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J J Diwan

Publications and source records attributed to J J Diwan.

At least 19 recordsLinked to original sources
PubMed1998

Two high conductance channels of the mitochondrial inner membrane are independent of the human mitochondrial genome.

Patch-clamp techniques were used to characterize the channel activity of mitochondrial inner membranes of two human osteosarcoma cell lines: a mitochondrial genome-deficient (rho0) line and its corresponding parental (rho+) line. Previously, two high conductance channels, mitochondrial Centum picoSiemen (mCS) and multiple conductance channels (MCC), were detected in murine mitochondria. While MCC was assigned to the protein import in yeast mitochondria, the role of mCS is unknown. This study demonstrates that mCs and MCC activities from mouse mitochondria are indistinguishable from those of human mitochondria. The channel activities and their functional expression levels are not altered in cells lacking mtDNA. Hence, rho0 cells may provide a model system for elucidating the role of mitochondrial channels in disease processes and apoptosis.

Animals↗
PubMed1992

Purification and patch clamp analysis of a 40-pS channel from rat liver mitochondria.

Patch clamp analysis of membranes reconstituted with a fraction isolated from detergent-solubilized mitochondrial membranes by affinity chromatography on immobilized quinine earlier indicated the presence of two classes of ion channels, of about 40- and 140-pS conductance in medium including 150 mM KCl. Now a 57-kDa constituent of the quinine-affinity column eluate has been identified as the 40-pS channel. Protein fractions derived from the quinine-affinity column eluate by preparative isoelectric focusing with a Rotofor cell have been reconstituted into phospholipid vesicle membranes by detergent dialysis, and vesicles have been enlarged for patch clamping by dehydration and rehydration. Voltage clamp analysis has been carried out on excised patches bathed symmetrically in buffered medium containing 150 mM KCl and 100 microM CaCl2. Patches of membrane incorporating the 57-kDa protein exhibit 40-pS conductance transitions. The magnitude of conductance transitions is similar when Na+ replaces K+ in the bathing medium, indicating little selectivity of the 40-pS channel for K+ relative to Na+. Another fraction derived from the quinine-affinity column eluate is found to contain the larger channel, now estimated to have an average conductance of about 130 pS. Patches of control membrane prepared in the same way but without protein exhibit no channel activity.

Animals↗
PubMed1991

Patch clamp analysis of a partially purified ion channel from rat liver mitochondria.

A protein fraction isolated from detergent-solubilized mitochondrial membranes by affinity chromatography on immobilized quinine was reconstituted into phospholipid vesicles by detergent dialysis. Vesicles were fused to a diameter of 10 microns or larger by dehydration and rehydration. Patch clamp recordings carried out in detached mode with a symmetrical solution of 150 mM KCl, 5 mM HEPES, and 0.1 mM CaCl2 revealed conductance increments of 140 pS. Transitions of 40 pS were less frequently observed. Control vesicles which lacked protein showed no channel activity. The probability for the 140 pS channel to be open increased with increasing voltage in the range from 20 to 80 mV (positive potentials relative to what was the vesicle interior prior to excision), while the single channel conductance remained essentially constant. The 140 pS channel did not open at negative voltages. The voltage dependence suggests asymmetric incorporation of the 140 pS channel into vesicle membranes during reconstitution.

Animals↗
PubMed1990

A mitochondrial protein fraction catalyzing transport of the K+ analog T1+.

A protein fraction has been obtained from detergent-solubilized mitochondrial membranes by its affinity for quinine, an inhibitor of K+ transport. A peptide derived from the predominant 53 kDa protein in this fraction is found to be identical in sequence to a portion of aldehyde dehydrogenase. Antigenically unrelated bands at 97, 77, 57, and 31 kDa are also seen on polyacrylamide gels. Observations utilizing a fluorescent probe entrapped in the lumen of membrane vesicles indicate that the reconstituted protein fraction imparts permeability to the K+ analog Tl+. These and other findings suggest that the affinity purified fraction includes a cation transport catalyst.

Aldehyde Dehydrogenase↗
PubMed1990

Effects of some protein-reactive compounds on K+ flux into mitochondria.

The pathway of unidirectional K+ flux into respiring mitochondria is sensitive to the protein reactive compounds mersalyl and dicyclohexylcarbodiimide (DCCD). When treated with either of these reagents, mitochondria retain sensitivity to other reagents which affect K+ flux into untreated mitochondria. The present studies show that the K+ influx mechanism modified by pretreatment with DCCD remains sensitive to inhibition by quinine. K+ influx stimulated by mersalyl, in the absence of exogenous Ca++, retains sensitivity to inhibition by quinine and to some extent by Mg++. The results support the conclusion that K+ uptake by mitochondria modified by mersalyl or DCCD occurs via the same proteinaceous pathway as that which mediates K+ uptake by untreated mitochondria.

Animals↗
PubMed1988

Reconstitution of transmembrane K+ transport with a 53 kilodalton mitochondrial protein.

A 53 kDa protein has been purified from a Triton X-100 extract of liver mitochondrial membranes, by affinity chromatography on immobilized quinine, a K+ transport inhibitor. KCl-containing lipid vesicles reconstituted with this protein lose K+ to a medium low in K+ faster than vesicles lacking protein. With bacteriorhodopsin reconstituted in vesicles containing K+, light induces faster development of a pH gradient if the 53 kDa protein is included during vesicle preparation. This effect is like that of valinomycin, which catalyzes K+ efflux, dissipating the membrane potential arising from H+ entry. Evidence that vesicles containing the 53 kDa protein are permeable to K+, but exhibit low permeability to H+, indicates that this protein acts as a K+ uniporter.

Animals↗
PubMed1988

Enhanced uptake of spermidine and methylglyoxal-bis(guanylhydrazone) by rat liver mitochondria following outer membrane lysis.

Isolated rat liver mitochondria rapidly bound the 14C-labeled organic cations spermidine, a physiologically important polyamine, and methylglyoxal-bis(guanylhydrazone) (MGBG), an anticancer drug. This rapid, Mg2+-sensitive, respiration-independent binding is assumed to involve adsorption to anionic surface groups. A slower progressive uptake of the organic cations exhibited respiration dependence, indicating that it involves transport across the inner mitochondrial membrane into the matrix compartment. Addition of digitonin, to lyse the outer mitochondrial membrane, caused an increase in the mitochondrial content of the organic cations and enhanced the rate of progressive, respiration-dependent cation uptake. The data are consistent with the interpretation that the outer mitochondrial membrane limits access of the organic cations, spermidine and MGBG, to the inner mitochondrial membrane. This conclusion is supported also by published data indicating that outer membrane lysis enhances inhibitory effects of the organic cations on mitochondrial respiration. The uptake of spermidine by mitochondria was inhibited by MGBG.

Animals↗
PubMed1988

Sensitivity of mitochondrial Mg++ flux to reagents which affect K+ flux.

Effects on Mg++ transport in rat liver mitochondria of three reagents earlier shown to affect mitochondrial K+ transport have been examined. The sulfhydryl reactive reagent phenylarsine oxide, which activates K+ flux into respiring mitochondria, also stimulates Mg++ influx. The K+ analog Ba++, when taken up into the mitochondrial matrix, inhibits influx of both K+ and Mg++. The effect on Mg++ influx is seen only if Mg++, which blocks Ba++ accumulation, is added after a preincubation with Ba++. Thus the inhibition of Mg++ influx appears to require interaction of Ba++ at the matrix side of the inner mitochondrial membrane. Added Ba++ also diminishes observed rates of Mg++ efflux but not K+ efflux. This difference may relate to a higher concentration of Ba++ remaining in the medium in the presence of Mg++ under the conditions of our experiments. Pretreatment of mitochondria with dicyclohexyl-carbodiimide (DCCD), under conditions which result in an increase in the apparent Km for K+ of the K+ influx mechanism, results in inhibition of Mg++ influx from media containing approximately 0.2 mM Mg++. The inhibitory effect of DCCD on Mg++ influx is not seen at higher external Mg++ (0.8 mM). This dependence on cation concentration is similar to the dependence on K+ concentration of the inhibitory effect of DCCD on K+ influx. Although mitochondrial Mg++ and K+ transport mechanisms exhibit similar reagent sensitivities, whether Mg++ and K+ share common transport catalysis remains to be established.

Animals↗
PubMed1986

Effect of quinine on mitochondrial K+ and Mg++ flux.

Quinine decreases rates of unidirectional K+ flux into and out of respiring rat liver mitochondria. K+ efflux is more sensitive to quinine than K+ influx. The data are consistent with the proposal that two separate mechanisms may mediate K+ influx, only one of which is sensitive to quinine. Effects on K+ flux of the stereoisomer quinidine are similar to effects of quinine. The smaller quinuclidine causes at most a slight inhibition of K+ efflux under the same conditions. Mg++ flux exhibits a pattern of inhibition by quinine similar to that of K+ flux. Mg++ efflux is more sensitive to quinine than is Mg++ influx. These and earlier findings indicate marked similarities between liver mitochondrial transport mechanisms for K+ and Mg++.

Animals↗
PubMed1986

Stimulation of K+ flux into mitochondria by phenylarsine oxide.

The dithiol-reactive reagent phenylarsine oxide causes a pH-dependent stimulation of unidirectional K+ flux into respiring rat liver mitochondria. This stimulation is diminished by subsequent addition of either the dithiol 2,3-dimercaptopropanol or the monothiol 2-mercaptoethanol. In contrast, uncoupling by phenylarsine oxide is reversed by 2,3-dimercaptopropanol but not by 2-mercaptoethanol. The data suggest separate sites of interaction of phenylarsine oxide with mechanisms of K+ entry and ATP synthesis. Stimulatory effects of mersalyl and phenylarsine oxide on K+ influx are not additive. Thus PheASO and mersalyl may affect K+ influx at a common site. Pretreatment of the mitochondria with DCCD, which inhibits K+ influx, fails to alter sensitivity to PheAsO or mersalyl. Thus the DCCD binding site associated with the K+ influx mechanism appears to be separate from and independent of the sulfhydryl group(s) which mediate stimulation of K+ influx by PheAsO and mersalyl. PheAsO, like mersalyl, also increases the rate of unidirectional K+ efflux from respiring mitochondria. The combined presence of PheAsO plus mersalyl causes a greater stimulation of K+ efflux than is observed with either reagent alone.

Animals↗
PubMed1985

Ba2+ uptake and the inhibition by Ba2+ of K+ flux into rat liver mitochondria.

Rapid uptake of Ba2+ by respiring rat liver mitochondria is accompanied by a transient stimulation of respiration. Following accumulation of Ba2+, e.g. at a concentration of 120 nmol per mg protein, the mitochondria exhibit reduced rates of state 3 and uncoupler-stimulated respiration. ADP-stimulated respiration is inhibited at a lower concentration of Ba2+ than is required to affect uncoupler-stimulated respiration, suggesting a distinct effect of Ba2+ on mechanisms involved in synthesis of ATP. Ba2+, which has an ionic radius similar to that of K+, inhibits unidirectional K+ flux into respiring rat liver mitochondria. This effect on K+ influx is observable at concentrations of Ba2+, e.g. 23 to 37 nmol per mg protein, which cause no significant change in state 4 or uncoupler-stimulated respiration. The accumulated Ba2+ decreases the measured Vmax of K+ influx, while having little effect on the apparent Km for K+. The inhibition of K+ influx by Ba2+ is seen in the presence and absence of mersalyl, an activator of K+ influx. In contrast, under the conditions studied, Ba2+ has no apparent effect on the rate of unidirectional K+ efflux. These data are consistent with the idea that K+ may enter and leave mitochondria via separate mechanisms.

Animals↗
PubMed1984

Activation of potassium ion transport in mitochondria by cadmium ion.

Low levels of Cd2+ (1-5 microM) produce rapid swelling of mitochondria, which is respiration-dependent and uncoupler-sensitive. No cation requirement is apparent, since the swelling occurs in a medium containing only sucrose and the respiratory substrate. The swelling is inhibited by ruthenium red, suggesting that this effect of Cd2+ requires its entry into mitochondria. In medium containing 9 mM K+, addition of Cd2+ along with ruthenium red increases the rate of K+ influx threefold. In the presence of K+, Rb+ or Li+, but not of Na+, addition of Cd2+ produces first efflux of H+ into the medium followed by discharge of the pH gradient or uncoupling. Only the latter effect is inhibited by ruthenium red, showing that the efflux and influx of H+ are independent reactions. The H+ efflux appears to be an antiport response to the induced K+ entry. Its activation by Cd2+ is similar to the known effect of p-chloromercuriphenyl sulfonate. The H+ influx or uncoupling appears to result from binding of Cd2+ to some matrix-facing membrane site, perhaps the dithiol group on coupling factor B, and may relate to apparent permeability changes associated Cd2+-induced swelling.

Animals↗
PubMed1982

K+ transport in mitoplasts.

K+ transport into mitoplasts, prepared by digitonin disruption and removal of the outer membranes from rat liver mitochondria, has been studied. Unidirectional K+ influx has been measured by means of 42K, in the presence of the respiratory substrate succinate. K+ influx is inhibited by CN-, antimycin A and dicyclohexylcarbodiimide, but is insensitive to oligomycin. A linear dependence of the reciprocal of the K+ -influx rate on the reciprocal of the external K+ concentration is observed. Under the conditions studied, the apparent Km for K+ of the transport mechanism is approx. 6 mM, while the Vmax of K+ influx is approx. 5 mu mol K+/g protein per min. The rate of K+ influx increases with increasing external pH over the range from 6.8 to 8.0. The observed kinetics, pH dependence and inhibitor sensitivity are essentially similar to previously reported characteristics of K+ transport into intact rat liver mitochondria. It is concluded that the outer mitochondrial membrane does not not have a role in controlling K+ flux into rat liver mitochondria.

Adenosine Diphosphate↗
PubMed1982

Some effects of dibutylchloromethyltin chloride and other reagents on mitochondrial K+ flux.

Respiration-dependent K+ fluxes across the limiting membranes of isolated rat liver mitochondria, measured by means of 42K, are stimulated by the oxidative phosphorylation inhibitor dibutylchloromethyltin chloride (DBCT). A lack of effect of Cl- concentration indicates that the stimulation of K+ flux by DBCT is not attributable to Cl-/OH- exchange activity. The mercurial mersalyl was previously shown to stimulate respiration-dependent K+ influx. The combined presence of mersalyl plus DBCT results in a greater stimulation of K4 influx than is caused by either DBCT or mersalyl alone. The oxidative phosphorylation inhibitor oligomycin, which alone has no effect on respiration-dependent K+ influx, enhances the stimulatory effect of mersalyl on K+ influx. The data are consistent with, although not proof of, a direct interaction of the K+ transport mechanism with the mitochondrial energy transduction apparatus.

Animals↗
PubMed1981

Uptake of ornithine by rat liver mitochondria.

Uptake of [14C]-L-ornithine by rat liver mitochondria has been measured by using the silicone sampling technique. The uptake of ornithine measured after 20-45 s of incubation exhibits stereospecificity, pH dependence, and a lack of dependence on respiratory energy. A slower subsequent increase in [14C]-L-ornithine counts associated with the mitochondria, which is blocked by the transaminase inhibitor aminooxyacetate, is attributed to metabolism of the labeled ornithine. Each of the reagents N-ethylmaleimide, Tris (HCl) buffer, Tl2+SO42(-), Mg2+SO42(-), and choline chloride inhibits ornithine accumulation. A lack of inhibition by mersalyl is interpreted as indicating that ornithine uptake does not require transmembrane Pi flux. Uptake of ornithine to levels in excess of the concentration in the medium can largely be accounted for by an osmotically insensitive fraction of the ornithine taken up, which is assumed to be adsorbed to solid structures of the mitochondria.

Aminooxyacetic Acid↗
PubMed1980

Effect of mersalyl on mitochondrial Mg++ flux.

The mercurial mersalyl has little effect either on rapid Mg++ binding by isolated rat liver mitochondria or on the total Mg++ content of these organelles measured after 0.75 min of incubation at 20 degrees C. The data do not support the previous suggestion that the increased permeability to K+ of mitochondria treated with mersalyl results from release of endogenous Mg++. An increased pH-dependence of unidirectional Mg++ flux into respiring rat liver mitochondria is suggested to arise indirectly from inhibition by mersalyl of pH shifts associated with exchanges of endogenous phosphate. In addition, mersalyl appears to have a stimulatory effect on Mg++ influx. Mersalyl also increases the average rate of unidirectional efflux of endogenous Mg++. The stimulatory effects of mersalyl on Mg++ flux are similar to, although quantitatively less than, the previously reported effects of mersalyl on mitochondrial K+ flux.

Animals↗