Proteins of the Golgi apparatus. Purification to homogeneity, N-terminal sequence, and unusually large Stokes radius of the membrane-bound form of UDP-galactose:N-acetylglucosamine beta 1-4galactosyltransferase from rat liver.
The Golgi marker enzyme, UDP-galactose:N-acetylglucosamine beta 1-4galactosyltransferase (beta 1-4GalT) was purified 44300-fold in its intact, membrane-bound form from rat liver membranes. The protein was isolated from detergent extracts as a high-M(r) form, having a Stokes radius approximating a globular protein of M(r) 440,000. It is comprised of a single protein component as observed on SDS/polyacrylamide gels, having an M(r) near 51,000, and does not have intermolecular disulfide cross-links. N-terminal sequencing of the enzyme demonstrated that it contains an N-terminal hydrophobic stretch deduced previously from cDNA encoding for the enzyme. Previous studies have indicated that the protein may be translated at either of two AUG sites near the 5' end of the mRNA [Russo, R. N., Shaper, N. L. & Shaper, J. H. (1990) J. Biol. Chem. 265, 3324-3331], giving rise to two polypeptides, one appended with 13 amino acids. In the work described here, evidence was only found for the sequence of the short form, missing a single methionine at the N-terminus. Mild proteolytic treatment cleaved the enzyme, giving rise to low-M(r) forms which were fully catalytically active and which, upon sequencing, were missing a 66-amino-acid stretch from the N-terminus (as compared to the mouse cDNA). Proteolytic treatment was accompanied by conversion of the form having a large Stokes radius to one approximating a globular protein with M(r) near 50,000. The N-terminal stretch appears to contribute to maintenance of the form having a large Stokes radius. This may be the result of interaction with a detergent micelle, dimerization or oligomerization, or interaction with some other large, non-protein molecule, although a detergent exchange still resulted in a form having a large Stokes radius.
Department of Enzymology, University of Washington, Seattle.
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B
Bendiak
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Ward
R J
RJ
Simpson
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004247:DNA; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006056:Golgi Apparatus / Q000201:enzymology*; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase / Q000737:chemistry / Q000302:isolation & purification*
D003902:Detergents; D008565:Membrane Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; D004247:DNA; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase