Norepinephrine:adenosinetriphosphate ratios in purified adrenergic vesicles.

Norepinephrine (NE):adenosinetriphosphate (ATP) ratios were studied in a highly purified fraction of large dense core vesicles isolated from the bovine splenic nerve. Vesicles prepared from nerves chilled approximately 10 and 30 min post mortem were compared. The NE:ATP molar ratio decreased from 6.3 to 4.8, p less than 0.005; NE decreased from 61 to 42 nmol, while ATP decreased only from 9.6 to 8.8 nmol/mg protein. Animals weighing 180-360 kg were compared with heavier ones weighing 400-700 kg. NE increased from 42 to 68 nmol and ATP increased from 5.9 to 13.2 nmol/mg protein, while the NE:ATP molar ratio decreased from 7.2 to 5.2, p less than 0.005. Changes during vesicle maturation were studied by comparing vesicles identically prepared from equal weights of a proximal nerve segment close to the coeliac ganglion and a distal, intrasplenic segment. NE increased from 45 to 70 nmol while ATP remained unchanged at 10.0 nmol/mg protein and the NE:ATP molar ratio increased from 4.5 to 7.0, p less than 0.005. It was interpreted that vesicle ATP content, like dopamine beta-hydroxylase, was established early in the cell body and remained unchanged during axoplasmic transport. ATP was in a complex which was relatively stable to post mortem hydrolysis at least between 10 and 30 min prior to chilling the nerves. The addition of newly synthesized NE into a readily releasable pool during axoplasmic transport occurs without ATP and can account for the increased ratio above 4:1 in the distal segment vesicles.     

Newly synthesized norepinephrine accumulation in the readily releasable pool of a purified adrenergic vesicle fraction

A highly purified fraction of large dense core adrenergic vesicles was studied after isolation from bovine splenic nerve chilled within 10 to 12 minutes post mortem. In a standard medium containing 5 mM each of Mg⁺⁺ and ATP and 6 μM norepinephrine (NE), this vehicle fraction contained NE in a readily releasable and a more stable pool. When vesicle dopamine β-hydroxylase was activated with 1.33 mM ascorbic acid using 6 μM ¹⁴C-dopamine as substrate at 30°C, ¹⁴C-NE was synthesized at a linear rate during the 45 minute incubation. Net accumulation of NE (p < 0.01) and a proportional net retention of newly synthesized ¹⁴C-NE occurred only when the readily releasable pool could still be demonstrated. The halftime for the fast release pool was doubled from 3 to 6 minutes (p < 0.01) with no effect on the slower released, ATP-facilitated uptake pool. Thus, both during axoplasmic transport and induced NE synthesis $\text{in vitro}$, there is evidence that newly synthesized NE preferentially accumulates in the readily releasable pool, a property also characteristic of the physiologically active pool $\text{in vivo}$.     

ATP-dependent calcium transport in plasma membrane vesicles from neutrophil leukocytes

Plasma membrane vesicles were prepared from guinea pig peritoneal exudate neutrophils, using nitrogen cavitation to rupture the plasma membrane and differential centrifugation to separate the vesicles. The vesicles were enriched 13.2-fold in (Na+, K+)-ATPase activity and had a cholesterol:protein ratio of 0.15, characteristic of plasma membranes. Contamination of the vesicle preparation with DNA or marker enzyme activities for intracellular organelles was very low. Studies designed to determine vesicle sidedness and integrity indicated that 33% were sealed, inside-out; 41% were sealed, right side-out, and 26% were leaky. The vesicles accumulated 45Ca2+ in a linear fashion for 45 min. The uptake was dependent on the presence of oxalate and MgATP in the incubating medium. Uptake showed a Ka for free Ca2+ of 164 nM and a Vmax of 17.2 nmol/mg . min (based on total protein). GTP, ITP, CTP, UTP, ADP, or AMP supported uptake at rates less than or equal to 11% of ATP. Ca2+ uptake was maximal at pH 7-7.5. Calcium stimulated the hydrolysis of ATP by the vesicles with a Ka for free Ca2+ of 440 nM and Vmax of 17.5 nmol/mg . min (based on total protein). When the Ca2+ uptake rate was based upon those vesicles expected to transport Ca2+ (33% sealed, inside-out vesicles) and Ca2+-stimulated ATPase activity was based upon those vesicles expected to express that activity (26% leaky + 33% sealed, inside-out vesicles), the molar stoichiometry of Ca2+ transported:ATP hydrolyzed was 2.12 +/- 0.12. Calmodulin did not increase either Vmax or Ka for free Ca2+ of the uptake system in the vesicles, even when they were treated previously with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The high affinity of this system for Ca2+, specificity for ATP, physiological pH optimum, and stoichiometry of Ca2+ transported:ATP hydrolyzed suggest that it represents an important mechanism by which neutrophils maintain low levels of cytoplasmic free Ca2+.     

ON THE DEVELOPMENT OF SYMPATHETIC NERVE TRUNK VESICLES DURING AXONAL TRANSPORT: DENSITY GRADIENT ANALYSIS OF DOPAMINE β-HYDROXYLASE IN BOVINE SPLENIC NERVE

—Dopamine β-hydroxylase was used as a marker enzyme for sympathetic nerve vesicles which were studied by density gradient technique in bovine splenic nerves. The enzyme analyses were complicated by the occurrence of inhibitors which had to be carefully neutralized with copper. The inhibitor was mainly found in the soluble fraction and no evidence for the occurrence of endogenous inhibitors in the nerve vesicles was obtained. A great variation in density of the dopamine β-hydroxylase containing particles was observed. This was probably mainly due to the variation in vesicle maturation since dopamine β-hydroxylase was distributed more towards the lighter gradient fractions in the proximal nerve segment preparations compared with intrasplenic nerve segment preparations. Noradrenaline/protein and noradrenaline/dopaminc β-hydroxylase ratios were found to be increased about 1·7-fold in the vesicle fraction isolated from the proximal nerve segments to those from the intrasplenic segments. A further increase of the noradrenaline/dopamine β-hydroxylase ratio was observed in a fraction with the same density isolated from the spleen. On the basis of these findings the noradrenaline/protein ratio was calculated to be about 500-600 nmol/mg in the nerve terminal vesicles.     

Acetylcholine, ATP, and Proteoglycan Are Common to Synaptic Vesicles Isolated from the Electric Organs of Electric Eel and Electric Catfish as Well as from Rat Diaphragm

Cholinergic synaptic vesicles were isolated from the electric organs of the electric eel (Electrophorus electricus) and the electric catfish (Malapterurus electricus) as well as from the diaphragm of the rat by density gradient centrifugation followed by column chromatography on Sephacryl-1000. This was verified by both biochemical and electron microscopic criteria. Differences in size between synaptic vesicles from the various tissue sources were reflected by their elution pattern from the Sephacryl column. Specific activities of acetylcholine (ACh; in nmol/mg of protein) of chromatography-purified vesicle fractions were 36 (electric eel), 2 (electric catfish), and 1 (rat diaphragm). Synaptic vesicles from all three sources contained ATP in addition to ACh (molar ratios of ACh/ATP, 9-12) as well as binding activity for an antibody raised against Torpedo cholinergic synaptic vesicle proteoglycan. Synaptic vesicles from rat diaphragm contained binding activity for the monoclonal antibody asv 48 raised against a rat brain 65-kilodalton synaptic vesicle protein. Antibody asv 48 binding was absent from electric eel and electric catfish synaptic vesicles. These antibody binding results, which were obtained by a dot blot assay on isolated vesicles, directly correspond to the immunocytochemical results demonstrating fluorescein isothiocyanate staining in the respective nerve terminals. Our results imply that ACh, ATP, and proteoglycan are common molecular constituents of motor nerve terminal-derived synaptic vesicles from Torpedo to rat. In addition to ACh, both ATP and proteoglycan may play a specific role in the process of cholinergic signal transmission.     

Impaired function of alpha2-adrenergic autoreceptors on sympathetic nerves associated with mesenteric arteries and veins in DOCA-salt hypertension

The present study tested the hypothesis that there is impaired function of alpha(2)-adrenergic autoreceptors and increased transmitter release from sympathetic nerves associated with mesenteric arteries and veins from DOCA-salt rats. High-performance liquid chromatography was used to measure the overflow of ATP and norepinephrine (NE) from electrically stimulated mesenteric artery and vein preparations in vitro. In sham arteries, nerve stimulation evoked a 1.5-fold increase in NE release, whereas in DOCA-salt arteries there was a 3.9-fold increase in NE release over basal levels (P < 0.05). In contrast, stimulated ATP release was not different in DOCA-salt arteries compared with sham arteries. In sham veins, nerve stimulation evoked a 2.9-fold increase in NE release, whereas in DOCA-salt veins there was a 8.4-fold increase in NE release over basal levels (P < 0.05). In sham rats NE release, normalized to basal levels, was greater in veins than in arteries (P < 0.05). The alpha(2)-adrenergic receptor antagonist yohimbine (1 microM) increased ATP and NE release in sham but not DOCA-salt arteries. The alpha(2)-adrenergic receptor agonist UK-14304 (10 microM) decreased ATP release in sham but not DOCA-salt arteries. In sham veins, UK-14304 decreased, but yohimbine increased, NE release; effects that were not observed in DOCA-salt veins. These data show that nerve stimulation causes a greater increase in NE release from nerves associated with veins compared with arteries. In addition, impairment of alpha(2)-adrenergic autoreceptor function in sympathetic nerves associated with arteries and veins from DOCA-salt rats results in increased NE release.     

Preparation and Characterization of Gas-filled Liposomes: Can They Improve Oil Recovery?

Although well known for delivering various pharmaceutical agents, liposomes can be prepared to entrap gas rather than aqueous media and have the potential to be used as pressure probes in magnetic resonance imaging (MRI). Using these gas-filled liposomes (GFL) as tracers, MRI imaging of pressure regions of a fluid flowing through a porous medium could be established. This knowledge can be exploited to enhance recovery of oil from the porous rock regions within oil fields. In the preliminary studies, we have optimized the lipid composition of GFL prepared using a simple homogenization technique and investigated key physico-chemical characteristics (size and the physical stability) and their efficacy as pressure probes. In contrast to the liposomes possessing an aqueous core which are prepared at temperatures above their phase transition temperature (T_c), homogenization of the phospholipids such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocoline (DSPC) in aqueous medium below their T_c was found to be crucial in formation of stable GFL. DSPC based preparations yielded a GFL volume of more than five times compared to their DPPC counter part. Although the initial vesicle sizes of both DSPC and DPPC based GFL were about 10 μm, after 7 days storage at 25°C, the vesicle sizes of both formulations significantly (p < 0.05) increased to 28.3 ± 0.3 μm and 12.3 ± 1.0 μm, respectively. When the DPPC preparation was supplemented with cholesterol at a 1:0.5 or 1:1 molar ratio, significantly (p < 0.05) larger vesicles were formed (12–13 μm), however, compared to DPPC only vesicles, both cholesterol supplemented formulations displayed enhanced stability on storage indicating a stabilizing effect of cholesterol on these gas-filled vesicles. In order to induce surface charge on the GFL, DPPC and cholesterol (1: 0.5 molar ratio) liposomes were supplemented with a cationic surfactant, stearylamine, at a molar ratio of 0.25 or 0.125. Interestingly, the ζ potential values remained around neutrality at both stearylamine ratios suggesting the cationic surfactant was not incorporated within the bilayers of the GFL. Microscopic analysis of GFL confirmed the presence of spherical structures with a size distribution between 1–8 μm. This study has identified that DSPC based GFL in aqueous medium dispersed in 2% w/v methyl cellulose although yielded higher vesicle sizes over time were most stable under high pressures exerted in MRI.     

Dialysate dihydroxyphenylglycol as a window for in situ axoplasmic norepinephrine disposition

To examine basal axoplasmic norepinephrine (NE) kinetics at the in situ cardiac sympathetic nerve ending, we applied a dialysis technique to the heart of anesthetized cats and performed the dialysate sampling with local administration of a pharmacological tool through a dialysis probe. The dialysis probe was implanted in the left ventricular wall, and dihydroxyphenylglycol (DHPG, an index of axoplasmic NE) levels were measured by liquid chromatogram-electrochemical detection. Control dialysate DHPG levels were 161+/-19 pg/ml. Pargyline (monoamine oxidase inhibitor, 1 mM) decreased the dialysate DHPG levels to 38+/-10 pg/ml. Further alpha-methyl-para-tyrosine, omega-conotoxin GVIA, desipramine (NE synthesis, release and uptake blockers) decreased the dialysate DHPG levels to 64+/-19, 106+/-15, 110+/-22 pg/ml, respectively. In contrast, reserpine (vesicle NE transport inhibitor, 10 microM) increased the dialysate DHPG levels to 690+/-42 pg/ml. Thus, NE synthesis, metabolism and recycling (release, uptake and vesicle transport) affected basal intraneuronal NE disposition at the nerve endings. Measurement of DHPG levels through a dialysis probe provides information about basal intraneuronal NE disposition at the cardiac sympathetic nerve endings. Yohimbine (alpha(2)-adrenoreceptor blocker, 10 microM) and U-521 (catechol-O-methyltransferase blocker, 100 microM) did not alter the dialysate DHPG levels. Furthermore, there were no significant differences in the reserpine induced DHPG increment between the presence and absence of desipramine (10 microM) or alpha-methyl-para-tyrosine (100 mg/kg i.p.). These results may be explained by the presence of two axoplasmic pools of NE, filled by NE taken up and synthesized, and by NE overflow from vesicle. The latter pool of NE may be closed to the monoamine oxidase system in the axoplasma.     

Characteristics and regulation of active calcium transport in inside-out red cell membrane vesicles

Sealed, inside-out human red cell membrane vesicles, prepared by a modified method of Steck (Steck T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed.), Vol 2, pp. 245–281, Plenum Press, New York), accomplish an ATP and Mg²⁺-dependent uphill calcium uptake with a reproducible maximum rate of 12–15 nmol/mg vesicle protein per min under physiological conditions. This maximum rate is increased by about 60–70% in the presence of a heatstable cytoplasmic activator protein (calmodulin) obtained from red cells. Calcium efflux from inside-out vesicles is smaller than 0.01 nmol/mg vesicle protein per min at intravesicular calcium concentrations between 0.1 and 20.0 mM.In the presence of Mg²⁺, active calcium uptake is supported by ATP, ITP, or UTP, but not by ADP, AMP, or p-nitrophenyl phosphate. The optimum pH for the process is 7.4–7.6, and the activation energy is 19–20 kcal/mol, irrespective of the presence or absence of calmodulin. Calcium uptake in inside-out vesicles is unaffected by ouabain or oligomycin, but blocked by low concentrations of lanthanum, ruthenium red, quercetin and phloretin. K⁺ and Na⁺, when compared to choline⁺ or Li⁺, significantly increase active calcium uptake. This stimulation by K⁺ and Na⁺ is independent of that by calmodulin.Concentrated red cell cytoplasm activates calcium uptake at low soluble protein:membrane protein ratios, while a ‘deactivation’ of the transport occurs at high cytoplasm: membrane protein ratios. A heat-labile cytoplasmic protein fraction antagonizing calmodulin activation, can be separated by DEAE-Sephadex chromatography. Based on these findings the regulation of active calcium transport in human red cells is discussed.     

A vesicular sequestration to oxidative deamination shift in myocardial sympathetic nerves in Parkinson's disease

In Parkinson's disease (PD), profound putamen dopamine (DA) depletion reflects denervation and a shift from vesicular sequestration to oxidative deamination of cytoplasmic DA in residual terminals. PD also involves cardiac sympathetic denervation. Whether PD entails myocardial norepinephrine (NE) depletion and a sequestration–deamination shift have been unknown. We measured apical myocardial tissue concentrations of NE, DA, and their neuronal metabolites 3,4‐dihydroxyphenylglycol (DHPG), and 3,4‐dihydroxyphenylacetic acid (DOPAC) from 23 PD patients and 23 controls and ascertained the extent of myocardial NE depletion in PD. We devised, validated in VMAT2‐Lo mice, and applied 5 neurochemical indices of the sequestration–deamination shift—concentration ratios of DOPAC:DA, DA:NE, DHPG:NE, DOPAC:NE, and DHPG:DOPAC—and used a kinetic model to estimate the extent of the vesicular storage defect. The PD group had decreased myocardial NE content (p < 0.0001). The majority of patients (70%) had severe NE depletion (mean 2% of control), and in this subgroup all five indices of a sequestration–deamination shift were increased compared to controls (p < 0.001 for each). Vesicular storage in residual nerves was estimated to be decreased by 84–91% in this subgroup. We conclude that most PD patients have severe myocardial NE depletion, because of both sympathetic denervation and decreased vesicular storage in residual nerves.

     

Interaction of cationic vesicle with ribonucleotides (AMP, ADP, and ATP) and physicochemical characterization of DODAB/ribonucleotides complexes

The interaction between ribonucleotides (AMP, ADP, and ATP) and cationic vesicles prepared from dioctadecyldimethylammonium bromide (DODAB) were investigated in detail. The physicochemical properties of ribonucleotides/cationic lipid complexes were present. Gel exclusion-UV spectroscopic results showed that all the charge ratios of DODAB/ribonucleotides (AMP, ADP, and ATP) are 2:1 when the maximal ribonucleotides were adsorbed onto DODAB, while the molar ratios were different, e.g., 2:1 for DODAB/AMP, 4:1 for DODAB/ADP and 6:1 for DODAB/ATP. These differences may be attributed to the different anion charges of AMP, ADP and ATP. The results demonstrated that ribonucleotides combined with DODAB vesicles with the electrostatic attraction in the complexation of DODAB and ribonucleotides. Transmission electron microscopic results revealed the different extents of aggregation of cationic vesicles in the complexation process of ribonucleotides with cationic lipid. The variation dependence of zeta-potentials or electrophoretic mobilities on vesicle size was also different. The zeta-potentials and electrophoretic mobilities of the DODAB vesicles (0.01 and 0.02 mM) gradually decreased when the ribonucleotide concentration increased. However, the mean diameters of the DODAB vesicles (0.1 and 0.5 mM) gradually increased when the ribonucleotide concentration increased.     

Rapid changes in synaptic vesicle cytochemistry after depolarization of cultured cholinergic sympathetic neurons

Sympathetic neurons taken from rat superior cervical ganglia and grown in culture acquire cholinergic function under certain conditions. These cholinergic sympathetic neurons, however, retain a number of adrenergic properties, including the enzymes involved in the synthesis of norepinephrine (NE) and the storage of measurable amounts of NE. These neurons also retain a high affinity uptake system for NE; despite this, the majority of the synaptic vesicles remain clear even after incubation in catecholamines. The present study shows, however, that if these neurons are depolarized before incubation in catecholamine, the synaptic vesicles acquire dense cores indicative of amine storage. These manipulations are successful when cholinergic function is induced with either a medium that contains human placental serum and embryo extract or with heart-conditioned medium, and when the catecholamine is either NE or 5-hydroxydopamine. In some experiments, neurons are grown at low densities and shown to have cholinergic function by electrophysiological criteria. After incubation in NE, only 6% of the synaptic vesicles have dense cores. In contrast, similar neurons depolarized (80 mM K+) before incubation in catecholamine contain 82% dense-cored vesicles. These results are confirmed in network cultures where the percentage of dense-cored vesicles is increased 2.5 to 6.5 times by depolarizing the neurons before incubation with catecholamine. In both single neurons and in network cultures, the vesicle reloading is inhibited by reducing vesicle release during depolarization with an increased Mg++/Ca++ ratio or by blocking NE uptake either at the plasma membrane (desipramine) or at the vesicle membrane (reserpine). In addition, choline appears to play a competitive role because its presence during incubation in NE or after reloading results in decreased numbers of dense-cored vesicles. We conclude that the depolarization step preceding catecholamine incubation acts to empty the vesicles of acetylcholine, thus allowing them to reload with catecholamine. These data also suggest that the same vesicles may contain both neurotransmitters simultaneously.     

Effect of cytochrome b5 on the size, density, and permeability of phosphatidylcholine vesicles.

Cytochrome b5, isolated from rabbit liver by a procedure using detergent, was incubated with phosphatidylcholine bilayer vesicles at 37 degrees for 30 min. A comparison of a number of physical properties was made between the cytochrome b5-phosphatidylcholine complex (at a molar ratio of 1:1000) and the phosphatidylcholine vesicles. The binding of the protein to the vesicle caused no aggregation and no detectable change in Stokes radius of the vesicle as monitored by gel filtration. Only small increases in s20 (from 2.67 up to 3.82 X 10(-13) s) and density (from 1.025 up to 1.042 g ml(-1)) were observed upon binding of the cytochrome b5 to phosphatidylcholine vesicles. At molar ratios of 5:1000, and above, two types of complexes could be detected by sucrose density gradient centrifugation: one had a molar ratio of approximately 1.066 g ml(-1)) the other, a more constant ratio of 20:1000 (density greater than 1.107 g ml(-1)). Cytochrome b5 was also incubated with phosphatidylcholine vesicles prepared with ferricyanide trapped inside. The leakage of the ferricyanide from inside the vesicles was increased when cytochrome b5 was present, but the vesicles, although leaking, were not completely depleted of their ferricyande, and so must still be intact. It is suggested that at molar ratios of cytochrome b5 to phosphatidylcholine below 5:1000, the binding of the protein causes minimal change in vesicle structure.     

Effect of lipid composition on the calcium/adenosine 5'-triphosphate coupling ratio of the Ca2+-ATPase of sarcoplasmic reticulum

The Ca2+-ATPase of sarcoplasmic reticulum was purified and depleted of proteolipids by solubilization in Triton X-100 and by fractionation on a DE-52 column. The protein reconstituted by deoxycholate-cholate dialysis at low lipid to protein ratios (2-5 mg of lipid/mg of protein), with either dioleoylphosphatidylethanolamine or monogalactosyldiglyceride, exhibited high initial rates of ATP-dependent Ca2+ uptake [300-900 nmol min-1 (mg of protein)-1] and coupling ratios (Ca2+ transported/ATP hydrolyzed) up to 1.2. Ca2+-ATPase reconstituted with lipids of increasing degrees of methylation (dioleoylphosphatidylethanolamine, dioleoylmonomethylphosphatidylethanolamine, dioleoyldimethylphosphatidylethanolamine and dioleoylphosphatidylcholine) or increasing degrees of glycosylation (monogalactosyldiglyceride and digalactosyldiglyceride) revealed a progressive decrease in both ATP-dependent Ca2+-uptake and coupling ratios. The rate and extent of Ca2+ uptake decreased as the dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine or monogalactosyldiglyceride/dioleoylphosphatidylcholine molar ratios in the reconstituted vesicles were reduced. Vesicles reconstituted with high molar ratios of dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine or monogalactosyldiglyceride/dioleoylphosphatidylcholine and at a high lipid to protein ratio became leaky and released the Ca2+ accumulated inside the vesicles when the temperature of the incubation mixture was increased (e.g., from 20 to 37 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine triphosphate. A constituent of cholinergic synaptic vesicles

1. Synaptic vesicles separated by density-gradient centrifugation from extracts of the cholinergic nerve terminals of the electric organ of Torpedo marmorata were found to contain appreciable amounts of ATP as well as acetylcholine. 2. Vesicular ATP was stable in the presence of concentrations of apyrase and myokinase that rapidly destroyed equivalent amounts of endogenous or added free ATP; pre-treatment of cytoplasmic extracts of electric tissue with these enzymes destroyed endogenous free ATP, but did not affect the vesicular ATP. 3. When [U-(14)C]ATP was added to electric tissue at the time of comminution and extraction of the vesicles, all the radioactivity was associated with soluble components in the subsequent fractionation: none was associated with vesicles or membrane fragments; thus it is unlikely that vesicular ATP can be accounted for by the sequestration of endogenous free ATP within any vesicles formed during comminution and extraction of the tissue. 4. When synaptic vesicles were passed through iso-osmotic columns of Bio-Gel A-5m, which separates vesicles from soluble proteins and small molecules, all the recovered ATP and acetylcholine passed through together in the void volume. 5. Regression analysis showed that vesicular ATP content was highly correlated with vesicular acetylcholine content in different experiments, the molar ratio acetylcholine/ATP being 5.32+/-(s.e.m.) 0.45 (21 expts.) for the peak density-gradient fraction. The ratio varied, however, somewhat across the density-gradient peak suggesting some degree of chemical heterogeneity in the vesicle population.     

Reaction centers fromRhodopseudomonas sphaeroides in reconstituted phospholipid vesicles. I. Structural studies

Reaction centers (RCs) fromRhodopseudomonas sphaeroides were reconstituted into asolectin vesicles by cosonication. Equilibrium centrifugation on sucrose gradients showed that the vesicles were homogeneous in density (i.e., lipid-to-protein ratio) when reconstituted at a molar lipid-to-protein ratio between 500 to 1000. At lower ratios, a considerable fraction of RCs was not incorporated into closed vesicles, while at higher ratios, an increasing population of liposomes was protein-free. The average vesicle size decreased with increasing lipid-to-protein ratio, exhibiting considerable size heterogeneity within a sample. The average diameter of the largest and smallest population of vesicles, reconstituted at a molar lipid-to-protein ratio of 560, was 1200 and 400 nm, respectively. The orientation of reconstituted RCs with respect to the plane of the membrane was determined from the flash-induced rereduction kinetics of the special-pair bacteriochlorophyll dimer in the presence of reduced cytochromec. The predominant orientation of RCs was such that the cytochromec binding sites faced the external medium. The net orientation of RCs in reconstituted vesicles decreased with vesicle size and was strongly influenced by the ionic strength during reconstitution.Abbreviations RC reaction center - LDAO lauryldimethylamine-N-oxide - UQ₀/UQ₀H₂ oxidized and reduced form of 2,3-dimethoxy-5-methyl-1,4-benzoquinone - CCCP carbonyl-cyanide-trichloromethoxy phenylhydrazone - D/D⁺ reduced and oxidized form of the primary electron donor of the reaction centers. During the course of this study K. J. H. was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). This research was supported by grants from the National Institutes of Health (EY-02084) and from the Office of Naval Research (ONR-NOOO 14-79-C 0798) to M. Montal.     

Stable complexes of axoplasmic vesicles and microtubules: protein composition and ATPase activity

Fast transport of axonal vesicles and organelles is a microtubule-associated movement (Griffin, J. W., K. E. Fahnestock, L. Price, and P. N. Hoffman, 1983, J. Neuroscience, 3:557-566; Schnapp, B. J., R. D. Vale, M. P. Sheetz, and T. S. Reese, 1984, Cell, 40:455-462; Allen, R. D., D. G. Weiss, J. H. Hayden, D. T. Brown, H. Fujiwake, and M. Simpson, 1985, J. Cell Biol., 100:1736-1752). Proteins that mediate the interactions of axoplasmic vesicles and microtubules were studied using stable complexes of microtubules and vesicles (MtVC). These complexes formed spontaneously in vitro when taxol-stabilized microtubules were mixed with sonically disrupted axoplasm from the giant axon of the squid Loligo pealei. The isolated MtVCs contain a distinct subset of axoplasmic proteins, and are composed primarily of microtubules and attached membranous vesicles. The MtVC also contains nonmitochondrial ATPase activity. The binding of one high molecular mass polypeptide to the complex is significantly enhanced by ATP or adenyl imidodiphosphate. All of the axoplasmic proteins and ATPase activity that bind to microtubules are found in macromolecular complexes and appear to be vesicle-associated. These data allow the identification of several vesicle-associated proteins of the squid giant axon and suggest that one or more of these polypeptides mediates vesicle binding to microtubules.     

ATP-dependent and drug-inhibited vesicle enlargement reconstituted using synthetic lipids and recombinant proteins

A recombinant ECTO-NOX (tNOX) and a recombinant plasma membrane associated AAA-ATPase (ATPase Associated with Different Cellular Activities) were combined in stoichiometric proportions into liposomes together with albumin as a source of protein thiols. Large lamellar vesicles were formed from phosphatidylcholine, cholesterol and dicetyl phosphate in a molar ratio of 50:45:5, where the phosphatidylcholine was a 2:1 mixture of synthetic dimyristoyl and dipalmitoyl phosphatidylcholines. The lipids were dried to a film and reconstituted into vesicles by resuspension in buffer containing the recombinant proteins in equimolar ratios of 0.04 nmoles/mg lipid. In the presence of ATP, these vesicles enlarged in an ATP-dependent manner based on light-scattering measurements. Because the drug-inhibited ECTO-NOX protein, tNOX was utilized, the enlargement was inhibited by capsaicin, a quinone site tNOX inhibitor specific for tNOX. With the lipid vesicle systems, the recombinant ECTO-NOX, the recombinant AAA-ATPase, a source of protein thiols and ATP all were required. In control experiments, no ATP-dependent vesicle enlargement was observed with the AAA-ATPase or the ECTO-NOX protein alone. Also addition of ATP was without any effect when only the single proteins were incorporated into the lipid vesicles. A model has been developed whereby the plasma membrane AAA-ATPase is linked via disulfide bonds, formed and broken by the ECTO-NOX protein, to membrane structural proteins. Binding of ATP and subsequent hydrolysis and release of ADP would advance the ATPase hexamer ratchet thereby both thinning the membrane and increasing the vesicle surface.     

Effects of dietary amylose and amylopectin ratio on growth performance,meat quality,postmortem glycolysis and muscle fibre type transformation of finishing pigs

The present study was conducted to investigate the effects of dietary amylose and amylopectin ratio on growth performance, meat quality, postmortem glycolysis and muscle fibre type transformation of finishing pigs. Twenty-four barrows (Duroc × Landrace × Yorkshire) with an average initial body weight of 61.7 ± 2.01 kg were randomly assigned to four dietary treatments with amylose: amylopectin ratios of 1:1 (HD), 1:2 (MD), 1:3 (CD) and 1:4 (LD). The results showed that the average daily weight gain of finishing pigs tended to reduce with the ratio of amylose and amylopectin decreased (p = 0.09). Diet LD increased the pH_24h value and decreased the shear force in longissimus dorsi (LM) compared with diet HD (p < 0.05). Diet LD decreased the lactate content and the HK-2 mRNA abundance and increased the mRNA abundance of ATP5B in LM compared with diet HD (p < 0.05). Higher mRNA abundance of MyHC I and lesser abundance of MyHC IIb in LM were found in pigs fed diet CD and LD than those fed diet HD (p < 0.05). Furthermore, pigs fed diet LD had higher mRNA abundances of PGC-1α and PPAR δ in LM than other groups (p < 0.05). These results suggested that diet with low amylose and amylopectin ratio could improve meat quality of finishing pigs via delaying muscle glycolysis capacity and shifting muscle fibre types.     

Ovarian steroidogenic enzyme activities during the rat estrous cycle

We have correlated the concentrations of serum LH, estradiol and progesterone with the activities of 2 ovarian steroid biosynthetic enzymes during the rat estrous cycle. Ovarian 3 β-hydroxysteroid dehydrogenase isomerase (3-βHSD) activity decreased from 29 ± 6 nmol/mg protein/ min (mean ± SEM) in diestrus, to 7 ± 0.4 nmol/mg protein/min in late proestrus (p < 0.005), and subsequently increased to 36 ± 9 nmol/mg protein/min in metestrus (p < 0.01). Ovarian 17-hydroxylase (17-OH) activity decreased from early to late proestrus (3.3 ± 0.2 vs 2.2 ± 0.2 nmol/mg protein/min, p <0.0025), and subsequently increased to 3.9 ± 0.2 in metestrus (p<0.001). Serum LH, estradiol and progesterone peaked during proestrus, and reached a nadir during estrus. We conclude that the activities of 3-βHSD and 17-OH in the rat ovary vary markedly during the estrous cycle. These changes may underlie the pattern of steroid secretion characteristic of this process.