Ceramide glycanase (CGase) activities have been detected in different human
tumor cells (colon,
carcinoma Colo-205;
neuroblastoma, IMR-32;
breast cancer lines, SKBr3 and MCF7). However, the level of enzymatic activity is lower in these cells compared to that present in other mammalian tissues reported before (Basu, M., Kelly, P., Girzadas, M. A., Li, Z., and Basu, S. Methods Enzymol. (in press)). The majority of CGase activity was found in the 100,000 g soluble supernatant fraction isolated from all these cell lines and tissues. Using the soluble
enzyme, the requirement for optimum CGase activity was found to be consistent with previous observations found for rat and rabbit tissues (Basu, M., Dastgheib, S., Girzadas, M. A., O'Donnell, P. H., Westervelt, C. W., Li, Z., Inokuchi, J. I., and Basu, S. (1998) Acta Pol. Biochim. 42:327). The CGase activities from both Colo-205 and IMR-32 cells are optimum at a
protein to
detergent ratio of one. All the mammalian CGases, including human
cancer cells, show an optimum pH between 5.5 and 5.8 in
sodium acetate buffer. The CGase activities from
cancer cells are found to be
cation-independent; however,
mercury,
zinc, and
copper ions seem to inhibit the
enzyme activity substantially in both
tumor cells lines. The
mercury ion inhibition of CGase activities from all different sources indicates a possible structural homology in the CGase
proteins. Radiolabeled substrates, labeled at the
sphingosine double bond or at the 3-position of
sphingosine without modifying double bond of
sphingosine were used in this investigation. Both were active substrates with all
enzyme preparations isolated from different
cancer cells (apparent Km, 500 microM for nLcOse5[3H-DT]Cer and 350 microM for GgOse4[sph-3-3H]Cer with Colo-205
enzyme). Structural analogues of
ceramide and
sphingosine (L-
PPMP. L-
PDMP, alkylamines, and
Tamoxifen) inhibited
cancer cell CGase activities in vitro.