CERAMIDE GLYCANASE FROM RAT MAMMARY TISSUES - INHIBITION BY PPMP(D- L-) AND ITS PROBABLE ROLE IN SIGNAL-TRANSDUCTION/

A ceramide glycanase (CGase activity has been characterized from lacta ting rat mammary tissue which cleaves the glycosidic bond between sphi ngosine and the glycose chain of a glycosphingolipid (GSL) thus libera ting the intact oligosaccharide chain from a GSL. The majority (65%) o f the hydrolase activity was detected in the supernatant fraction when the rat mammary tissue homogenate was centrifuged at 100,000xg. Attem pts to purify the enzyme indicated that the CGase protein is of hydrop hobic nature as it binds to hydrophobic columns. The enzyme has been p artially purified using hydrophobic columns in tandem. The partially p urified protein was found to be immunoreactive to the antibody raised against the purified clam CGase. The immunostained band corresponded t o a 64 kDa protein as also found with the clam enzyme. This immune cro ss-reactivity indicated probable structural similarities between CGase proteins isolated from widely separated species in the evolutionary t ree. The rat CGase was found to have a specific detergent requirement for optimal activity, and the pH optimum was found to be between 5 and 6. The enzyme activity is partially heat stable. It is not a divalent cation requiring enzyme; however, the activity is totally inhibited i n the presence of mercury, indicative of a sulfhydryl group in the act ive site of the enzyme. The rat mammary CGase activity is inhibited in the presence of both D- and L-PPMP henyl-2-hexadecanoylamino-3-morpho lino-1-propanol. HCl), homologs of PDMP (1-phenyl-2-decanoylamino-3-mo rpholino-1-propanol. HCl), a well-known inhibitor of GlcT-1 (Ceramide: UDP-Glc Glucosyltransferase), an enzyme in the glycolipid synthetic p athway. The inhibition seems to be of a competitive nature and the sam e type of inhibition is also observed with clam CGase. The CGase activ ity was found to be highest in lactating tissue compared to the activi ty found in either pregnant or post-lactating rat mammary tissues. Tis sue survey indicated the presence of high levels of CGase in lactating rat liver, uterus, and ovary; moderate activity was detected in kidne y and spleen. Both virgin and male rat mammary tissue also indicated a basic level of CGase activity. However, newborn spleen and mammary ti ssue showed a comparable level of activity to that found in lactating rat tissues. This report is mainly concerned with the characterization of CGase activity from a mammalian source and its importance in cellu lar processes.