ABSTRACT
A key component of the recently described bioluminescent system of higher fungi is luciferase, a new class of proteins. The properties of fungal luciferase and their relationship with its structure are interesting both for improving autoluminescent systems already created on its basis and for creating new ones. Therefore, it is extremely important to understand the spatial structure of this protein. We have performed heterologous expression and purification of Neonothopanus nambi luciferase, obtained a protein suitable for subsequent crystallization, and also determined some biochemical properties of the recombinant luciferase.
Subject(s)
Agaricales/metabolism , Luciferases/biosynthesis , Luciferases/chemistry , Circular Dichroism , Detergents , Escherichia coli/metabolism , Fungal Proteins/biosynthesis , Fungal Proteins/chemistry , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Industrial Microbiology , Kinetics , Luminescence , Magnetic Resonance Spectroscopy , Protein Conformation , Protein Domains , Recombinant Proteins/chemistry , Saccharomycetales/metabolism , TemperatureABSTRACT
This is the first study to obtain a high-purity luciferase from the fungus Neonothopanus nambi biomass that is suitable for subsequent sequencing.
Subject(s)
Basidiomycota/enzymology , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Luciferases/chemistry , Luciferases/isolation & purificationABSTRACT
The structure of fungal oxyluciferin was determined, the enzymatic bioluminescence reaction under substrate saturation conditions with discrete monitoring of formed products was conducted, and the structures of the end products of the reaction were established. On the basis of these studies, the scheme of oxyluciferin degradation to the end products was developed. The structure of fungal oxyluciferin was confirmed by counter synthesis.
Subject(s)
Fungi/chemistry , Indoles/chemistry , Luminescent Agents/chemistry , Pyrazines/chemistry , Fungi/metabolism , Luminescent Measurements , Molecular StructureABSTRACT
We show that an enzyme exists in rat brain capable of cleaving the caspase-3 specific peptide substrate Ac-DEVD-AMC at low pH. The enzyme shows properties of a cysteine protease and is localized, predominantly, in lysosomes. We have purified this enzyme from rat brain and identified it by MALDI-TOF MS. The enzyme possessing "acidic" DEVDase activity in rat brain appears to be cathepsin B. It remains obscure, whether cathepsin B participates in cleavage of caspase-3 substrates in vivo. We suggest that under certain conditions (e.g. in hypoxia) cathepsin B participates in cleavage of caspase-3 substrates in brain cells.
