Hsp60 may serve as a regulatory protein bound to N-SMase2. Our data further showed that Hsp60 serves as a negative regulator for N-SMase2�Cinduced DA re-uptake by inducing a decrease in the protein N-SMase2, thereby reducing ceramide generation. In this study, a neuronal form of Butenafine hydrochloride N-SMase from salt extracts of the membrane fractions of bovine brain, termed N-SMase e, was purified to near homogeneity. On two-dimensional gel electrophoresis, the purified enzymatic activity was represented as different pI values with identical molecular mass. MALDI-TOF analysis of the tryptic peptides revealed that these four protein species were identical, and they were commonly identified as Hsp60. Although Hsp60 had a considerable chromatographic profile, Hsp60 is not a structural gene for novel N-SMase. In other purification procedure, the purified N-SMase preparation had components of apparent molecular weight 57, 71 and 82-kDa. We have found significant variation in different preparation of purified enzymes; in extreme cases, gel electrophoresis revealed only the 60-kDa band. Many groups have attempted to purify, identify, and characterize a membrane-bound N-SMase from different sources, including rat brain, liver, hepatoma cells, and human brain. Thus far, purification efforts have not been very successful, probably due to the highly hydrophobic nature of the integral membrane proteins. Even in this study, N-SMase e appeared to be highly hydrophobic, since it was only slightly eluted until distilled water or Triton X-100 was used. This hydrophobic property was further confirmed in the gel filtration and ionexchange column chromatographies; that is, N-SMase e activity was eluted from the Superose 12 gel filtration column near 
void volume, regardless of the presence of Triton X-100. Moreover, the enzyme was insufficiently eluted until Triton X-100 was used as a component of the elution buffer in some of the ion-exchange columns. Our results revealed that purified N-SMase e appears to migrate as a complex on a gel filtration column, suggesting that the enzyme may form a large complex with other proteins. The known functions of chaperonins include folding, assembly, and translocation of other proteins. In addition to these Oxysophocarpine housekeeping and cytoprotective roles, signal transduction functions of Hsps have been revealed. Other functions of Hsps have also been shown to include involvement in several processes during synaptic transmission; for example, Hsc70 is essential for uncoating synaptic vesicles at presynaptic terminals, and Hsp90 is involved in the synaptic cycling of AMPA receptors. Moreover, constitutively expressed Hsp90, Hsc70, Hsp40, Hsp60, and a range of neurotransmitter receptors are associated with lipid rafts isolated from the rat forebrain and cerebellum. The observations presented herein suggest that N-SMase2 interacts with the chaperone protein Hsp60 in the brain synaptosome and in PC12 cells and that this interaction is important in the maintenance of N-SMase2 protein levels. In our results, while N-SMase2 knockdown effectively decreased NSMase activity, ceramide levels, and N-SMase2 mRNA levels, Hsp60 knockdown increased Mg2+-dependent N-SMase activity and ceramide production in PC12 cells. However, Hsp60 siRNA transfection did not alter N-SMase2 mRNA levels, also suggesting that the observed increase in N-SMase activity may be due to the reduction in protein degradation rather than to an increase in transcription/translation. This conclusion is further supported by similar results from HEK293 cells. Hsp60 siRNA treatment significantly increased N-SMase activity and the corresponding band in western blotting analysis in N-SMase2overexpressed HEK293 cells.