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note
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148-213 was >70% pure.
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26
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0345610480
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note
-
12, 106 μM ribonuclease A, 54 μM BSA, 23 μM aldolase (Bio-Rad and Pharmacia gel filtration standards) or 54 μM bovine serum albumin alone was prepared in 50 mM tris-HCl containing 0.2 M NaCl (pH 8.0). The aggregation reaction mixtures (126 μl) containing 13 μM Ure2p (17) or 0.16 volume of protein mixture or both in 50 mM tris-HCl (pH 8.0) containing 0.2 M NaCl were filter-sterilized and peptides were diluted into the protein solutions to a final concentration of 13 μM. Controls included 0.14 M guanidine and 13 μM Ure2p without the peptide. Reaction mixtures were gently rocked continuously at 8 °C for 20 hours. The aggregated protein was collected by centrifugation for 1 min at 8500g and washed with buffer. Pellets were boiled for 5 min in 10 μl of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and 30 μl of 8 M urea and then analyzed by SDS-PAGE.
-
-
-
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27
-
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0344748294
-
-
note
-
6-Ure2p according to SDS-PAGE and immunoblot analysis were pooled and concentrated. Concentrated protein was applied and eluted on a Superdex 200 HR 10/30 column (Pharmacia) in 50 mM tris-HCl (pH 8.0) containing 0.2 M NaCl.
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28
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0345178439
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Data not shown
-
Data not shown.
-
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31
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0345178437
-
-
note
-
2-terminal region of Ure2p (1). Proteinase K digestion of mixed fibers for electron microscopy was done for 15 min under similar conditions with 3.75-fold higher protease and protein concentrations.
-
-
-
-
33
-
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0345178435
-
-
note
-
Aggregated protein or peptide suspensions were mixed with 0.1 volume of 2% (w/v) CR (Sigma) and, after 1 hour at 20 °C, were centrifuged at 8500g for 30 s. The aggregates were washed twice with 100 μl of water. Aggregates were suspended in an equal volume of water and 10 μl was placed on a glass slide and allowed to dry. Excess CR was removed by washing with 90% ethanol. Samples were viewed by polarization microscopy using a Zeiss Axialfold microscope equipped with optimally aligned cross-polarizers.
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0015150725
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G. G. Glenner et al., ibid. 174, 712 (1971).
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Glenner, G.G.1
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40
-
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0344748289
-
-
note
-
After the Ni-NTA step, Ure2p aggregates were formed on dialysis against <0.15 M NaCl in 50 mM tris-HCl (pH 7.5). However, these aggregates were amorphous and had no filament structure.
-
-
-
-
41
-
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0344316024
-
-
note
-
6-URE2 fusion) did not take up ureidosuccinate, showing that the URE2 fusion was functional. [URE3] could be cytoduced from strain 3310 [URE3-1] (MATa kar1 arg1 [URE3]) into strain 3947 containing pKT18 showing that the fusion protein could assume the prion form. These clones, made [ure-o] by growth to single colonies on minimal medium containing 5 mM CuHCl, were used for purification of Ure2p.
-
-
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42
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0021095444
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R. W. Williams, J. Mol. Biol. 166, 581 (1983); in Enzyme Structure, C. H. W. Hirs and S. N. Timasheff, Eds. (Academic Press, New York, 1986), vol. 130, pp. 311-331.
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Hirs, C.H.W.1
Timasheff, S.N.2
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44
-
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0344316023
-
-
note
-
We thank P. McPhie for help with spectroscopy, G. Poy for synthetic peptides, L. Pannell for mass spectrometry, and H. Edskes for plasmid pH7.
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