Interference No. 103,586
performed by Reiss on September 25, 1989.
The following paragraphs comprise the complete testimony of Reiss with respect to
his activities on the above dates.
13. On September 11, 1989, I conducted another filter assay, this time using
membrane fractions from the previous rat brain preparation (Ex 32; page 0018). Though
the results were inconclusive, there appeared to be some transfer of radioactively labeled
14
FPP to ras. By this point, we had begun to realize that background radioactivity from C-
FPP, in this type of study, was going to be a problem and that we needed to lower the
background in order to observe any difference between the amount of radioactivity in a
labeled versus unlabeled ras substrate. (AR -48)
21. On September 20, 1989, I performed yet another assay using whole cell
extracts and supernatants from the rat brain preparation of September 18,(Exhibit 32;
pages 0028 to 0031). The format of this assay was different in that it did not employ a
filter. The rat brain extract supernatant, recombinant ras, reaction mixture (1 M Tris, pH
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7.5; 2M KCL; 0.25 M MgCl ; 0.1 M DTT; 0.1 M ATP; 1 mg/ml PK) and C-labeled FPP
2
were mixed together and incubated for various times. The samples where then separated
by gel electrophoresis. The resulting gel was dried and x-ray film used to detect
radiolabeled proteins. As can be seen from the autoradiograph of the gel (Exhibit 32;
page 0032), a band migrating between 14.3 and 30 kilodaltons could be observed for
several of the reactions including a ras substrate, but not in any lane lacking ras. This
clearly indicated a ras-specific reaction in which a molecule, in the approximate molecular
14
size range ras, incorporated C-FPP. This was the first study actually demonstrating
farnesyl transferase activity in vitro. (AR 50-51)
24. On September 25, 1989, I conducted an assay to determine the pH
dependence of the farnesyl transferase preparation currently under use (Exhibit 32; pages
0035 to 0039). This study also employed a peptide considered to be a potential inhibitor
of ras farnesylation. This peptide comprised the carboxy-terminal ten amino acids of the
ras molecule. The format of this assay was the gel electrophoresis format, described
above in paragraph 20[sic]. The autoradiograph developed from the corresponding gel
(Exhibit 32; page 0038) clearly shows that inclusion of peptide at 10
and 20 Fg (lanes 14 and 15, respectively) inhibited farnesyl transferase-mediated labeling
14
of ras by C-FPP, as determined by the reduction/absence of ras-specific bands in these
-10-
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