably more radioactivity was recovered in the 4 S region when samples from ventral prostate cytosol were analysed by sucrose density gradient centrifugation compared with samples from liver or muscle cytosols. Similar results were found using Sephadex gel filtration. However, when cytosols were incubated with tritated estrogen alone only unspecific binding was registered. After in vitro tissue incubation with the tritiated hormone-cytostatic complexes, radioactivity was found in purified nuclei of both ventral prostate and liver tissue. The possible nature of the macromolecules which bind hormone-cytostatic complexes is discussed.

Animals↗
PubMed1975

Relaxation complexes of plasmid DNA and protein. II. Characterization of the proteins associated with the unrelaxed and relaxed complexes of plasmid ColE1.

The proteins of the DNA-protein relaxation complex of plasmid ColE1 were labeled with [3H]leucine by growth of ColE1 containing Escherichia coli cells in the presence of this radioactive labeled amino acid. Three major [3H]leucine-labeled proteins are found associated with the supercoiled DNA in the ColE1 relaxation complex. The molecular weights of these proteins, determined by sodium dodecyl sulfate-acrylamide gel electrophoresis, are 60,000, 16,000, and 11,000, respectively. Induction of relaxation of the supercoiled DNA by treatment of the complex with sodium dodecyl sulfate results in a dissociation of the two smaller proteins from the DNA. The 60,000 protein, however, remains associated specifically with the nicked strand of the open circular DNA. The strand-specific association of this protein with the relaxed DNA resists heat denaturation of the DNA, sedimentation through an alkaline (pH 12.5) sucrose gradient, and centrifugation to equilibrium in an alkaline (pH 12.5) CsCl gradient.

Bacterial Proteins↗
PubMedDate not supplied

[Application of rare-earth elements as labels in studying biologically active compounds. III. Natural and magnetic circular dichroism of complexes of rare-earth elements with pyridoxalidene amino acid complexes].

Circular dichroism of pyrodoxalidenaminoacid complexes with rare earths and other metals and magnetic circular dichroism of several neodimium complexes are investigated. CD spectra of the complexes of the rare earths with azometines made of different amino acids could be classified into four groups. Some of the CD spectra are shown to be individual. The dependence of the CD spectra on the rare earch ion dimension is considered. Coordination of the rare earth ions is shown to be the same as in the case of zinc, cobalt, iron or copper.

Amino Acids↗
PubMed1975

Trypsin-activated complex of human factor B with cobra venom factor (CVF), cleaving C3 and C5 and generating a lytic factor for unsensitized guinea pig erythrocytes. II. Physico-chemical characterization of the activated complex.

A complex, CVF-B, between cobra venom factor (CVF) and human factor B(B) showed weak, short-lived enzymatic activity against the third component of human complement (C3). Once activated with trypsin, it showed strong, stable activity against C3 and C5. CVF-B, an activated form of CVF-B complex, was not affected by the trypsin inhibitor, diisopropylfluorophosphate and neuraminidase. Heating at 56 C for 30 min completely destroyed its activity and heating at 50 C for 30 min destroyed approximately half its activity. The activity of DVF-B decrease markedly at pH 6.0 but was stable at pH 6.5 to 8.5. CVF-B lost 90% of its activity on reduction with 1 mM dithiothreitol, and was completely adsorbed on a cellulose acetate membrane. CVF-B was found to be a complex of CVF and glycine-rich gamma-glycoprotein, with a molecular weight of 340,000. The CVF-B molecule consisted of 4-polypeptide chains, 3 of which were derived from CVF and one from GGG. Hemolytically active CVF-B may be formed from 2 molecules with four-polypeptide chains linked by unknown bonds. Human, rat and guinea pig sera could react with CVF-B to generate a lytic factor. Human and sheep erythrocytes were not sensitive to the lytic factor generated by CVF-B, whereas liposomes prepared from their membrane lipids were equally sensitive to the lytic factor.

ABO Blood-Group System↗
PubMed1976

Activator activities of the transient forms of the human plasminogen-streptokinase complex during its proteolytic conversion to the stable activator complex.

When human plasminogen and the bacterial protein streptokinase are mixed, a tight equimolar complex is formed in which an active center of well defined hydrolytic activity developes; this event precedes the cleavage of the plasminogen chain, i.e. the conversion to plasmin. Immediately after the formation of the complex, a series of proteolytic transformations occurs which, within a few minutes, results in at least two cleavages in the plasminogen, and at least five cleavages in the streptokinase peptide chains. None of the fragments so created seem to dissociate from the main body of the complex, but the activator activity, when measured by a rapid bovine clot-lysis system, undergoes a characteristic pattern of fluctuation coincident with the fragmentation of the two components. When the latter process is followed by sodium dodecyl sulfate gel electrophoresis, the state of fragmentation of the activator can be correlated with the measured activator activities. By manipulating the temperature, and by the introduction of inhibitors, it was possible to slow down, or temporarily arrest, the fragmentation at certain stages, allowing the identification in a number of cases of the predominant activator species, and the determination of a characteristic relative activator activity for it. By the use of such relative activities, it was possible to carry out a calculation, based on electrophoretic analysis alone, which predicted reasonably successfully the kinetics of activator fluctuation.

Electrophoresis, Polyacrylamide Gel↗
PubMedDate not supplied

[Circular dichroism of DNA--dye complexes. II. Anisotropy of the long-wave circular dichroism effect and structure of the complex].

Anisotropy of torsional strength of the splitted electronic transition in the case of chromophore-chromophore interaction of dye molecules situated on the helical matrix was considered theoretically and as analytical expression for the value Rperpendicular/Rparallel was obtained. These theoretical results were compared with the experimental data obtained with DNA-proflavine, DNA-pyronine and DNA-acridine orange complexes oriented in multicappilar flow-cell. Studies of the optical effects (optical density and CD changes) due to orientation of these complexes showed that the acridine chromophores are not perpendicular with respect to the DNA axis (alpha D = 19--22 degrees). The DNA base pairs in complexes as assumed also are not perpendicular to the DNA axis, the inclination angle of their transition moments (for the band near 260 nm) being bigger than that of dye chromophores (24 degrees). These results indicate that under experimental conditions used by us no intercalation can be observed.

Acridines↗
PubMedDate not supplied

[Oriented complexes of nucleic acids with low-molecular weight ligands. I. Anisotropy of the absorbance of DNA complexes with distamycin A and its analogs].

The anisotropy of absorbance (near 320 and 258 nm) of the DNA complexes with distamycin A and two analogues have been studied. Measurements were made along the direction of the flow oreintation of the complexes. The comparison of two orientation effects: deltaA320=(Astop--Aflow)320 and deltaA258=(Astop--Aflow)258, gave the opportunity to estimate the inclination angle of distamycin chromophores with respect to the DNA axis: for all three analogues the angle was lying between 40 and 45 degrees. These results strongly support the model of the complex where the antibiotic is inserted into the narrow grove of the DNA helix.

Chemical Phenomena↗
PubMedDate not supplied

[Molecular organization of the long-wave complexes of purple photosynthesizing bacteria. Effect of pronase on the B890 complex of Chromatium minutissium and Rhodopseudomonas palustris].

The pronase action on the long-wave complexes B890 from two different purple bacteria has been investigated. Differences in the kinetics of decrease of the reaction center photochemical activity of electron-donor activity of cytochromes and of destruction of Bx890 (875) forms have been discovered. Different rates of the proteolysis of RC proteins were revealed by SDS-gel-electrophoresis. The heavy protein of RC was the first to degrade. The photochemical transformations deltaA890(875) in B890 complexes was observed during formation of peptides with molecular weight about 17 000 from two other RC proteins. On the basis of obtained data the model of molecular organization of B890 complexes from purple bacteria is discussed.

Bacterial Chromatophores↗
PubMedDate not supplied

[Circular dichroism of DNA complexes with dyes. III. Effect of latent optical activity and the structure of the complexes].

Circular dichroism anisotropy was studied both theoretically and experimentally for the complexes of DNA and dsRNA with dyes (proflavine, 2,7-di-t-butyl proflavine, "Hoechst-33258") and antibiotics (distamycin A, netropsin and olivomycin). Theoretical analysis showed that general features of CD anisotropy, revealed in the previous studies (CD components--delta epsilon parallel to and delta epsilon perpendicular--are ten times or more bigger than the CD-effect without orientation, and delta epsilon parallel to approximately 2 delta epsilon perpendicular) are due to the existence of a specific effect named "latent" optical activity (LOA). This effect can be observed in many cases of non-chiral symmetrical chromophores if they are oriented. The effect is due to the excitation of an electrical dipole transition and a perpendicular magnetic dipole transition (or quadrupole transition) of a molecule. The amplitude and the sign of the LOA-effect depends on the orientation of the chromophores with respect to the light beam; with a random orientation the mutual compensation of LOA-effects of different chromophores happens and no LOA-effect appears. The analitycal expressions relating the value of LOA-effect of the system with electronical characteristics of the chromophores and the geometrical parametra of their arrangement was obtained. The experimental data obtained for the oriented complexes of DNA and dsRNA with proflavine made it possible to determine an angle between the chromophore and the plane perpendicular to the DNA axis--gamma. For the calf thymus DNA gamma = = + 1.8 +/- 0.4 degrees, for the phage T2 DNA gamma = + 2.2 +/- 0.4 degrees, and for phage f2 dsRNA gamma=--3.5 +/- +/- 0.5 degrees. These results, obtained at relatively low concentrations of the bound proflavine (r approximately 0.01), are in accordance with the intercalating mode of the dye binding. A study of CD anisotropy of DNA complexes with other ligands showed that many different chromophores possess LOA-effect. This phenomenon can be used to obtain both spectroscopic and structural information about the systems similar to those reported here.

Acridines↗
PubMed1979

Experimental chronic serum sickness in rats. A model of immune complex glomerulonephritis and systemic immune complex deposition.

This article describes a method of immunization that produces chronic serum sickness in rats within a relatively short time. Fisher rats, which were immunized subcutaneously three times with bovine serum albumin (BSA) in adjuvant, responded with high titers of antibodies to BSA. 2 weeks after the third subcutaneous immunization, daily increasing amounts of BSA were injected either intraperitoneally or intravenously. When an intravenous dose of 2mg of BSA was reached, the rats were given daily intravenous injections of BSA for several weeks. This procedure, which avoided death from anaphylaxis, induced severe proliferative glomerulonephritis in all the rats and produced deposition of antigen-antibody complexes in many other organs besides the kidney. This highly reproducible model of experimental chronic serum sickness in inbred animals may have applications for the study of the mechanisms of immune complex disease.

Animals↗
PubMed1979

Complexed rheumatoid factor measurements in sera, synovial fluids and in immune complex fractions.

Mild acidic treatment increases the rheumatoid factor titre of some sera and synovial fluids (SF) in rheumatoid arthritis (RA), juvenile RA (JRA) and most frequently in rheumatoid vasculitis. This unmasking of 'hidden' RF in serum and SF samples correlated with the RF-immune complexes (RF-IC) and complexed C4 present in the 3% polyethylene glycol (PEG) precipitates, indicating that by means of 'hidden' RF measurements RF-ICs are possibly detected. This method seems to provide a diagnostic tool for detecting RF-ICs in RA and other related diseases.

Antigen-Antibody Complex↗
PubMed1975

Immune complexes in cancer: demonstration of complexes in mice bearing neuroblastomas.

Immune complexes occurred in a large proportion of mice with syngeneic neuroblastomas. Circulating immune complexes formed in all mice, whereas almost 50% had deposits of host IgG and complement in a granular pattern along the mesangia and basement membranes of the renal glomeruli. The bound IgG was eluted and recovered from the glomeruli and demonstrated immunologic specificity to neuroblastoma cell-surface antigens.

Animals↗
PubMed1978

Viral immune complexes in systemic lupus erythematosus: C-type viral complex deposition in skin.

Punch biopsies were examined by indirect immunofluorescence for immune complex deposits containing C-type viral antigen. Antisera specific for immunoglobulins and HEL-12 virus mediated fluorescence at the dermal-epidermal junction and in vessel walls of 16 of 16 biopsies involved skin from patients with systemic lupus erythematosus (SLE). Preimmune sera did not mediate fluorescence and gradient purified HEL-12 virus, simian sarcoma virus and baboon endogenous virus but not Rous sarcoma virus blocked the reaction of anti-HEL-12 virus serum with SLE tissue. Ten biopsies from uninvolved skin of the patients with SLE did not react with the antiviral serum, nor did tissue from 9 patients with discoid lupus erythematosus, psoriasis, bullous pemphigoid or normal skin. These data support the hypothesis that C-type viral immune complexes participate in the pathogenesis of SLE.

Animals↗
PubMed1976

Cryoprecipitates and immune complexes: differentiation between cold insoluble immune complexes and nonspecific cryoprecipitates.

Cold insoluble circulating immune complexes occurred in 77% of rabbits immunized with 125I BSA. 9.8% of the total cryoprotein was 125I BSA. This represented 43% of the serum antigen. Tripling the antigen concentration in vitro decreased the amount of cold insoluble antigen by as much as 83%. In nonspecific cryoprecipitates only 0.4% of the total cryoprotein was 125I BSA. This represented only 0.028% of the serum antigen. Tripling of the antigen concentration in vitro increased the amount of cold insoluble antigen. Cold insoluble circulating immune complexes occur at a critical antigen-antibody ratio and can be differentiated from nonspecific cryoprecipitates.

Animals↗
PubMed1979

Circulating immune complexes in acute uveitis: a possible association with the histocompatibility complex locus antigen B27.

39 patients with acute anterior uveitis were investigated for the presence of circulating immune complex (IC) and correlation with the major histocompatibility complex antigen B27 (HLA-B27). ICs were demonstrated by a number of techniques including rheumatoid factor, complement (C) activation, anticomplementary activity, cryoglobulins, inhibition of IgG-EA rosette formation and neutrophil chemotactic index (NCI) in plasma. ICs were most frequently detected in HLA-B27-negative patients. These results indicate that deposition of circulating ICs may be involved in the pathogenesis of acute anterior uveitis, especially in HLA-B27-negative patients.

Acute Disease↗
PubMed1978

Studies into the occurrence of soluble antigen-antibody complexes in disease. VIII. Fractionation of rheumatoid samples containing immune complex-like material.

Fractionation studies were performed of an immune complex-like material measured in rheumatoid synovial fluid using a biological assay based on histamine release from guinea pig lung. This material was eluted from Sepharose 6B in the excluded fraction, from DEAE cellulose predominantly in the third buffer, and from polyacrylamide electrophoresis in a cathodal position. Heterogeneity of the complexes was evident on iso-electric focussing, with one peak of activity at a pH of 4.2--5.6 and the other at pH 6.8--8.6. Purification of the material was limited by a loss of activity which occurred on using three fractionation procedure in sequence. This instability was avoided in the case of Sepharose 6B by adding 0.1% bovine or rabbit albumin to the elution buffer.

Animals↗
PubMed1978

Virus-induced immune complex disease: identification of specific viral antigens and antibodies deposited in complexes during chronic lymphocytic choriomeningitis virus infection.

Structural proteins of LCMV were identified and their role in the immune complex glomerulonephritis of LCMV carrier mice was examined. Purified LCMV contained three major polypeptides, a single nonglycosylated nucleoprotein with an estimated m.w. of 63,000, and two surface glycoproteins of 54,000 and 35,000. Deposition of nucleoprotein antigen in the glomeruli of LCMV carrier mice of several strains was demonstrated by immunofluorescent staining with a monospecific antibody. In addition, Ig eluted from kidneys of three strains of LCMV carrier mice was shown by immune precipitation to react against all of major viral polypeptides of LCMV. Antibody from normal mice, and from mice with immune complex disease unrelated to LCMV did not show deposition of LCMV antigen in glomeruli, and Ig eluted from the kidneys of these mice did not react against LCMV antigens. Hence, mice infected at birth with LCMV and persistently infected throughout their life make antibodies to all the known structural polypeptides of the virus.

Animals↗