Low-Km mannose-6-phosphatase as a criterion for microsomal integrity

Area of Science:

• Biochemistry
• Cell Biology
• Enzymology

Background:

• Mannose-6-phosphatase (Man-6-Pase) low-Km activity has historically served as a measure of microsomal structural integrity.
• Histone II-A activates Man-6-Pase and glucose-6-phosphatase (Glc-6-Pase) without disrupting microsomal vesicles, challenging previous assumptions.
• This suggests Man-6-Pase latency can be abolished without compromising microsomal integrity, implying normal microsomes might exhibit some activity.

Purpose of the Study:

• To further investigate the relationship between Man-6-Pase and microsomal integrity.
• To measure the latency of various endoplasmic reticulum lumenal enzymes.
• To correlate enzyme latency with the permeability of microsomal substrates.

Main Methods:

• Measurement of enzymatic latency for lumenal enzymes within endoplasmic reticulum.
• Assessment of microsomal permeability using osmotically induced light scattering changes.
• Enzymatic assays for glucose-6-phosphate (Glc-6-P) and mannose-6-phosphate (Man-6-P) hydrolysis under varying detergent concentrations.
• Kinetic analysis of mannose-6-phosphate hydrolysis in untreated microsomes.

Main Results:

• Lumenal enzymes exhibit distinct degrees of latency.
• Microsomal permeability does not consistently correlate with enzymatic latency.
• Hydrolysis of Glc-6-P and Man-6-P increased non-parallelly with increasing detergent concentrations.
• Kinetic studies indicate multiple mechanisms for mannose-6-phosphate hydrolysis in untreated microsomes.

Conclusions:

• Mannose-6-phosphatase (Man-6-Pase) is not a reliable index for assessing microsomal integrity.
• Microsomal integrity is complex and influenced by factors beyond simple permeability.
• Multiple enzymatic pathways may contribute to Man-6-Pase activity.