Philip D. Bragg

Subunit composition, function, and spatial arrangement in the Ca2+-and Mg2+-activated adenosine triphosphatases of Escherichia coli and Salmonella typhimurium.

Abstract The Ca2+- and Mg2+-activated ATPases of Escherichia coli NRC 482 and Salmonella typhimurium LT2 were purified to homogeneity. Both enzymes consisted of five polypeptides (α-ϵ). The molecular weights of the α, β, and ϵ polypeptides were 56,800, 51,800 and 13,200 for both enzymes. The molecular weights of the γ and δ polypeptides of the E. coli and S. typhimurium ATPases were 32,000 and 20,700, and 30,900 and 21,500, respectively. In both ATPases the stoichiometry of the subunits was α3β3γδϵ as determined with the 14C-labeled enzymes. The ATPases of either organism reacted with equal effectiveness with ATPase-deficient particles of the other organism to reconstitute energy-dependent transhydrogenase activity. Treatment of the homogeneous ATPases of both organisms with TPCK-trypsin stimulated ATPase activity but resulted in destruction of coupling factor activity. Trypsin treatment completely digested the δ and ϵ polypeptides, and removed up to 70% of the γ polypeptide. In the presence of the bifunctional cross-linking reagent dithiobis(succinimidyl propionate) ATPase activity was lost and cross-linking of α to β polypeptides occurred. Crosslinking of α to α or β to β polypeptides was not detected. The function of the individual polypeptides of the ATPase is discussed and a model for their spatial arrangement in the enzyme is presented.

Organization of proteins in the native and reformed outer membrane of Escherichia coli

1. 1. The proteins of the outer membrane of Escherichia coli were characterized by polyacrylamide gel electrophoresis and their molecular weights determined. 2. 2. A buffer system is described which permits reproducible resolution of three proteins which do not resolve on polyacrylamide gel electrophoresis using conventional phosphate-sodium dodecyl sulfate buffer systems. 3. 3. Pronase digestion of spheroplast membranes, Triton-treated cell envelopes and isolated outer membranes of E. coli showed that only two major proteins were accessible to the enzyme in spheroplast membranes, while Triton treatment revealed several others to pronase attack. Several other proteins were not attacked by pronase under the conditions employed. An asymmetric arrangement of proteins within the membrane is suggested. 4. 4. Pronase digestion of reformed outer membrane gave a result similar to that obtained for the original outer membrane preparation. It is suggested that reaggregation of the components of the solubilized outer membrane has occurred in a specific manner with the proteins organized as in the native membrane.

Purification of a factor for both aerobic‐driven and ATP‐driven energy‐dependent transhydrogenases of Escherichia coli

We previously reported that a soluble protein fraction from Escherichiu coli extracts would enhance the ATP-driven energy-dependent transhydrogenase of this organism [ 11. Subsequently, Fisher et al. [2] stimulated the aerobic-driven reaction in this organism with a factor prepared from rat liver. In this paper we report the isolation from E. coli of a factor in near homogeneous form which stimulates both the aerobicand the ATP-driven energy-dependent transhydrogenase reactions.

Function of energy-dependent transhydrogenase in Escherichia coli

Abstract The activity of the energy-dependent transhydrogenase of membrane particles of E. coli varied markedly with growth conditions The activity of the enzyme was not related to the efficiency of oxidative phosphorylation. The enzyme was not subject to catabolite repression but was repressed by mixtures of amino acids. It is suggested that the transhydrogenase has a role in generating NADPH for biosynthesis.

Effect of removal or modification of subunit polypeptides on the coupling factor and hydrolytic activities of the Ca2+ and Mg2+-activated adenosine triphosphatase of Escherichia coli

Abstract The effect of removal or modification of the polypeptide subunits (α, β γ, δ, and ϵ) of the Ca 2+ and Mg 2+ -activated ATPase of Escherichia coli was investigated. Removal of the δ-polypeptide, although giving some decrease in ATPase activity, resulted in complete loss of coupling activity, where coupling activity was measured by the restoration of the energy-dependent transhydrogenase activity of ATPase-stripped respiratory particles. Modification of the γ-polypeptide, as found in the ATPase of an energy transfer coupling mutant ( etc-15 ), resulted in diminution of the ATPase and coupling activities. The diminished coupling activity could be overcome by using more of the enzyme which suggested that this enzyme may not be able to bind to the membrane as firmly as the enzyme from the wild type.

Properties of a soluble Ca2+- and Mg2+-activated ATPase released from Escherichia coli membranes

Abstract 1. 1. Spheroplasts of Escherichia coli NRC 482 were prepared by lysozyme digestion alone without the addition of EDTA. Subsequent osmotic lysis in 0.2 mM MgCl 2 gave less than 0.1 % survival of viable cells. 2. 2. The spheroplast membranes from the lysate were put through a washing procedure similar to that used by Abrams 1 . During the 5th washing a Ca 2+ - and Mg 2+ -activated ATPase (ATP phosphohydrolase, EC 3.6.1.4) was released. 3. 3. On gel filtration through Sepharose 6B the enzyme eluted as a sharp peak corresponding to a molecular weight of 365 000–390 000. 4. 4. The ATPase activity was dependent on the addition of a divalent metal ion. With Ca 2+ and Mg 2+ the pH optimum was at 9.5 and the optimum ratio of ion to substrate was less than 1. The Ca 2+ - and Mg 2+ -dependent activities appeared to be catalysed by the same enzyme. 5. 5. In the presence of Ca 2+ the enzyme was specific for ATP but with Mg 2+ there was some hydrolysis of ADP. 6. 6. There was no stimulation of ATPase activity by Na + or K + or by a mixture of the two ions. 7. 7. The enzyme was cold labile at 0° but the addition of glycerol (20 % v/v) overcame this instability. 8. 8. The Arrhenius plot for the ATPase reaction was linear over the range 10–36° and from this an activation energy of 20.7 kcal/mole was determined.

Inhibition by cyanide of the respiratory chain oxidases of Escherichia coli

The kinetics of inhibition by KCN of NADH oxidation in respiratory particles from Escherichia coli could be related to the relative amounts of cytochromes d and o which were present. Particles which contained higher levels of cytochrome d relative to cytochrome o were less sensitive to inhibition by cyanide. When cyanide reacted with the respiratory particles, the absorption bands of reduced cytochrome d at 442 and 628 nm in the reduced plus cyanide minus reduced difference spectrum were eliminated, as also were the bands at 423, 428, and 555 nm of b- and/or c-type cytochromes. Cyanide appeared to react with the oxidized form of cytochrome d to eliminate its α-band absorption with a second-order rate constant of 0.011 m−1 sec−1 for the rate of formation of cyanocytochrome d in the absence of added substrate. Under turnover conditions using NADH as substrate, the rate constant was 0.58 m−1 sec−1. This value is close to that determined from cyanide inhibition of NADH oxidase activity. The magnitude of the second-order rate constant for the formation of cyanocytochrome d was directly related to the rate of electron flux through cytochrome d. It is suggested that an intermediate species formed during the normal oxidation-reduction cycle of cytochrome d reacts with cyanide.

Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes

The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha 2 beta 2 structure (alpha: Mr, 54,000; beta: Mr, 48,700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha subunit and the N-terminal 20-25 kDa region of the beta subunit are composed of transmembranous alpha-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the alpha subunit. The beta subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both alpha and beta subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The beta subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the beta subunit. Digestion of the beta subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the alpha and beta subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the alpha subunit and the C-terminal 30 kDa region of the beta subunit are exposed on the cytoplasmic side of the inner membrane of E. coli. Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.

Crosslinking and radiation inactivation analysis of the subunit structure of the pyridine nucleotide transhydrogenase of Escherichia coli

The pyridine nucleotide transhydrogenase of Escherichia coli consists of two types of subunit (alpha: Mr 53,906; beta: Mr 48,667). The purified and membrane-bound enzymes were crosslinked with a series of bifunctional crosslinking agents and by catalyzing the formation of inter-chain disulfides in the presence of cupric 1,10-phenanthrolinate. Crosslinked dimers alpha 2, alpha beta and beta 2, and the trimer alpha 2 beta were obtained. A small amount of tetramer, probably alpha 2 beta 2, was also formed. Radiation inactivation was used to determine the molecular size of the transhydrogenase. The radiation inactivation size (217,000) and chemical crosslinking are consistent with the structure (Mr 205,146) being the oligomer that is responsible for biological activity.

Reduced nicotinamide adenine dinucleotide dependent reduction of fumarate coupled to membrane energization in a cytochrome deficient mutant of Escherichia coli K12

Escherichia coli SASX76 does not form cytochromes unless supplemented with 5-aminolevulinic acid. It can grow anaerobically on glycerol and DL-glycerol 3-phosphate in the absence of 5-aminolevulinic acid with fumarate but not with nitrate as the terminal electron acceptor. Cytochrome-independent NADH oxidase, glycerol 3-phosphate- and NADH-fumarate oxidoreductase activities are induced by anaerobic growth on a glycerol-fumarate medium. The pathway of electrons from substrate to fumarate involves menaquinone. The NADH-fumarate oxidoreductase and cytochrome-independent NADH oxidase systems are inhibited by piericidin A, 2-heptyl-4-hydroxyquinoline N-oxide, and iron chelating agents. Both systems can energize the membrane particles as indicated by quenching of atebrin fluorescence.