Properties of solubilized microsomal lipase from germinating Brassica napus
1989
Weselake, R.J. | Thomson, L.W. | Tenaschuk, D. | MacKenzie, S.L.
Lipase (triacylglycerol acylhydrolase [EC 3.1.1.3.]) was extracted from the microsomal fraction of cotyledons of dark grown seedlings of Canola (Brassica napus L. cv Westar) by treatment with Triton X-100. The enzyme was partially purified by chromatography on Sephacryl S-300 and DEAE Bio-Gel and was stable when stored at -20 degrees C in 50% (v/v) glycerol. The lipase aggregated readily but the distribution of species present in solution could be controlled by nonionic detergents. A species with an apparent Mr of about 250,000 was obtained by gel filtration chromatography in the presence of 1% (v/v) Triton X-100. Lipase activity was optimal near neutral pH, and the reaction approached maximum velocity at a concentration of 0.5 to 1 millimolar emulsified triolein. The reaction rate responded linearly to temperature up to about 40 degrees C and the hydrolytic process had an activation energy of 18 kilocalories per mole. Microsomal lipase lost about 20% and 80% activity when heat-treated for 1 hour at 40 degrees C and 60 degrees C, respectively. At appropriate concentrations, the detergents Triton X-100, n-octyl-beta-D-glucopyranoside, 3-[3-cholamidopropyl- O-dimethylammonio]-1-propanesulfonate, cetyl trimethylammonium bromide, and sodium dodecyl sulfate all inhibited lipase activity. n-Octyl-beta-D-glucopyranoside, however, was stimulatory in the 2 to 8 millimolar concentration range. The inhibitory effects of Triton X-100 were reversible.
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