Sex-determining genes on mouse autosomes identified by linkage analysis of C57BL/6J-YPOS sex reversal.

A powerful approach for identifying mammalian primary (gonadal) sex determination genes is the molecular genetic analyses of sex reversal conditions (that is, XX individuals with testicular tissue and XY individuals with ovarian tissue). Here we determined the number and chromosomal location of autosomal and X-linked genes that cause sex reversal in C57BL/6J (B6) mice carrying a Y chromosome of Mus domesticus poschiavinus origin (YPOS). B6 XYPOS mice develop either as females with exclusively ovarian tissue or as true hermaphrodites with ovarian and testicular tissue. In contrast, the YPOS chromosome is fully masculinizing on most other inbred strain backgrounds. B6-YPOS sex reversal appears to result from the incompatibility of the Sry (sex determining region, Y chromosome) allele carried on the YPOS chromosome with B6-derived autosomal or X-linked loci. We found strong evidence for the location of one gene, designated tda1 (testis-determining, autosomal 1), at the distal end of Chromosome (Chr) 4 and a second gene, tda2, in the central region of Chr 2. A third gene, tda3, on Chr 5 is implicated, but the evidence here is not as strong. We suggest that B6 alleles at these loci predispose XYPOS fetuses to ovarian tissue development, but no single locus or combination of loci is necessary and sufficient to cause sex reversal. The TDA proteins may regulate Sry expression or form complexes with the SRY protein to regulate other genes, or the tda genes may be activated or repressed by the SRY protein.

Appropriate maximum holding times for analysis of total suspended solids concentration in water samples taken from open-channel waterways.

Many scientific laboratories follow, as standard practice, a relatively short maximum holding time (within 7 days) for the analysis of total suspended solids (TSS) in environmental water samples. In this study we have subsampled from bulk water samples stored at ∼4 °C in the dark, then analysed for TSS at time intervals up to 105 days after collection. The nonsignificant differences in TSS results observed over time demonstrates that storage at ∼4 °C in the dark is an effective method of preserving samples for TSS analysis, far past the 7-day standard practice. Extending the maximum holding time will ease the pressure on sample collectors and laboratory staff who until now have had to determine TSS within an impractically short period.

Correlation of general anesthetic potency with solubility in membranes.

Recently (Franks, N.P. and Lieb, W.R. (1978) Nature 274, 339-342) it has been claimed that the traditional correlation between anesthetic potency and vegetable oil solubility breaks down when the alkanols are compared to other volatile anesthetics. Lately, however, new information on the partitioning of anesthetics into lipid bilayers has become available. In this report the potency of twenty-one structurally diverse anesthetic agents is shown to correlate well with their ability to partition into phosphatidylcholine bilayers. Thus the original Meyer-Overton oil solubility hypothesis accommodates a wider range of anesthetics, including alkanols, volatile and gaseous agents, and barbiturates, when lipid bilayer solubility is substituted for oil solubility.

Diamide blocks H+ conductance in mitochondrial H+-ATPase by oxidizing FB dithiol.

Effects of diamide on proton conductance of electron transport particles (ETPH), purified H+-ATPase (F1-F0), F0 of the H+-ATPase from beef heart mitochondria and binding of cadmium (109Cd) to the H+-ATPase have been examined in the present paper. When ETPH and purified H+-ATPase are treated with 1 mM diamide, ATP-dependent generation of membrane potential, monitored by the absorbance change produced by the redistribution of oxonol VI, is consistently inhibited. Diamide also blocks passive H+ conductance driven by a K+ diffusion potential in the membrane sector, F0, of H+-ATPase. Furthermore, diamide treatment drastically reduces the binding of 109Cd2+ to H+-ATPase, showing competition for the FB dithiol group.

ATP synthase complex from bovine heart mitochondria. Passive H+ conduction through mitochondrial coupling factor 6-depleted F0 complexes.

Submitochondrial particles prepared by treatment of mitochondria with ammonia and silicotungstic acid were found to be deficient in coupling factor 6 according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting and had reduced ATP-Pi exchange activity. Requirement of coupling factor 6 for passive proton conductance through mitochondrial F0 was investigated by assaying the ability of depleted particles to sustain NADH-induced proton fluxes as measured by the quenching of 9-amino-6-chloro-2-methoxyacridine fluorescence. The depleted particles themselves showed negligible quenching, but the quenching increased markedly after treating the particles with oligomycin. The data show for the first time that coupling factor 6-depleted complexes have an active proton channel that can be blocked by oligomycin. Therefore, coupling factor 6 is not essential for inhibitor-sensitive proton conductance through mitochondrial F0.

Fluorescent analogues of N,N'-dicyclohexylcarbodiimide as structural probes of the bovine mitochondrial proton channel.

N-Cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]carbodiimide (NCD-4) and N-cyclohexyl-N'-(1-pyrenyl)carbodiimide (NCP) are two novel fluorescent analogues of the mitochondrial inhibitor dicyclohexylcarbodiimide (DCCD). Although nonfluorescent in aqueous media, both compounds form fluorescent conjugates with mitochondrial electron transport particles (ETPH) or purified H+-ATPase (F1-F0) vesicles. DCCD prevents the reaction of ETPH with both NCD-4 and NCP. The fluorescent probes are effective inhibitors of ATPase activity and ATP-driven membrane potential, although their reaction rates are considerably slower than that of DCCD. The fluorescence of NCD-4- or NCP-treated H+-ATPase is quenched by hydrophobic spin-label nitroxide derivatives of stearic acid (chi-NS) in the order 16-NS greater than 12-NS greater than 7-NS approximately equal to 5-NS, whereas membrane-impermeant iodide ions have negligible effect. The quenching behavior of 16-NS (the most effective quencher) suggests that a small fraction of labels remain inaccessible to the quencher. It is concluded that the DCCD-binding sites are oriented toward the membrane lipids and are located in the lipid bilayer ca. 18 A from the membrane surface.

Effects of Cd2+ on ATP-driven membrane potential in beef heart mitochondrial H+-ATPase: a study using the voltage-sensitive probe oxonol VI.

Beef heart mitochondrial H+-ATPase (F1-F0) vesicles were prepared by lysolecithin extraction of ETPH. ATP-driven membrane potential was monitored indirectly by following absorbance changes of the potential-sensitive dye oxonol VI. The steady-state potential was discharged by oligomycin and/or Cd2+ (a dithiol reagent). At 13 degrees C, the agents appeared to act synergistically; at 24 degrees C the data were equivocal. When Cd2+ was added before energization, the membrane potential was markedly attenuated. Both effects of Cd2+ were inhibited by dithiothreitol. The activation energy for oligomycin-sensitive ATPase exhibited a discontinuity at 16 degrees C. However, the temperature dependence of the rate of potential discharge by oligomycin showed no such discontinuity. The results are discussed in terms of the involvement of thiol groups in proton translocation and the thermotropic behavior of the membrane vesicles.

Effects of delipidation on proton translocation and ATPase activity in beef heart electron transport particles.

Delipidation of beef heart electron transport particles with phospholipase A2 has been examined. When the particles were treated with the lipase and subjected to a low bovine serum albumin wash, ATPase activity was unaffected as was the lipid/protein ratio of the particles. However, energisation by ATP/Mg2+ was abolished. Furthermore, unsaturated but not saturated fatty acids discharged the steady-state ATP-driven membrane potential of control samples. When the phospholipase A2 hydrolysis products were removed, inhibition of energy-linked reactions in the lipid-depleted particles was still observed and was interpreted in terms of non-specific leaks in the vesicle membranes, and 'specific' leaks through impaired H+-ATPase complexes. ATPase activity was less susceptible to delipidation than energisation but was, nevertheless, strongly inhibited at 50 percent lipid depletion. Spin label studies indicated a decrease in the fluidity of particle membranes accompanying delipidation. Moreover, the discontinuity seen in Arrhenius plots of ATPase activity was shifted from 17 degrees C (control) to 22 degrees C at 50 percent phospholipid depletion. The data are consistent with a release of unsaturated fatty acids by phospholipase A2 rendering the transport particles both leakier and the membranes less fluid than controls.

Lipid-protein interactions in sarcoplasmic reticulum are not perturbed by ionophore A23187. An EPR and fluorescence study.

The divalent-cation ionophore A23187 at micromolar concentrations prevents the ATP-dependent accumulation of calcium into sarcoplasmic reticulum vesicles. Under the same conditions and throughout the temperature range of 4 degrees-37 degrees C, A23187 has no effect on either the rotational motion of the Ca2+ -ATPase in the membrane, or on the mobility of the lipid acyl chains. The steady-state fluorescence polarization of a polyene fluorescent probe incorporated into the membrane lipids was similarly unaffected by the ionophore. These results show conclusively that the mechanism of action of the ionophore does not involve significant of lipid-protein interactions.

ATP synthase complex from bovine heart mitochondria. Passive H+ conduction through F0 does not require oligomycin sensitivity-conferring protein.

Oligomycin sensitivity-conferring protein (OSCP) is a water-soluble subunit of bovine heart mitochondrial H(+)-ATPase (F1-F0). In order to investigate the requirement of OSCP for passive proton conductance through mitochondrial F0, OSCP-depleted membrane preparations were obtained by extracting purified F1-F0 complexes with 4.0 M urea. The residual complexes, referred to as UF0, were found to be deficient with respect to OSCP, as well as alpha, beta, and gamma subunits of F1-ATPase, but had a full complement of coupling factor 6 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. These UF0 complexes had no intrinsic ATPase activity and were able to bind nearly the same amount of F1-ATPase in the presence of either OSCP or NH4+ ions alone, or a combination of the two. However, the preparations exhibited an absolute dependency on OSCP for conferral of oligomycin sensitivity to membrane-bound ATPase. The passive proton conductance in UF0 proteoliposomes was measured by time-resolved quenching of 9-amino-6-chloro-2-methoxyacridine or 9-aminoacridine fluorescence following a valinomycin-induced K(+)-diffusion potential. The data clearly establish that OSCP is not a necessary component of the F0 proton channel nor is its presence required for conductance blockage by the inhibitors oligomycin or dicyclohexylcarbodiimide. Furthermore, OSCP does not prevent or block passive H+ leakage. Comparisons of OSCP with the F1-F0 subunits from Escherichia coli and chloroplast lead us to suggest that mitochondrial OSCP is, both structurally and functionally, a hybrid between the beta and delta subunits of the prokaryotic systems.

Topology and function of "stalk" proteins in the bovine mitochondrial H+-ATPase.

Proton translocating ATPases comprise a hydrophilic sector F1, a membrane sector F0, and, in the case of bovine mitochondria, a connecting "stalk" which is believed to contain the oligomycin sensitivity-conferring protein (OSCP) and coupling factor 6 (F6). The present study was undertaken to verify the accessibility of F6 and OSCP to trypsin and to examine the functional consequences of such treatment. Our data show that F1 binds equally to trypsin-treated F0 and untreated F0, but the former complexes exhibit cold lability and only partial sensitivity to oligomycin. Furthermore, these complexes fail to exhibit ATP-driven proton translocation or ATP-32Pi exchange activity. Trypsinization of F0 does not, however, inhibit passive proton conductance through the membrane sector but actually enhances it. Immunological data indicate extensive degradation of OSCP under conditions where F6 proteolysis is insignificant. Intact H+-ATPase complexes are relatively resistant to both the structural and functional effects of trypsin. We conclude that OSCP is predominantly an extrinsic protein which is shielded by F1 in the native membrane. F6 may also be an extrinsic protein but is shielded from trypsinization by OSCP and/or other F0 polypeptides. The exposed, trypsin-sensitive segments of OSCP are not required for passive proton conductance through F0 but may be required for ATP-driven reactions. We propose that bovine mitochondrial OSCP is a functional analogue of subunit b in the Escherichia coli H+-ATPase.

On the functional role of coupling factor B in the mitochondrial H+ -ATPase.

Evidence for the presence of a functionally important vicinal dithiol in mitochondrial coupling factor B (FB) has been presented earlier (Sanadi, D. R. (1982) Biochim. Biophys. Acta 683, 39-56). FB was completely inactivated by 38 micron of copper o-phenanthroline or 0.63 mM iodosobenzoate, and the kinetics were consistent with intramolecular disulfide formation as were polyacrylamide gel patterns which showed that FB which had been treated with copper o-phenanthroline had a different mobility from that of untreated FB. ATP-Pi exchange activity and ATP-induced binding of bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxonol (oxonol VI) to H+ -ATPase were also inhibited by the thiol oxidizing reagents, although oligomycin-sensitive ATPase activity was unaffected. F0 isolated from H+ -ATPase rebinds purified F1 with the restoration of ATP-induced oxonol-binding activity. Prior treatment of F0 (but not of F1) with copper o-phenanthroline abolished the oxonol-binding activity of reconstituted F0-F1. 115Cd binds tightly to H+ -ATPase and the bound protein can be recovered by gel electrophoresis in phosphate buffer in the presence of sodium dodecyl sulfate at a position corresponding to FB. Prior treatment of the H+ -ATPase with copper o-phenanthroline abolished 115Cd binding. The results indicate that the major effect of these inhibitors is on FB dithiol and leave little doubt that Cd2+ is indeed bound to a vicinal dithiol group.

Environment of the sulfhydryl groups in bovine heart mitochondrial H+-ATPase.

Electron transport particles and purified H+-ATPase (F1-F0) vesicles from beef heart mitochondria have been treated with two classes of thiol reagent, viz. membrane-impermeable organomercurials and a homologous series of N-polymethylene carboxymaleimides (Mal-(CH2)x-COOH or AMx). The effect of such treatment on ATP-driven reactions (ATP-Pi exchange and proton translocation) has been examined and compared to the effects on rates of ATP hydrolysis. The organomercurials inhibited ATP-Pi exchange and one of them (p-chloromercuribenzoate) inhibited ATPase activity. Of the maleimide series (AMx), AM10 and AM11 inhibited both ATP-Pi exchange and ATP-driven membrane potential, but not ATPase activity. The other members of the series were essentially inactive. N-Ethylmaleimide was intermediate in its efficacy. Passive H+ conductance through the membrane sector F0 was 50% blocked by AM10, slightly blocked by AM2 and N-ethylmaleimide, and unaffected by the other members of the AMx series. The data imply that one -SH near the membrane surface and one -SH about 12 A from the surface are functional in proton translocation through the H+-ATPase.