Purification of diacylglycerol:acyltransferase from rat liver to near homogeneity.
A method to isolate a protein related to the diacylglycerol:acyltransferase (DGAT) activity in rat liver microsomes has been developed. The microsomes were treated with sodium deoxycholate (DOC; 0.1 mg/mg protein) at a concentration of 1 mM, i.e., below the critical micellar concentration (CMC), to remove luminal and loosely bound proteins. Three percent of the DGAT activity and all of the acylCoA hydrolyse activity were present in the supernatant, i.e., among the extracted loosely bound proteins. The insoluble material, recovered as a pellet, was suspended in DOC (1.6 mg/ml and mg protein in the original microsomes), and subjected to multiple, short (1-2 sec) sonications. CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; 5 mg/ml) was then added, and the sonication was repeated. The detergent-treated microsomal membranes were filtered through a 0.22-micron filter and chromatographed on a Superose 6 column from which the DGAT activity was recovered in a high molecular mass fraction. A monoclonal antibody that reacted with this fraction was raised and used in immunoaffinity experiments. This antibody removed 93 +/- 6% (mean +/- SD, n = 4) of the DGAT activity present in solution and 44 +/- 6% (mean +/- SD, n = 5) of the applied activity could be recovered after desorption. The antibody recognized a 60 kDa protein upon Western blot of rat liver microsomal proteins as well as of the DGAT-containing fraction from the Superose 6 column. A 60 kDa protein was highly enriched in the DGAT-containing retained fraction from the immunoaffinity chromatography. This 60 kDa protein reacted with the monoclonal antibody on Western blot. In addition to the 60 kDa protein, the retained fraction from the immunoadsorber contained a 77 kDa protein. This protein did not react with the monoclonal antibody on Western blots. Neither the 60 nor the 77 kDa protein reacted with antibodies to mouse immunoglobulins or showed any unspecific reaction with immunoglobulins.
Andersson
M
M
Department of Medical Biochemistry, University of Göteborg, Sweden.
Wettesten
M
M
Borén
J
J
Magnusson
A
A
Sjöberg
A
A
Rustaeus
S
S
Olofsson
S O
SO
D000217:Acyltransferases / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D002793:Cholic Acids; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003840:Deoxycholic Acid; D051048:Diacylglycerol O-Acyltransferase; D007163:Immunosorbent Techniques; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility; D013010:Sonication
D000911:Antibodies, Monoclonal; D002793:Cholic Acids; D003840:Deoxycholic Acid; D000217:Acyltransferases; D051048:Diacylglycerol O-Acyltransferase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate