Purification and Characterization of Alanine Carrier Isolated from H-Proteins of Bacillus subtilis

Kusaka, Iwao; Kanai, Keiko

doi:10.1111/j.1432-1033.1978.tb12095.x

Cited by 19 publications

(2 citation statements)

References 10 publications

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“…Regarding the unique ability of a carrier to couple with a proton motive force or chemical energy (4), it now seems attractive to find out what the intrinsic attributes are that make a carrier function as a chemosmotic pump. Recent findings of a protonation-dependent mechanism of substrate binding by carrier (5) and possible cooperative control of the uptake rate by sodium ion (6), as well as improved methods for purifying alanine translocating membrane proteins (7,8), may offer important clues to answer this question.…”

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confidence: 99%

Amplification and characterization of the proline transport carrier of Escherichia coli K-12 by using proT+ hybrid plasmids.

Motojima¹,

Yamato²,

Anraku³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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A previous report [Motojima, K., Yamato, I. & Anraku, Y. (1978) J. Bacteriol. 136, 5-9) described the characteristics of mutants (proT-) of Escherichia coli K-12 that are defective in proline transport carrier activity. Two hybrid plasmids from the Clarke and Carbon colony bank were found to complement the mutation by conjugation and transformation. Analysis with restriction endonucleases showed that both plasmid DNAs carried the same fragment of the E. coli chromosome. A polypeptide with a molecular weight of 24,000, specifically coded for by the proT+ plasmid, was identified in the cytoplasmic membrane by using a double-isotope labeling method in a minicell system. The strain harboring the proT+ plasmid has 6 times as much proline transport carrier as the wild strain. This amplification of the carrier makes it possible to measure proline binding to the carrier by microequilibrium dialysis. Detailed analysis of binding indicated that the maximal amount of proline bound to the carrier is 0.70 nmol/mg of protein of the cytoplasmic membrane of the amplified strain. From this value and the assumption that the carrier has one binding site per minimal molecular weight of 24,000, the content of the proline transport carrier in the cytoplasmic membrane was estimated to be 1.7%.

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confidence: 99%

Amplification and characterization of the proline transport carrier of Escherichia coli K-12 by using proT+ hybrid plasmids.

Motojima¹,

Yamato²,

Anraku³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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“…In this organism, intrinsic membrane proteins were released from the membrane and accumulated in the cells as a waterinsoluble protein aggregate (H protein) during membrane autolysis [7]. H protein contained protein components which are responsible for active transport of certain nutrients [8,9]. Using H protein as a starting material, we have now purified the antiporter to homogeneity and reconstituted this protein into proteoliposomes.…”

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confidence: 99%

Purification and characterization of Ca2+/H+ antiporter from Bacillus subtilis

1986

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Caz+ was accumulated in inside-out membrane vesicles of Bacillus subtilis when NADH was used as an energy source. A dpH (acid interior) could also drive Ca2+ accumulation in the membrane vesicles and the accumulation was inhibited by carbonylcyanide p-trifluoromethoxyphenylhydrazone and nigericin plus K + . These results indicate the presence of a Ca2 +/H + antiporter (exchanger) in this organism.The antiporter was isolated and purified to homogeneity from the membrane proteins by chromatography on hydroxyapatite, diethylaminoethyl(DEAE)-Toyopearl650 M and butyl-Toyopearl650 M. The purified antiporter has a molecular mass of about 45000 daltons and an isoelectric point of 5.0.The fluorescence quenching of a cyanine dye (3,3'-dipropylthiodicarbocyanine iodide [diS-C3-(5)] during Ca2 ' accumulation in proteoliposomes by the purified antiporter showed the generation of a membrane potential (interior negative) suggesting a H+/Ca2' stoichiometry above 2 in the transport. This was also supported by the result that the K+-diffusion potential, interior positive, stimulated the Ca2+ uptake in the presence of a ApH. The apparent K,,, for CaZ+ of the antiporter was about 40 pM and La3+ inhibited the transport. Amino acid analysis of the purified antiporter indicated the presence of large amounts of glutamic and aspartic acids and small amounts of histidine, lysine and arginine. This is consistent with the low isoelectric point (about 5.0) of the protein. [6] reported that Ca2+/H+ exchange in E. coli is electrogenic: the stoichiometry of H+/Ca2+ > 2. They also suggested that the antiporter functions as a system for Caz+ extrusion from the cells.For the elucidation of the molecular mechanism of the Ca2 +/H+ antiporter, it is important to purify and reconstitute the protein(s) responsible for Ca2 +/H+ exchange into proteoliposomes.In this paper, firstly, we have studied Ca2+ transport system in the membrane vesicles of Bacillus subtilis and found that the vesicles prepared by lysis with a French pressure cell, which appeared to be predominantly inside-out, accumulated Ca2+ by externally supplied NADH. An artificially imposed H + gradient in these membrane vesicles, acid inside, was also found to drive Ca2+ uptake. These results indicated the presence of a Ca2+/H+ antiporter in B. subtilis. Secondly, we have tried to isolate and purify the antiporter from B. subtilis. In this organism, intrinsic membrane proteins were released from the membrane and accumulated in the cells as a waterinsoluble protein aggregate (H protein) during membrane autolysis [7]. H protein contained protein components which are responsible for active transport of certain nutrients [8,9]. Using H protein as a starting material, we have now purified the antiporter to homogeneity and reconstituted this protein into proteoliposomes. MATERIALS AND METHODS Preparation of inside-out membrane vesiclesBacillus subtilis W23 was grown in GPY medium (IYo glucose, 0.25% Difco yeast extract and 0.5% Daigoeoyo polypeptone) and the cells were harvested in mid-log phas...

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Methodological aspects of purification and reconstitution of transport proteins from mammalian plasma membranes

Koepsell

Reviews of Physiology, Biochemistry and Pharmacology

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Purification and Characterization of Alanine Carrier Isolated from H-Proteins of Bacillus subtilis

Cited by 19 publications

References 10 publications

Amplification and characterization of the proline transport carrier of Escherichia coli K-12 by using proT+ hybrid plasmids.

Amplification and characterization of the proline transport carrier of Escherichia coli K-12 by using proT+ hybrid plasmids.

Purification and characterization of Ca2+/H+ antiporter from Bacillus subtilis

Methodological aspects of purification and reconstitution of transport proteins from mammalian plasma membranes

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