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Relationship between microsomal membrane permeability and the inhibition of hepatic glucose-6-phosphatase by
Abstract:
Arion et al; (Arion, W. J., Wallin, B. K., Lange A. J., and Ballas, L. M. (1975) Mol. Cell. Biochem. 6, 75-83) propsed a model for glucose-6-phosphatase in which the substrate was transported across the microsomal membrane by a carrier before hydrolysis on the cisternal side. Evidence to support this model has been obtained by studying the inhibition of the enzyme by pyridoxal-P. Pyridoxal-P was a linear noncompetitive inhibitor of glucose-6-phosphatase (EC 3.1.3.9) in freshly isolated ("intact") microsomes from rat liver. Pyridoxol-P was a much less effective inhibitor and no inhibition was observed with pyridoxamine-P. When microsomes were subjected to nitrogen cavitation, treatment with solium deoxycholate, or glutaraldehyde fixation, the Km of glucose-6-phosphatase for glucose-6 P decreased from approximately 6 mM to approximately 2.5 mM; the corresponding change in the Vmax ranged from-10% to +40%. The same procedures decreased the inhibition of glucose-6-phosphatase by pyridoxal-P several-fold. No inhibition by pyridoxal-P was observed in a preparation of glucose-6-phosphatase purified approximately 20 fold (on the basis of Vmax) from micoromes. A nondialyzable inhibitor was apparently formed when intact microsomes were reacted with pyridoxal-P and NaBH4; this inhibition was also reversed by procedures which changed the kinetic properties of glucose-6-phosphatase.
Insights
Pyridoxal-phosphate inhibits glucose-6-phosphatase in rat liver microsomes, supporting a carrier-mediated transport model. Disrupting the microsomal membrane reduced inhibition, indicating carrier involvement in substrate transport.
Area of Science:
- Biochemistry
- Enzymology
- Membrane Transport
Background:
- The glucose-6-phosphatase system is crucial for glucose homeostasis.
- A proposed model suggests substrate translocation across the microsomal membrane by a carrier protein prior to hydrolysis.
- Investigating enzyme inhibitors can provide evidence for such transport mechanisms.
Purpose of the Study:
- To investigate the role of a carrier-mediated transport in glucose-6-phosphatase activity.
- To evaluate pyridoxal-phosphate as an inhibitor to support the proposed model.
- To characterize the kinetic changes in glucose-6-phosphatase upon membrane perturbation.
Main Methods:
- Enzyme inhibition assays using pyridoxal-P, pyridoxol-P, and pyridoxamine-P on intact rat liver microsomes.
- Kinetic analysis of glucose-6-phosphatase (Km, Vmax) after treatments like nitrogen cavitation, deoxycholate solubilization, and glutaraldehyde fixation.
- Purification of glucose-6-phosphatase and subsequent inhibition studies.
Main Results:
- Pyridoxal-P acted as a linear noncompetitive inhibitor of glucose-6-phosphatase in intact microsomes.
- Membrane disruption treatments significantly decreased the inhibition by pyridoxal-P and altered kinetic parameters (Km decreased).
- Purified glucose-6-phosphatase showed no inhibition by pyridoxal-P, and a nondialyzable inhibitor formed upon reaction with pyridoxal-P and NaBH4 was reversible.
Conclusions:
- The findings support a model where glucose-6-phosphatase activity involves carrier-mediated translocation of the substrate across the microsomal membrane.
- Pyridoxal-P's inhibitory effect is dependent on the integrity of the microsomal membrane, suggesting interaction with the transport component.
- These results highlight the importance of membrane structure for enzyme function and substrate access.