Nature of proton cycling during gramicidin uncoupling of oxidative phosphorylation

Addition of gramicidin D to liver mitochondria, incubated in low- or high-salt media, results in stimulation of respiration in the absence or presence of depression of delta muH, respectively. Gramicidin D concentrations 2 orders of magnitude higher are required in the low-salt media with full uncoupling at 1 nmol of gramicidin.mg-1. The stimulation of respiration is not accompanied by increased passive proton influx in low-salt media. In high-salt media, the extent of respiratory stimulation and the extent of delta muH depression differ according to the nature and concentration of cation. The flow-force relationship is very steep when gramicidin D induced uncoupling occurs in low-salt media and much less steep in high-salt media. A multiplicity of flow-force relationship, respiratory rate vs delta muH, is obtained, the slope of which depends on the nature and concentration of cation, and which can be reproduced by computer simulation by introducing a variable extent of proton cycling either in the membrane or in the pump. The apparent proton conductance, as analyzed in the relationship of Je/delta muH vs delta muH, increases in the so-called ohmic and nonohmic regions according to whether gramicidin D is added in high-salt or low-salt media, respectively. Titration with antimycin of the respiratory control ratio (RCR) in gramicidin D treated mitochondria leads to a depression of the RCR in high-salt but not in low-salt media. The view is discussed that in low-salt media the gramicidin D induced uncoupling is due to a cycling of protons within a proton domain operationally located at or near the proton pump.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum

A rapid and simple method for the isolation of membranes from subcellular organelles is described. The procedure consists of diluting the organelles in ice-cold 100 mM Na2CO3 followed by centrifugation to pellet the membranes. Closed vesicles are converted to open membrane sheets, and content proteins and peripheral membrane proteins are released in soluble form. Here we document the method by applying it to various subfractions of a rat liver microsomal fraction, prepared by continuous density gradient centrifugation according to Beaufay et al. (1974, J. Cell Biol. 61:213-231). The results confirm and extend those of previous investigators on the distribution of enzymes and proteins among the membranes of the smooth and rough endoplasmic reticulum. In the accompanying paper (1982, J. Cell Biol. 93:103-110) the procedure is applied to peroxisomes and mitochondria.     

Isolation of the major subcellular organelles from mouse liver using Nycodenz gradients without the use of an ultracentrifuge

Commonly, subcellular organelles such as nuclei, mitochondria, lysosomes, and Golgi membranes are isolated first by differential centrifugation in low-speed or high-speed centrifuges and then purified by gradient centrifugation in ultracentrifuges. We have prepared these organelles using a new high-speed centrifuge (28,000 rpm max) which allows the generation of higher radial centrifugal forces (rcfs) than are available in standard machines. We have shown that most subcellular organelles can be purified by using low-viscosity Nycodenz gradients at rcfs lower than those normally used in ultracentrifuges, without increasing the time of centrifugation. Use of Nycodenz also allows rapid harvesting of material from gradients and we have adapted a number of enzyme assays to facilitate gradient analysis.     

Two fractions of rough endoplasmic reticulum from rat liver. I. Recovery of rapidly sedimenting endoplasmic reticulum in association with mitochondria

Low-speed centrifugation (640 g) of rat liver homogenates, prepared with a standard ionic medium, yielded a pellet from which a rapidly sedimenting fraction of rough endoplasmic reticulum (RSER) was recovered free of nuclei. This fraction contained 20-25% of cellular RNA and approximately 30% of total glucose-6-phosphatase (ER marker) activity. A major portion of total cytochrome c oxidase (mitochondrial marker) activity was also recovered in this fraction, with the remainder sedimenting between 640 and 6,000 g. Evidence is provided which indicates that RSER may be intimately associated with mitochondria. Complete dissociation of ER from mitochondria in the RSER fraction required very harsh conditions. Sucrose density gradient centrifugation analysis revealed that 95% dissociation could be achieved when the RSER fraction was first resuspended in buffer containing 500 mM KCl and 20 mM EDTA, and subjected to shearing. Excluding KCl, EDTA, or shearing from the procedure resulted in incomplete separation. Both electron microscopy and marker enzyme analysis of mitochondria purified by this procedure indicated that some structural damage and leakage of proteins from matrix and intermembrane compartments had occurred. Nevertheless, when mitochondria from RSER and postnuclear 6,000-g pellet fractions were purified in this way fromanimals injected with [35S]methionine +/- cycloheximide, mitochondria from the postnuclear 6,000-g pellet were found to incorporate approximately two times more cytoplasmically synthesized radioactive protein per milligram mitochondrial protein (or per unit cytochrome c oxidase activity) than did mitochondria from the RSER fraction. Mitochondria-RSER associations, therefore, do not appear to facilitate enhanced incorporation of mitochondrial proteins which are newly synthesized in the cytoplasm.     

Local protons and uncoupling of aerobic and artificial delta muH-driven ATP synthesis

Gramicidin D causes inhibition of ATP synthesis either in the absence or in the presence of depression of delta muH, in low-salt and in high-salt media, respectively, at concentrations 2 orders of magnitude higher in the former with respect to the latter case. When the number of active redox pumps is reduced by increasing the antimycin concentration, the P/O ratio of respiring, gramicidin-treated mitochondria either is slightly increased in low-salt media or is first decreased and then constant in high-salt media. Addition of gramicidin D in low-salt media to mitochondria synthesizing ATP by means of artificially imposed delta muH gradients results in (a) no effect on the K+ efflux ratio +/- ADP (equivalent to the aerobic respiratory control ratio) and (b) no effect on the ATP/K+ ratio (equivalent to the P/O ratio) except at the low gramicidin D concentrations where there is also a slight enhancement of the rate of ATP hydrolysis. During respiration-driven ATP synthesis, addition of valinomycin plus K+ causes depression of delta muH with little inhibition of ATP synthesis while addition of gramicidin D causes inhibition of ATP synthesis with little depression of delta muH. The view is discussed that the gramicidin-accessible protons which uncouple aerobic ATP synthesis in a delta muH-independent manner are of a different class from the gramicidin-inaccessible protons which uncouple diffusion potential driven ATP synthesis in a delta muH-dependent manner. The gramicidin-accessible protons are suggested to be pump associated and to reflect primary events in energy transduction.     

Lithium nuclear magnetic resonance measurements in halotolerant bacterium Ba1

Measurements of relaxation times T₁ and T₂, were carried out on a high-salt- and low-salt-grown bacterial pellets of halotolerant bacterium B_a1. In our measurements, T₁ ? T₂ and both were frequency-independent. In the high-salt-grown pellet the relaxation time values were much shorter than in the case of low-salt growth medium. Intensity measurements show that only 55% of the lithium in the high-salt pellet is detected; for the low-salt pellet almost all the lithium is detected. Growth measurements were carried out on the B_a1. It is suggested that there is some form of adaptation of the bacteria to the growth medium. The adaptation is reflected in the lithium NMR results.     

A rapid method for the preparation of microvilli from rabbit kidney

A simple method for the isolation of microvilli from kidney brush border is described. The method depends on the preferential aggregation of other subcellular structures by bivalent metal ions. MgCl(2) is added to a homogenate of cortical tissue prepared from frozen rabbit kidneys. Aggregated material is removed by a low-speed centrifugation and the supernatant centrifuged at 15000g to yield a pellet enriched in microvilli. This is resuspended and given a second treatment with Mg(2+). The purified preparation is obtained after four short differential centrifugations. The six brush-border enzymes that were monitored were enriched 11-17-fold compared with the original homogenate and were obtained in about 10% yield. Marker enzymes for other subcellular components showed the preparation to be essentially free of mitochondria and to be less contaminated with endoplasmic reticulum and baso-lateral plasma membranes than are conventional brush-border preparations. The main contamination was of lysosomal origin, about half of which was attributable to adsorbed acid hydrolases rather than to intact lysosomes. The aggregated components in the low-speed pellet bound less Mg(2+) than did the microvillus fraction. A possible mechanism for the role of Mg(2+) is discussed.     

Studies with digitonin-treated rat hepatocytes (nude cells)

Isolated rat hepatocytes were treated with digitonin to strip the plasma membrane. The effect of digitonin concentration and exposure time on the recovery of marker enzymes for cell organelles was examined. Hepatocytes treated at room temperature for 1-2 min with 1 mg/ml of digitonin lose some 40% of their protein but retain over 95% of their intact mitochondria and peroxisomes, 90-95% of their endoplasmic reticulum, and about 80% of their lysosomal enzymes. There is little loss of the mitochondrial intermembrane content, and both oxygen uptake and phosphorylation are unimpaired by the treatment. Electron microscopy reveals a complete loss of the plasma membrane, in spite of limited loss of marker enzymes for this membrane. Scanning electron microscopy revealed the interior of the cells to be made up of a dense network of fibers and lamellae attached to the nucleus, mitochondria, and small organelles. The treated cells were stable for many hours when kept in 0.25 M sucrose containing 25 mM monovalent salts. In salt-free sucrose the cells broke up very rapidly into nuclei and other single organelles. Addition of 5 mM NaCl or KCl retards breakup, and 15-20 min were required for dissolution. Intermediate stages, illustrated by scanning electron micrographs, show structure and chains made up mainly of mitochondria held together by a lamellar network. The rapid breakdown occurred at a pH above 7.5 in an oxygen atmosphere and in the presence of phosphate and apparently is an energy-requiring process. It is slow below a pH of 7.2, and at a pH of 6.8 the treated cells remain completely stable in salt-free sucrose. Our results suggest that endoplastic reticulum is a major component of the cytostructure holding together nuclei and organelles.     

Localization of calcium in nerve fibers

Using the desheathed nerve preparation, a pyroantimonate precipitation method was used to examine the distribution of electron-dense particles seen in various organelles of the nerve fibers following exposure of nerve to various levels of Ca2+ in vitro. The presence of Ca2+ in the electron-dense particles was indicated by their extraction with EGTA and by the use of energy-dispersive X-ray microanalysis. In normal Ringer or in a Ca2+ -free medium, electron-dense particles were seen associated with the outer membrane of the mitochondria, with the smooth endoplasmic reticulum (SER), along the axolemma and yet others scattered throughout the axoplasm. When nerves were incubated in media containing higher than normal concentrations of 20-60 mM Ca2+, an increase in the number of such electron-dense particles was seen in the axoplasm and within the mitochondrial matrix. Nerves loaded with a high concentration of 60mM Ca2+ could be depleted of these particles after transfer to a Ca2+ -free or low Ca2+ Ringer medium. The sequestration of Ca2+ in axonal organelles is discussed with respect to Ca2+-regulatory mechanisms in the axon needed to maintain a low level of Ca2+ which is optimal for the support of axoplasmic transport.     

Analytical subcellular fractionation of needle-biopsy specimens from human liver.

1. Fragments (2-20 mg wet wt.) of closed needle-biopsy specimens from human liver were disrupted in iso-osmotic sucrose and subjected to low-speed centrifugation. The supernatant was layered on a linear sucrose-density gradient in the Beaufay small-volume automatic zonal rotor. The following organelles, with equilibrium densities (g/ml) and principal marker enzyme shown in parentheses, were resolved: plasma membrane (1.12-1.14; 5'-nucleotidase); lysosomes (1.15-1.20; N-acetyl-beta-glucosaminidase); mitochondria (1.20; malate dehydrogenase); endoplasmic reticulum (1.17-1.21; neutral alpha-glucosidase); peroxisomes (1.22-1.24; catalase). 2. The distribution of particulate alkaline phosphatase and, to a lesser degree, leucine 2-naphthylamidase followed that of 5'-nucleotidase. gamma-Glutamyltransferase was associated with membranes of significantly higher equilibrium density than was 5'-nucleotidase. 3. The distribution of 12 acid hydrolases was determined in the density-gradient fractions. beta-Glucosidase had a predominantly cytosolic localization, but the other enzymes showed a broad distribution of activity throughout the gradient. Evidence was presented for two populations of lysosomes with equilibrium densities of 1.15 and 1.20 g/ml, but containing differing amounts of each enzyme. Further evidence of lysosomal heterogeneity was demonstrated by studying the distribution of isoenzymes of hexosaminidase and of acid phosphatase. 4. The resolving power of the centrifugation procedure can be further enhanced with membrane perturbants. Digitonin (0.12 mM) selectively disrupted lysosomes, markedly increased the equilibrium density of plasma-membrane components and lowered the density of the endoplasmic reticulum, but did not affect the mitochondria or peroxisomes. Pyrophosphate (15 mM) selectively lowered the equilibrium density of the endoplasmic reticulum.     

Association of maternal and newly synthesized ribosomes with membranous noncytoskeletal structures in Xenopus laevis embryonic cells

In Xenopus laevis embryos a high concentration of both KCl and 0.5% DOC (sodium deoxycholate) is needed for maximal extraction of ribosomes and polysomes. We studied the nature of the structures that keep ribosomes and polysomes immobilized within the cytoplasm of embryonic cells at cleavage through tailbud stages, using various combinations of a low-salt buffer (20 mM KCl), a high-salt buffer (500 mM KCl), 0.5% DOC, and 0.5% Triton X-100. With a low-salt buffer and 0.5% DOC, but not Triton X-100, 80S ribosomal monomers and polysomes were liberated from the cytoplasmic rapidly sedimenting structures (RSS) to the soluble fraction. With a high-salt buffer (500 mM KCl), ribosomes were solubilized as 60S and 40S subunits together with about one-half of the total polysomes. When cells were homogenized in a low-salt buffer with added inhibitors of the cytoskeleton (cytochalasin B or colchicine), the majority of polysomes but not ribosomes were solubilized. These results provide evidence for the following conclusions. 1) Polysomes are bound to cytoskeletal structures in Xenopus embryos, but ribosomes, both maternal and newly synthesized, are associated with membranous noncytoskeletal structures. 2) The membranous structures consist of two compartments, one high-salt sensitive and the other high-salt resistant. 3) Ribosomes of the high-salt resistant group increase in amount with developmental stage and appear to be the precursor to the ribosomes of the high-salt sensitive group.     

The Preparation of Subcellular Organelles from Mouse Liver in Self-Generated Gradients of Iodixanol

This paper reports the use of a new density gradient compound, Iodixanol, for the resolution of the major organelles from mouse liver. A major advantage of Iodixanol over other iodinated density gradient media is its ready ability to form self-generated gradients. Gradient-forming conditions have been modulated to provide optimal recoveries of Golgi membranes, lysosomes, mitochondria, and peroxisomes. The organelles were isolated in high yield (80-90% of gradient input) and high purity. Nycodenz and Iodixanol were compared using preformed gradients. Iodixanol provided resolution superior to that of Nycodenz, notably of peroxisomes and mitochondria and the separation of lysosomes from endoplasmic reticulum. Because Iodixanol does not interfere significantly with marker enzyme activities, gradient fractions can be analyzed without removal of the gradient medium.     

Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate

Chicken liver plasma membranes, minimally contaminated with Golgi apparatus-derived vesicles, were prepared from a low-speed (400 g) pellet by means of flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous sucrose gradient. Based on the analysis of suitable marker enzymes, alkaline phosphatase and alkaline phosphodiesterase, two plasma membrane fractions were isolated with enrichments, depending on the equilibrium density and marker of 28-97 and with a total yield of 4-5%. Golgi apparatus fractions were prepared by flotation of microsomes, obtained from the same homogenate as the low-speed pellet, in a discontinuous sucrose gradient. The trans-Golgi marker galactosyltransferase was 27-fold enriched in a fraction of intermediate density (d=1.077-1.116 g/ml). Approximately 12% of galactosyltransferase was recovered in the membranes equilibrating d=1.031-1.148 g/ml. Contamination with plasma membrane fragments was low in the light (d=1.031-1.077 g/ml) and intermediate density Golgi vesicles. The isolation of purified plasma membranes and Golgi vesicles from one liver homogenate will enable future studies on receptor cycling between these cell organelles.     

Stimulation of mitochondrial pyruvate metabolism and citrulline synthesis by dexamethasone. Effect of isolation and incubation media.

Hepatic mitochondria isolated in 0.3 M-sucrose or 0.3 M-mannitol from rats treated for 3h with dexamethasone displayed stimulated rates of pyruvate carboxylation and decarboxylation and citrulline synthesis when compared with organelles from control animals. Mitochondria isolated in mannitol also displayed elevated rates of pyruvate carboxylation and decarboxylation when compared with those isolated in sucrose, and this stimulation was shown to be independent of the lengthy isolation procedure. Citrulline synthesis proceeded at similar rates in mitochondria isolated in either sugar. The concentration of exchangeable adenine nucleotides was identical in mitochondria isolated in sucrose or mannitol, suggesting that those prepared in the former sugar are not more permeable to metabolites than those prepared in the latter. The matrix volume of mitochondria isolated in mannitol was greater than that of mitochondria isolated in sucrose, and the effect of mannitol on pyruvate metabolism was mimicked by swelling the organelles in hypo-osmotic sucrose. Measurements of the extra-matrix volume by using [14C]sucrose or [14C]mannitol suggest that mannitol can permeate mitochondria to a greater extent than can sucrose. The possibility that mannitol elicits its effect by entering the mitochondrial matrix and so initiating swelling is discussed.     

Secondary and quaternary structural transition of the halophilic archaeon nucleoside diphosphate kinase under high- and low-salt conditions

Most halophilic enzymes from extremely halophilic archaea are denatured immediately after transfer from high-salt to low-salt medium. However, nucleoside diphosphate kinase (HsNDK) from the extremely halophilic archaeon Halobacterium salinarum seems to be exceptional, since the enzyme exhibited catalytic activity even under the low-salt condition. Here we show the mechanism how HsNDK is active under both high- and low-salt conditions that the HsNDK hexamer in high-salt medium dissociates into a dimer in the low-salt medium without denaturation. The observed change of the subunit structure was accompanied by a large decrease of alpha-helical content and lowered thermal sensitivity, yet keeping the conformations. This novel hexamer to dimer conversion under high- and low-salt conditions, respectively, seems to be the mechanism by which HsNDK is avoided from the irreversible denaturation.     

ISOLATION OF CILIATED OR UNCILIATED BASAL BODIES FROM THE RABBIT OVIDUCT

Techniques have been developed for the isolation of basal bodies with cilia attached or for the isolation of only basal bodies from the rabbit oviduct. Oviducts are removed, cut open, and placed in an extraction medium composed of 0.25 M sucrose, 0.001 M EDTA, 0.025 M KCl, 0.02 M Hepes buffer pH 7.5, and 0.05% Triton X-100. After the oviduct is agitated in this medium on a Vortex mixer for ½ h, the lumenal cortex of each ciliated cell, containing 200–300 basal bodies with cilia attached, is released as a unit. The cortices and the intact nuclei, which are also released from the disrupted cells, form a pellet when the extraction medium is centrifuged at 600 g for 10 min. When cortices which contain only basal bodies are to be isolated, the oviduct is subjected to conditions which remove the cilia prior to being processed as above. The cilia are removed when the oviduct is placed in a medium of 0.25 M sucrose, 0.01 M CaCl₂, 0.02 M Pipes buffer pH 5.5, and 0.05% Triton X-100 and continuously agitated for 15 min on a Vortex mixer. The low pH and Ca⁺⁺ solubilize the transition region of the cilium and also prevent the cell from being disrupted. The cortices can be partially purified if the 600-g pellet is resuspended in 2.2 M sucrose pH 6.5 and centrifuged at 40,000 g for 2 h. Under these conditions, 85% of the nuclei form a pellet and the cortices float to the surface of the sucrose. In addition to the basal bodies or basal bodies with cilia, the cortices contain some adherent cytoplasm, a few fibers, and a few vesicles which may be remnants of mitochondria or endoplasmic reticulum. The structure of the cilia and the basal bodies isolated with either procedure is normal.     

Nonuniform distribution of sodium in the rat hepatocyte

The volume of the nucleus, endoplasmic reticulum (including Golgi complex), mitochondria, and cytoplasmic ground substance was measured in rat hepatocytes by stereological methods. The Na content was also measured by flame photometry. Variations in Na content correlated significantly with variations in volume of nucleus and endoplasmic reticulum. From the correlation parameters, Na concentrations were estimated as follows: nucleus, 108 mM; endoplasmic reticulum (ER) (including Golgie complex) 27 mM; cytoplasm (including and remaining organelles) 16 mM.     

Partial characterization of the androgen receptor of the newborn rat gubernaculum

Androgen receptors were measured by sucrose density gradient techniques in low-salt and high-salt extracts of gubernaculum, urogenital sinus, and bladder from 3- to 5-day-old male rats. Specific 5 alpha-[3H] dihydrotestosterone binding was present in both the low-salt and high-salt extracts of gubernacular tissue. The total amount of receptor in gubernaculum was approximately one-fifth the total amount of androgen binding measured in the urogenital sinus (119 fmol/g tissue in gubernaculum vs. 562 fmol/g tissue in urogenital sinus). No androgen receptor was detected in bladder. A 10-fold excess of nonradioactive 5 alpha-dihydrotestosterone competed well for 5 mM 5 alpha-[3H]-dihydrotestosterone binding, whereas 10-fold molar excesses of testosterone, estradiol, and progesterone were ineffective. These results suggest that the gubernaculum may be a target tissue for androgens.     

3-hydroxy-3-methylglutaryl coenzyme A reductase from rat intestine: subcellular localization and in vitro regulation

The subcellular localization of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in rat intestine was reinvestigated. Highly enriched fractions of endoplasmic reticulum and mitochondria were prepared from mucosal cells. The highest specific activity of HMG-CoA reductase was located in the endoplasmic reticulum fraction with recovery of 25% of the total activity. The mitochondria had low specific activity and low recovery of reductase activity relative to whole homogenate (2-5%). Despite attempts to maximize cell lysis, much of the activity (about 60%) was recovered in a low speed pellet which consisted of whole cells, nuclei, and cell debris as determined by light microscopy. Taken together, the evidence strongly suggests that much of the cellular HMG-CoA reductase activity is present in the endoplasmic reticulum fraction and that mitochondria have little or no intrinsic HMG-CoA reductase. The in vitro regulation of intestinal microsomal HMG-CoA reductase was studied. The intestine possesses a cytosolic HMG-CoA reductase kinase-phosphatase system which appears to be closely related to that present in the liver. Intestinal reductase activity in microsomes prepared from whole mucosal scrapings was inhibited 40-50% by the presence of 50 mM NaF in the homogenizing buffer. It was less susceptible to the action of the kinase than liver reductase. The effects of NaF were reversed by incubation with partially purified intestinal or liver phosphatases. These results suggest that the kinase-phosphatase system could play a role in the regulation of intestinal sterol and isoprene synthesis in vivo.