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J J Lemasters

Publications and source records attributed to J J Lemasters.

At least 217 records · Page 12Linked to original sources
PubMed1980

Relationships between bilayer lipid, motional freedom of oxidoreductase components, and electron transfer in the mitochondrial inner membrane.

The relationships between bilayer lipid, diffusional and conformational activities of oxidoreduction components, and electron transfer activity in the mitochondrial inner membrane are considered. Using a new, low pH method to fuse liposome phospholipid (asolectin) with the isolated mitochondrial inner membrane, the membrane bilayer is enriched up to 700% with exogenous phospholipid. During such enrichment, ultrastructural analysis reveals that integral proteins diffuse freely and randomly into the expanding bilayer. Kinetic analysis reveals that a diffusion limited step occurs between succinate- and NADH dehydrogenase and cytochromes bc1, and that the dehydrogenases, ubiquinone, and cytochromes bc1 are free to diffuse independently of one another in the membrane plane. Whether cytochromes bc1 and cytochrome c oxidase codiffuse in the membrane plane, or diffuse independently of one another remains unclear. The specific activities of succinate- and NADH-dehydrogenase as well as cytochrome c oxidase are affected by bilayer enrichment. This most likely occurs through the direct modulation by the newly incorporated phospholipid on conformational activity required in the oxidoreductases for electron transfer.

Animals↗
PubMed1980

Ethanol-induced changes in the intralobular oxygen gradient of perfused rat liver.

A new tissue fluorometric method was developed to estimate the intralobular oxygen gradient in hemoglobin-free perfused rat liver. The method employs a two-branch micro-light guide with a tip diameter of 170 mu. With this light guide, it was possible to measure pyridine nucleotide fluorescence (366 nm leads to 450 nm) from periportal and pericentral regions of the liver lobule. By measuring inflow PO2 values at which pyridine nucleotide fluorescence increased in the pericentral regions of the liver lobule, the mean intralobular oxygen gradient was estimated. The measured gradient was approximately 180 torr in livers from sucrose-treated control rats. Chronic treatment with ethanol increased both the mean intralobular oxygen gradient and the rate of hepatic oxygen uptake by 30%. The antithyroid drug, 6-propyl-2-thiouracil, completely reversed the effects of ethanol on both the intralobular oxygen gradient and the rate of oxygen uptake. These data present direct physical evidence that the increased tissue respiration induced by chronic ethanol treatment indeed accentuates the intralobular oxygen gradient and thus support the hypothesis that selective depletion of oxygen in the pericentral region of the liver lobule may underlie ethanol-induced cellular injury confined to this site.

Animals↗
PubMed1980

Fusion of liposomes with mitochondrial inner membranes.

A procedure is outlined for the fusion of mixed phospholipid liposomes (small unilamellar vesicles) with the mitochondrial inner membrane, which enriches the membrane lipid bilayer 30-700% in a controlled fashion. Fusion was initiated by manipulation of the pH of a mixture of freshly sonicated liposomes and the functional inner membrane/matrix fraction of rat liver mitochondria. During the pH fusion procedure, liposomes became closely apposed with and sequestered by the inner membranes as revealed by freeze-fracture electron microscopy. After the pH fusion procedure, a number of ultrastructural, compositional, and functional characteristics were found to be proportionally related: the membrane surface area increased; the lateral density distribution of intramembrane particles (integral proteins) in the plane of the membrane decreased whereas the particles remained random; the membrane became more buoyant; the ratio of membrane lipid phosphorus to total membrane protein increased; the ratio of membrane lipid phosphorus to heme a of cytochrome c oxidase increased; and the rate of electron transfer between some interacting membrane oxidoreduction proteins decreased. These data reveal that liposomal phospholipid was incorporated into the membrane bilayer (not simply adsorbed to the membrane surface) and that integral membrane proteins diffused freely into the laterally expanding bilayer. Furthermore, the data suggest that the rate of electron transfer may be limited by the rate of lateral diffusion of oxidoreduction components in the bilayer of the mitochondrial inner membrane.

Animals↗
PubMed1977

Kinetics of product inhibition during firefly luciferase luminescence.

A theoretical and experimental analysis is made of the kinetics of product inhibition during firefly luciferase luminescence. Equations for competitive, noncompetitive, and uncompetitive inhibition are derived which are useful in determining inhibitory mechanism when the product inhibitor, or its concentration, is unknown and not subject to direct experimental manipulation. Comparisons of experimental data with predictions based upon the three inhibitory models show that product inhibition during luciferase luminescence is noncompetitive with respect to both luciferin and ATP as substrates. The competitive and uncompetitive models are inconsistent with experimental data. These findings provide the basis for using luminescence to measure ATP concentration continuously in in vitro biological systems such as isolated mitochondria.

Animals↗
PubMed1976

Continuous measurement and rapid kinetics of ATP synthesis in rat liver mitochondria, mitoplasts and inner membrane vesicles determined by firefly-luciferase luminescence.

Oxidative phosphorylation in rat liver mitochondria, the mitoplast or inner membrane-matrix fraction, and inverted inner membrane vesicles was studied with firefly luciferase luminescence. All preparations showed relatively high ATP/O ratios under initial reaction conditions in the presence of Mg2+, but only intact mitochondria demonstrated significant respiratory control. Mitochondria and mitoplasts, but not vesicles, catalyzed nearly complete conversion of ADP to ATP. Vesicles converted between 50% and 70% of added ADP to ATP establishing a phosphate potential of 9.8 to 10.5 kcal/mol (43.9 kJ/mol). In "loosely coupled" submitochondrial systems which do not display respiratory control, the phosphate potential appears to be a useful parameter of the integrity of the phosphorylating membrane, while ATP/O ratios may be dependent on the relative concentrations of ADP and ATP in the reaction medium. The rapid kinetics of oxidative synthesis of ATP by inverted inner membrane vesicles was investigated in stopped-flow rapid-mixing experiments. After an oxygen pulse to reduced vesicles, ATP synthesis commenced within 20 ms. In antimycin-inhibited vesicles, ATP synthesis resulting from the rapid oxidation of the terminal portion of the respiratory chain commenced within 20ms and took approximately 100 ms for half completion. Since half oxidation of the terminal portion of the respiratory chain occurs within a few milliseconds, ATP synthesis resulting from such redox reactions must occur over a significantly longer time course than the redox reactions themselves.

Adenosine Diphosphate↗
PubMed1971

Oxidative phosphorylation and ultrastructural transformation in mitochondria in the intact ascites tumor cell.

We have examined the ultrastructure of mitochondria as it relates to energy metabolism in the intact cell. Oxidative phosphorylation was induced in ultrastructurally intact Ehrlich ascites tumor cells by rapidly generating intracellular adenosine diphosphate from endogenous adenosine triphosphate by the addition of 2-deoxyglucose. The occurrence of oxidative phosphorylation was ascertained indirectly by continuous and synchronous monitoring of respiratory rate, fluorescence of pyridine nucleotide, and 90 degrees light-scattering. Oxidative phosphorylation was confirmed by direct enzymatic analysis of intracellular adenine nucleotides and by determination of intracellular inorganic orthophosphate. Microsamples of cells rapidly fixed for electron microscopy revealed that, in addition to oxidative phosphorylation, an orthodox --> condensed ultrastructural transformation occurred in the mitochondria of all cells in less than 6 sec after the generation of adenosine diphosphate by 2-deoxyglucose. A 90 degrees light-scattering increase, which also occurs at this time, showed a t (1/2) of only 25 sec which agreed temporally with a slower orthodox --> maximally condensed mitochondrial transformation. Neither oxidative phosphorylation nor ultrastructural transformation could be initiated in mitochondria in intact cells by the intracellular generation of adenosine diphosphate in the presence of uncouplers of oxidative phosphorylation. Partial and complete inhibition of oxidative phosphorylation by oligomycin resulted in a positive relationship to partial and complete inhibition of 2-deoxyglucose-induced ultrastructural transformation in the mitochondria in these cells. The data presented reveal that an orthodox --> condensed ultrastructural transformation is linked to induced oxidative phosphorylation in mitochondria in the intact ascites tumor cell.

Adenine Nucleotides↗
PubMedDate not supplied

Early midzonal cell death during low-flow hypoxia in the isolated, perfused rat liver: protection by allopurinol.

Trypan blue uptake and lactate dehydrogenase release were measured as indices of irreversible cell damage in isolated, perfused rat livers during low-flow hypoxia. In livers from fasted rats perfused in the anterograde direction, trypan blue uptake took place beginning at about 45 min of hypoxia. Cells which took up trypan blue first were located in narrow bands at the border between anoxic pericentral areas and normoxic periportal regions of the liver lobule. After longer periods of hypoxia, trypan blue uptake progressed towards the central vein until after 120 min virtually all cells in the pericentral regions were stained. Under these conditions, cells in periportal regions were spared. In perfusions in the retrograde direction, cell death began in midzonal regions and spread towards the portal vein. Release of lactate dehydrogenase into the effluent paralleled trypan blue uptake, beginning at about 40 min of low-flow hypoxia and peaking at 80 min. In contrast to livers from fasted rats, trypan blue was not taken up, and lactate dehydrogenase was not released in livers from fed rats exposed to low-flow hypoxia for as long as 120 min. To test the hypothesis that xanthine oxidase-mediated oxygen-free radical formation was involved in cell injury at the border between anoxic and normoxic regions (anoxic edge), allopurinol, an inhibitor of xanthine oxidase, was studied. Allopurinol (0.2 to 5 mM) delayed the release of lactate dehydrogenase during low-flow hypoxia in a dose-dependent fashion (e.g., 5 mM allopurinol delayed hypoxia-induced lactate dehydrogenase release by about 30 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Allopurinol↗
PubMedDate not supplied

Inhibition of Na+/H+ exchange preserves viability, restores mechanical function, and prevents the pH paradox in reperfusion injury to rat neonatal myocytes.

Rat neonatal myocytes exposed to 2.5 mM CaCN and 20 mM 2-deoxyglucose at pH 6.2 (chemical hypoxia) quickly lose viability when pH is increased to 7.4, with or without washout of inhibitors--a 'pH paradox'. In this study, we evaluated the effect of two Na+/H+ exchange inhibitors (dimethylamiloride and HOE694) and a Na+/Ca2+ exchange inhibitor (dichlorobenzamil) on pH-dependent reperfusion injury. Intracellular free Ca2+ and electrical potential were monitored by laser scanning confocal microscopy of rat neonatal cardiac myocytes grown on coverslips and co-loaded with Fluo-3 and tetramethylrhodamine methylester. After 30-60 min of chemical hypoxia at pH 6.2, mitochondria depolarized and Ca2+ began to increase uniformly throughout the cell. Free Ca2+ reached levels estimated to exceed 2 microM by 4 h. Washout of inhibitors at pH 7.4 (reperfusion), with or without dichlorobenzamil, killed most cells within 60 min, despite a marked reduction of Ca2+ in dichlorobenzamil-treated cells. Reperfusion at pH 7.4 in the presence of 75 microM dimethylamiloride or 20 microM HOE694, or at pH 6.2, prevented cell death. HOE694-treated cells placed into culture medium recovered mitochondrial membrane potential. In most cells, this occurred before normal Ca2+ was restored. Contracted myocytes re-extended over a 24-h-period. By 48 hours, most cells contracted spontaneously and showed normal Ca2+ transients. Our results indicate that Na+/H+ exchange inhibition protects against pH-dependent reperfusion injury and facilitates full recovery of cell function.

Amiloride↗
PubMedDate not supplied

Assessment of Fura-2 for measurements of cytosolic free calcium.

Fura-2 has become the most popular fluorescent probe with which to monitor dynamic changes in cytosolic free calcium in intact living cells. In this paper, we describe many of the currently recognized limitations to the use of Fura-2 in living cells and certain approaches which can circumvent some of these problems. Many of these problems are cell type specific, and include: (a) incomplete hydrolysis of Fura-2 acetoxymethyl ester bonds by cytosolic esterases, and the potential presence of either esterase resistant methyl ester complexes on the Fura-2/AM molecule or other as yet unidentified contaminants in commercial preparations of Fura-2/AM; (b) sequestration of Fura-2 in non-cytoplasmic compartments (i.e. cytoplasmic organelles); (c) dye loss (either active or passive) from labeled cells; (d) quenching of Fura-2 fluorescence by heavy metals; (e) photobleaching and photochemical formation of fluorescent non-Ca2+ sensitive Fura-2 species; (f) shifts in the absorption and emission spectra, as well as the Kd for Ca2+ of Fura-2 as a function of either polarity, viscosity, ionic strength or temperature of the probe environment; and (g) accurate calibration of the Fura-2 signal inside cells. Solutions to these problems include: (a) labeling of cells with Fura-2 pentapotassium salt (by scrape loading, microinjection or ATP permeabilization) to circumvent the problems of ester hydrolysis; (b) labeling of cells at low temperatures or after a 4 degrees C pre-chill to prevent intracellular organelle sequestration; (c) performance of experiments at lower than physiological temperatures (i.e. 15-33 degrees C) and use of ratio quantitation to remedy inaccuracies caused by dye leakage; (d) addition of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to chelate heavy metals; (e) use of low levels of excitation energy and high sensitivity detectors to minimize photobleaching or formation of fluorescent non-Ca2+ sensitive forms of Fura-2; and (f) the use of 340 nm and 365 nm (instead of 340 nm and 380 nm) for ratio imaging, which diminishes the potential contributions of artifacts of polarity, viscosity and ionic strength on calculated calcium concentrations, provides a measure of dye leakage from the cells, rate of Fura-2 photobleaching, and can be used to perform in situ calibration of Fura-2 fluorescence in intact cells; however, use of this wavelength pair diminishes the dynamic range of the ratio and thus makes it more sensitive to noise involved in photon detection. Failure to consider these potential problems may result in erroneous estimates of cytosolic free calcium.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗