Changes in a protein-nucleic acid complex from synaptic plasma membrane of scrapie-infected mouse brain.

The infective agent of scrapie is characterized by unusual physicochemical properties and has not as yet been purified (Millson etal., 1976). This has prompted suggestions that the infective agent may not possess a conventional virion structure, and its association with the host may be different from that found in other infectious diseases. A close association of infectivity with cell membranes has been demonstrated (Millson et al., 1971) and fractionation of a cell line in which scrapie replicates has shown that most of the infectivity is found with the plasma membrane (Clarke & Millson, 1976). The infective scrapie agent has been purified to only a limited extent, but most successfully by treating scrapie-infected brain with mild detergents, such as lysophosphatidylcholine, followed by high-speed sedimentation (Millson et al., 1976). In the present study, an attempt has been made to identify components of the plasma membrane which may form part of the infectious agent and to identify changes taking place in membranes as a consequence of the development of the disease. Fractions rich in synaptic plasma membrane were prepared from groups of 12 mouse or three hamster brains by the procedures of Whittaker et al. (1964). The mice and hamsters were inoculated intracerebrally with either the Chandler strain of scrapie or control homogenates of normal brain as described by Hunter et al. (1971). The resulting membrane fraction was incubated with lysophosphatidylcholine (5 mglml) buffered with 5m~-Hepes* (pH8.5) for 1 h at 37°C and then centrifuged at 150000g for 1 h at 25°C. On some occasions the resulting pellet was labelled with lZ5I with chloramine-T as a catalyst (Greenwood et al., 1963). The pellet was resuspended in 1 % Triton X-100 and centrifuged as before except at 4°C. If iodination had been performed then the treatment with Triton X-100 was repeated after dialysis overnight against 0.1 %Triton X-100. The pellet was resuspended in 20% (w/w) sucrose (1.0ml) and overlaid on a discontinuous sucrose gradient of 30% (w/w) sucrose (lSml), 40% (w/w) sucrose (2.5ml) and 50 % (w/w) sucrose (1 .Oml). All sucrose solutions contained 0.1 %Triton X-100,l M-KCl and 10mwHepes (pH 8.0). After centrifugation at 4OOOOOg for 16h, 0.25ml fractions were collected by dripping. Labelled protein-containing material can be observed near the bottom of the gradient from both normal (fraction N1) and scrapie-infected (fraction S1) brain (Fig. 1). However, synaptic plasma membrane derived from clinically scrapie-infected mice shows another peak of protein-containing material (fraction S2) in the middle of the gradient, which is not present in normal brain. Similar observations were made with synaptic plasma membrane derived from normal hamsters and those infected with scrapie. As the clinical phase of the disease progresses, fraction S1 gets smaller, and increasingly more material is found in fraction S2. The nucleic acid composition of each peak (see Corp & Somerville, 1976) shows that an RNA with a molecular weight of about lo7 is found in peaks N1 and S1 but not in peak S2. However, the S2 peak contains a small single-stranded DNA (mol.wt. approx. 6x lo5) which is absent from the other peaks (N1 and Sl). The polypeptide composition of the material from each peak was examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, the gel patterns obtained being complex, with more than 10 polypeptides observed from each peak; the profiles obtained from each of the peaks N1, S1 and S2 are essentially similar. * Abbreviation : Hepes, 2-(N-2-hydroxyethylpiperazin-N’-yl)ethanesulphonic acid.