BIOCHEMICAL-EVIDENCE FOR A NOVEL LOW-MOLECULAR-WEIGHT 2-5A-DEPENDENT RNASE-L IN CHRONIC-FATIGUE-SYNDROME

Previous studies from this laboratory have demonstrated a statisticall y significant dysregulation in several key components of the 2',5'-oli goadenylate (2-5A) synthetase/RNase L and PKR antiviral pathways in ch ronic fatigue syndrome (CFS) (Suhadolnik et al, Clin Infect Dis 18, S9 6-104, 1994; Suhadolnik et al, In Vivo 8, 599-604, 1994), Two methodol ogies have been developed to further examine the upregulated RNase L a ctivity in CFS, First, photoaffinity labeling of extracts of periphera l blood mononuclear cells (PBMC) with the azido 2-5A photoaffinity pro be, [P-32]pApAp(8-azidoA), followed by inmunoprecipitation with a poly clonal antibody against recombinant, human 80-kDa RNase L and analysis under denaturing conditions, A subset of individuals with CFS was ide ntified with only one 2-5A binding protein at 37 kDa, whereas in extra cts of PBMC from a second subset of CFS PBMC and from healthy controls , photolabeled/immunoreactive 2-5A binding proteins were detected at 8 0, 42, and 37 kDa, Second, analytic gel permeation HPLC was completed under native conditions, Extracts of healthy control PBMC revealed 2-5 A binding and 2-5A-dependent RNase L enzyme activity at 80 and 42 kDa as determined by hydrolysis of poly(U)-3'-[P-32]pCp. A subset of CFS P BMC contained 2-5A binding proteins with 2-5A-dependent RNase L enzyme activity at 80, 42, and 30 kDa, However, a second subset of CFS PBMC contained 2-5A binding and 2-5A-dependent RNase L enzyme activity only at 30 kDa, Evidence is provided indicating that the RNase L enzyme dy sfunction in CFS is more complex than previously reported.