Human erythrocyte galactosyltransferase. Characterization, membrane association and sidedness of active site |
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Authors: | Francis J Hesford Eric G Berger |
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Institution: | Medizinisch-Chemisches Institut der Universität Bern, Bühlstrasse 28, Postfach, CH-3000 Bern 9 Switzerland |
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Abstract: | Human erythrocyte UDPgalactose : 2-acetamido-2-deoxy-α-d-galactopyranosylpeptide galactose
transferase (Galactosyltransferase) has been characterized in terms of detergent and metal ion requirements, Michaelis constants for donor and acceptor substrates, inhibition constant for N-acetylgalactosamine, pH optimum and ionic strength effects. The assay thus optimized permits initial velocity measurements. Galactosyltransferase was shown to be membrane-bound by demonstrating its association with erythrocyte ghosts after high and low ionic strength treatments to remove weakly-associated proteins. In the absence of detergents, no activity was detectable in sealed ghosts and inside-out vesicles derived from erythrocyte membranes. Enzyme activation by detergents paralleled solubilization of membrane proteins. Both latency and solubilization studies indicated a substrate-inaccessible active site for the enzyme in situ in the membrane. Galactosyltransferase activity in resealed ghosts, leaky ghosts and inside-out vesicles was resistant to the action of trypsin, chymotrypsin or pronase applied as single agents. A mixture of these proteases, however, strongly reduced the enzyme activity in inside-out vesicles and leaky ghosts, indicating a cytosolic orientation for the active site of the galactosyltransferase. |
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Keywords: | Galactosyltransferase Active site Sidedness (Human erythrocyte membrane) |
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