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K K Gunter

Publications and source records attributed to K K Gunter.

13 recordsLinked to original sources
PubMed1992

Mn2+ sequestration by mitochondria and inhibition of oxidative phosphorylation.

Manganese is known to accumulate in mitochondria and in mitochondria-rich tissues in vivo. Although Ca2+ enhances mitochondrial Mn2+ uptake, ATP-bound Mn2+ is not sequestered by suspended rat brain mitochondria, and ATP binds Mn2+ even more tightly than it binds Mg2+. Physiological levels of the polyamine spermine enhanced 54 Mn2+ uptake at the low [Ca2+]s characteristic of unstimulated cells (approximately 100 nM). With succinate as substrate, Mn2+ inhibited oxygen consumption by suspensions of rat liver mitochondria after the addition of ADP but not after the addition of uncoupler. With glutamate/malate as substrate, Mn2+ inhibited ADP-stimulated respiration and also slightly inhibited uncoupler-stimulated respiration. State 4 (resting) respiration was unchanged in all cases, indicating that the inner membrane retained its impermeability to protons. These results suggest that Mn2+ was not oxidized and that it can interfere directly with oxidative phosphorylation, most likely by binding to the F1 ATPase. Mn2+ may also bind to the NADH dehydrogenase complex, but not strongly enough to affect electron transport in vivo. It is suggested that accumulation of manganese within the mitochondria of globus pallidus may help explain the distinctive pathology of manganism.

Adenosine Triphosphate↗
PubMed1991

The Na(+)-independent Ca2+ efflux mechanism of liver mitochondria is not a passive Ca2+/2H+ exchanger.

Whether the Na(2+)-independent Ca2+ efflux mechanism of liver mitochondria is a Ca2+/2H+ exchanger and whether this exchanger is a passive mechanism have been controversial since shortly after the discovery of this mechanism. Here, a new approach to determining if the mechanism is passive is developed based on the energy available to a passive Ca2+/2H+ exchanger. Conditions are identified in which the Na(+)-independent Ca2+ efflux mechanism transports Ca2+ out of mitochondria against a Ca2+ gradient many times greater than that possible for a passive Ca2+/2H+ exchanger, thus ruling this out as a possible mechanism.

Animals↗
PubMed1991

Mn2+ transport across biological membranes may be monitored spectroscopically using the Ca2+ indicator dye antipyrylazo III.

The metallochromic indicator antipyrylazo III can be used for the rapid and convenient monitoring of Mn2+ transport in biological systems. The apparent KD of the Mn-antipyrylazo III complex in buffered 150 mM KCl (pH 7.2 at 20 degrees C) is approximately 2.5 x 10(-5) M. The sensitivity of antipyrylazo III to Mn2+ is comparable to that of arsenazo III to Ca2+. Mn2+ can be measured without interference from Ca2+, by using dual-wavelength spectrophotometry at the wavelength pair 510-590 nm, or 530-565 nm in cell or mitochondrial suspensions. Ca2+ can be monitored at the wavelength pair 720-790 nm without interference from Mn2+. This paper represents the first application of this technique, here used to characterize mitochondrial efflux kinetics of Mn2+. We report that Mn2+ is transported out of liver mitochondria with a Vmax of 1-2 nmol/(mg.min) and a Km of about 12 nmol/mg. These results are in close agreement with results of measurements using 54Mn.

Animals↗
PubMed1990

Manganese and calcium efflux kinetics in brain mitochondria. Relevance to manganese toxicity.

Manganese shares the uniport mechanism of mitochondrial calcium influx, accumulates in mitochondria and is cleared only very slowly from brain. Using dual-label isotope techniques, we have investigated both Mn2+ and Ca2+ mitochondrial efflux kinetics. We report that (1) there is no significant Na(+)-dependent Mn2+ efflux from brain mitochondria; (2) Mn2+ inhibits both Na(+)-dependent and Na(+)-independent Ca2+ efflux in brain, in a mode that appears to be primarily competitive and with apparent Ki values of 5.1 and 7.9 nmol/mg respectively; and (3) Ca2+ does not appear to inhibit Mn2+ efflux from brain mitochondria. Findings (1) and (2) suggest the possibility of mitochondrial accumulation of both Mn2+ and Ca2+ in Mn2(+)-intoxicated brain.

Animals↗
PubMed1990

Cytosolic free calcium concentrations in avian growth plate chondrocytes.

Isolated avian growth plate chondrocytes convert the acetoxymethyl ester (AM) form of Fura-2 quickly and efficiently to the Ca2(+)-sensitive pentacarboxylic acid (FA) form. Control experiments indicate that the Kd for intracellular Fura-2/FA is very close to that of extracellular Fura-2/FA at the same ionic strength and pH and that the Fura-2/FA fluorescence from indicator converted by intracellular organelles is quite small. Correcting for the effects of extracellular Fura-2/FA and partial hydrolysis products has improved the accuracy of determination of intracellular [Ca2+] over earlier measurements in chondrocytes. Cytosolic [Ca2+] in isolated growth plate chondrocytes (containing cells from each maturational stage) is found to require approximately 9 hours to recover from the isolation process. After this recovery period, cytosolic [Ca2+] in these cells converges to approximately 70 nM regardless of the [Ca2+] of the recovery medium, suggesting regulation of cytosolic [Ca2+] to a set point. Chondrocytes that are separated into maturationally distinct fractions using countercurrent centrifugal elutriation show an increase in cytosolic [Ca2+] with cellular maturation. The least mature resting cells have a [Ca2+] near 57 nM, while the most mature hypertrophic cells are around 95 nM.

Animals↗
PubMed1988

Mechanisms of mitochondrial calcium transport.

Mitochondria are known to possess a rapid calcium uptake mechanism or uniport and both sodium-dependent and sodium-independent efflux mechanisms. Whether sodium-independent calcium efflux is mediated and whether sodium-dependent calcium efflux can be found in liver mitochondria have been questioned. Kinetics results relevant to the answers of these questions are discussed below. A slow, mediated, sodium-independent calcium efflux mechanism is identified which shows second order kinetics. This mechanism, which shows "nonessential activation" kinetics, has a Vmax around 1.2 nmol calcium per mg protein per min and a half maximal velocity around 8.4 nmol calcium per mg protein. A slow, sodium-dependent calcium efflux mechanism is identified, which is first order in calcium and second order in sodium. This mechanism has a Vmax around 2.6 nmol of calcium per mg protein per min. The sodium dependence is half saturated at an external sodium concentration of 9.4 mM, and the calcium dependence is half saturated at an internal calcium concentration of 8.1 nmol calcium per mg protein. The cooperativity of the sodium dependence effectively permits a terreactant system to be fit by a bireactant model in which [Na] only appears as the square of [Na]. This liver system shows simultaneous, as opposed to ping-pong, kinetics. It is also found to be sensitive to inhibition by tetraphenyl phosphonium, magnesium, and ruthenium red. A model is proposed in which mitochondrial calcium transport could function to "shape the pulses" of cytosolic calcium. Simultaneously, mitochondria may mediate a "calcium memory" coupled perhaps to activation of cytosolic events through calmodulin or perhaps to activation of electron transport through the activation of specific dehydrogenases by intramitochondrial calcium.

Animals↗
PubMed1988

Conversion of esterified fura-2 and indo-1 to Ca2+-sensitive forms by mitochondria.

Rat liver mitochondria are shown to convert the acetoxymethyl ester forms of fura-2 and indo-1 into Ca2+-dependent forms of these indicators. The excitation spectrum of the Ca2+-dependent conversion product of fura-2 acetoxymethyl ester is shown to be similar to that of the pentacarboxylic acid form of fura-2. A systematic investigation of the ionic strength and pH dependences of the fluorescence of the pentacarboxylic acid forms of these indicators shows small changes within the ranges thought to obtain within the mitochondrial matrix after Ca2+ uptake. Intramitochondrial free Ca2+ levels are studied both before and after Ca2+ sequestration by mitochondria, and a rough estimate is made of the mitochondrial contribution to the Ca2+-dependent fura-2 fluorescence of a hepatocyte suspension.

Animals↗
PubMed1986

The efficiencies of the component steps of oxidative phosphorylation. II. Experimental determination of the efficiencies in mitochondria and examination of the equivalence of membrane potential and pH gradient in phosphorylation.

In the accompanying article (T.E. Gunter and B.D. Jensen, 1986 Arch. Biochem. Biophys. 248, 289-304), a method is described for measuring the efficiencies of individual steps of the process of oxidative phosphorylation. The results of applying this method to the case of state 3 phosphorylation in rat liver mitochondria are reported here. The rate of energy use (or power use) at the gradient generation, leakage, and phosphorylation steps are reported as efficiencies and energy use factors in tabular form. The limits of the degrees of coupling of the gradient generation and phosphorylation steps are also determined and under the current conditions of measurement these degrees of coupling are found to be quite close to unity. The data can be used to show that the only sets of the stoichiometric parameters noH (the charge/2e- ratio in this case from succinate to oxygen), nPH (the H+/ATP ratio), and nTH (number of protons translocated during substrate-product transport) which are simultaneously consistent with both the laws of thermodynamics and with the current data are 8, 3, 1, and 6, 3, 0. The The efficiency of the phosphorylation step which is independent of noH and nTH averages 80% for the control data analyzed. If noH is 8 (succinate to oxygen), the average value of the efficiency of generation of the electrochemical proton gradient is approximately 91 percent. Since very little power (energy) would then be left over to be coupled in parallel to phosphorylation through some other means of coupling, this would place the electrochemical proton gradient in the direct path of power flow and identify it as "an" intermediate in the process. This would suggest that any other intermediate should be considered as being "in series" with the electrochemical proton gradient. The agents butyrate and propionate have been employed to permit investigation over a range of pH gradient and membrane potential. Both butyrate and propionate decrease the efficiency of generation of the electrochemical proton gradient and increase proton leakage. In addition, butyrate activates electron transport whereas propionate inhibits it. By using butyrate to modify the values of pH gradient and membrane potential, it can be shown that the ratio of the efficiency with which the pH gradient is used in phosphorylation to that with which the membrane potential is used is 1.08 +/- 0.38.

Adenosine Diphosphate↗
PubMed1976

Evidence for more than one Ca2+ transport mechanism in mitochondria.

The active transport and internal binding of the Ca2+ analogue Mn2+ by rat liver mitochondria were monitored with electron paramagnetic resonance. The binding of transported Mn2+ depended strongly on internal pH over the range 7.7-8.9. Gradients of free Mn2+ were compared with K+ gradients measured on valinomycin-treated samples. In the steady state, the electrochemical Mn2+ activity was larger outside than inside the mitochondria. The observed gradients of free Mn2+ and of H+ could not be explained by a single "passive" uniport or antiport mechanism of divalent cation transport. This conclusion was further substantiated by observed changes in steady-state Ca2+ and Mn2+ distributions induced by La3+ and ruthenium red. Ruthenium red reduced total Ca2+ or Mn2+ uptake, and both inhibitors caused release of divalent cation from preloaded mitochondria. A model is proposed in which divalent cations are transported by at least two mechanisms: (1) a passive uniport and (2) and active pump, cation antiport or anion symport. The former is more sensitive to La3+ and ruthenium red. Under energized steady-state conditions, the net flux of Ca2+ or Mn2+ is inward over (1) and outward over (2). The need for more than one transport system inregulating cytoplasmic Ca2+ is discussed.

Acetates↗