ATP synthesis of submitochondrial particles driven by proton gradient

l) The submitochondrial particle system can synthesize ATP in the early phase (220 seconds after the accition of ADP) in the presence of sodium succinate and Pi, in spite of the absence of the hexokinase-glucose system, and this phosphorylation is inhibited by oligomycin. 2) The submitochondrial particle system can synthesize ATP by the base-acid transition (proton pulse) only in the presence of ADP and Pi, in spite of the absence of oxidizing substrates and the hexokinase-glucose system, and this phosphorylation is dependent on the span of pH change, and is inhibited by oligomycin and 2, 4-dinitrophenol. 3) The role of the proton vector in the oxidative phosphorylation and the proton ejection was discussed from the stand point of a new hypothesis.</p

Correlation of structure and function in the oxidative phosphorylation system of submitochondrial particles

1. After the centrifugation of sonicated heavy beef heart mitochondria at 75, 000 &#215; g for 10 minutes, the supernatant was centrifuged at 144, 000 &#215; g for 30 minutes. The residue was revealed being composed of vesicular inner membrane fragments (ETPH), about 600 to 1000 &#197;. in diameter, showing a morphological homogeneity and a high capacity of oxidative phosphorylation. 2. The Pia ratio of the ETPH in the presence of succinate and of NADH2 was 1.68 and 2.54, respectively, and the corrected Pia value for O2 gas equilibrium was 1. 01 and 1.40, respectively. 3. The capacity of oxidative phosphorylation in ETPH fraction was parallel to the activity of the oligomycin. sensitive ATPase in these fractions. 4. The P/0 ratio of ETPH was decreased to about 50 % by hypotonic treatment. The decrease of P/0 ratio was restored to the level of about 90 % by incubating the ETPH with ATP and BSA. In the instance where the P/0 ratio was low level in the hypotonic medium, the surface structure of ETPH was observed as a swollen form and the head pieces of the elementary particles were clearly observed in contrast to the solid surface structure of ETPH in the isotonic medium. 5. The P/0 ratio of ETPH was decreased to about 60 % by relatively severe sonication, and after separating the residue from the supernatant, that of the residue decreased further to about 40 %. The P/0 ratio of the residue was restored to the level before the separation on the addition of the supernatant containing oligomycin-insensitive ATPase. 6. A discussion was made on the correlation between the surface structure and the activities at terminal phosphorylation step of ETPH after the simple physico-chemical treatment.</p

The presence of phosphate-binding protein in inner mitochondrial membrane

Phosphate-binding protein(s) was found in the inner mitochondrial membrane of calf heart by Sephadex G-200 and G-25 gel filtration. The binding activity was inhibited by N-ethylmaleimide and competed by a large amount of cold phosphate. The amount of phosphate bound to the fraction was 29 nmoles per mg of protein. Affinity chromatography with phosphate-bound Sepharose 4B confirmed the presence of phosphate-binding protein(s) in the active fraction of mitochondrial membrane fractionated by gel filtration.</p

Purification and fine structure of reduced coenzyme Q-cytochrome C reductase in the mitochondrial membrane

For the purpose of revealing the molecular organization of the mitochondrial membrane the authors attempted to clarify the fine structure of reduced coenzyme Q-cytochrome c reductase and also studied how the CoQH2-cyt. c reductase is arranged in the mitochondrial membrane by systematic analyses of fractions from the purification process of CoQH2-cyt. c reductase. 1. Purified CoQH2-cyt. c reductase contained high concentration of cyt. b (9.5 m&#956;moles per mg protein) and cyt. Cl (4.5 m&#956;moles per mg protein), and was almost free from cyt. c, a, flavoproteins, primary dehydrogenases and ATPase. The enzyme complex also showed a high specific activity (48 &#956;moles of cyt. c reduced per mg protein per min at 30°). 2. CoQH2-cyt. c reductase was composed of particles of about 120 &#197; in diameter with irregular form, some time exhibiting electron opaque cores. In the loose aggregates of the particles, the size of each particle was about 95 &#197; in diameter. 3. An intimate correlation was observed between the particles of CoQH2cyt. c reductase and those on the surface of the NADH-cyt. c reductase fraction. 4. Regular arrays of uniform particles (about 82 &#197; in diameter with a center to center distance of about 100 &#197;) were observed on the surface of the submitochondrial membrane (brown membrane) obtained from beef heart mitochondria by treatment with deoxycholate (0.1 mg / mg protein) and KCl (72 g/l). The correlation between these particles and CoQH2-cyt. c reductase was discussed.</p

Role of hydrophobic interaction in hapten-antibody binding

The precipitation reaction of bovine serum albumin coupled with p-azophenylleucine with homologous antibody was inhibited by several structurally related haptens. The isobutyl group substituent on alpha-carbon atom of the leucine residue contributed more than -5.8 Kcal/mol to the free energy of binding. This value was consistent with the free energy change expected from the transfer of n-butane from an aqueous environment to liquid n-butane. The observed contribution was explained, in terms of the hydrophobic interaction of the isobutyl group with the antigen binding site of the antibody molecule. These results were also compared with other hapten-antibody systems.</p

Avian myeloblastosis virus-induced lymphosarcoma producing erythroblastic leucosis in chicks

Acute myeloblastosis and several forms of tumor, including one case of lymphosarcoma occurred when avian myeloblastosis virus (BAI-A strain) was inoculated into newly hatched chicks (SPF). The homogenate of lymphosarcoma inoculated intraperitoneally into other newly hatched chicks induced a high incidence of erythroblastic leucosis. Electron microscopy did not reveal the presence of C-type virus particles in the tumor tissue. The relationship between avian myeloblastosis virus, lymphosarcoma and erythroblastic leucosis is discussed.</p

Plural growth factors in the supernatant of embryos and adult muscles of chickens

An attempt was made to isolate the cell proliferation stimulation factors in the supernatant of embryo carcases and adult muscles of chickens. Evidence was obtained for the presence of at least two or more stimulating factors in both the embryonic and adult muscular supernatants. These factors did not require a supplement of sera or other supporting agents. Furthermore, the use of the salting-out method with ammonium sulfate revealed two or more growth stimulants in the supernatant of chick cells.</p

Partial purification and properties of bovine heart catalase.

Catalase was partially purified (about 380-fold purification) from the post-mitochondrial supernatant of bovine heart and compared with catalases from bovine erythrocytes and bovine liver. The electrophoretic mobility in polyacrylamide gel (pH 8.0) of heart catalase was the same as that of erythrocyte catalase and was smaller than that of the liver enzyme. The heart catalase was indistinguishable from erythrocyte catalase in regard to the molecular weights of subunit polypeptides, the inhibition patterns produced by several catalase inhibitors, and specific activity. The pH-activity curve of heart catalase consisted of a characteristic biphasic pattern with a peak at pH 7.5 and a shoulder at pH 10.</p

Modulation of optical density by sulfhydryl reagents in microbiuret method: a modified method for protein determination in the presence of sulfhydryl reagents.

2-Mercaptoethanol increases the optical density of assay solutions at wavelengths between 280 to 400 nm, and therefore interferes with the measurement of protein concentration by the microbiuret method. Protein concentration can be determined in the presence of 2-mercaptoethanol up to 6 mM by modification of the method as follows: after the precipitation of protein by trichloroacetic acid in the presence of deoxycholate, the precipitate is resolubilized with NaOH solution. Dithiothreitol interfered with the protein determinations could by made in the presence of 4 mM of dithiothreitol with the modified microbiuret method. This modified method is time-saving and more reliable than other methods for protein determination, such as Lowry's method, in the presence of sulfhydryl reagents.</p