PubMed Health⌕ Search

Biomedical subjects

L Corazzi

Publications and source records attributed to L Corazzi.

At least 19 recordsLinked to original sources
PubMed2004

Binding and release of cytochrome c in brain mitochondria is influenced by membrane potential and hydrophobic interactions with cardiolipin.

Factors influencing the release and anchorage of cytochrome c to the inner membrane of brain mitochondria have been investigated. Metabolic activity of mitochondria caused a decrease in the membrane potential delta psi(m), accompanied by detachment of the protein from the inner membrane. In a model system of cytochrome c reconstituted in cardiolipin (CL) liposomes, phosphate was used to breach the hydrophilic lipid-protein interactions. About 44% cytochrome c was removable when heart CL (80% 18:2n-6) was employed, whereas the remaining protein accounted for the tightly bound conformation characterized by hydrophobic lipid-protein interactions. Cytochrome c release from brain CL liposomes was higher compared to heart CL, consistent with lower polyunsaturated fatty acid content. The release was even higher with CL extracted from metabolically stressed mitochondria, exhibiting more saturated fatty acid profile compared to control (30% vs. 17%). Therefore, weakening of the hydrophobic interactions due to saturation of CL may account for the observed cytochrome c release from mitochondria following metabolic stress. Moreover, mitochondria enriched with polyunsaturated CL exhibited higher delta psi(m), compared to less unsaturated species, suggesting that CL fatty acid composition influences delta psi(m). Mitochondria incorporated exogenous cytochrome c without protease-sensitive factors or delta psi(m). The internalized protein anchored to the inner membrane without producing swelling, as monitored by forward and side light scattering, but produced delta psi(m) consumption, suggesting recovery of respiratory activity. The delta psi(m) decrease is ascribed to a selected mitochondrial population containing the incorporated cytochrome c.

Animals↗
PubMed2001

Acidic pH generated by H+-ATPase pumps triggers the activity of a fusogenic protein associated with rat liver endoplasmic reticulum.

Fusogenic protein (FP) is a glycoprotein ( approximately 50 kDa), previously purified by us from rat liver endoplasmic reticulum, which explicates fusogenic activity at acidic pH in vitro. To suggest a possible role of FP in membrane fusion, the topology of the protein in the membrane and the conditions in which FP is operating in microsomes have been investigated. Anti-FP polyclonal antibodies inhibited pure FP activity, but not the protein activity in microsomes, suggesting interaction of antibodies with a part of FP concealed in intact membranes. FP activity in microsomes was lost after treatment with Pronase. Western blot analysis of Pronase-treated microsomes showed that the proteolysis removed a fragment ( approximately 5 kDa). This fragment is exposed on the outer surface of microsomes and involved in fusogenic activity, whereas the largest part of FP is embedded in microsomal vesicles. Therefore, FP can be affected by modifications on the cytosolic and luminal sides of microsomal membranes. Indeed, when microsomal lumen was acidified by H+-ATPase activity, binding and fusion of fluorescent labelled liposomes to microsomes occurred. Direct involvement of FP in the fusogenic event was observed by reconstituting pure FP in liposomes with a preformed H+ gradient. FP triggered a fusion process in response to the acidic interior of liposomes, despite an exterior 7.4 pH unable to promote fusogenic protein activity. As intracellular membrane fusion occurs at neutral pH involving the cytosolic sides of membranes, FP may participate in this event by exploiting the acidic pH formed in the lumen of endoplasmic reticulum through H+-translocating ATPase activity.

Adenosine Triphosphate↗
PubMed2000

Respiratory state and phosphatidylserine import in brain mitochondria in vitro.

The mechanism of phosphatidylserine (PS) movement from donor membranes into rat brain mitochondria was investigated. Mitochondria were incubated with liposomes and subjected to density gradient centrifugation. The energized state was monitored by flow cytometry measuring the fluorescence of membrane-potential-sensitive rhodamine-123 dye. Mitochondria density decreased upon increase of the respiratory rate, as a consequence of their association with liposomes. After interaction of mitochondria with (14)C-PS containing liposomes, (14)C-PS became a substrate of PS decarboxylase, as monitored by the formation of (14)C-phosphatidylethanolamine (PE), indicating translocation of (14)C-PS to the inner membrane. The kinetics of (14)C-PE formation showed a high rate upon addition of ADP, malate and pyruvate (state 3) compared to control (state 1). In state 3, (14)C-PE formation decreased in the presence of NaN(3). Mitochondria-associated membranes (MAM) are the major site of PS synthesis. However, their role in the translocation of PS to mitochondria has not been completely elucidated. A crude mitochondrial fraction (P(2)) containing MAM, synaptosomes and myelin was prelabeled with (14)C-PS and incubated in different respiratory states. At a high respiratory rate, low-density labeled mitochondria, whose band overlaps that of synaptosomes, were obtained by centrifugation. A parallel decrease of both radioactivity and protein in MAM fraction was observed, indicating that the association of MAM and mitochondria had occurred. Synthesis and translocation of (14)C-PS in P(2) membranes were also studied by incubating P(2) with (14)C-serine. In the resting state (14)C-PS accumulated in MAM, indicating that the transfer to mitochondria was a limiting step. In state 3 both the transfer rate of (14)C-PS and its conversion to (14)C-PE increased. Respiratory mitochondrial activity modulated the association of MAM and mitochondria, triggering a mechanism that allowed the transport of PS across the outer mitochondrial membrane.

Adenosine Diphosphate↗
PubMed1998

A glycoprotein from rat liver endoplasmic reticulum promotes both aggregation and fusion of liposomes at acidic pH.

Low-pH-induced fusion of liposomes with rat liver endoplasmic reticulum was evidenced. Fusion was inactivated by treatment of microsomes with trypsin or EEDQ (N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline), indicating the involvement of a protein. The protein was purified 555-fold by chromatographic steps. The identification and purification to homogeneity was obtained by electroelution from a slab gel, which gave a still active protein of about 50 kDa. The protein promoted the fusion of liposomes; laser light scattering showed an increase of mean radius of vesicles from 60 up to about 340 nm. Fusion was studied as mass action kinetics, describing the overall fusion as a two-step sequence of a second order aggregation followed by a first order fusion of liposomes. For phosphatidylcholine containing liposomes aggregation was not rate-limiting at pH 5.0 and fusion followed first order kinetics with a rate constant of 13 . 10(-3) sec-1. For phosphatidylethanolamine/phosphatidic acid liposomes aggregation was rate-limiting; however, the overall fusion was first order process, suggesting that fusogenic protein influences both aggregation and fusion of liposomes. The protein binds to the lipid bilayer of liposomes, independently of pH, probably by a hydrophobic segment. Exposed carboxylic groups might be able to trigger pH-dependent aggregation and fusion. It is proposed that the protein inserted in the lipid bilayer bridges with an adjacent liposome forming a fused doublet. Since at endoplasmic reticulum level proton pumps are operating to generate a low-pH environment, the membrane bound fusogenic protein may be responsible for both aggregation and fusion of neighboring membranes and therefore could operate in the exchange of lipidic material between intracellular membranes.

Animals↗
PubMed1997

Phosphatidylserine translocation into brain mitochondria: involvement of a fusogenic protein associated with mitochondrial membranes.

Data reported in the literature indicate that lipid movement between intracellular organelles can occur through contacts and close physical association of membranes (Vance, J.E. 1990. J Biol Chem 265: 7248-7256). The advantage of this mechanism is that the direct interaction of membranes provides the translocation event without the involvement of lipid-transport systems. However, pre-requisite for the functioning of this machinery is the presence of protein factors controlling membrane association and fusion. In the present work we have found that liposomes fuse to mitochondria at acidic pH and that the pre-treatment of mitochondria with pronase inhibits the fusogenic activity. Mixing of 14C-phosphatidylserine (PS) labeled liposomes with mitochondria at pH 6.0 results in the translocation of 14C-PS into mitochondria and in its decarboxylation to 14C-phosphatidylethanolamine through the PS decarboxylase activity localized on the outer surface of the inner mitochondrial membrane. Incorporation of 14C-PS is inhibited by the pre-treatment of mitochondria with pronase or with EEDQ, a reagent for the derivatization of the protonated form of carboxylic groups. These results indicate the presence of a protein associated with mitochondria which is able to trigger the fusion of liposomes to the mitochondrial membrane. A partial purification of a mitochondrial fusogenic glycoprotein is described in this work. The activity of the fusogenic protein appears to be dependent on the extent of protonation of the residual carboxylic groups and is influenced by the glucidic moiety, as demonstrated by its interaction with Concanavalin A. The purified protein is able to promote the recover of the 14C-PS import from liposomes to pronase-treated mitochondria. Therefore, the protein is candidate to be an essential component in the machinery for the mitochondrial import of PS.

Animals↗
PubMed1995

Import of phosphatidylethanolamine for the assembly of rat brain mitochondrial membranes.

Mitochondria can synthesize phosphatidyl-ethanolamine (PE) through phosphatidylserine decarboxylase (PS decarboxylase) activity or can import this lipid from the endoplasmic reticulum. In this work, we studied the factors influencing the import of PE in brain mitochondria and its utilization for the assembly of mitochondrial membranes. Incubation of rat brain homogenate with [1-3H]ethanolamine resulted in the synthesis and distribution of 3H-PE to subcellular fractions. Twenty-one percent of labeled PE was recovered in purified mitochondria. The import of PE in mitochondria was studied in a reconstituted system made of microsomes (donor particles) and purified mitochondria (acceptor particles). Ca2+ and nonspecific lipid transfer protein purified from liver tissue (nsL-TP) enhanced the translocation process. 3H-PE synthesized in membrane associated to mitochondria (MAM) could also translocate to mitochondria in the reconstituted system. Exposure of mitochondria to trinitrobenzensulfonic acid (TNBS) resulted in the reaction of more than 60% of 3H-PE imported from endoplasmic reticulum and of about 25% of 14C-PE produced in mitochondria by decarboxylation of 14C-PS. Moreover, the removal of the outer mitochondrial membrane by digitonin treatment, resulted in the loss of 3H-PE, but not 14C-PE. These results indicate that labeled PE imported in mitochondria is mainly localized in the outer mitochondrial membrane, whereas PE produced by PS decarboxylase activity is confined to the inner mitochondrial membrane. Phospholipase C hydrolyzed 25-30% of both PE radioactivity and mass of the outer mitochondrial membrane indicating an asymmetrical distribution of this lipid across the membrane.

Animals↗
PubMed1995

[3H]aniracetam binds to specific recognition sites in brain membranes.

[3H]Aniracetam bound to specific and saturable recognition sites in membranes prepared from discrete regions of rat brain. In crude membrane preparation from rat cerebral cortex, specific binding was Na+ independent, was still largely detectable at low temperature (4 degrees C), and underwent rapid dissociation. Scatchard analysis of [3H]aniracetam binding revealed a single population of sites with an apparent KD value of approximately 70 nM and a maximal density of 3.5 pmol/mg of protein. Specifically bound [3H]aniracetam was not displaced by various metabolites of aniracetam, nor by other pyrrolidinone-containing nootropic drugs such as piracetam or oxiracetam. Subcellular distribution studies showed that a high percentage of specific [3H]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at least some [3H]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37 degrees C under fast shaking (a procedure that substantially reduced the amount of glutamate trapped in the membranes) and could be restored after addition of either glutamate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [3H]aniracetam binding in unwashed membranes. High levels of [3H]aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A fusogenic protein from rat brain microsomal membranes: partial purification and reconstitution into liposomes.

The procedures for purification and reconstitution of rat brain microsomal membrane protein that causes fusion of liposomes at acidic pH are described. A 1,860-fold purification was achieved, starting from the detergent-solubilized microsomal membranes. The fusion process was assayed spectrofluorimetrically by monitoring the formation of terbium-dipicolinic acid complex (Wilschut, J. et al. 1980. Biochemistry 19:6011-6021) evoked by the protein after mixing of two populations of liposomes. The fusogenic activity of the protein inserted into the membrane of Tb3+-containing vesicles was found to be strongly dependent on phospholipid composition and was higher in vesicles enriched with exogenous phosphatidylserine, phosphatidylglycerol and phosphatidylethanolamine than in those prepared with an excess of phosphatidylcholine. The vesicles enriched in negatively charged phospholipids were bound to Concanavalin A coupled to Sepharose-4B and could be released from this column only in the presence of a high concentration of alpha-methylmannopyranoside and detergent, indicating a glycoprotein nature of the fusogenic protein. Furthermore, these data show that protein inserted into membrane has its oligosaccharide chains exposed to the environment.

Animals↗
PubMed1993

Rat brain microsome fluidity as modified by prenatal ethanol administration.

The fluorescence anisotropy (r) of diphenylhexatriene (DPH) and of trimethylamino-diphenylhexatriene (TMA-DPH) as a function of temperature (10 degrees to 54 degrees C) was measured in brain microsomes of newborn rats prenatally exposed to ethanol. In this temperature range, the relationship between r and T was linear. The addition of ethanol in vitro to microsomal suspensions influenced the slope of the line of r versus T only when DPH was used as a probe and with high concentrations of the alcohol (> or = 0.3 M). The administration of ethanol (18% of total energy intake) in vivo to pregnant dams affected the slope of the lines of r versus T of the microsomes of pups, either using DPH or TMA-DPH as probes. The slope was also affected in brain microsomes obtained from dams, yet, only with TMA-DPH and in the opposite sense than in pups. We conclude that the effect of prenatal exposure to ethanol depended on metabolic alterations induced by the alcohol and not on its detergent properties for the following reasons: (a) The effects in vitro and in vivo were different and (b) in vitro effects could be obtained only with high concentrations (> or = 0.3 M), whereas in vivo effects were produced by small doses of ethanol. Besides, the effects of the administration of the alcohol in vivo were different in adult and intrauterine life.

Animals↗
PubMed1993

Transport of phosphatidylserine from microsomes to the inner mitochondrial membrane in brain tissue.

Phosphatidylserine was labeled by incubating rat brain homogenates with [3-14C]serine in the presence of Ca2+ (base-exchange conditions). Some labeled phosphatidylethanolamine also forms, in spite of the inhibition of Ca2+ on phosphatidylserine decarboxylase. Phosphatidylserine labeling and decarboxylation also occur on incubating a mixture of purified mitochondria and microsomes, suggesting that no soluble factors are necessary for the synthesis and the decarboxylation of phosphatidylserine. Ca2+ favors the transfer of phosphatidylserine from microsomes (where it forms) to mitochondria (where it is decarboxylated). The specific radioactivity of the phosphatidylserine transferred to mitochondria is higher than that of microsomal phosphatidylserine. This finding supports the hypothesis that the lipid is compartmentalized in microsomes and that radioactive, newly synthesized phosphatidylserine is much better exported than the bulk of microsomal phospholipid.

Animals↗
PubMed1993

The fate of phosphatidylethanolamine formed by decarboxylation in rat brain mitochondria.

In the liver, the synthesis and the decarboxylation of phosphatidylserine (PS) are a part of a metabolic cycle forming choline from serine. Yet, in the brain, the methylation of PE to phosphatidylcholine is a minor path. Therefore it is possible that the decarboxylation of PS has different biological meanings in the two organs. In this paper, we follow the formation and the fate of radioactive PE; to this purpose, we load brain mitochondria with PS by incubating cell homogenates with labelled serine in base-exchange conditions. Then, we isolate mitochondria and incubate them for PS decarboxylase. We also subfractionate mitochondria to investigate the distribution of PE in inner and outer membranes. We conclude that newly formed PE slowly moves from its site of synthesis (inner mitochondrial membranes). Besides, it reacts better with TNBS and with FLA than the pre-existing PE. This suggests that the newly-synthesised lipid does not mix quickly with the rest of mitochondrial PE. Therefore, our data demonstrate that the fate of PE formed through the decarboxylation path in the brain is different from that reported for the liver where newly-formed PE is rapidly exported to outer mitochondrial membranes.

Animals↗
PubMed1992

Acetyl-L-carnitine influences the fluidity of brain microsomes and of liposomes made of rat brain microsomal lipid extracts.

The fluorescence anisotropy (r) of diphenylhexatriene (DPH) was measured in different preparations (bovine spinal cord phosphatidylserine liposomes, rat brain microsomes, liposomes made with rat brain microsomal lipid having different phospholipid:cholesterol ratios) at temperatures ranging from 10 degrees to 55 degrees C. Phosphatidylserine liposomes exhibited an exponential relationship of r versus temperature, whereas the relationship shown by microsomes and liposomes prepared with microsomal lipid extracts was a linear one. The removal of protein and high phospholipid:cholesterol ratios decreased the slope of the lines (fluidity increased), although the intercept was unaffected. This means that differences were better appreciated at high temperatures and were well evident at 37 degrees C. Acetyl-L-carnitine decreased r in rat brain microsomes and in liposomes made with microsomal lipids with different phospholipid:cholesterol ratios. The fluidifying effect of acetyl-L-carnitine was mild but statistically significant and could explain, at least in part, the data reported in the literature of acetyl-L-carnitine acting on some parameters affected by ageing. Besides, acetyl-L-carnitine seemed to oppose the changes of viscosity due to lipid peroxidation, which has been reported to increase in ageing and dementia. L-carnitine shares the properties of its acetyl ester, but only in part.

Acetylcarnitine↗
PubMed1992

The transfer of phosphatidylserine from rat brain microsomes to mitochondria is regulated by microsomal lipid pattern.

We label the phosphatidylserine of rat brain microsomes through the base-exchange reaction and study the export of this lipid to mitochondria in a reconstituted system. We fuse microsomes to liposomes to vary the lipid composition of donor membranes and investigate the effect of membrane lipid pattern on phosphatidylserine movement. The specific radioactivity of the phosphatidylserine transferred to mitochondria is higher than that of microsomal phosphatidylserine. This finding supports the hypothesis that the lipid is compartmented in microsomes and that the radioactive, newly synthesized phosphatidylserine is much better exported than the bulk of microsomal phospholipid. The transfer of phosphatidylserine from microsomes, where it forms through the base-exchange reaction, to mitochondria, where it decarboxylates to phosphatidylethanolamine, is enhanced by phosphatidylserine itself, and by other lipid classes. This is proposed as a part of a possible mechanism for regulating phosphatidylserine metabolism in the brain.

Animals↗
PubMed1992

Serine base-exchange in rat liver microsomes: effect of phospholipids.

We enriched liver microsomes in lipid classes and molecular species disrupting membranes with octyl glucoside and reassembling them by detergent removal. Phosphatidylethanolamine incorporated into membranes better than phosphatidylserine or phosphatidylcholine. In addition, the degree of incorporation depended on the unsaturation of fatty acyl-chains. The enrichment of the membranes with phosphatidylserine or phosphatidylcholine inhibited serine base-exchange, whereas the addition of phosphatidylethanolamine usually stimulated it. The effect of exogenous lipids also depended on molecular species; egg yolk phosphatidylcholine and dipalmitoyl phosphatidylcholine inhibited base-exchange whereas the effect of palmitoyl-oleoyl phosphatidylcholine depended on the incorporated amount. The degree of unsaturation also modulated the effect of phosphatidylethanolamine.

Animals↗
PubMed1991

Regulation of liver base-exchange activity by acidic phospholipids.

Liver microsomes were enriched in liposomal acidic lipids by Ca(2+)-dependent fusion of liposomes at pH 7.0. The extent of fusion was monitored by the transfer of radioactive cholesteryl oleate. The enrichment of membranes in phosphatidylserine inhibited ethanolamine base-exchange, whereas the fusion with phosphatidylinositol inhibited both ethanolamine and serine base-exchange reactions. In contrast, these two phospholipids had scarce effects on choline base-exchange. Phosphatidic acid did not suppress any of the three base-exchange activities. Possible functional implications are discussed.

Animals↗
PubMed1991

The fusion of liposomes to rat brain microsomal membranes regulates phosphatidylserine synthesis.

Rat brain microsomal membranes were fused to liposomes prepared with several pure lipids, namely, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and mixtures of phosphatidic acid and phosphatidylcholine or phosphatidylethanolamine. The fusion between liposomes and microsomes was measured by the octadecyl rhodamine B chloride method. The extent and other properties of fusion largely depend on the lipid used to prepare liposomes; phosphatidic acid and phosphatidylinositol fuse more extensively than other lipid classes. The activity of serine base exchange is affected by the fusion between rat brain microsomes and lipids. It is strongly inhibited by phosphatidylserine, but it is activated by phosphatidic acid. The inhibition produced by phosphatidylserine on its own synthesis is proposed as a mechanism for controlling the formation of phosphatidylserine in rat brain microsomes.

Animals↗
PubMed1990

The effect of membrane lipid molecular species on rat brain base-exchange reactions: an appraisal of phosphatidylserine and of polyunsaturated phosphatidylcholine.

Rat brain microsomal membranes were solubilized with octyl glucoside and reassembled by removing the detergent. The composition of reconstituted membranes was modified by adding exogenous lipid to the detergent-membrane mixtures. The base-exchange reactions were sensitive to these procedures. The inhibiting or activating capacity of added lipid was related to the charge of polar head groups; indeed, the addition of phosphatidylserine always resulted in inhibition and that of phosphatidylcholine very often in activation. The addition of phospholipid possessing unsaturated acyl chains inhibited less (or activated more) serine and choline base-exchange than the addition of saturated species. It is concluded that phosphatidylserine and polyunsaturated phosphatidylcholine have opposite effects on base-exchange.

Animals↗