Gaucher disease is a heterogeneous disease characterized by impaired activity of 
the lysosomal enzyme glucocerebrosidase. This heterogeneity is attributed to a 
large number of mutations in the corresponding gene. In order to test the 
biochemical properties of some mutations prevalent among Israeli populations, 
the normal human glucocerebrosidase cDNA and cDNAs carrying mutations N370S, 
L444P, D409H, recTL, recNcil, P415R and 84GG were coupled to the T7 RNA 
polymerase promoter in a vaccinia virus-derived expression vector (pTM-1). 
Recombinant viruses were produced and used to infect human tissue culture cells. 
RNA and protein stability, recognition by anti-glucocerebrosidase monoclonal 
antibodies and intracellular enzymatic activity were measured. The results 
demonstrated that the D409H allele directed synthesis of cytoplasmic RNA with 
decreased stability compared with its normal counterpart or other mutated forms. 
The D409H and L444P mutated proteins had lower stability than that of their 
normal counterpart, while the recNcil-mutated protein was more stable. Only 
glucocerebrosidase forms harboring leucine at position 444 were recognized by 
the anti-glucocerebrosidase monoclonal antibodies used (8E4 and 2C7). 
Measurements of enzymatic activity of the recombinant proteins in cells loaded 
with a fluorescent glucosylceramide demonstrated that the N370S mutated enzyme 
had activity similar to that of the normal enzyme. The other mutated enzymes 
exhibited varying degrees of activities, generally corresponding to the 
phenotypes with which they are associated. The results presented demonstrate the 
use of the vaccinia virus-derived expression system and of loading living cells 
with fluorescent substrate as efficient tools for studying mutants in Gaucher 
disease and in other lysosomal diseases.