High energy irradiation of bacterial membrane vesicles

Abstract

The interactions of membrane components and two well-defined .' transport systems in the!. coli ML 308-225 membrane vesicles with 60Co gamma radiation were investigated. The phosphoeno1pyruvate:g1ucose phosphotransferase system (PEP-PTS) was more sensitive to radiation than the D-1actate dehydrogenase (D-LDH)-coup1ed amino acid transport system. Both systems were inactivated by the direct and indirect effects of radiation, the latter being more effective than the former. The free radical scavenger, cysteamine, exerted more radioprotection on the PEP-PTS than on the amino acid transport system. The differential radiosensitivities of the two transport systems might be attributed to their relative accessibility to the radiation-generated free radicals presumably from water. Phosphorylation of a-methyl glucoside, which is an integral step in the transport of sugar by the PEP-PTS, was as radiosensitive as the active transport itself indicating that gamma radiation did not induce changes in the membrane permeability. This observation was supported by data from the spin label study using fatty acid spin labels. Gamma radiation did not affect the order parameter of membrane-bound 1(12,3) suggesting that the fluidity of the membrane was unchanged upon irradiation. The ascorbate-induced reduced of membrane-bound 1(12,3) depended, among other factors, on the permeability of the membrane to ascorbate. The finding that the rate of reduction of 1(12,3) by ascorbate was not affected by radiation indicated that radiation did not change the permeability properties of the membrane. The radiation effect was quite different from the effect of Triton X-100 on the PEP-PTS. At a concentration of 0.05%, Triton X-100 stimulated phosphorylation but decreased active transport of a-methyl glucoside showing that the decrease in active transport was due to leakage of accumulated sugar. A decrease in the order parameter of 1(12,3) bound to Triton X-100-treated membranes further showed that the detergent affected the fluidity of the membrane bilayer. Hence, the data suggest that the decrease in active transport of a-methyl glucoside across irradiated membranes might be due to inactivation of the components that comprise the transport system rather than to leakage of accumulated sugar. The active transports of proline and glutamic acid were more radiosensitive than the D-lactate:DC1 reductase system and the 02 uptake of the electron transport chain. This observation shows that a more sensitive component located outside of the main flow of electron transport, possibly in a shunt, might account for the sensitivity of the active transport. Possible identities and locations of the radiation-sensitive components of the D-LDH-linked amino acid transport system were discussed. The spin label study revealed the presence of a radiosensitivity gradient along the membrane matrix. The partially immobilized (P~ sites) NEM spin labels were more radiosensitive than the strongly immobilized (SI sites) NEM spin labels. The PI sites were protected by free radical scavengers such as cysteamine and dithiotreito1 while the SI sites were hardly protected by these compounds. The 81 sites were as radiosensitive as the membrane-bound 1(12,3) and the cysteamine-treated transport systems. The difference in the radiosensitivities of the NEM spin labels indicated that the -SH groups spin-labelled by NEM are located differently in the membrane, and that their radiosensitivities vary according to their proximity to the aqueous medium. The -SH groups on the surface of the membrane are more affected by radiation than groups embedded in the membrane. The results presented show that gamma radiation can monitor membrane components and functions of varying radiosensitivities. The possible application of high-energy radiation as a physical probe of membrane structure and functions is indeed promising.