Selective chemical modification of plasma membrane ectoenzymes

Area of Science:

• Cell Biology
• Neuroscience
• Biochemistry

Background:

• Cell surface macromolecular assemblies are crucial for cellular function.
• Understanding the organization of these assemblies requires specific chemical probes.
• Enzymes like ecto-ATPases, ecto-5'-nucleotidases, and cholinesterases are key cell surface components.

Purpose of the Study:

• To investigate the organization of cell surface macromolecular assemblies.
• To identify selective chemical probes for studying cell surface proteins and lipids.
• To characterize the enzymatic activities of ecto-ATPases, ecto-5'-nucleotidases, and cholinesterases in response to chemical modification.

Main Methods:

• Treatment of intact central nervous system cells with covalent chemical probes.
• Selective inactivation of enzymatic activities using specific reagents like difluorodinitrobenzene and trinitrobenzenesulfonic acid.
• Protection studies using appropriate substrates to prevent enzyme inactivation.

Main Results:

• 1,5-difluoro-2,4-dinitrobenzene potently inactivated ecto-ATPase in various cell lines (C6 glioblastoma, IMR-32 neuroblastoma, RB astroblasts).
• Ecto-5'-nucleotidase and acetylcholinesterase showed less sensitivity to difluorodinitrobenzene.
• 2,4,6-trinitrobenzenesulfonic acid selectively inactivated ecto-5'-nucleotidase but not ecto-ATPase.
• Enzyme inactivation by probes was prevented by the presence of specific substrates.
• Isotopic probe fluorodinitro[3H]-benzene showed decreased reactivity with proteins and lipids in the presence of protecting nucleotide substrates.

Conclusions:

• Chemical probes can selectively target and inactivate specific cell surface enzymes.
• The differential sensitivity of ecto-ATPases, ecto-5'-nucleotidases, and cholinesterases to various probes provides insights into their distinct molecular environments.
• Substrate protection studies confirm the specificity of probe interactions and enzyme active sites.
• These findings contribute to understanding the organization and function of cell surface macromolecular assemblies.