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Oligonucleotide-directed, site-specific mutagenesis has been utilized to modify the lac Y gene of Escherichia coli such that Cys148 in the lac permease is converted to Ser. A mutagenesis protocol is used that significantly improves the efficiency of mutant recovery by in vitro methylation of closed-circular heteroduplex DNA containing the mutation, followed by nicking with HindIII in the presence of ethidium bromide and heat denaturation prior to transfection. In contrast to Gly148 permease (Trumble, W.R., Viitanen, P.V., Sarkar, H.K., Poonian, M.S., and Kaback, H. R. (1984) Biochem. Biophys. Res. Commun. 119, 860-867), permease containing Ser at position 148 catalyzes active lactose transport at a rate comparable to wild-type permease. Like Gly148 permease, however, transport activity is less sensitive to inactivation by N-ethylmaleimide, and galactosyl-1-thio-beta-D-galactopyranoside affords no protection against inactivation. The observations provide strong support for the contention that Cys148 is obligatory for substrate protection against inactivation by sulfhydryl reagents, but does not play an essential role in lactose:H+ symport.
H K Sarkar, and
D R Menick, and
P V Viitanen, and
M S Poonian, and
H R Kaback
January 1986,
Methods in enzymology,
H K Sarkar, and
D R Menick, and
P V Viitanen, and
M S Poonian, and
H R Kaback
October 1985,
Proceedings of the National Academy of Sciences of the United States of America,
H K Sarkar, and
D R Menick, and
P V Viitanen, and
M S Poonian, and
H R Kaback
November 1988,
Biochemistry,
H K Sarkar, and
D R Menick, and
P V Viitanen, and
M S Poonian, and
H R Kaback
July 1989,
Biochemistry,
H K Sarkar, and
D R Menick, and
P V Viitanen, and
M S Poonian, and
H R Kaback
October 1994,
Biochemistry,
H K Sarkar, and
D R Menick, and
P V Viitanen, and
M S Poonian, and
H R Kaback
February 1987,
Biochemistry,
H K Sarkar, and
D R Menick, and
P V Viitanen, and
M S Poonian, and
H R Kaback
January 1990,
Research in microbiology,
H K Sarkar, and
D R Menick, and
P V Viitanen, and
M S Poonian, and
H R Kaback
October 1994,
Biochemistry,
H K Sarkar, and
D R Menick, and
P V Viitanen, and
M S Poonian, and
H R Kaback
July 1995,
Biochemistry,
H K Sarkar, and
D R Menick, and
P V Viitanen, and
M S Poonian, and
H R Kaback
July 1994,
Biochemistry,
