Showing papers on "Gel electrophoresis published in 1973"

The Subunit Structure of Rabbit‐Skeletal‐Muscle Phosphorylase Kinase, and the Molecular Basis of Its Activation Reactions

Abstract: Phosphorylase kinase was isolated from rabbit skeletal muscle in a state approaching homogeneity as judged by the criteria of ultracentrifugal analysis, ion-exchange chromatography, and antigen-antibody precipitation in agar. The purified enzyme showed four protein-staining components upon acrylamide gel electrophoresis in the presence of sodium dodecylsulphate, termed α, α′, β and γ. A variety of precautions designed to eliminate the possible action of proteases and ensure complete reduction and denaturation of the protein had no influence on the gel pattern. The molecular weights of the four components determined by gel electrophoresis, and supported by sedimentation equilibrium in the presence of 6 M guanidinium chloride, following partial separation of the chains by gel filtration on Sephadex G-200 in the presence of sodium dodecylsulphate, were:–α= 145000, α′= 140000, β= 128000 and γ= 45000. The α' component was present only in trace amounts and the evidence suggests it may be derived from the α component. The three subunits α, β, and γ, were found to exist in equimolar quantities by densitometric analysis of acrylamide gels, by gel filtration on Sephadex G-200 in the presence of sodium dodecylsulphate, and by carboxymethylation with iodo[14C]acetate, suggesting a minimal binding molecular weight, αβγ, of 318000. The molecular weight of the native enzyme was determined as 1.28 × 106, demonstrating that phosphorylase kinase is composed of 4.0 αβγ units. Activation of the enzyme by incubation with adenosine-3′:5′-phosphate-dependent protein kinase, adenosine 3′:5′-phosphate and Mg-ATP, was accompanied by the phosphorylation of one site on the β subunit, although a second site on the α subunit was phosphorylated at a slower rate. Activation of the enzyme by proteolysis resulted from a limited cleavage of the α subunit. The products of proteolytic attack suggest that the α and β subunits may be structurally related. The γ subunit was not phosphorylated, was resistant to proteolysis and distinct from the (α+β) subunits in its amino acid composition. The possible functions of the chains, and the implications of the activation reactions to the nervous and hormonal control of glycogenolysis in vivo are discussed.

Purification and characterization of adenosine triphosphate: ribonucleic acid adenyltransferase from Escherichia coli.

Abstract: A primer-dependent poly (A)-polymerizing activity using ATP as substrate (ATP: RNA adenyltransferase) was isolated in high yield from Escherichia coli and purified to apparent homogeneity. For this purpose an assay system had to be used which restricted a variety of infering enzyme activities. Since the enzyme aggregates to macromolecular cell components or precipitates when kept in low salt conditions (<0.4 M NaCl), it was necessary to perform the entire purification procedure in high salt conditions. This was accomplished by using a high salt ribosomal supernatant, a polyethylenglycol-dextran-NaCl phase partition step and a very efficient final high salt phosphocellulose chromatography. At 0.5 M NaCl the enzyme has a molecular weight of approximately 58000. Dodecylsulfate gel electrophoresis shows a single polypeptide chain of molecular weight × 50000. The enzyme has a high preference for ATP as substrate. Manganese ions show higher activity as cofactors than magnesium ions. All classes of natural RNAs are used as primers. The free 3′terminal hydroxyl group of the RNA is required. The enzyme is unable to catalyze a pyrophosphorolysis or a phosphorolysis reaction. It is shown that ATP: RNA adenyl-transferase preferentially synthesizes rather long chains of poly(A) attached to the RNA primers. No relation between the enzyme protein and subunits of the DNA-dependent RNA polymerase could be detected.

Gel Electrophoresis of Avian Leukosis and Sarcoma Viral RNA in Formamide: Comparison with Other Viral and Cellular RNA Species

Abstract: Class a and class b 30 to 40S RNA subunits obtained by heat dissociation from the 60 to 70S RNA complex of avian tumor viruses were compared with several RNA standards by electrophoresis in formamide-polyacrylamide gels. Class a RNA was found to have a lower electrophoretic mobility and hence probably a higher molecular weight than class b RNA. The absolute molecular weight of class a and b RNA could not be determined with accuracy, because the relationship between logarithm of molecular weight and mobility of the RNA standards was not linear. The size of class a RNA fell into the range of 2.4 × 106 to 3.4 × 106 daltons and that of class b into the range of 2.2 × 106 to 2.9 × 106 daltons, depending on the standards used. The possible biological significance of this difference is discussed.

Antibodies to a specific surface antigen of t cells in human sera inhibiting mixed leukocyte culture reactions

Abstract: Surface radioiodination of lymphocytes by the lactoperoxidase procedure has permitted demonstration of an assortment of different antibodies to lymphocytes in the sera of patients with systemic lupus erythematosus. One type of antibody proved of special interest because it appeared to be associated with inhibition of the responder cells in the mixed leukocyte culture reactions. This reacted with an antigen on T cells and thymocytes which on sodium dodecyl sulfate acrylamide gel electrophoresis showed a molecular weight of approximately 15,000 daltons. The possible relation of this antigen to T cell receptors and products of immune response genes is discussed.

The duck-globin messenger-ribonucleoprotein complex. Resistance to high ionic strength, particle gel electrophoresis, composition and visualisation by dark-field electron microscopy.

Abstract: The duck globin messenger ribonucleoprotein complex which contains the biologically active globin mRNA and two major protein components associated with it, has been further characterized. When analysed on gradients of increasing ionic strength, the mRNA · protein particle showed resistance to 0.25 M LiCl, a condition which fully dissociates artificial RNA · protein complexes, and was partially resistant to 0.75 M LiCl. By polyacrylamide gel electrophoresis the particle was shown to consist of one major component A and two minor components B and C of increasing mobility. Component A and C were ribonucleoproteins, B consisted of free proteins; no free RNA could be detected. Component A, when analysed on dodecylsulfate gels contained all the proteins of the complex: the two major bands of molecular weight 49000 and 73000, diffuse minor bands in the 52000 to 64000 molecular weight range and three more sharp bands in the 86000 to 120000 range. Components B and C contained only some of the protein bands. It is concluded that the composition of the mRNA protein particle is more complex than was originally thought. The protein bands with molecular weights of 73000, 64000 and 49000 were found to be phosphorylated, containing phosphoserine. Using the method of dark-field electron miroscopy, we were able to resolve for the first time internal structural details of the mRNA · protein complex and of the mRNA. The proteins seem to be attached all along the mRNA chain in 4 to 7 distinct blobs, probably in regions of high secondary mRNA structure. Taken together, these results strongly suggest that mRNA · protein particles are not artefacts formed during cell lysis and probably have a biological significance.

Structural characteristics of some murine rna tumor viruses studied by lactoperoxidase iodination.

Abstract: Iodination by the noninvasive enzymatic lactoperoxidase technique has been used to study the enzyme-accessible and enzyme-inaccessible proteins of three oncorna viruses (radiation leukemia virus, Moloney leukemia virus, and mouse mammary tumor virus). The number and relative molecular weight of proteins associated with virion preparations purified on sucrose gradients were characterized by scans of Coomassie blue-stained bands after dodecyl sulfate-polyacrylamide gel electrophoresis. Gel scans from the leukemia viruses are similar, each showing six distinct major protein bands on stained gels. The mammary tumor virus proteins by this analysis are not similar to those of the leukemia viruses. Enzymatic iodination of intact virion preparations led to the solitary labeling of one of the major proteins of each virus-the 80,000-dalton protein of the leukemia viruses and the 52,500-dalton protein of the mammary tumor virus. These are tentatively positioned as surface (enzyme-accessible) moieties in the virions. Disruption of each virus with a nonionic nonionic detergent before enzymatic iodination led to the labeling of the remaining stainable bands. The four lower molecular weight bands of the leukemia viruses are tentatively positioned as internal (enzyme-inaccessible) components.

Homogeneity of Envelope Proteins of Escherichia coli Separated by Gel Electrophoresis in Sodium Dodecyl Sulfate

Abstract: Some envelope proteins of Escherichia coli show variable behavior in acrylamide gel electrophoresis in 1% sodium dodecyl sulfate, depending upon the conditions of the solubilization. When solubilized in 1% sodium dodecyl sulfate at 70 C for 20 min, three distinct peaks (peaks 4, 6, and 7) are seen at molecular weights of 57,800, 44,300, and 38,400, respectively. However, when the envelope fractions are solubilized in 1% sodium dodecyl sulfate at 100 C for 5 min, or when they are treated with N, N-dimethylformamide at acidic pH before solubilization by our method, only a single peak at 48,000 molecular weight is observed in the molecular weight range mentioned above. That is, peaks 4 and 7 disappear and a new peak appears at the position overlapping with peak 6. Proteins isolated from peaks 4 and 7 show the similar molecular weight shifts to the new peak by the treatment at 100 C. No other peaks show any change by the heat treatment. The increase at the new peak is completely accounted for by the decrease at peaks 4 and 7, indicating that the new peak is composed of proteins from peaks 4, 6, and 7. However, it is concluded that these three peaks consist of distinctly different proteins for the following reasons: (i) they have different amino acid compositions, (ii) they show different solubilities in the nonionic detergent, Nonidet P-40, and as shown previously, (iii) peak 6 (protein Y) is related to deoxyribonucleic acid synthesis, and (iv) proteins in peaks 4, 6, and 7 have different resistance to proteolytic enzymes. Although the reasons for the anomalous molecular weight shifts of these peaks are not well understood at present, it is important to solubilize the E. coli envelope proteins by the standard method in order to investigate their properties and functions of the envelope proteins.