Pituitary protein lipolytic factor(s): Partial purification by isoelectric focusing (IEF)

Tests were made on highly purified bovine peptidal pituitary factors for lipolytic activity on rat adipose tissue. The lipolytic protein factor from bovine pituitary glands was isolated and characterized by ethanol precipitation, gel filtration, and isoelectric focusing. The supernatant of the homogenized tissue was precipitated with ethanol, the precipitate was lyophilized and resuspended in HCl 1 mmole/liter, the insoluble was discarded, and the supernatant lyophilized. A solution of the lyophilized fraction was fractionated by gel filtration on Sephadex G-75. The bands with lipolytic activity were pooled and lyophilized. Lipolytic activity was determined by incubation of epididimal rat adipose tissue and by successive measurements of free fatty acids and glycerol released in the incubation medium. The gel filtration with the highest lipolytic activity (20.4 µequiv/g per 4 hr and 17.0 µmole/g per 4 hr glycerol) was submitted to isoelectric focusing. The gel filtration still appeared highly heterogeneous, but most of the lipolytic activity was concentrated in the protein band at pH 8.6.