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Wheat Germ DNA-dependent RNA Polymerase II: Purification and Properties
Abstract
INTRODUCTION
Detailed studies of the chemical and physical properties of enzymes require that large amounts be conveniently prepared in high purity. The low RNA polymerase content and unavailability and/or expense of the starting material prohibit purification of large amounts of this enzyme from most eukaryotic sources, and most of the procedures currently used are not amenable for scaling up. Wheat germ is an excellent source for the purification of RNA polymerase. The material is inexpensive, easily stored, available in virtually unlimited quantities from the grain milling industry, ready for use with no preliminary tissue isolation, and is rich in the α-amanitin-sensitive RNA polymerase (II or B). An efficient method for the purification of this enzyme will be presented which utilizes Polymin P to remove nucleic acids and much of the protein. The large purification achieved at the initial stages of this procedure allows more efficient use of subsequent column chromatographic steps and results in the complete purification of RNA polymerase II from several kilograms of starting material in 2–3 days at a yield of 20–30 mg/kg. Using the large amounts of enzyme made available from wheat germ by this method, we have investigated various physical and chemical properties of the enzyme, including subunit structure and metal content.
Detailed studies of the chemical and physical properties of enzymes require that large amounts be conveniently prepared in high purity. The low RNA polymerase content and unavailability and/or expense of the starting material prohibit purification of large amounts of this enzyme from most eukaryotic sources, and most of the procedures currently used are not amenable for scaling up. Wheat germ is an excellent source for the purification of RNA polymerase. The material is inexpensive, easily stored, available in virtually unlimited quantities from the grain milling industry, ready for use with no preliminary tissue isolation, and is rich in the α-amanitin-sensitive RNA polymerase (II or B). An efficient method for the purification of this enzyme will be presented which utilizes Polymin P to remove nucleic acids and much of the protein. The large purification achieved at the initial stages of this procedure allows more efficient use of subsequent column chromatographic steps and results in the complete purification of RNA polymerase II from several kilograms of starting material in 2–3 days at a yield of 20–30 mg/kg. Using the large amounts of enzyme made available from wheat germ by this method, we have investigated various physical and chemical properties of the enzyme, including subunit structure and metal content.
EXPERIMENTAL PROCEDURES
Materials
Raw wheat germ was obtained from the VioBin Corporation (Monticello, Illinois) in 100-kg batches and was stored in a cold room (4°C) for over 1 year with no loss in extractable RNA polymerase activity. Polymin P was kindly...
DOI: http://dx.doi.org/10.1101/0.779-791