Rat intestinal ceramidase: purification, properties, and physiological relevance

Area of Science:

• Biochemistry
• Enzymology
• Gastroenterology

Background:

• Neutral ceramidase activity is known in the intestine, important for sphingolipid digestion.
• This enzyme's specific characteristics and role in the gut have not been fully elucidated.

Purpose of the Study:

• To purify and characterize neutral ceramidase from rat intestine.
• To understand its enzymatic properties, stability, and potential role in sphingolipid metabolism.

Main Methods:

• Purification of rat intestinal neutral ceramidase using a combination of techniques including salt perfusion, acetone precipitation, and various chromatographies.
• Enzymatic assays using radiolabeled substrates and analysis of enzyme kinetics and inhibition.
• Mass fragmentographic analysis for sequence identification and comparison with kidney ceramidase.

Main Results:

• A homogenous 116 kDa neutral ceramidase protein was purified.
• The enzyme efficiently hydrolyzes ceramides in the presence of specific bile salts but is inhibited by others.
• It is a glycosylated protein, stable to proteases, and influenced by specific divalent cations (inhibited by Zn2+, Cu2+).
• Identified fragments match neutral/alkaline ceramidase 2, suggesting intestinal ceramidase is a form of this enzyme.

Conclusions:

• Intestinal neutral ceramidase is identified as neutral/alkaline ceramidase 2, released by bile salts and resistant to pancreatic proteases.
• The enzyme is well-suited for metabolizing dietary and brush border sphingolipids, generating bioactive sphingolipid messengers.