Binding of [α-³²P] NTP to Cholera Toxin and Light Signal Transmission Stimulating the Binding of [α-³²P] GTP via Pea Plasma Membrane

Abstract

Cholera toxin is composed of a catalytic subunit (29 kDa) called the A protomer and binding subunits (pentamer of 11.6 kDa subunit) called B oligomer. The binding of [α-³²P] ATP, [α-³²P] GTP, [α-³²P] CTP and [α-³²P] UTP at 1.6×10^-7 M was detected in the binding subunits, in which the binding of [α-³²P] GTP was most efficient. The binding of [α-³²P] ATP to the binding subunits was stimulated in the presence of 10^-4 M ATP, GTP, CTP or UTP. The binding of [α-³²P] GTP was strongly inhibited in the presence of 10^-3 M ATP, GTP or UTP. Similar results were observed in the binding of [α-³²P] CTP. The binding of [α-³²P] UTP was strongly inhibited in the presence of 10^-4 M ATP, GTP, CTP or UTP. The binding of [α-³²P] GTP to the B oligomer was stimulated in the presence of the A protomer. The [³²P] ADP-ribosylation of the A protomer (29 kDa) and A-2 protomer (24 kDa) lacking A-1 (5 kDa) was unaffected by ATP, GTP, CTP or UTP at a concentration of 10^-4 or 10^-3 M. At a concentration of 10^-4 M NAD stimulated the binding of [α-³²P] ATP and [α-³²P] GTP but at a concentration of 10^-3 M it inhibited it.
The plasma membrane was purified from the red light irradiated third internode of etiolated pea seedlings. The purified plasma membrane was mixed with cholera toxin in the presence of 1.6×10^-7 M [α-³²P] ATP, [α-³²P] GTP, [α-³²P] CTP or [α-³²P] UTP. The binding of [α-³²P] GTP was stimulated partly proportional to the fluence rates of red light irradiation of the third internodes.