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lmmunoprecipitate Pellet
1 3
3M Guanidine HCI O-3M Guanidine HCI
\ /
1. Mix and dilute out guanidine
2. Incubate
3. Proteinase K digest
4. SDS-PAGE + fluorography to
detect protease-resistant 3%PrP
Fig. 1. Flow diagram of the cell-free reaction for converting PrP-sento PK-resrs-
tant forms

to be useful m conversron reactrons. The eluates are stored on Ice and, other
than radroactrve decay, are often stable for weeks.
The Cell-free Conversion Reaction
3.1 2.
The converston of 35S-PrP-sen to protemase K-resrstant forms can be
achieved by incubating 35S-PrP-sen with brain-derived PrP-res that has been
pretreated with 0-3M guanidme hydrochlorrde (GdnHCl) at 37°C for 0.25-24 h
(Fig. 1) (52-54). The optrmal GdnHCl concentration varies slightly with the
PrP-res preparatron. For instance, the converting actrvtty of many preparations
of hamster PrP-res (263K strain) 1senhanced by a 3M GdnHCl pretreatment
(52), whereas the activity of some other preparations of 263K PrP-res have
been optima1 with pretreatments of 2-2SMGdnHCl. The reason for this varia-
tion in GdnHCl sensitivity is unclear but it may be influenced in part by the
purity of the preparation or repeated freeze-thaw cycles. A l-3 ug/pL suspen-
sion of PrP-res in the desired GdnHCl concentratron IS mixed with an equal
volume of 35S-PrP-seneluate prediluted to the same GdnHCl concentratron.
The mixture is then diluted further to 0.75M GdnHCl using TN supplemented
with cetylpyridinium chloride (CPC) to give a final concentration of 1.5 mM
CPC and incubated at 37°C for 216 h.
The PK-resistance of the 35S-PrPis then tested after a further dilution of the
GdnHCl to 0.075 - 0.4M by digesting with 20-500 ug/mL of PK for 1 h at
37OC (54). The PK IS inhibited using Pefabloc SC (Boehringer Mannheim) as
recommended by the manufacturer. Twenty mrcrograms of a carrier protein
(thyroglobulin) is added, and the proteins are precipitated with methanol. The
294 Caughey et al.
resulting pellet IS boiled m SDS-PAGE sample buffer and electrophoresed.
Radiolabeled PrP-res was visualized by fluorography wtth Entenslfy (DuPont,
Wilmington, DE) or Phosphorlmager analysis (Molecular Dynamics, Sunny-
vale, CA)
In summary, a typical reaction would be*
I 2 pL of 1 mg/mL hamster PrP-res m 3MGdnHCl
2 2 pL hamster 35S-PrP-sen with 30,000 cpm/pL m 3M GdnHCl
3 12pLTN+CPC
4 MIX these constituents, somcate bnefly, and Incubate for 1 d at 37°C
5 Dilute with 64 pL TN and then add 4 pL of 1 mg/mL proteinase K
6.After the 37°C Incubation, add 20 pL of 5 mM Pefabloc SC, 4 pL 5 mg/mL
thyroglobulm, and 4 vol methanol
7 After 1 h at -2O”C, the tube 1s centrifuged for 15 mm at 11 ,OOOg
8 Sonicate and boil the pellet mto 20 pL of sample buffer Analyze by SDS-PAGE-
fluorography
Usmg these condltlons, newly converted PK-reslstant 35S-PrP bands often
can be detected with an overnight fluorographic exposure of the gel Smce PK
treatment of scraple brain-derived PrP-res generally results m a 6-7 kDa
reduction in apparent molecular mass, we look for PK-resistant 35S-PrPbands
that are 6-7 kDa smaller than the corresponding untreated 35S-PrP-sen precursor
(52-H). The presence of 35S-PrPbands that are the same size as the 35S-PrP-sen
precursor would suggest that some nonspecific trapping or protection of
35S-PrP-sen precursor from PK may have occurred. We have also observed
PK-resistant 35S-PrP bands that are >6-7 kDa smaller than the 35S-PrP-sen
precursors, which has suggested that a partial conversion occurred (52,53).
The relationship between these incomplete cell-free conversion products and
brain-derived PrP-res molecules IS not clear. To control for the role of PrP-res
m the conversions, comparisons can be made to samples of 35S-PrP-senmcu-
bated without PrP-res or samples to which PrP-res IS added immediately before
the PK dtgestion (52,53) The efficiency of conversion of hamster 35S-PrP-sen
to PK-resistant species using the above protocol with hamster PrP-res has been
variable m our hands. Usually 5-30% of the 35S-PrP-senprecursor m the reac-
tlon becomes PK-resistant but, on occasion, as much as 100% of the label IS
converted. The reason for this varlabihty is not yet understood.
3 1.3. Specres and Strain Speaficities
in the Cell-Free Conversion Reaction
The cell-free conversion reaction can be used to investigate the species
specificity of PrP-res-PrP-sen mteractlons. For instance, mouse and hamster
PrP-res have been combined in conversion reactions with mouse, hamster, and
chimeric PrP-sen molecules (53). The species speclficitles in the conversion
PrP Metabolrsm and PrP-res Formation 295

reacttons correlated with the relative transmtsstbrlmes of these scrapte strains
m VIVO.The hamster PrP-res (263K strain) would not convert mouse PrP-sen
to PK-resistant forms just asthe hamster scrapte ISnot infectious to mice. Chan-
dler mouse PrP-res converted both mouse and hamster PrP-sen molecules to
PK-resistant forms that correlated with the ability of the Chandler mouse
scrapte strain to infect both mice and hamsters.
Conversion experiments performed with chtmertc mouse/hamster PrP-sen
precursors identified certain species-specific residues that affected the conver-
sion efficiency and the size of the resultant protease-resistant PrP species
These studies indicated that mcompatibtlittes m direct PrP-res-PrP-sen mter-
actions may be a molecular basis for the barriers to mterspectes transmtssrons
of TSEs m vtvo.
The observatron of this type of specificity m the cell-free converston reac-
tion and its correlation with in vivo species barrier effects suggests that this
experimental system may be useful for quick m vitro tests of the suscepttbtllty
of various species to TSE agents of other species. Of particular interest at
present IS the issue of whether humans are likely to be susceptible to bovine
spongtform encephalopathy (BSE) agent. Tests of the abthty of PrP-res from
BSE cattle brain to convert human PrP-sen to PK-resistant forms might shed
light on this subject. However, given the newness of the cell-free converston
reaction and the likelihood that other factors profoundly influence TSE species
barrier effects m vtvo, caution should be used in extrapolatmg between the m
vitro and in vivo srtuatrons. The condmons for optrmal cell-free conversron
may differ from those described herem when PrP-res and PrP-sen derived from
vartous species and strains of TSEs are used.
The scrapie-strain specificity of the cell-free converston reaction also can be
investigated under ctrcumstances m which there IS no difference m the ammo
acid sequence between PrP-res and PrP-sen (54) This type of experiment can
be used to model the strain-specific differences m PrP-res formation that occur
in a single host species. For example, two hamster adapted strains of transmts-
stble mmk encephaiopathy (TME) lead to the in vivo formation of PrP-res
forms that are cleaved differently by PK. When these two forms of PrP-res
were reacted with the same labeled PrP-sen precursor in the cell-free conver-
sion reaction, the resulting conversron products exhibited a similar difference
m PK cleavage. These data provided evidence that the stram-specific proper-
ties (or phenotypes) of PrP-res can be transmitted to PrP-sen as a result of
direct PrP-res-PrP-sen interacttons.
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296 Caughey et al.
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protem to protease-resrstant forms. a model for the scrapre specres barrrer. Proc

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