Mixed protein gels, complex

It was found that a water-insoluble complex can be formed between purified lysozyme and flavoprotein, both of which are constituents of hen egg white, and that the optimal pH of the complex formation was about 7. When the mixing ratio of lysozyme and flavoprotein in molar concentration was 2 1 or above 3 1, the molar ratio of lysozyme and flavoprotein of the complex was 2 1 or 3 1, respectively. However, the changes of molar ratio of the complex were observed in the presence of other egg white proteins. The complex obtained directly from egg white by treatment with cold acetone and by dialysis consisted of lysozyme, flavoprotein, conalbumin and ovomucin-like substance, and the molar ratio of the former three components in the complex was calculated from the gel-filtration pattern to be 16 4 1. 

The saturated HpHb complex of type 1-1 was formed in solution and mixed with a pure solution of globin. The proteins were then separated by starch gel electrophoresis with a neutral phosphate buffer. The procedure was afterward repeated, but with a solution of Hp-globin complex mixed with an Hb solution. 

Before crystallization trials, the protein was subjected to gel filtration on Superdex-75 (Pharmacia) in 50 mM sodium/potassium phosphate buffer, pH 7.4, containing 1 mM EDTA, 50 mM 2-mercaptoethanol, 150 mM sodium chloride, 5% glycerol and 5% 2-propanol, as described previously (12). The statine-based inhibitor, LP-149 (Ac-Nal-Val-Sta-Glu-Nal-NH2 e Nal is naphtylalanine and Sta is statine) (Fig. 1), was prepared at Lilly Research Laboratories (K. Hui, unpublished results). Crystallization was carried out at 4 °C using the hanging-drop vapor diffusion method as follows 2.5 //I of the FIV PR(D30N) at 7 mg/ml complexed with LP-149 (1 4 molar ratio) in 50 mM imidazole-HCl pH 7.0 containing ImM EDTA and 1 mM dithiothreitol were mixed with an equal volume of 2 M ammonium sulfate, 0.1 M sodium acetate, pH 4.6 (Hampton Crystal Screen, solution 47). Crystals appeared within a few days and reached the size of 0.2 x 0.2 X 0.4 mm in one week. 

Figure 12-4 Differential binding of IRPl and IRP2 to natural IREs (Iron Responsive Elements). P-5 -RNAs (n = 29-30 nucleotides) were melted and annealed before mixing with recombinant IRP proteins (The proteins were kindly provided by E. A. Leibold, University of Utah, and W. E. Walden, University of Illinois). RNA-protein complexes were separated from RNA by electrophoresis in non-denatuiing polyacrylamide gels [20]. Per contains an internal loop/bulge (Figure 12-3), and TfR, eALAS, and m-aconitase IREs have C-bulges. Per mutation AU6 converts the Per internal loop/bulge to a C-bulge. Per ferritin TfR transferrin receptor eALAS erythroid amino-levulinate synthase and m-aconitase. No IRE/IRP complex was detectable.

Thylakoid membranes were first treated with 2M NaBr in order to remove the CF1 complex. The treated membranes were washed once with an SMN solution (0.4 M sucrose/50 mM MES (pH 6.0)/10 mM NaCl) in order to remove the NaBr, and were subsequently resuspended in SMN to a final Chi concentration of 2 mg Chl/ml. A part of the washed thylakoid membranes was mixed well with an equal volume of a solution containing 50 mM MES (pH 6.0), 1 M sucrose, 10 mM NaCl and 25 mM dodecylmaltoside and the solubilized thylakoid membranes were centrifuged for 3 hrs at 40.000xg. The pellet and the supernatant which were isolated were examined by gel electrophoresis and it was found that the pellet contains most of Photosystem I and the light harvesting complex (LHC) whereas the supernatant contains the PS II core and traces of PS I and LHC. Figure 1a. shows the protein content of the thylakoid extract a comparison with the polypeptides of the PSII core (1b), isolated by the method described in ref. 4, clearly demonstrates that the solubilization step described above results in a selective isolation of the PS II core. The clean PSII core complex was isolated from... 

Truncation of the large subunit is associated with a dramatic decline in the catalytic activity. The specific activity of the Lg mutant is reduced to 1% in comparison of the wild type enzyme Verification that the Lg mutant mdergoes carbamylation was provided by trapping the carbamate with CABP. The quaternary complex (E.CO. Mg. CABP) was isolated by gel filtration of the mutant protein pre-mixed with u02, MgCl and CABP. The binding of X02 (0.83 mole/ mole of protomer) is close to the 1 1 stochiometry obtained with the wild type enzyme. However, the stability of the complex is greatly reduced relative to that of the native... 

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