Hajime Hirata

ATP synthesis catalyzed by purified DCCD-sensitive ATPase incorporated into reconstituted purple membrane vesicles

Purified dicyclohexylcarbodiimide-sensitive ATPase from a thermophilic bacterium (TF0·F1) and purple membranes from Halobacterium halobium were incorporated into P-lipid vesicles. The reconstituted vesicles took up protons dependent on either illumination or addition of ATP. Net formation of ATP was observed when the vesicles were illuminated in the presence of ADP and Pi and this was completely abolished by addition of an uncoupler or energy transfer inhibitor. These results indicate that purified DCCD-sensitive ATPase, consisting of 8 kinds of polypeptides, was capable of ATP synthesis coupled with proton translocation.

Respiration dependent transport of proline by electron transport particles from Mycobacterium phlei

Abstract Respiration dependent transport of proline was demonstrated in electron transport particles from Mycobacterium phlei . The uptake proceeded against the concentration gradient with succinate, generated NADH, exogenous NADH or ascorbate-TPD as substrate. The latter two electron donors were most effective for the transport of proline and for oxidation, but they were least efficient for oxidative phosphorylation. Glucose, Dlactate, fumarate and ATP had no effect on the transport process. The transport of proline was sensitive to certain uncoupling agents and respiratory inhibitors. It was independent of oxidative phosphorylation since it proceeded in the absence of coupling factors or phosphate and was not inhibited by arsenate. The transport of proline appears to occur with membranes orientated as in the intact cell.

Membrane orientation and active transport of proline

Abstract Active transport of proline with ascorbate-TPD as the electron donor has been demonstrated in both the protoplast ghosts and the electron transport particles (ETP) from Mycobacterium phlei . The steady state level of proline uptake in the ETP, generated with ascorbate-TPD, was found to decrease on the further addition of succinate. The succinate induced efflux of proline from the ETP was inhibited by malonate. In contrast, with ghost preparations, in which the membrane orientation is right side out, no efflux of proline was observed on addition of succinate. Evidence was obtained that indicates that succinic dehydrogenase is located on the outer surface of the membrane in the ETP and on the inner surface of the ghost membrane. It would appear that the direction of active transport is influenced by the location of specific respiratory components.

Partial purification of alanine carrier from rabbit small intestine brush border membrane and its functional reconstitution into proteoliposomes.

An alanine transport carrier was partially purified from brush border membranes of rabbit small intestine. The alanine carrier activity was not solubilized with 0.4% deoxycholate but recovered in the detergent-insoluble fraction. The detergent-insoluble proteins were reconstituted into proteoliposomes with soybean phospholipids. The reconstituted proteoliposomes were capable of uptake of alanine driven by an electrochemical potential of Na+. The initial rate of alanine uptake into the proteoliposomes was 90 pmoles/mg protein/sec, which was 15-fold higher than that observed with the native membrane vesicles. The uptake of alanine was effectively suppressed by various neutral amino acids but not by either cationic or anionic amino acids.

Gene Structure and Function of Thermophilic ATP Synthase

An operon for thermophilic ATP synthase (TF0F1)was sequenced and mutated. The advantage of using TF0F1 for mechanistic studies is its reconstitutability without MgATP. All genes for TF0F1 were arranged in the order of promotors, structural genes coding for the I, TF0 subunits (a, c, b) and TF1 subunits (δ, α, γ, β and e), and a terminator. The cause of the stability of these subunits was deduced from their sequence. The site-directed mutagenesis of the α and β subunits revealed that the 4 ionizable residues corresponding to Lys 21 and Asp 119 in the MgATP binding segments of adenylate kinase, are essential for the normal catalytic activity of this enzyme. The resulting βI164 and βN252 mutant subunits were both noncatalytic after reassembly into the αβγ subunit complex, even though both subunits bound significant amounts of ADP. The resulting αI175 reassembled weakly into an oligomer, while the αN261 was reconstituted into an αβγ subunit complex that showed no intersubunit cooperativity.

A factor(s) required for activation of oxidative phosphorylation in protoplast ghosts of Mycobacterium phlei

Summary Oxidative phosphorylation in protoplast ghosts of M. phlei was found to be cryptic. This latent activity was activated by the addition of soluble components found in the supernatant fraction. The synthesis of ATP from oxidation was shown to occur within the membrane structure; however, the ghost preparation failed to transfer high energy phosphate across the membrane to exogenous nucleotides and was impermeable to added nucleotide. In contrast, phosphorylation of exogenous nucleotides was found to occur on addition of a soluble protein factor(s) to the ghost preparation. This system with respect to its ability to interact with exogenous nucleotides is similar to the electron transport particle in which the membrane orientation is inside out. Inhibition of phosphorylation of the activated system by uncoupling agents or by antibody to the membrane bound coupling factor was found to be similar to that observed with the electron transport particles.