Agarose Gel Chromatography Research Articles

Photoregulation of K+-ATPase in vitro by Red and Far Red Light in Extracts from Cucumber Hypocotyls

Particulate material from cucumber hypocotyls was purified by differential centrifugation and agarose gel chromatography. The fraction was heterogeneous in nature and contained both phytochrome and ATPase activity. The latter consisted of at least two different components, a non-specific acid phosphatase and a potassium stimulated ATPase (K + -ATPase). The K + -ATPase appeared to be strongly inhibited by Ca +2 and was subject to photoregulation by red and far red light (R and F) in vitro . F caused an increase in Km for ATP and this was reversible by subsequent R. No significant effect on Vmax was caused by the light treatments. Photoregulation of ATPase in vitro was only seen if R was given in vivo prior to extraction. The effect of R in vivo was reversible by F indicating that this also was a phytochrome response. The acid phosphatase activity was not subject to photoregulation but its presence prevented further characterisation of the K + -ATPase and its photoregulation. The results are interpreted as an indirect effect of phytochrome on the K + -ATPase possibly by affecting membrane permeability and substrate availability.

Structural polypeptide composition of a murine myeloma (MOPC-315) type C retrovirus

Summary The polypeptide composition of an endogenous NB-tropic type C retrovirus of murine myeloma MOPC-315 cells was analyzed by agarose gel chromatography in 6 M guanidine hydrochloride and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Seven polypeptides were reproducibly resolved: five with estimated molecular weights of 110,000, 70,000, 45,000, 15,000 and 10,000 daltons, and two with molecular weights of 30,000 daltons. The 110,000-, 70,000- and 45,000-dalton polypeptides were found to be glycosylated, and of these the 70,000-dalton protein was the major glycoprotein, while the 45,000-dalton protein was minor and poorly glycosylated. The two 30,000-dalton polypeptides of MOPC-315 virus which were found to be identical with regard to their peptide profile share antigenic determinants with the Mo-MuLV-derived 30,000 protein but differ from it in their peptide composition. The findings of two 30,000-dalton proteins in an additional member of the myeloma viruses coupled with the presence of unusual high molecular weight glycoproteins, provides further evidence in support of the notion that the myeloma type C viruses comprise a distinct group within the murine leukemia viruses.

Monoglyceride hydrolase activities of rat plasma and platelets. Their properties and roles in the activity of lipoprotein lipase.

Rat plasma contains monoglyceride hydrolase activities against both 1(3)- and 2-monoglycerides. These activities are present as lipoprotein complexes recovered by either density flotation or by agarose gel chromatography with plasma high density lipoprotein. However neither activity is complexed with the major apoproteins (apo-A-I, apo-E) of this lipoprotein class. 2-Monoglyceride hydrolase (but not 1(3)-monoglyceride hydrolase) activity associates with the triglyceride-rich lipoprotein class. The two activities are also noncompetitive with respect to substrate, and differ in pH- and cofactor-dependence and sensitivity to inhibition by diethyl p-nitrophenyl phosphate. Rat platelets also contain both 1(3)- and 2-monoglyceride hydrolase activities. These differ in reactivity with antiesterase and after solubilization and electrophoretic migration with each other and with the corresponding plasma activities. Studies with the isolated perfused rat heart suggest that a major role in the catabolism of 2-monoglyceride generated from lipoprotein lipase activity at the coronary bed is played by the plasma 2-monoglyceride hydrolase activity.

Comparison of different methods for the detection of rubella-specific IgM antibodies.

The rubella specific IgM titer in the serum specimens originating from healthy persons and from patients with clinical signs of rubella infection was determined by hemagglutination inhibition or hemagglutination reduction after IgM separation with the following methods: (a) density gradient centrifugation; (b) polyacrylamide agarose gel chromatography; (c) ion exchange chromatography with diethylaminoethyl cellulose columns; (d) solid-phase immunosorbent technique using microplates; (e) solid-phase immunosorbent technique using polyacrylamide microimmunobeads. Alternatively, we removed IgG and IgA by the use of protein A, anti-IgG, and anti-IgA, covalently coupled to controlled-pore glass (f). The titers obtained by the different methods showed qualitatively good correlations when combined with mercaptoethanol reduction. The quantitative measurement of specific IgM titers, however, revealed a lower sensitivity of column chromatography and methods of removal of IgG/IgA.

Transformation of the estrogen-receptor complex from chick oviduct in 2 steps

Salt treatment of the cytoplasmic estradiol-receptor complex from chick oviduct induces a strong affinity of the complex for DNA-cellulose and phenyl-sepharose. This process is called activation. Binding to heparin- and lysozyme-sepharose is also observed with the untreated complex. But, after salt treatment, additional binding of the complex to these adsorbents is seen. The increased ability of the complex to bind to polyanions and polycations is destroyed by mild trypsination. The binding to the hydrophobic adsorbent is not affected by this treatment. Neither a change of the sedimentation constant nor of the size of the receptor protein is observed after salt treatment in the cold. After binding of the salt-activated estradiol-receptor complex to DNA-cellulose in the cold, an increase of its sedimentation constant and its size, as measured by density-gradient centrifugation and agarose gel chromatography, resp., becomes apparent. A similar phenomenon is observed after binding to DEAE-cellulose and to some extent after binding to heparin-sepharose. The nuclear complex seems to have the same sedimentation constant as the cytoplasmic complex eluted from DNA-cellulose. The sedimentation constant of the nuclear complex is not changed after DNA-cellulose chromatography. The cytoplasmic progesteronereceptor complex from the same tissue i.e. the oviduct, does not show any change of size. Thus the well-known process of transformation can now be separated into 2 steps. (1) Activation of the estradiol-receptor complex for its binding to various adsorbents in vitro and probably to its acceptor site(s) in vivo. (2) Increase of receptor size. This second step seems to be a special property of the estradiol-receptor complex. Its physiological significance is unclear.

Purification and characterization of a nuclear non-histone basic protein (Mi-1) which reacts with anti-immunoglobulin sera and the sera of patients with dermatomyositis

Mi-1, a soluble nuclear non-histone basic protein, was purified from calf thymus nuclear extract. The nucleoplasmic fraction was prepared and subjected to ammonium sulfate fractionation, DEAE-cellulose chromatography and filtration on agarose gel columns, resulting in a 1563-fold purification. The Mi protein migrated as a single band of gamma globulin mobility in polyacrylamide gel electrophoresis and many of its physical and chemical properties have been determined. Agarose gel chromatography on a column calibrated with appropriate markers revealed that the molecular weight of the Mi-1 antigen was 150,000 daltons. Determination of the molecular weight in dissociating conditions of sodium dodecyl sulfate-mercaptoethanol demonstrated that a subunit of 75,000 daltons was generated. Gel diffusion and immunoaffinity column studies revealed that the Mi-1 antigen bears immunoglobulin determinants, notwithstanding its nuclear localization and absence of demonstrable light chains. The antigen has been radioiodinated and a double antibody radioimmunoassay developed. A clinical survey reveals that only 2 of 19 patients with dermatomyositis had high binding of the Mi-1 antigen, while a large number of sera from patients with polymyositis, other connective tissue diseases, and normal persons failed to bind this antigen.

Microcomplement fixation and particle size of chicken lipoproteins

Conformational changes of the lipoproteins of chicken serum and egg yolk were studied with the use of microcomplement fixation. Egg yolk very low density lipoproteins and serum total low density lipoproteins were separated by 2% agarose gel chromatography into four and five fractions, respectively, with different average particle diameters. A correlation between percent complement fixed at equivalence and particle diameter was noted. Thus, smaller particles fixed less complement. The shortening of the radius of curvature could alter the steric fit of apolipoprotein with antibody and, as a consequence, reduce complement fixation. The patterns obtained after delipidation of very low density lipoproteins indicate a general distortion of apolipoprotein conformation supporting the view that lipids are essential for maintaining the conformation of these apolipoproteins. On the other hand, the general conformation of the high density apolipoproteins does not appear to depend on lipid content. It has been reported that lipids restrain the free movement of these apolipoproteins. After delipidation, the greater degree of freedom allowed the apolipoprotein may increase the strength of the antigen-antibody union and therefore an increase in complement fixation is observed. These studies demonstrate the applicability of quantitative microcomplement fixation to the eludication of lipoprotein structure and function.

Kinetic mechanism of rabbit muscle glycogen synthase I.

The kinetic mechanism of rabbit muscle glycogen synthase I was investigated by determining isotope-exchange rates at chemical equilibrium between uridine diphosphoglucose (UDPG) and glycogen and between UDPG and uridine 5'-diphosphate (UDP). The rates were followed simultaneously by use of UDPG labeled with 14C in the glucose moiety and with 3H in the uracil group. They were found to be independent of the concentrations of glycogen and the UDPG-UDP pair, averaging 6 X 10(-9) mol min-1 mg-1, with a ratio of UDPG-glycogen exchange to UDPG-UDP exchange of 0.85-0.95. The conclusion is that glycogen synthase has a rapid equilibrium random bi bi mechanism. The previously reported slow activation of glycogen-free synthase in the presence of glycogen was examined kinetically. The activation rate appears to be independent of glycogen concentration over a wide range, while the maximum activation is related to the third or fourth root of the glycogen concentration. This suggest that the slow bimolecular reaction mechanism proposed for human polymorphonuclear leucocyte glycogen synthase I [Sølling, H., & Esmann, V. (1977) Eur. J. Biochem. 81, 129] does not apply to rabbit muscle synthase I. The rate of exchange of glycogen molecules in the complex between glycogen and rabbit muscle synthase I under conditions where the enzyme is catalytically active was estimated by a novel method. The enzyme-glycogen complex was treated with [glucose-14C]UDPG and glycogen of different molecular weight. The distribution of isotope between the two forms of glycogen was determined after their separation by agarose gel chromatography. A rate constant of 0.3 min-1 was estimated for the exchange. It can be calculated, on the basis of the specific activity of the enzyme (20 mumol min-1 mg-1) and its action pattern, that hundreds of individual chains in the glycogen molecule must be available to the enzyme during the average lifetime of the complex. A mechanism is proposed for this process.

Products of delipidation of intestinal chylomicrons with diethyl ether

To investigate potential modes of dissociation of their surface constituents, chylomicrons, obtained from human subjects with chyluria or from rat mesenteric lymph, were delipidated with diethyl ether. The ether dissolved most of the chylomicron apolar lipids. The polar surface constituents remaining in the acqueous phase were incorporated into discrete “low” and “high density” lipoproteins, as judged by preparative ultracentrifugation or agarose gel chromatography. The density 1.006–1.063 particles consisted primarily of phospholipid, apoA-I, apoA-IV and C-apoproteins and the density 1.063–1.21 particles of phospholipid, ApoA-I and apoA-IV. The density 1.006–1.063 particles appeared as phospholipid vesicles when examined by negative stain electron microscopy. When chylomicrons were delipidated in the presence of [ 3H]glucose, radioactivity was associated with the vesicles, consistent with trapping of [ 3H]glucose in their internal aqueous space. In further experiments human plasma high density lipoproteins (HDL) were added to the aqueous phase after delipidation of chylomicrons with diethyl ether. Following incubation for 1h, vesicle phospholipids, apoA-I and apoA-IV were largely incorporated into the column HDL region, with release of vesicle-associated [ 3H]glucose. Following incubation for 16 h, part of this HDL fraction was transformed into a larger lipoprotein, enriched in apoE and apoA-II. Solvent delipidation of the chylomicron core leads to dissociation of the surface polar lipids primarily as lipid bilayer vesicles containing adsorbed water-soluble apoproteins. Upon interaction with plasma HDL, there is disruption of the vesicle structure and incorporation of phospholipid and probably apoA-I and apoA-IV into HDL-like particles.

Studies on the transfer of phosphatidylcholine from unilamellar vesicles into plasma high density lipoproteins in the rat.

To investigate the metabolic disposition of phosphatidylcholine vesicles, unilamellar vesicles of egg phosphatidylcholine or cholesterol/phosphatidylcholine were injected intravenously into rats. Ten minutes after injection of 0.5 to 3.0 mg phosphatidylcholine vesicles, a major fraction of their radioactivity and mass had been incorporated into high density lipoproteins (HDL). Agarose gel chromatography showed that the phospholipid of vesicles was incorporated into the plasma HDL region, in association with release of a trapped marker, [3H]inulin. Density gradient ultracentrifugtion showed that the highest specific activity of phospholipid was present in HDL of d 1.08-1.12 g/ml, consisting of homogenous spherical particles by negative stain electron microscopy. With increasing cholesterol/phosphatidylcholine ratio of injected vesicles, there was progressively less incorporation of phospholipid into HDL and vesicles retaining [3H]inulin were re-isolated from plasma. Sixty minutes after injection of cholesterol/phosphatidylcholines vesicles, phospholipid appeared to be partly transferred into HDL and partly taken up by the liver. In summary, injection of unilamellar egg phosphatidylcholine vesicles results in a rapid incorporation of vesicle phospholipid into plasma HDL, primarily as a result of insertion of phospholipid into pre-existing HDL. This process is inhibited by a high content of vesicle unesterified cholesterol. These studies may have relevance to the mechanisms of transfer of phosphatidylcholine from chylomicrons into plasma.

DNA-binding ability and partial purification of the cytoplasmic estrogen receptor from chicken oviduct

1. 1. Large chicken oviducts containing an optimal amount of the cytoplasmic estrogen receptor can be obtained by the application and short-term withdrawal of diethylstilbestrol implants to chickens. 2. 2. The cytoplasmic estrogen-receptor complex from chicken oviduct is able to bind to DNA. The efficiency of the binding (maximal 60%) is dependent on the temperature and the presence of the hormone. 3. 3. The strogen-receptor complex eluted from the DNA-cellulose by high salt is different from that of untreated cytosol. The observed increase in sedimentation coefficient, molecular weight and assymetry of the receptor molecule resembles the ‘transformation’ of the strogen-receptor complex from rat uterus. 4. 4. Using DNA-cellulose and agarose gel chromatography, the cytoplasmic estrogen-receptor complex from chicken oviduct cna be purified 4000-fold.

The effect of avian retroviruses on limb bud chondrogenesis in vitro

Mesenchymal cells isolated from stage 24 embryonic chicken limb buds were infected with the temperature-sensitive transformation mutants of Rous sarcoma virus tsNY68, tsNY10 and tsLA25 at the nonpermissive temperature for transformation (41°C). Virus infection greatly inhibited subsequent limb bud chondrogenesis under nontransforming conditions, as indicated by a reduction in the rate of 35SO 4 incorporation into cell-associated proteoglycans. The inhibition of chondrogenesis was directly related to the percentage of cells infected with tsNY68 at 41°C. The observed inhibition of chondrogenesis was independent of src gene expression since this effect was also caused by many viruses which lack the src gene, including the leukosis viruses RAV-1, RAV-2 and MAV-2(O); the src deletion mutant RSVtd107; and the reticuloendotheliosis viruses REV-T and SNV. Infection of mesenchymal cells with tsNY68 under nontransforming conditions did not cause changes in parameters such as the rate of thymidine incorporation, total cell DNA and total cell protein. Infection with tsNY68 at 41 °C resulted in altered kinetics of 35SO 4 incorporation into cell-associated proteoglycans and a corresponding reduction in 35SO 4-labeled proteoglycans extracted from the cell layer. There were no apparent quantitative effects on the rate of accumulation of proteoglycans in the culture medium. The proteoglycans extracted from the cells and the collected medium of tsNY68-infected cultures were smaller than those of uninfected cultures, as shown by agarose gel chromatography.

Electrophoretic Characterization of Bovine Lipoprotein Subfractions Isolated by Agarose Gel Chromatography

Lipoproteins isolated from whole bovine serum were applied to agarose gel columns and fractionated by molecular size. Three distinct lipoprotein absorbance peaks (280nm) emerged from the columns (Peaks I, II, and III). A typical elution profile exhibited lipoprotein distribution of approximately 11% Peak I, 6% Peak II, and 83% Peak III. Characterization of Peak I lipoproteins on agarose gel electrophoresis and polyacrylamide gel electrophoresis suggested that Peak I consisted of very low density lipoproteins. Comparison of polyacrylamide gel electrophoresis patterns of successive tube fractions in Peak II demonstrated the progressive emergence of three bands. The slowest migrating band appeared in fractions from the ascending portion of Peak II. Two additional bands progressively emerged from fractions in the central portion of the peak. These bands may represert low density lipoprotein and its metabolic precursors (intermediate density lipoproteins). Successive tube fractions in the area between Peaks II and III displayed a progressive change from β to α migration on both polyacrylamide gel electrophoresis and paper electrophoresis. This heterogeneous group of lipoproteins is probably equivalent to high density lipoprotein-subfraction 1. Lipoproteins from the center of Peak III demonstrated a electrophoretic migration patterns.

Detection of fragment D-fibrin complex by agarose chromatography

Bovine fibrin monomer-fragment D mixtures, after prolonged incubation, were assayed by agarose gel chromatography. The elution peak of fragment D was found to be symmetrical with the constant elution volume, but fibrin elution profile changed depending on fragment D concentration. At high fragment D concentrations a part of it eluted together with fibrin, apart from the free fragment D, revealing fibrin-fragment D complex formation. Of the two fragment D subspecies differing in molecular weight the light one couples with fibrin preferably. In similar experiments performed with fibrinogen substituting for fibrin no coupling occured.

Cryoprecipitation- a more efficient method than agarose gel chromatography for removal of contaminating soluble fibrin in human fibrinogen preparations

Cryoprecipitation- a more efficient method than agarose gel chromatography for removal of contaminating soluble fibrin in human fibrinogen preparations

Purification and insolubilization of reptilase for the preparation of human des-AA fibrin monomers in urea

Reptilase was extensively purified from crude Bothrops atrox venom, and insolubilized on CNBr-activated Sepharose 4 B. When human fibrinogen, I-125-labelled or unlabelled, was exposed to insolubilized highly purified reptilase in the presence of 2.5 M urea, pH 7.4, at +20°C for 1 h, complete transformation of fibrinogen to des-AA fibrin monomers (labelled or unlabelled) as judged by N-terminal amino acid analysis and FPA-RIA quantitation, occurred without gelation. Subunit chain pattern of mercaptolysed, carboxymethylated des-AA fibrin monomers in acid polyacrylamide gel electrophoresis substantiated that only fibrinopeptides A had been proteolysed, and that the B beta chain had a mobility similar to that of control fibrinogen. Agarose gel chromatography of the des-AA fibrin monomer preparation in 2.5 M urea demonstrated a symmetrical peak. When the urea concentration was reduced by dilution, the fibrin monomers had the same clottability and polymerization speed as control fibrinogen exposed to large amounts of reptilase. Storage for 4 weeks at +4°C or quick freezing in liquid nitrogen with subsequent thawing and testing, caused only slightly prolonged polymerization times. Identical results were obtained with both labelled and unlabelled des-AA fibrin monomers.

Separation of transcriptively active and inactive chromatin Agarose gel chromatography

Sheared chromatin fractionated by currently accepted methods of agarose gel exclusion chromatography, undergoes a limited and non-specific aggregation resulting from the high ionic strength and divalent cation concentration of the column elution buffer. Such aggregation causes the artifactual appearance of radioactively labeled, newly synthesized RNA within the column exclusion volume, erroneously suggesting an enrichment for actively transcribed chromatin. Claims for the efficacy of agarose gel exclusion as a method for separating template-active and -inactive chromatin are based largely on assays for active chromatin which rely on localization of specific molecular complexes of chromatin and nascent RNA. Under the conditions employed, the present studies invalidate this assay and thus cast considerable doubt on the agarose gel exclusion method itself.

Characterization of proteins and other macromolecules by agarose gel chromatography

A number of proteins and proteoglycans were chromatographed on a Sepharose 4B column at various ionic strengths and after application of various amounts of samples. K av was negligibly affected by the chromatographic conditions. When K av was plotted against Stokes′ radius, it was found that molecules with different frictional ratios followed relationships. These results are discussed in relation to the effects of molecular asymmetry. Physical parameters for Sepharose 4B and 6B gels were calculated from chromatography of two protein mixtures.

Characterization of the collagen chains synthesized by cultured smooth muscle cells derived from rhesus monkey thoracic aorta

Five different collagen chains and one smaller collagenous fragment have been isolated from the collagens found in the combined cell layer and medium of rhesus monkey aortic smooth muscle cell cultures. The collagen chains which can be identified are alpha1 (III), alpha1(I), alpha2, A and B. The smaller collagenous peptide exhibits an apparent molecular weight of 45 000 and has been designated CP45 (Mayne, R., et al. (1977), Arch. Biochem. Biophys. 181, 462). Smooth muscle cells continue to synthesize the collagens from which these components are derived for at least eight passages in culture. At each passage the alpha1 (III) chain consistently represents about one-half of the total collagen which is recovered after initial fractionation by agarose gel chromatography. The results show that smooth muscle cells derived from rhesus monkey thoracic aorta are phenotypically stable for many generations in vitro.

Polyacrylamide gel electrophoresis of visna virus polypeptides isolated by agarose gel chromatography

The proteins of visna are separated into nine major peaks by agarose gel chromatography in 6 M guanidine hydrochloride (GuHCl). The polypeptides in eack peak were isolated by acid precipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The patterns of SDS-PAGE show that the excluded material from the GuHCl column contains an aggregate of 10 non-glycosylated polypeptides. It is shown that this aggregate represents virus substructures that are not completely solubilized by GuHCl. Two glycoproteins, gp175 and gp115, were isolated from the column eluate. The major glycoprotein gp115 was coeluted with P90, P68, and P61 in GuHCl 4. Each of the four major peaks (GuHCl 5 to 8) contains more than one nonglycosylated polypeptide. However, a small polypeptide, P12, can be isolated in a homogeneous form in the last peak, GuHCl 9. Analysis of the virus proteins (100 microgram) by SDS-PAGE shows that 20 radioactive bands can be recognized. During fractionation of the protein on agarose gel columns followed by analysis with SDS-PAGE, a number of minor polypeptides that were not detected before became clearly recognizable. Thus, the combined use of column chromatography and SDS-PAGE shows that visna virus is composed of 25 proteins.