Preparation of intact microsomes from cultured mammalian H4IIE cells.
BACKGROUND
Mammalian cell culture is widely used for the cloning and expression of insoluble proteins. The established methods of sub-cellular fractionation of tissues are not always directly suitable for the sub-cellular fractionation of cultured cells. In this study we have optimized the conditions for the preparation of microsomal fractions from cultured cells with the aim of isolating intact vesicles that are suitable for the assay of transport proteins and lumenal enzymes.


METHODS
H4IIE cell cultures were used as a convenient model with high latency of internal endoplasmic reticulum enzyme glucose-6-phosphatase towards mannose-6-phosphate. Also 7-ethoxyresorufin O-deethylase (EROD) activity was determined as a reflection of the state of monooxygenase system.


RESULTS
The variations in a number of homogenization strokes and buffer composition revealed that one homogenization stroke in glass homogenizer with 0.25 M sucrose, 5 mM HEPES, pH 7.4 buffer provides the best latency/activity ratio for homogenates, but for the isolation of microsomes the higher number of strokes (10) as well as low-osmotic buffer (5 mM HEPES, pH 7.4) are needed. However EROD activity is largely reduced in the preparations using buffers containing sucrose, so 5 mM HEPES buffer is recommended as the most suitable to study the microsomal reactions in H4IIE cells.


CONCLUSIONS
The isolation of microsomes was followed by the significant proteolytic breakdown of the glucose-6-phosphatase enzyme. It is recommended to use cell culture homogenates for assays when possible.
Sukhodub
Andrey L
AL
School of Biological Sciences, University of Portsmouth, St. Michaels Building, White Swan Road, Portsmouth, Hampshire, PO1 2DT, England, UK.
Burchell
Ann
A
D000818:Animals; D002021:Buffers; D002458:Cell Fractionation / Q000379:methods*; D045744:Cell Line, Tumor; D002499:Centrifugation, Density Gradient; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D005952:Glucose-6-Phosphatase / Q000378:metabolism; D006531:HEPES; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D009994:Osmolar Concentration; D051381:Rats; D022161:Transport Vesicles / Q000378:metabolism
D002021:Buffers; D019363:Cytochrome P-450 CYP1A1; D005952:Glucose-6-Phosphatase; D006531:HEPES