Reduction in mitochondrial respiratory capacity in Saccharomyces cerevisiae induced by expression of hepatitis B virus surface antigen.

Transformants of Saccharomyces cerevisiae strain TL154 (MATalpha, trp1, leu2) expressing hepatitis B virus surface antigen showed reduced rates of cell growth compared with those of nontransformed cells. The rates of phosphorylative, nonphosphorylative, and uncoupled respiration in mitochondria isolated from the transformants were reduced relative to those of mitochondria derived from nontransformed cells, regardless of whether the cells were cultured in rich or minimal medium. The electrophoretic protein profiles of cell and mitochondrial extracts did not differ substantially between transformed and nontransformed cells. These results suggest that the reduced rate of mitochondrial respiration in the transformants may be due to impairment of metabolic function rather than to inhibition of the expression of components of the respiratory chain.

The effect of chloroform on mitochondrial energy transduction.

The effect of chloroform on mitochondrial respiration with succinate was investigated by applying the method of Brand, Chien and Diolez [(1994) Biochem. J. 297, 27-29] to examine whether chloroform causes redox slip (fewer protons pumped per electron transferred) during mitochondrial electron transport. N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD), which lowers H+/O (the number of protons pumped to the external medium by the electron transport complexes per oxygen atom consumed) by altering the electron flow pathway, was investigated for comparison. Non-phosphorylating mitochondria that had been treated with 350 microM TMPD or 30 mM chloroform were titrated with malonate in the presence of submaximal concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Linear relations between CCCP-induced extra respiration and protonmotive force were obtained. These results showed that there was no measurable protonmotive force-dependent or rate-dependent slip in mitochondria treated with either TMPD or chloroform. However, both TMPD and chloroform seemed to decrease H+/O in a manner independent of protonmotive force and rate. The relationship between non-phosphorylating respiration and protonmotive force was simulated in mitochondria of which 25% of the total population were assumed to have been broken. The simulation showed that the apparent decrease in H+/O on the addition of TMPD or chloroform to mitochondria could be in principle accounted for by breakage. Assays of mitochondrial breakage (ATP hydrolysis in the presence of atractyloside and oxidation of exogenous NADH) showed that chloroform broke mitochondria but TMPD did not. We conclude that chloroform changes the measured H+/O as an artifact by causing mitochondrial breakage and does not cause measurable redox slip, whereas TMPD genuinely lowers H+/O.

The causes and functions of mitochondrial proton leak.

The non-linear relationship between respiration rate and protonmotive force in isolated mitochondria is explained entirely by delta p-dependent changes in the proton conductance of the mitochondrial inner membrane and is not caused by redox slip in the proton pumps. Mitochondrial proton leak occurs in intact cells and tissues: the futile cycle of proton pumping and proton leak accounts for 26% +/- 7% of the total oxygen consumption rate or 33% +/- 7% of the mitochondrial respiration rate of isolated hepatocytes (mean +/- S.D. for 43 rats); 52% of the oxygen consumption rate of resting perfused muscle and up to 38% of the basal metabolic rate of a rat, suggesting that heat production may be an important function in the proton leak in homeotherms. Together with non-mitochondrial oxygen consumption, it lowers the effective P/O ratio in cells from maximum possible values of 2.33 (palmitate oxidation) or 2.58 (glucose oxidation) to as low as 1.1 in liver or 0.8 in muscle. The effective P/O ratio increases in response to ATP demand; the ability to allow rapid switching of flux from leak to ATP turnover may be an even more important function of the leak reaction than heat production. The mitochondrial proton conductance in isolated mitochondria and in hepatocytes is greatly modulated by thyroid hormones, by phylogeny and by body mass. Usually the reactions of ATP turnover change in parallel so that the coupling ratio is not greatly affected. Changes in proton leak in tissues are brought about in the short term by changes in mitochondrial protonmotive force and in the longer term by changes in the surface area and proton permeability of the mitochondrial inner membrane. Permeability changes are probably caused by changes in the fatty acid composition of the membrane phospholipids.

TSH and TSH receptor antibody-binding sites in fibroblasts of pretibial myxedema are related to the extracellular domain of entire TSH receptor.

The role of TSH receptor antibodies in the pathogenesis of pretibial myxedema is still unclear. This study was designed to determine whether patients with pretibial myxedema had higher serum titers of TSH receptor antibodies, and whether there were TSH and TSH receptor antibody-binding sites on plasma membranes of fibroblasts derived from the skin of pretibial myxedema. If there were, were the binding sites similar to the TSH receptor? The TSH receptor antibodies were determined with radioreceptor assay in 20 normal subjects, 18 hyperthyroid Graves' disease patients without ophthalmopathy, 26 hyperthyroid Graves' disease patients with ophthalmopathy, and 11 patients with pretibial myxedema associated with Graves' ophthalmopathy. TSH and TSH receptor antibody-binding sites were studied on plasma membranes of fibroblasts cultured from the skin of pretibial myxedema with radioreceptor assay. RNA was also extracted from the fibroblasts of pretibial myxedema and reverse transcribed using random primers as the primers for cDNA synthesis. The resulting cDNAs were subjected to amplification by polymerase chain reaction with the use of a set of primers spanning the 5' region (+256/+275 and +616/+635) and the 3' region (+1819/+1838 and +2405/+2424) of the TSH receptor cDNA (+1 transcription start codon). They were further identified by Southern blot hybridization, with the probe spanning the 5' region (+272/+612) and the 3' region (+1908/+2268) of the TSH receptor cDNA (+ 1 transcription start codon), and sequencing. The results showed that patients with pretibial myxedema had higher titers of TSH receptor antibodies in the serum. TSH and TSH receptor antibody-binding sites were present on plasma membranes of fibroblasts derived from the skin of pretibial myxedema patients and related to the extracellular domain of the TSH receptor. These data suggest a common antigenic site in the skin and in the thyroid as a putative target for TSH receptor antibodies or lymphocytes of Graves' disease.

Experimental discrimination between proton leak and redox slip during mitochondrial electron transport.

By measuring the relationship between protonmotive force and the increment in oxygen consumption by mitochondria treated with submaximal amounts of uncoupler, we have experimentally tested four different models of imperfect coupling of oxidative phosphorylation. The results show that the increased rate of oxygen consumption at high protonmotive force is explained entirely by the dependence on protonmotive force of the passive proton leak conductance of the mitochondrial inner membrane. There is no measurable contribution from redox-slip reactions in the proton pumps caused by high protonmotive force. Neither is there any contribution from increased proton conductance of the membrane or increased redox slip in the respiratory chain caused by high turnover rates of the complexes.

Pentoxifylline inhibits the proliferation and glycosaminoglycan synthesis of cultured fibroblasts derived from patients with Graves' ophthalmopathy and pretibial myxoedema.

Excessive amounts of glycosaminoglycans accumulate in the extraocular muscles of patients with Graves' ophthalmopathy and in the affected skin of patients with pretibial myxoedema. It is widely accepted that fibroblasts are the sources of glycosaminoglycan synthesis. Pentoxifylline, an analogue of the methylxanthine theobromine, inhibits the proliferation and certain biosynthetic activities of fibroblasts derived from normal human skin and from skin of patients with some fibrotic disorders. Our objective was to determine whether pentoxifylline has similar effects on fibroblasts derived from patients with Graves' ophthalmopathy and pretibial myxoedema and could serve as a candidate for the treatment of these manifestations. Fibroblasts from the extraocular muscles of two patients with Graves' ophthalmopathy and normal extraocular muscles of two subjects with strabismus, as well as the affected skin of two patients with pretibial myxoedema were cultured in vitro in the presence and absence of pentoxifylline to assay its effect on the proliferation of fibroblasts and their production of glycosaminoglycans. In subconfluent fibroblast cultures, pentoxifylline treatment caused a dose-dependent inhibition of serum-driven fibroblast proliferation. In confluent fibroblast cultures both in the presence and absence of serum, exposure to pentoxifylline similarly resulted in a dose-dependent inhibition of glycosaminoglycan synthesis for all these different kinds of fibroblasts. These findings may form the rationale for a clinical trial using pentoxifylline for the treatment of Graves' ophthalmopathy and pretibial myxoedema.

ATPase of Rhodospirillum rubrum requires three functional copies of beta subunit as determined by radiation inactivation analysis.

Radiation inactivation analysis yielded a functional unit of 170 +/- 26 kDa as beta subunit of ATPase was irradiated and then reconstituted to beta-depleted chromatophores of Rhodospirillum rubrum. A functional size of 132 +/- 17 kDa for the beta-depleted ATPase moiety involved in ATP hydrolysis reaction was also determined. When both purified beta subunit and beta-depleted chromatophore were irradiated separately, reconstituted, and then activity measured, the functional mass was 312 +/- 50 kDa. Our compelling evidence directly indicates that three functional copies of beta subunits were required for ATP hydrolysis.

Radiation inactivation analysis of chloroplast CF0-CF1 ATPase.

Radiation inactivation technique was employed to measure the functional size of adenosine triphosphatase of spinach chloroplasts. The functional size for acid-base-induced ATP synthesis was 450 +/- 24 kilodaltons; for phenazine methosulfate-mediated ATP synthesis, 613 +/- 33 kilodaltons; and for methanol-activated ATP hydrolysis, 280 +/- 14 kilodaltons. The difference (170 +/- 57 kilodaltons) between 450 +/- 24 and 280 +/- 14 kilodaltons is explained to be the molecular mass of proton channel (coupling factor 0) across the thylakoid membrane. Our data suggest that the stoichiometry of subunits I, II, and III of coupling factor 0 is 1:2:15. Ca2+- and Mg2+-ATPase activated by methanol, heat, and trypsin digestion have a similar functional size. However, anions such as SO3(2-) and CO3(2-) increased the molecular mass for both ATPase's (except trypsin-activated Mg2+-ATPase) by 12-30%. Soluble coupling factor 1 has a larger target size than that of membrane-bound. This is interpreted as the cold effect during irradiation.

Functional size of photosynthetic electron transport chain determined by radiation inactivation.

Radiation inactivation technique was employed to determine the functional size of photosynthetic electron transport chain of spinach chloroplasts. The functional size for photosystem I+II (H(2)O to methylviologen) was 623 +/- 37 kilodaltons; for photosystem II (H(2)O to dimethylquinone/ferricyanide), 174 +/- 11 kilodaltons; and for photosystem I (reduced diaminodurene to methylviologen), 190 +/- 11 kilodaltons. The difference between 364 +/- 22 (the sum of 174 +/- 11 and 190 +/- 11) kilodaltons and 623 +/- 37 kilodaltons is partially explained to be due to the presence of two molecules of cytochrome b(6)/f complex of 280 kilodaltons. The molecular mass for other partial reactions of photosynthetic electron flow, also measured by radiation inactivation, is reported. The molecular mass obtained by this technique is compared with that determined by other conventional biochemical methods. A working hypothesis for the composition, stoichiometry, and organization of polypeptides for photosynthetic electron transport chain is proposed.