Phosphatidylcholine transfer protein regulates size and hepatic uptake of high-density lipoproteins

Phosphatidylcholine transfer protein (PC-TP) is a steroidogenic acute regulatory-related transfer domain protein that is enriched in liver cytosol and binds phosphatidylcholines with high specificity. In tissue culture systems, PC-TP promotes ATP-binding cassette protein A1-mediated efflux of cholesterol and phosphatidylcholine molecules as nascent pre-beta-high-density lipoprotein (HDL) particles. Here, we explored a role for PC-TP in HDL metabolism in vivo utilizing 8-wk-old male Pctp(-/-) and wild-type littermate C57BL/6J mice that were fed for 7 days with either chow or a high-fat/high-cholesterol diet. In chow-fed mice, neither plasma cholesterol concentrations nor the concentrations and compositions of plasma phospholipids were influenced by PC-TP expression. However, in Pctp(-/-) mice, there was an accumulation of small alpha-migrating HDL particles. This occurred without changes in hepatic expression of ATP-binding cassette protein A1 or in proteins that regulate the intravascular metabolism and clearance of HDL particles. In Pctp(-/-) mice fed the high-fat/high-cholesterol diet, HDL particle sizes were normalized, whereas plasma cholesterol and phospholipid concentrations were increased compared with wild-type mice. In the absence of upregulation of hepatic ATP-binding cassette protein A1, reduced HDL uptake from plasma into livers of Pctp(-/-) mice contributed to higher plasma lipid concentrations. These data indicate that PC-TP is not essential for the enrichment of HDL with phosphatidylcholines but that it does modulate particle size and rates of hepatic clearance.     

A TEST FOR DIFFUSIBLE IONS : II. THE IONIC NATURE OF PEPSIN.

1. The distribution of pepsin between particles of gelatin or coagulated egg albumin and the outside solution has been found to be equal to the distribution of chloride or bromide ion under the same conditions. This is the case from pH 1 to 7, and in the presence of a variety of salts. 2. Pepsin is therefore probably a monovalent anion. 3. Under certain conditions the enzyme may be adsorbed on the surface of the protein particles. This reaction is irreversible and is markedly influenced by the presence of low concentrations of electrolytes.     

A study of the dicyclohexylcarbodiimide-binding component of the mitochondrial ATPase complex from beef heart

1. The binding of [14C]-dicyclohexylcarbodiimide to membrane proteins of beef heart mitochondria has been investigated using dodecylsulphate/polyacrylamide gel electrophoresis. Upon incubation of submitochondrial particles with low concentrations of dicyclohexylcarbodiimide (5 nmol/mg protein) radioactivity was incorporated into three components with apparent molecular weights of 30000, 18000 and less than 6500. Only the two smaller components were found to be extracted into chloroform/methanol. The same two components were labelled when the isolated ATPase complex or a reconstituted F0F1 system was incubated with low concentrations of dicyclohexylcarbodiimide. High concentrations of dicyclohexylcarbodiimide (20-100 nmol/mg protein) resulted in binding to several mitochondrial proteins. 2. The maximal amount of dicyclohexylcarbodiimide which can bind to submitochondrial particles, the isolated ATPase complex, and the reconstituted F0F1 system was found to exceed the amount required for maximal inhibition of the ATPase activity by several-fold. The distribution of the bound [14C]dicyclohexylcarbodiimide between the different dicyclohexylcarbodiimide-binding components was investigated as a function of dicyclohexylcarbodiimide concentration. The smallest and largest components revealed a high affinity for dicyclohexylcarbodiimide-binding which paralleled the inhibition of ATPase activity. The intermediate component had a markedly lower affinity for dicyclohexylcarbodiimide-binding. 3. The larger dicyclohexylcarbodiimide-binding component of the isolated ATPase complex can be converted into the smaller component by treatment of the ATPase complex with performic acid. Partial conversion can also be achieved by extraction of the band from the dodecylsulphate-polyacrylamide gel after electrophoresis, followed by re-electrophoresis. The observations suggest that the larger component may be an oligomer of the smaller one. 4. Using concentrations of oligomycin and dicyclohexylcarbodiimide which were equal to or greater than those required for maximal inhibition of the ATPase activity, oligomycin was found to diminish the binding of [14C]dicyclohexylcarbodiimide to both dicyclohexylcarbodiimide-binding components of the isolated ATPase complex.     

Rubber elongation factor from Hevea brasiliensis. Identification, characterization, and role in rubber biosynthesis

The presence of a protein, rubber elongation factor (REF), which is tightly bound to serum-free rubber particles purified from Hevea brasiliensis latex, is necessary for prenyltransferases from a number of sources to add multiple cis-isoprene units to rubber molecules. These prenyltransferases show normal farnesyl pyrophosphate synthase activity (two trans additions of isopentenyl pyrophosphate to dimethylallyl pyrophosphate) in the absence of REF bound to rubber particles. REF bound to rubber molecules can be highly purified from all other proteins in whole latex by treatment of rubber particles with low concentrations of detergent. Treatment of rubber particles with trypsin which hydrolyzes bound REF, removal of REF with high concentrations of various detergents, or treatment of whole latex with polyclonal antibodies specific for REF all prevent prenyltransferase from adding [14C]isopentenyl pyrophosphate to rubber molecules. However, we have not been successful using detergent-solubilized REF in the reconstitution of in vitro rubber biosynthesis with either REF-depleted rubber particles or allylic pyrophosphate primers. REF has a molecular mass of 14,600 Da and is associated specifically with rubber particles in whole latex. It makes up between 10-60% of the total protein in whole latex but is absent in C-serum, the supernatant fluid obtained when rubber particles are removed by centrifugation. The amount of REF in whole latex is proportional to the rubber content. Based on a number average molecular mass of 500,000 Da for rubber and the content of rubber and REF in whole latex or serum-free rubber particles, the stoichiometry of REF molecules to rubber molecules is 1:1 in both cases. There is sufficient REF to form a monomolecular protein layer coating large rubber particles (700-1,000 nm). In the electron microscope, serum-free rubber particle preparations contain particles with diameters from 800 to as small as 10 nm. In the presence of 1% sodium dodecyl sulfate no particles smaller than 100 nm are observed. We suggest that the smaller particles may be mainly composed of REF molecules.     

Restoration of ATP synthesis in urea-treated membranes prepared from pea cotyledon mitochondria

Submitochondrial particles were prepared from pea cotyledon mitochondria by sonication in a medium containing 5 mM MgCl₂. The resulting particles (Mg²⁺-submitochondrial particles) catalyzed oxidative phosphorylation at the rate of 100–200 nmol ATP formed / min per mg protein. Treatment of Mg²⁺-submitochondrial particles with 3.0 M urea resulted in a preparation of highly resolved particles with low ATPase activity and no capacity for oxidative phosphorylation. However, the resulting membranes were not capable of reconstitution of oxidative posphorylation with the purified mitochondrial F₁-ATPase. Urea particles capable of reconstitution of oxidative phosphorylation could be prepared by extracting Mg²⁺-submitochondrial particles with concentrations of urea ranging from 1.7 to 2.0 M. We have used 1.9 M urea for large-scale preparation of urea particles that could be stored in liquid nitrogen without any loss of reconstitution capacity. The residual oxidative phosphorylation rate of these particles was 6–8 nmol ATP / min per mg protein and this rate could increase to 60–70 nmol ATP / min per mg protein on incubation with saturating amounts of purified mitochondrial F₁-ATPase. In contrast to the mitochondrial F₁, purified activated pea chloroplast CF₁ was unable to stimulate ATP synthesis in 1.9 M urea particles.     

Some viscoelastic properties of human erythrocyte spectrin networks end-linked in vitro

We have succeeded in making macroscopic networks of end-linked human erythrocyte spectrin. The network junctions were made using erythrocyte protein 4.1 irreversibly attached to 5 nm (diameter) colloidal gold particles. Rotary shadowing electron microscopy verifies that the protein 4.1-labelled colloidal gold particles bind only to the tail end of the spectrin molecules. Electron micrographs of protein 4.1-labelled colloidal gold particles incubated at 4 degrees C with spectrin dimers reveal that 1-5 spectrin dimers attach to each protein 4.1-labelled colloidal gold particle yielding a spider-like appearance of these complexes. Incubation with a low concentration of spectrin tetramers instead of dimers leads to extensive formation of spectrin microaggregates whereas use of spectrin concentrations higher than 3 mg/ml and a molar ratio between spectrin tetramers and protein 4.1/Au of 4 leads to formation of macroscopic spectrin networks. We have quantitated the viscoelastic properties of such end-linked macroscopic spectrin networks using a gravitational pendulum viscoelastometer. We find that in vitro end-linked spectrin networks can be described by linear viscoelastic theory. The dynamic storage modulus increases almost linearly with the spectrin-protein 4.1/gold particle concentration when the spectrin concentration exceeds about 3 mg/ml and the molar ratio between spectrin tetramers and protein 4.1/Au is 4. At a spectrin concentration of 6 mg/ml and the same ratio between spectrin and protein 4.1/Au, we find a dynamic storage modulus at low frequency of about 80 dyn/cm2. This is in adequate agreement with what is predicted by simple elastomer theory.     

Further studies on F1-ATPase inhibition by local anesthetics

We have measured the inhibitory potencies of several local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and related compounds (chlorpromazine, procainamide and propranolol) on the ATPase activities of bovine heart submitochondrial particles and purified F₁ extracted from these particles. All of these agents cause inhibition of ATPase in F₁ as well as in submitochondrial particles. A linear relationship is found between the log of the octanol/water partition coefficients and the log of the concentrations required for 50% inhibition of F₁. Sedimentation velocity ultracentrifugation and polyacrylamide gel electrophoresis showed that 1.0 mM tetracaine caused partial dissociation of the F₁ complex. Complete reversibility of the enzyme inhibitory effects was demonstrated, however. This work shows that local anesthetics can affect protein structure and enzyme activity without the mediation of lipid.     

Interaction of F1-ATPase, from ox heart mitochondria with its naturally occurring inhibitor protein. Studies using radio-iodinated inhibitor protein

The ox heart mitochondrial inhibitor protein may be iodinated with up to 0.8 mol 125I per mol inhibitor with no loss of inhibitory activity, with no change in binding affinity to submitochondrial particles, and without alteration in the response of membrane-bound inhibitor to energisation. Tryptic peptide maps reveal a single labelled peptide, consistent with modification of the single tyrosine residue of the protein. A single type of high-affinity binding site (Kd=96 . 10 (-9)M) for the inhibitor protein has been measured in submitochondrial particles. The concentration of this site is proportional to the amount of membrane-bound F1, and there appears to be one such site per F1 molecule. The ATp hydrolytic activity of submitochondrial particles is inversely proportional to the occupancy of the high-affinity binding site for the inhibitor protein. No evidence is found for a non-inhibitory binding site on the membrane or on other mitochondrial proteins. In intact mitochondria from bovine heart, the inhibitor protein is present in an approx. 1:1 ratio with F1. Submitochondrial particles prepared by sonication of these mitochondria with MgATP contain about 0.75 mol inhibitor protein per mol F1, and show about 25% of the ATPase activity of inhibitor-free submitochondrial particles. Additional inhibitor protein can be bound to these particles to a level of 0.2 mol/mol F1, with consequent loss of ATPase activity. If MgATP is omitted from the medium, or inhibitors of ATP hydrolysis are present, the rate of combination between F1 and its inhibitor protein is very much reduced. The equilibrium level of binding is, however, unaltered. These results suggest the presence of a single, high-affinity, inhibitory binding site for inhibitor protein on membrane-bound F1. The energisation of coupled submitochondrial particles by succinate oxidation or by ATP hydrolysis results in both the dissociation of inhibitor protein into solution, and the activation of ATP hydrolysis. At least 80% of the membrane-bound F1-inhibitor complex responds to this energisation by participating in a new equilibrium between bound and free inhibitor protein. This finding suggests that a delocalised energy pool is important in promoting inhibitor protein release from F1. Dissipation of the electrochemical gradient by uncouplers, or the binding of oligomycin or efrapetin effectively blocks energised release of the inhibitor protein. Conversely, the addition of aurovertin or adenosine 5'--[beta, lambda--imido]triphosphate enhances energy-driven release. The mode of action of various inhibitors on binding and energised release of the protein inhibitor is discussed.     

Equilibrium between monomeric and dimeric mitochondrial F1-inhibitor protein complexes.

Mg-ATP particles from bovine heart mitochondria have more than 95% of their F1 in complex with the inhibitor protein (IF1). The F1-IF1 complex was solubilized and purified. The question addressed was if this naturally occurring complex existed as monomers or dimers. Size exclusion chromatography and electron microscopy showed that most of the purified F1-IF1 complex was a dimer of two F1-IF1. As determined by the former method, the relative concentrations of dimeric and monomeric F1-IF1 depended on the concentration of protein that was applied to the column. Apparently, there is an equilibrium between the two forms of F1-IF1.     

Effects of naturally occurring polyols and urea on mitochondrial F0F1ATPase

We show that urea inhibits the ATPase activity of MgATP submitochondrial particles (MgATP-SMP) with Ki = 0.7 M, probably as a result of direct interaction with the structure of F0F1-ATPase. Counteracting compounds (sorbitol, mannitol or inositol), despite slightly (10-20%) inhibiting the ATPase activity, also protect the F0F1-ATPase against denaturation by urea. However, this protection was only observed at low urea concentrations (less than 1.5 M), and in the presence of three polyols, the Ki for urea shift from 0.7 M to 1.2 M. Urea also increases the initial activation rate of latent MgATP-SMP in a dose-dependent-manner. However, when the particles (0.5 mg/ml) were preincubated in the presence of 1 M, 2 M or 3 M urea, a decrease in the activation level occurred after 1 h, 30 and 10 min, respectively. At high MgATP-SMP concentration (3 mg/ml) a decrease in activation was observed after 2 h, 1 h and 20 min, respectively. These data indicate that the effect of urea on the activation of MgATP-SMP depends on time, urea and protein concentrations. It was also observed that polyols suppress the activation of latent MgATP-SMP in a dose-dependent manner, and protect the particles against urea denaturation during activation. We suppose that a decrease in membrane mobility promoted by interactions of polyols with phospholipids around the F0F1-ATPase may also increase the compactation of protein structure, explaining the inhibition of natural inhibitor protein of ATPase (IF1) release and the activation of the enzyme.     

Alpha-Calcitonin Gene-Related Peptide Can Reverse The Catabolic Influence Of UHMWPE Particles On RANKL Expression In Primary Human Osteoblasts

Background and purpose: A linkage between the neurotransmitter alpha-calcitonin gene-related peptide (alpha-CGRP) and particle-induced osteolysis has been shown previously. The suggested osteoprotective influence of alpha-CGRP on the catabolic effects of ultra-high molecular weight polyethylene (UHMWPE) particles is analyzed in this study in primary human osteoblasts. Methods: Primary human osteoblasts were stimulated by UHMWPE particles (cell/particle ratios 1:100 and 1:500) and different doses of alpha-CGRP (10^-7 M, 10^-9 M, 10^-11 M). Receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) mRNA expression and protein levels were measured by RT-PCR and Western blot. Results: Particle stimulation leads to a significant dose-dependent increase of RANKL mRNA in both cell-particle ratios and a significant down-regulation of OPG mRNA in cell-particle concentrations of 1:500. A significant depression of alkaline phosphatase was found due to particle stimulation. Alpha-CGRP in all tested concentrations showed a significant depressive effect on the expression of RANKL mRNA in primary human osteoblasts under particle stimulation. Comparable reactions of RANKL protein levels due to particles and alpha-CGRP were found by Western blot analysis. In cell-particle ratios of 1:100 after 24 hours the osteoprotective influence of alpha-CGRP reversed the catabolic effects of particles on the RANKL expression. Interpretation: The in-vivo use of alpha-CGRP, which leads to down-regulated RANKL in-vitro, might inhibit the catabolic effect of particles in conditions of particle induced osteolysis.     

Rapid directional translocations in virus replication

By viewing virus development in real time, the experiments reported here reveal novel processes--rapid directional translocations--that are likely to be important elements of virus replication. Herpes simplex virus type 1 (HSV-1) was labeled by the fusion of the green fluorescent protein to a structural protein of its tegument (VP11/12), the product of gene UL46. Infection of cultured cells with this recombinant virus (GHSV-UL46) produced fluorescent particles that were distributed throughout the cytoplasm with concentrations in the perinuclear region; they were absent from the nucleus. Viewing infected cells in real time by means of video microscopy produced a novel dynamic picture of virus development. Most strikingly, some of the fluorescent particles exhibited extremely rapid directional translocations at velocities as great as 5 microm/s. The trajectories and destinations of these particles suggest that the rapid directional translocations serve at least three functions: the rapid transport of viral components to and between cytoplasmic processing stations, the delivery of materials for functions specific to the perinuclear region, and the conveyance of maturing virus particles to the plasma membrane. These rapid directional translocations are novel elements of virus assembly that are likely to be critical for efficient replication.     

Plasmalemmal vesicle associated protein (PV1) modulates SV40 virus infectivity in CV-1 cells

Plasmalemmal vesicle associated protein (Plvap/PV1) is a structural protein required for the formation of the stomatal diaphragms of caveolae. Caveolae are plasma membrane invaginations that were implicated in SV40 virus entry in primate cells. Here we show that de novo Plvap/PV1 expression in CV-1 green monkey epithelial cells significantly reduces the ability of SV40 virus to establish productive infection, when cells are incubated with low concentrations of the virus. However, in presence of high viral titers PV1 has no effect on SV40 virus infectivity. Mechanistically, PV1 expression does not reduce the cell surface expression of known SV40 receptors such as GM1 ganglioside and MHC class I proteins. Furthermore, PV1 does not reduce the binding of virus-like particles made by SV40 VP1 protein to the CV-1 cell surface and does not impact their internalization when cells are incubated with either high or low VLP concentrations. These results suggest that PV1 protein is able to block SV40 infectivity at low but not at high viral concentration either by interfering with the infective internalization pathway at the cell surface or at a post internalization step.     

Non-haem iron and the dissociation of piericidin A sensitivity from site 1 energy conservation in mitochondria from Torulopsis utilis

1. The aerobic incubation of iron-deficient Torulopsis utilis cells for 12h under non-growing conditions results in the recovery by mitochondria of the previously absent site 1 energy conservation and sensitivity to piericidin A. 2. The recovery of piericidin A sensitivity but not site 1 is prevented by the presence of cycloheximide (100mug/ml) in the medium used for aerobic incubation of the cells. Rotenone sensitivity behaved similarly. 3. Chloramphenicol, erythromycin and tetracycline were without effect on the recovery of site 1 and piericidin A sensitivity. 4. Inclusion of (59)Fe in the growth medium can be used as the basis for a highly sensitive assay for non-haem iron. 5. Iron-limited growth of T. utilis lowers the concentration of both non-haem iron and acid-labile sulphide of submitochondrial particles by over 20-fold compared with the ;normal' situation with iron-supplemented glycerol-limited growth. 6. Increases in the non-haem iron and acid-labile sulphide concentrations of submitochondrial particles occur when site 1 and piericidin A sensitivity are recovered. The increase is approximately halved by the presence of cycloheximide. 7. The non-haem iron of T. utilis submitochondrial particles does not exchange with added iron. 8. Continuous culture of T. utilis at the transition between glycerol- and iron-limitation results in cells where mitochondria possess site 1 energy conservation but lack piericidin A sensitivity. 8. It is concluded, in contrast with widely held views to the opposite, that energy conservation at site 1 does not require electron flow to proceed through a piericidin A- or rotenone-sensitive route. 9. Restriction of the iron supplied to growing T. utilis to a concentration just above that required for growth limitation demonstrates that a 10- to 20-fold decrease of the ;normal' non-haem iron concentration of both cells and mitochondria is without effect on the growth yield per unit of carbon source. Submitochondrial particles prepared from such iron-restricted but otherwise functionally normal cells have a non-haem iron concentration of about 0.5-0.8ng-atom/mg of protein. It is concluded that the concentration of iron-sulphur protein required for normal function by the respiratory chain is close to the concentrations of cytochromes and flavoproteins.     

Strategies for manipulating the relative concentration of recombinant rotavirus structural proteins during simultaneous production by insect cells

Adequate production strategies of virus-like particles are among the challenges that must be addressed before such complex multimeric structures find practical applications as vaccines. Attainment of the correct stoichiometric relation between proteins that constitute virus-like particles should result in an increased productivity by maximizing the concentration of assembled proteins and preventing the accumulation of waste monomers. In this work, strategies for manipulating the relative concentration between two of the structural proteins that constitute rotavirus-like particles (VP2 and VP6) were explored using the insect cell baculovirus expression vector system. It was shown that multiplicity of infection is a useful tool for manipulating protein production rates and maximum concentrations in cultures expressing one or two recombinant proteins. Thus, multiplicity of infection can be employed for improving production of rotavirus-like particles. VP2 and VP6 production rates obtained during individual infections remained unchanged when both were simultaneously produced, indicating that such rates can be utilized for estimating protein concentrations during coexpression. Manipulation of the time of infection between the two recombinant baculoviruses, proposed here for the first time, also proved to be effective for controlling the relative protein concentrations. The use of such sequential infections constituted an effective production alternative that does not require high amounts of virus stocks and is easy to implement. In addition to VP2 and VP6, kinetic parameters for the individual production of the other two proteins (VP4 and VP7) that constitute rotavirus-like particles were also obtained.     

Interference of biogenic amines with the measurement of proteins using bicinchoninic acid

The use of bicinchoninic acid (BCA) to measure protein concentrations has received wide acceptance because the reagent is insensitive to many of the buffers, sucrose solutions and detergents used with various tissue and enzyme preparations. However, any compound capable of reducing Cu2+ in an alkaline medium such as biogenic amines will produce a color reaction. The primary objective of this study was to determine whether biogenic amines present in neuronal tissue would interfere with the measurement of protein using the BCA method. Catecholamines were found to produce a linear increase in color of the BCA reagent at concentrations between 1 and 100 nmol/2.1 ml assay volume. Catecholamines appeared to be more sensitive to the BCA reagent than either serotonin or ascorbic acid. Catecholamines at concentrations of 50 nmol/mg of protein or 1 nmol/2.1 ml assay volume or higher will produce significantly (P less than 0.0001) higher color reactions than protein alone. The BCA reagent is not ideal for measuring protein concentrations of intact synaptic vesicles and chromaffin granules since the catecholamine concentrations in these organelles are high enough to increase the color developed by 1.1 to 2.5 times that observed with protein alone. The linearity of the color development produced by catecholamines suggest that BCA could be used to quantitate catecholamine concentrations between 1 and 100 nmol. The BCA reagent will not distinguish between the different catecholamines.     

Messenger ribonucleoprotein complexes in human KB cells infected with adenovirus type 5 contain tightly bound viral-coded '100K' proteins.

Late after infection of KB cells with adenovirus 5 an extra protein becomes associated with messenger ribonucleoprotein particles present in the polysomes. This protein has a molecular weight of 100000 and is identical to the virus coded '100K' protein found previously. The extra protein is firmly attached to the messenger ribonucleoprotein complexes. Its binding resists exposure to high salt concentrations as used in puromycin/high-salt dissociation and equilibrium centrifugation in Cs2SO4 gradients. In this respect it resembles the binding of two other proteins of Mr 74000 and 48000 which are commonly found in messenger ribonucleoprotein particles of various eukaryotic cells. The identity between the messenger ribonucleoprotein protein of Mr 100000 and the "100K' protein present in the soluble part of the cytoplasm was established by sodium dodecylsulphate/polyacrylamide gel electrophoresis, isoelectric focusing and peptide mapping after limited proteolysis with Staphylococcus aureus protease.     

In vitro effect of Triton WR-1339 on canine plasma high density lipoproteins

We studied the effect in vitro of various concentrations of Triton WR-1339 on normolipidemic canine plasma and on the high density lipoproteins (HDL) isolated from this plasma by ultracentrifugation. As a preamble to this study, we established that Triton WR-1339 has a unimer molecular weight of 4,500, a micellar molecular weight of 180,000, and a critical micellar concentration (CMC) of 0.018 mM or 0.008 g/dl. Above its CMC, Triton WR-1339 in concentrations between 2 and 10 mg/ml induced concentration-dependent structural changes in HDL which were characterized by a progressive displacement of apoA-I from the HDL surface without loss of lipids. The addition of Triton WR-1339 to the HDL particles modified their electrophoresis mobility and caused an increase in size (95 +/- 5 A to 114 +/- 7 A). At the extreme Triton WR-1339 concentrations utilized in these studies (10 mg/ml) disruption of the HDL particles occurred; at this stage, the original, relatively homogeneous, spherical HDL particles were replaced by a heterogeneous population ranging in size between 50 and 250 A, representing complexes of Triton WR-1339 with lipids essentially free of apoA-I which could be sedimented by ultracentrifugation. The effects of Triton WR-1339 on whole plasma or isolated HDL were comparable. These studies indicate that Triton WR-1339 in vitro alters HDL in a concentration-dependent manner and that these changes vary from a displacement of apoA-I from the HDL surface to a state where all lipids are solubilized into the Triton WR-1339 micellar phase and are driven away from the protein moiety.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carrier-mediated urea transport across the mitochondrial membrane of an elasmobranch (Raja erinacea) and a teleost (Oncorhynchus mykiss) fish

In osmoregulating teleost fish, urea is a minor nitrogen excretory product, whereas in osmoconforming marine elasmobranchs it serves as the major tissue organic solute and is retained at relatively high concentrations ( approximately 400 mmol/l). We tested the hypothesis that urea transport across liver mitochondria is carrier mediated in both teleost and elasmobranch fishes. Intact liver mitochondria in rainbow trout (Oncorhynchus mykiss) demonstrated two components of urea uptake, a linear component at high concentrations and a phloretin-sensitive saturable component [Michaelis constant (K(m)) = 0.58 mmol/l; maximal velocity (V(max)) = 0.12 mumol.h(-1).mg protein(-1)] at lower urea concentrations (<5 mmol/l). Similarly, analysis of urea uptake in mitochondria from the little skate (Raja erinacea) revealed a phloretin-sensitive saturable transport (K(m) = 0.34 mmol/l; V(max) = 0.054 mumol.h(-1).mg protein(-1)) at low urea concentrations (<5 mmol/l). Surprisingly, urea transport in skate, but not trout, was sensitive to a variety of classic ionophores and respiration inhibitors, suggesting cation sensitivity. Hence, urea transport was measured in the reverse direction using submitochondrial particles in skate. Transport kinetics, inhibitor response, and pH sensitivity were very similar in skate submitochondrial particle submitochondrial particles (K(m) = 0.65 mmol/l, V(max) = 0.058 mumol.h(-1).mg protein(-1)) relative to intact mitochondria. We conclude that urea influx and efflux in skate mitochondria is dependent, in part, on a bidirectional proton-sensitive mechanism similar to bacterial urea transporters and reminiscent of their ancestral origins. Rapid equilibration of urea across the mitochondrial membrane may be vital for cell osmoregulation (elasmobranch) or nitrogen waste excretion (teleost).