Purification of protein fatty acyltransferase and determination of its distribution and topology.
Studies reported from this laboratory have demonstrated that O-glycosidic glycoproteins of salivary, pulmonary, and gastrointestinal origin are acylated by fatty acyltransferase residing in Golgi and microsome-enriched fraction (Slomiany, A., Liau, Y.H., Takagi, A., Laszewicz, W., and Slomiany, B.L. (1984) J. Biol. Chem. 259, 13304-13308). Here we report on the successful purification of this enzyme from rough microsomal membranes of rat gastric mucosa and its identification in a number of diverse tissues and organs, such as heart, liver, pancreas, lung, kidney, salivary glands, and lymphoblasts. The enzymatic activity has been released from the stripped and salt-extracted microsomes with 0.5% Triton X-100 and recovered from 100,000 x g supernatant by affinity chromatography on Cibacron blue F3GA column. The retained fatty acyltransferase protein was selectively displaced from the column with 50 microM palmitoyl-CoA. On nonreducing polyacrylamide gel electrophoresis, the enzymatic activity was associated with a 234-kDa complex, and on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the complex afforded 65- and 67-kDa protein bands. Incubation of microsomes with trypsin prior to enzyme extraction resulted in a 50% inactivation of the fatty acyltransferase and generation of 53- and 55-kDa protein bands, which also had affinity to Cibacron blue F3GA and were displaced from the column together with the active (intact) enzyme. We suggest that the fatty acyltransferase is an integral rough microsomal protein partially exposed to cytosol, which catalyzes the fatty acyl-CoA-protein reaction on the cytosolic site of the rough endoplasmic reticulum and that this enzyme is responsible for processing of the group of protein which are entering rough endoplasmic reticulum-Golgi secretory pathway.
Kasinathan
C
C
Research Center, University of Medicine and Dentistry of New Jersey, Newark 07103-2400.
Grzelinska
E
E
Okazaki
K
K
Slomiany
B L
BL
Slomiany
A
A
D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005753:Gastric Mucosa / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D017830:Octoxynol; D010449:Peptide Mapping; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D014357:Trypsin
D000214:Acyl Coenzyme A; D003902:Detergents; D011092:Polyethylene Glycols; D017830:Octoxynol; D000217:Acyltransferases; C043421:glycoprotein N-palmitoyltransferase; D014357:Trypsin