Lecithin biosynthesis in liver mitochondrial fractions

The pretreatment of rat liver mitochondrial fractions with phospholipase C preparations enhanced the incorporation of cytidine diphospho-[¹⁴C]-choline into phospholipids several-fold. Similar pretreatment of the microsomal fraction produced a similar stimulation. When the extent of microsomal contamination in the mitochondria was determined, and increments of pretreated microsomes were added to the mitochondria, the incorporation values extrapolated to zero for zero microsomal contamination. It was concluded that lecithin biosynthesis from endogenous diglycerides in the mitochondrial fractions could be ascribed to contaminating microsomes.     

Hepatic mitochondrial membrane lipid environment and protein nutrition

Effect of feeding rice diet with and without lysine and threonine supplementation on hepatic mitochondria and its inner and outer membrane proteins, enzymes and phospholipids has been studied. The exchange of phosphatidylcholine and phosphatidylethanolamine between microsomes and mitochondria has also been studied under these conditions. Deficient diet lead to significant decrease in proteins as well as activities of monoamine oxidase, succinate dehydrogenase, cytochrome a + a3 and cytochrome c in mitochondria and its inner and outer membranes. Feeding of the deficient diet also significantly reduced total phospholipids and PC in mitochondria and its outer mitochondrial membrane. In the inner mitochondrial membrane, only PE and cardiolipin were reduced. The incorporation (DPM/microgram PLP) of [methyl-3H]choline and [methyl-14C]methionine into PC of mitochondria and its outer membrane and that of 32Pi into PC and PE of outer mitochondrial membrane but only into PC of inner mitochondrial membrane were significantly reduced in the deficient group. The exchange rates of PC and PE between microsomes and mitochondria were reduced in the deficient group. Supplementation of the deficient diet with lysine and threonine profoundly improved the above biochemical lesions as compared to casein fed rats.     

The incorporation of glycerol into mitochondrial lipids during neonatal renal compensatory growth

[¹⁴C]glycerol incorporation into isolated inner and outer mitochondrial membrane is enhanced in the remaining kidney after unilateral nephrectomy. Serum from neonatal rabbits taken 24 hours after unilateral nephrectomy and added to tissue slice incubations appears to stimulate incorporation of [¹⁴C]glycerol into mitochondrial lipids of normal kidney cortex. Post-nephrectomy serum, however, depresses incorporation of [¹⁴C] glycerol and [³H]leucine into mitochondria when added to kidney cortex from animals in which uninephrectomy was performed 24 or 48 hours previously.     

Studies on the biosynthesis of protein and lipid components of rat liver mitochondria

1. Male rats were injected intraperitoneally with l-[³⁵S]methionine, [³²P]-phosphate and [2-¹⁴C]acetate. The animals were killed at various times up to 72hr. after injection, and liver mitochondria were prepared and fractionated into soluble protein, insoluble protein and lipid for assay of the radioactivity of each fraction. 2. The maximal specific radioactivity of total mitochondrial phospholipid with respect to both ³²P and ¹⁴C was attained after approx. 6hr. 3. ³²P was incorporated most rapidly into phosphatidylethanolamine, maximal incorporation being attained after approx. 6hr.; maximal incorporation into lecithin occurred after 6–12hr. The specific radioactivity of cardiolipin was still slowly increasing at the end of the experiment (72hr.). 4. There were no major differences between the rates of incorporation of ¹⁴C into the lecithin, phosphatidylethanolamine and cardiolipin fractions of mitochondrial phospholipid, maximal incorporation in each case occurring after approx. 6hr. 5. Maximal incorporation of ³⁵S into both soluble and insoluble protein fractions was attained less than 12hr. after injection, the maximal specific radioactivity of soluble protein being higher than that of insoluble protein.     

Studies on the flavins in rat liver mitochondrial outer membranes

The incorporation of radioactivity derived from [2-¹⁴C] riboflavin into the flavins of rat liver mitochondrial outer membranes was studied. These membranes were found to contain about 0.6 nmol of non-covalently bound flavins per mg protein; the majority is in the form of FAD (73%) and FMN (24%). The membranes also contain about 1.5 nmol per mg of covalently bound flavins.After labeling, radioactive flavins appeared in the non-covalently bound flavins for about 4 h. Most of this radioactivity was in FAD (77%). Neither the rate nor extent of this labelling was affected by cycloheximide (1 mg/kg) administered 30 min prior to the radioactive riboflavin. With the covalently bound flavins, radioactivity was incorporated into the coenzymes for at least 18 h, but the rate of incorporation was much slower. After cycloheximide, radioactive flavins continued to appear in covalently bound flavins for about 2 h, but then stopped. Labeling of both types of flavins after [¹⁴C] riboflavin was considerably slower than the incorporation of [³H] leucine into outer membrane proteins. These results suggest that with flavoproteins from the mitochondrial outer membranes, the incorporation of flavins occurs after synthesis of the various apoenyzmes is complete.     

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. T-wenty-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). Ca⁺² 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.Mr. Carlo Ricci is thanked for his skillful technical assistance. This work has been supported by a grant from the Ministry of Education, Rome, Italy.     

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-phosphatilyserine (PS) labeled liposomes with mitochondria at pH 6.0 results in the translocation of ¹⁴C-PS into mitochondria and in its decarboxylation to¹⁴ C-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 purifed protein is able to promote the recover of the¹⁴ C-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. (Mol Cell Biochem 175: 71–80, 1997)     

Phosphorylation/dephosphorylation of maize mitochondrial proteins with different localization

Phosphorylation and dephosphorylation of the proteins residing in the outer mitochondrial membrane, mitoplasts and whole mitochondria of maize (Zea mays L.) were investigated in order to reveal the possible participation of these processes in mitochondrial signaling. A mitochondrial protein of around 57 kD was identified by immunocytochemistry as α-subunit of the F₁-ATPase complex. In isolated mitochondria of maize, phosphorylation of this protein could be visualized only after treating mitochondria with endotholl, an inhibitor of the PP1a and PP2A protein phosphatases. A phosphorylated protein of 46.6 kD was identified as β-subunit of the F₁-ATPase complex. Ca²⁺ is the most common second messenger participating in mitochondrial signaling. We conclude that the transmission of the Ca²⁺ signal to the plant mitochondria occurs via proteins of the outer mitochondrial membrane. The systems perceiving this signal could include the protein phosphatases residing in the outer mitochondrial membrane, which preferentially dephosphorylate the proteins in the inner membrane.     

Action ofl-acetylcarnitine on age-dependent modifications of mitochondrial membrane proteins from rat cerebellum

Protein patterns of mitochondrial outer membrane, inner membrane, and matrix from nonsynaptic (free) mitochondria from rat cerebellum at different ages (4, 8, 12, 16, 20, and 24 months) were analyzed by gel electrophoresis. Acutel-acetylcarnitine treatment was per-formed by a single i.p. injection (100 mg/kg body weight) of the substance 60 min before the sacrifice of the animals. Different age-dependent changes were obtained for the proteins of the three fractions. The amount of some protein subunits increased and/or decreased after drug treatment. In particular, protein composition of the inner mitochondrial membrane showed significant age-related modifications. This result probably indicates differences in protein synthesis and/or turnover rates in the various mitochondrial compartments during aging. Acutel-acetylcarnitine treatment caused: a high increase in the amount of one inner membrane protein with Mw 16 kDa, at all the ages studied; a decrease in the amount of many other inner membrane proteins; modifications of some matrix proteins. Our results show that in vivo administration ofl-acetylcarnitine affects mainly the inner membrane protein composition of cerebellar mitochondria.     

DNA biosynthesis in rat liver mitochondria: Inhibition by sulfhydryl compounds and stimulation by cytoplasmic proteins

The sulfhydryl compounds, 2-mercaptoethanol, dithiothreitol, cysteine. and glutathione inhibit the incorporation of [³H]dTTP or [³H]dATP into mitochondrial DNA by rat liver mitochondria in vitro. The lack of inhibition by non-SH-containing analogs indicates that the SH group is responsible for the inhibition.The inhibition does not result from an effect of the sulfhydryl compounds on precursor permeability, ATP formation, or respiration, or the action of the thiol on the outer mitochondrial membrane. An intact inner membrane is not required for the action of the inhibitor. Furthermore, SH compounds do not appear to exert their effect by activation of a mitochondrial nuclease, chemical breakdown of high molecular-weight mitochondrial DNA or dissociation of membrane-bound DNA from the inner mitochondrial membrane. Incorporation of labeled precursor into DNA by mitochondrial DNA polymerase, when removed from the inner mitochondrial membrane, is not inhibited by SH compounds.Cytoplasmic extracts prepared from rat and mouse tumors and 22-h regenerating rat liver contain a protein(s) not detectable in normal rat liver which can reverse the inhibition by SH compounds of the synthesis of mitochondrial DNA in rat liver mitochondria in vitro.More importantly, when the stimulatory protein(s) is partially purified by affinity chromatography on DNA-cellulose, it is possible to demonstrate that this protein(s) also stimulates the synthesis of mitochondrial DNA by normal rat liver mitochondria in vitro in the absence of the sulfhydryl inhibitor.     

Synaptosomal protein synthesis in a cell-free system

—Synaptosomes were isolated from cerebral cortex of young rats and incubated with ¹⁴C-labelled l -leucine in vitro. Amino acid incorporation into proteins of the synaptosomal cytoplasm, mitochondria and membrane components was observed. There was no incorporation into proteins of the vesicles. The protein-synthesizing system was not stimulated by the addition of either ATP or an ATP-generating system. ATP at all concentrations was inhibitory. Two different protein-synthesizing systems operate in the synaptosome. One, sensitive to inhibition by chloramphenicol and related antibiotics, is found in the mitochondrial subfraction and the other, inhibited by cycloheximide, is located either in the membrane components or the synaptosomal cytoplasm. This second system resembles the eukaryotic ribosomal system in its sensitivity to cycloheximide. Both the synaptosomal soluble fraction and the synaptosomal membrane fractions were shown previously to contain RNA. This RNA could function in protein-synthesizing mechanisms in the synaptosome. These results deomonstrate that protein is synthesized in axonal components and show that it is unnecessary to postulate that all axonal protein is supplied by somato-axonal flow.     

LECITHIN SYNTHESIS, INTRACELLULAR TRANSPORT, AND SECRETION IN RAT LIVER : IV. A Radioautographic and Biochemical Study of Choline-Deficient Rats Injected with Choline-3H

Injection of choline-³H into choline-deficient rats resulted in an enhanced incorporation of the label into liver lecithin, as compared to the incorporation of label into liver lecithin of normal rats. The results obtained with the use of different lecithin precursors indicate that in the intact liver cell, both in vivo and in vitro, exchange of choline with phosphatidyl-choline is not significant. The synthesis and secretion of lecithins by the choline-deficient liver compare favorably with the liver of choline-supplemented rats, when both are presented with labeled choline or lysolecithin as lecithin precursors. Radioautography of the choline-deficient liver shows that 5 min after injection of choline-³H the newly synthesized lecithin is found in the endoplasmic reticulum (62%), mitochondria (13%), and at the "cell boundary" (20%). The ratio of the specific activity of microsomal and mitochondrial lecithin, labeled with choline, glycerol, or linoleate, was 1.53 at 5 min after injection, but the ratio of the specific activity of phosphatidyl ethanolamine (PE), labeled with ethanolamine, was 5.3. These results indicate that lecithin and PE are synthesized mainly in the endoplasmic reticulum, and are transferred into mitochondria at different rates. The site of a precursor pool of bile lecithin was studied in the intact rat and in the perfused liver. Following labeling with choline-³H, microsomal lecithin isolated from perfused liver had a specific activity lower than that of bile lecithin, but the specific activity of microsomal linoleyl lecithin was comparable to that of bile lecithin between 30 and 90 min of perfusion. It is proposed that the site of the bile lecithin pool is located in the endoplasmic reticulum and that the pool consists mostly of linoleyl lecithin.     

Biogenesis of mammalian mitochondria in the renoprival kidney. I. Amino acid incorporation and respiratory activity

Changes in the yield of mitochondrial protein, in the incorporation of leucine into mitochondrial proteins, and in the respiratory activity of isolated mitochondria were determined in the remaining kidney (renoprival kidney) of the rat during the first 72 hr postmononephrectomy. At 24, 48, and 72 hr the yield of mitochondrial protein isolated from the renoprival kidney increased 13, 23, and 34%, respectively, whereas renal mass increased 9, 14, and 19%. Incorporation of [³H]-leucine in vivo into total mitochondrial protein was increased 96 and 130% over control at 12 and 24 hr, respectively. Incorporation of leucine in vitro by mitochondria was increased 27% over control at 24 hr; chloroamphenicol, but not cycloheximide, inhibited the in vitro incorporation.     

Amino acid incorporation by nuclear membrane fraction of rat liver.

Nuclear membrane fraction of rat liver is able to incorporate ¹⁴C-leucine into its proteins $\text{in vitro}$. The incorporation of ¹⁴C-leucine into the nuclear membrane fraction was almost completely inhibited by chloramphenicol, but the inhibition by cycloheximide and puromycin was not so remarkable. RNase and DNase were not effective. The incorporation was also inhibited by several reagents known to interfere with energy metabolism. These characteristics of the incorporation of ¹⁴C-leucine by the nuclear membrane fraction are quite similar to those of the incorporation by nuclei isolated from rat liver and mitochondrial fraction, but seem to be different from those of the ordinary protein synthetic system in microsomal fraction. ¹⁴C-Leucine was preferentially incorporated into intrapolypeptide or C-terminal residues but not into N-terminal residues. Acrylamide gel electrophoresis showed that three protein species were mainly labelled. The incorporating activity of the nuclear membrane fraction obtained from regenerating liver 17 h after partial hepatectomy showed 220 % of the control. The possibility that the contaminated mitochondrial fraction might be responsible for the incorporation of ¹⁴C-leucine by the nuclear membrane fraction was ruled out.     

Energetic aspects of spore germination in filamentous fungi

The antifungal activity of substances interfering with the function and biogenesis of mitochondria was studied. Strict anaerobiosis, cyanide, azide, oligomycin, bongkrekic acid and ethidium bromide were found to prevent spore germination ofAspergillus niger andPenicillium italicum in liquid germination medium. The effect of azide, oligomycin and ethidium bromide was fungicidal. Cyanide and azide completely inhibited the incorporation of¹⁴C-leucine and¹⁴C-uracil into germinating conidia ofA. niger. Oligomycin and ethidium bromide reduced the extent of incorporation of both precursors in the first few hours of conidial germination and at later stages stopped it completely. The inhibition of both spore germination and macromolecules synthesis during the germination ofA. niger conidia were in relation to the specific inhibitory effect of the agents on respiratory activity of dormant conidia and mycelial cells. The results indicate that both the function of mitochondrial genetic and protein synthesizing systems and the function of oxidative phosphorylation are essential for normal spore germination and fungal growth.     

Apparent pyridine nucleotide synthesis in mitochondria: An artifact of NMN and NAD glycohydrolase activity?

Intact and Triton disrupted mitochondria incorporate [¹⁴C]nicotinamide into [¹⁴C]NMN and [¹⁴C]NAD. Dialyzed Triton extracts lose this activity. The ability to form [¹⁴C]NMN is restored by addition of a fraction of boiled mitochondrial extract or of NMN. PRPP and ATP are not required for [¹⁴C]NMN formation. The specific activity of [¹⁴C]NMN formation decreases with serial washing of mitochondria while that of an outer membrane enzyme (kynurenine-3-monooxygenase) remains about constant. These finding suggest that the previously reported synthesis of NMN and NAD by mitochondria may be due to exchange reactions catalyzed by active glycohydrolase(s) in contaminating microsomes.     

Asymmetric synthesis followed by transmembrane movement of phosphatidylethanolamine in rat liver endoplasmic reticulum

Studies with phospholipase C have indicated that two-thirds of the phosphatidylethanolamine of rat liver endoplasmic reticulum is located in the inner leaflet of the membrane bilayer. Phosphatidyl[¹⁴C]ethanolamine is synthesised in microsomes incubated with CDP[¹⁴C]ethanolamine. Using phospholipase C as a probe we have observed that the labelled phospholipid is initially (1–2 min) concentrated in the ‘outer leaflet’ of the membrane bilayer. The specific activity of this pool of phosphatidylethanolamine was 3.5 times that of the inner leaflet. If, however, the microsomes were opened with 0.4% taurocholate or the French pressure cell to make both sides of the bilayer available to phospholipase C, the phosphatidylethanolamine behaves as a single pool for hydrolysis. On longer incubation, up to 30 min, with CDP[¹⁴C]ethanolamine the specific activity of the outer leaflet phosphatidylethanolamine becomes close to that of the inner leaflet. In chase experiments, in which microsomal phosphatidylethanolamine was labelled by incubation with CDP[¹⁴C]ethanolamine for 1 min, the reaction stopped by addition of calcium, and the microsomes isolated by centrifugation and reincubated, labelled phosphatidylethanolamine was transferred from the ‘outer leaflet’ to the ‘inner leaflet’, so that both were equally labelled. These observations suggest that phosphatidylethanolamine is synthesised at the cytoplasmic leaflet of the endoplasmic reticulum and subsequently transferred across the membrane to the cisternal leaflet of the bilayer. Transmembrane movement is apparently temperature-dependent and independent of continued synthesis of phosphatidylethanolamine.     

Protein kinase activity and endogenous phosphorylation in subfractions of rat liver mitochondria

Rat liver mitochondria were subfractionated into outer membrane, intermembrane and mitoplast (inner membrane and matrix) fractions. Of the recovered protein kinase activity, 80–90% was found in the intermembrane fraction, while the rest was associated with mitoplast. The intermembrane prostimulated kinase was stimulated by cyclic AMP, while the mitoplast enzyme was stimulated by the nucleotide only after treatment with Triton X-100. Extracted protein kinase resolved into three peaks on DEAE-cellulose chromatography. All three peaks were present both in the intermembrane fraction and in mitoplast. One peak corresponded to the catalytic subunit of cyclic AMP-dependent protein kinase, one was a cyclic AMP-independent enzyme, and the third was the cyclic AMP-dependent type II enzyme. The endogenous incorporation of phosphate was particularly high in the outer mitochondrial membrane, and occurred also in the mitoplast fraction. The incorporation in mitoplasts was to a double band of Mr 47 500, and in outer membranes to apparently heterogeneous material of comparatively low molecular weight.     

Metabolism of phosphatidylcholine, phosphatidylinositol and palmityl carnitine in synaptosomes from rat brain

Abstract— Synthesis of phosphatidylcholine, phosphatidylinositol and palmityl carnitine in synaptosomes isolated from rat brain was investigated and compared with the synthesis of these compounds in microsomes and mitochondria. Electron microscopic and marker enzyme studies showed the contaminants in the synaptosomal preparation to consist of a few microsomes and almost no free mitochondria. In synaptosomes, addition of 1,2-diglyceride exerted no effect on the incorporation of [¹⁴C]choline into phosphatidylcholine or on the incorporation of [³H]myo-inositol into phosphatidylinositol, but it stimulated the incorporation of CDP[1,2-¹⁴C]choline into phosphatidylcholine by more than 50 per cent. The incorporation of the latter in intact synaptosomes, lysed synaptosomes and purified mitochondria was 15-6, 27 and 9-9 per cent, respectively, of that in the microsomes. The incorporation of [³H]myo-inositol into the phosphatidylinositol of synaptosomes and purified mitochondria was 15-8 and 11-1 per cent, respectively, of that in the microsomes. Maximal incorporation of [³H]myo-inositol occurred at pH 7–5 in a medium containing Mg²⁺ and CTP; it was linear with time and protein concentration and was inhibited by 1 mM Ca^{2 +} but unaffected by the presence of ATP. This incorporation of myo-inositol appeared to occur through the reversal of the CDP-diglyceride: inositol transferase reaction. The demonstration of carnitine palmityl transferase in synaptosomes indicated that, as in mitochondrial and erythrocyte membranes, fatty acids can be transported across the synaptosomal membrane. In contrast to mitochondria where maximal incorporation of [¹⁴C]carnitine into palmityl carnitine was observed after 20 min of incubation, the incorporation in synaptosomes increased as a function of time up to 60 min of incubation. We conclude that synaptosomes can carry on de novo synthesis of lipids, although at a limited rate. From the present data we cannot state with certainty how much of this synthesis is attributable to membranes originating from the endoplasmic reticulum.     

Subcellular distribution of rat liver porin

The subcellular distribution of rat liver porin was investigated using the immunoblotting technique and monospecific antisera against the protein isolated from the outer membrane of rat liver mitochondria. Subfractionation of mitochondria into inner membranes, outer membranes and matrix fractions revealed the presence of porin only in the outer membranes. Porin was also not detected in highly purified subcellular fractions, including plasma membranes, nuclear membranes, Golgi I and Golgi II, microsomes and lysosomes. Thus, liver porin is located exclusively in the outer mitochondrial membrane.