Functional lysosomal hydrolase size as determined by radiation inactivation analysis

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Lysosomal hydrolases are crucial enzymes involved in cellular catabolism.
• Determining the functional target size of enzymes is essential for understanding their structure-function relationships.
• Previous methods for determining protein molecular mass may not accurately reflect functional size.

Purpose of the Study:

• To determine the functional target size of various lysosomal hydrolases using electron inactivation analysis.
• To compare the functional target sizes across different lysosomal hydrolases and species.
• To assess the utility of lysosomal hydrolases as endogenous markers for determining protein molecular mass.

Main Methods:

• Electron inactivation analysis using 16 MeV electrons.
• Measurement of functional target sizes for beta-galactosidase, beta-glucuronidase, N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, alpha-galactosidase, beta-mannosidase, beta-glucosidase, arylsulphatase A, sphingomyelinase, alpha-glucosidase, and alpha-fucosidase.
• Analysis of freeze-dried cell material from mouse and human sources.

Main Results:

• Functional target sizes varied, with beta-galactosidase at 62,000 +/- 4000 Da and mouse beta-glucuronidase at 200,000 +/- 17,500 Da.
• A group of lysosomal hydrolases exhibited target sizes between 100,000-120,000 Da.
• Alpha-glucosidase and alpha-fucosidase were identified as complex multimers in the 150,000-160,000 Da range.
• Minimal species variation (mouse vs. human) was observed, except for beta-glucuronidase.

Conclusions:

• Electron inactivation analysis provides a reliable method for determining the functional target size of lysosomal hydrolases.
• Lysosomal hydrolases exhibit a range of functional target sizes, indicating diverse quaternary structures.
• These enzymes show potential as endogenous markers for determining the molecular mass of proteins with unknown sizes.