Figure 3 of
Tucker, Mol Vis 2004;
Figure 3. Extracellular domain mutations C105Y and L325P reduce RetGC-1 acivity by only 50%
1 bp del (5' UTR) has expression profile cyclase activity undistinguishable from wild-type, while C105Y and L325P substitutions have moderately reduced RetGC-1 activities. HEK293 cells were transfected with 5 μg of wild-type RetGC-1 or the indicated extracellular domain cyclase mutant. Membranes from transiently transfected HEK 293 cells were assayed for RetGC-1 activity for 20 min in GC buffer (described in methods). Equivalent total protein amounts of RetGC-1 in pRC-CMV, del G (1 bp deletion), GC-1 L325P, GC-1 C105Y or co-expressed L325P and C105Y were immunoblotted with an antibody to the KHD of RetGC-1 (inset). For GCAP-stimulated samples, 17 μM GCAP-1 or GCAP-2 and 1 mM EGTA was added. Mn/Tx100 labeled samples contained 1% Triton-X-100 and 10 mM MnCl2 instead of MgCl2. "GC-1" refers to RetGC-1 and "ΔG" refers to the 5' UTR -18 1 bp del homozygous mutation.