Individual molecular species of phosphatidylcholine and phosphatidylethanolamine in myelin turn over at different rates.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of the myelin membrane exhibit heterogeneity with respect to metabolic turnover rate (Miller, S. L., Benjamins, J. A., and Morell, P. (1977) J. Biol. Chem. 252, 4025-4037). To test the hypothesis that this is due to differential turnover of individual molecular species (which differ in acyl chain composition), we have examined the relative turnover of individual molecular species of myelin PC and PE. Phospholipids were labeled by injection of [2-3H]glycerol into the brains of young rats. Myelin was isolated at 1, 15, and 30 days post-injection, lipids were extracted, and phospholipid classes were separated by thin-layer chromatography. The PC and PE fractions were hydrolyzed with phospholipase C, and the resulting diacylglycerols were dinitrobenzoylated and fractionated by reverse-phase high performance liquid chromatography. The distribution of radioactivity among individual molecular species was determined. The labeled molecular species of myelin PC were 16:0-16:0, 16:0-18:0, 16:0-18:1, and 18:0-18:1, with most of the label present in 16:0-18:1 and 18:0-18:1. Changes in distribution of label with time after injection indicated that 16:0-18:1 turned over more rapidly than 18:0-18:1. The labeled molecular species of myelin PE were 18:0-20:4, 18:1-18:1, 16:0-18:1, 18:0-18:2, and 18:0-18:1. As with myelin PC, 16:0-18:1 (and 18:1-18:1) turned over more rapidly than 18:0-18:1. The relative turnover of individual molecular species of PC in the microsomal fraction from forebrain was also examined. The molecular species profile was different from myelin PC, but again, 16:0-18:1 turned over more rapidly than the other molecular species. Thus, within the same membrane, individual molecular species of a phospholipid class are metabolized at different rates. Comparison of our results with previous studies of turnover of molecular classes of phospholipids indicates that in addition to polar head group composition (Miller et al., 1977), fatty acid composition is very important in determining the metabolic fate of a phospholipid.     

Turnover rate of molecular species of sphingomyelin in rat brain

Turnover rate of individual molecular species of sphingomyelin of adult rat brain myelin and microsomal membranes was determined after an intracerebral injection of 100 Ci of [C³H₃]choline. Myelin and microsomal membrane sphingomyelins were isolated from the rest of the lipids. The individual molecular species of benzoylated sphingomyelin were separated and quantitated by reversed-phase high performance liquid chromatography. All individual major molecular species of microsomal and myelin sphingomyelin had maximum incorporation at 6 and 15 days, respectively, after the injection. The specific radioactivity of all the various molecular species of both myelin and microsomal sphingomyelin declined at a similar rate after reaching a maximum. There was no significant difference in the turnover rate of short chain (16:0, 18:0) and long chain (>22:0) fatty acid containing sphingomyelin. The average apparent turnover rate of myelin and microsomal sphingomyelin molecular species was about 14–16 days for the fast pool and about 45 days for the slow pool. It is concluded that individual molecular species of sphingomyelin of myelin and microsomal membranes turned over at a similar rate. Thus, turnover rate of sphingomyelin in myelin and microsomal membranes is not affected by the fatty acyl composition of the lipid.     

Turnover of Axonally Transported Phospholipids in Nerve Endings of Retinal Ganglion Cells

We have investigated the metabolic turnover of axonally transported phospholipids in myelinated axons (optic tract) and nerve endings (superior colliculus) of retinal ganglion cells. One week following intraocular injection of [2-3H]glycerol, turnover rates for individual phospholipid classes in the retina (which contains a number of other cell types in addition to the ganglion cells) were all very similar to each other, with apparent half-lives of approximately 7 days. Apparent half-lives of labeled phospholipids in superior colliculus (presumably primarily in retinal ganglion cell nerve endings) were 10 days for both choline and inositol phosphoglycerides and 13 days for both serine and diacylethanolamine phosphoglycerides. Subcellular fractionation data obtained from superior colliculus at various times after injection suggested that apparent turnover rates determined for nerve ending phospholipids probably were not significantly affected by transfer of axonally transported 3H lipids into myelin. Apparent half-lives for phospholipids in optic tract were somewhat longer than in superior colliculus, ranging from 11 to 18 days. The slower turnover rates in optic tract may, in part, reflect the transfer of some axonal lipids to the more metabolically stable pool of lipids in the myelin ensheathing the retinal ganglion cell axons. In both optic tract and superior colliculus, apparent half-lives for axonally transported phospholipids labeled with [32P]phosphate were only slightly longer than for [2-3H]glycerol, while those for [14C]choline and [3H]acetate were markedly longer, indicating differing degrees of metabolic conservation or reutilization of these precursors relative to glycerol.     

Relative degradation of different arachidonoyl molecular species of choline glycerophospholipids in opsonized zymosan-stimulated rabbit alveolar macrophages

The relative degradation of arachidonoyl molecular species of glycerophospholipids prelabeled with [3H]20:4 caused by opsonized zymosan was studied in rabbit alveolar macrophages using a recently developed high-performance liquid chromatographic method. The opsonized zymosan caused the release of [3H]20:4 only from choline glycerophospholipids, no significant changes being observed in the radioactivities of other glycerophospholipids and triacylglycerol. Choline glycerophospholipids were resolved into seven arachidonoyl molecular species, which differed as to the alkyl ether or acyl residue bound at the 1-position, by high-performance liquid chromatography. Arachidonate was predominantly located in the alkyl type having 16:0 at the 1-position which comprised more than half of the total arachidonoyl molecular species of choline glycerophospholipids. The radioactivities of all arachidonoyl molecular species of choline glycerophospholipids, except for the 18:2-20:4 and 18:1-20:4 species of diacylglycerophosphocholine, decreased to 80-85% of the control values as a result of the challenge with opsonized zymosan for 1 h. However, 50% of the released 20:4 came from the 16:0-20:4 species of alkylacylglycerophospholipids, which were the most predominant species of choline glycerophospholipids. The present results indicate that the 16:0-20:4 species of alkylacylglycerophosphocholine is a significant source of arachidonate and 1-O-alkyl-2-lysoglycerophosphocholine, the precursor of the platelet-activating factor, relative to other arachidonoyl species in activated alveolar macrophages.     

DIFFERENTIAL DISTRIBUTION OF [U-14C]GLUCOSE AND [U-14C]GLYCEROL AMONG MOLECULAR SPECIES OF PHOSPHATIDYL CHOLINE,PHOSPHATIDYL ETHANOLAMINE AND 1,2-DIACYLGLYCEROL IN RABBIT BRAIN12

Specific radioactivities of molecular species of phosphatidyl choline(PC), phosphatidyl ethanolamine(PE) and 1,2-diacylglycerol were determined in rabbit brain 15 and 30 min after intraventricular injection of 10OpCi of either [U-¹⁴C]glucose or [U-¹⁴C]glycerol. The rate of de nouo synthesis of glycerophospholipids and their molecular species could be determined after glycerol labelling, since 94.0–99.7% of ¹⁴C activity was recovered in glyceryl moieties of brain lipids. After injection of glucose radioactivity was measured in both glyccrol and acyl residues of lipids. High incorporation rates were measured in species of PC, PE and 1,2-diacylglycerol with oleic acid in position 2 and with palmitic, stearic or oleic acids in position 1. The conclusion may therefore be drawn that these molecular species were preferably synthesized de novo by selective acylation of glycerol 3-phosphate. The lowest specific activities were observed for 1,2-dipalmitoyl- and l-stearoyl-2- arachidonoyl-glycerol, -PC and -PE. These turnover rates point to incorporation of arachidonate, and probably also of palmitate in dipalmitoyl-PC, amounting to 20% of total PC, via deacylation-acylation- cycle.     

Phosphate groups modifying myelin basic proteins are metabolically labile; methyl groups are stable

Young and adult rats received intracranial injections of [33P]orthophosphoric acid. The time course of the appearance and decay of the radioactive label on basic proteins in isolated myelin was followed for 1 mo. Incorporation was maximal by 1 h, followed by a decay phase with a half-life of approximately 2 wk. However, radioactivity in the acid-soluble precursor pool (which always constituted at least half of the total radioactivity) decayed with a similar half-life, suggesting that the true turnover time of basic protein phosphates might be masked by continued exchange with a long-lived radioactive precursor pool. Calculations based on the rate of incorporation were made to more closely determine the true turnover time; it was found that most of the phosphate groups of basic protein turned over in a matter of minutes. Incorporation was independent of the rate of myelin synthesis but was proportional to the amount of myelin present. Experiments in which myelin was subfractionated to yield fractions differing in degree of compaction suggested that even the basic protein phosphate groups of primarily compacted myelin participated in this rapid exchange. Similar studies were carried out on the metabolism of radioactive amino acids incorporated into the peptide backbone of myelin basic proteins. The metabolism of the methyl groups of methylarginines also was monitored using [methyl-3H]methionine as a precursor. In contrast to the basic protein phosphate groups, both the peptide backbone and the modifying methyl groups had a metabolic half-life of months, which cannot be accounted for by reutilization from a pool of soluble precursor. The demonstration that the phosphate groups of myelin basic protein turn over rapidly suggests that, in contrast to the static morphological picture, basic proteins may be readily accessible to cytoplasm in vivo.     

Effect of a single dose of triiodothyronine on the uptake of (methyl-14C)choline into chick liver phospholipids

The effects of a single dose of 3,3'-5-triiodothyronine (T3) on the uptake of (methyl-14C) choline into liver phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) were studied in chicks as a function of time up to 6 h after injection of the radioactive precursor. In all cases, chicks received the T3 dose intraperitoneally 5 h before injection of the labelled compound. T3 enhances the incorporation of 14C-choline into liver PC, showing a biphasic response; the main uptake occurs between 2 and 3 h after administering the precursor. A smaller but significant hormone-dependent increase in incorporation of the labelled compound is observed in the case of LPC. Lipid P associated to PC and LPC remains constant throughout the experiment, and does not vary with hormone treatment. It is suggested that T3-injection increases, either directly or through other metabolic processes, PC and LPC turnover in chick liver cells.     

Heterogeneity in the metabolism of the arachidonoyl molecular species of glycerophospholipids of rabbit alveolar macrophages. The interrelationship between metabolic activities and chemical structures of the arachidonoyl molecular species

The relative incorporation of [3H]arachidonic acid (20:4) into individual molecular species containing 20:4 at the 2 position (18:1-20:4, 16:0-20:4 and 18:0-20:4 species) of diacyl and ether-linked glycerophosphocholine, glycerophosphoethanolamine and glycerophosphoinositol of rabbit alveolar macrophages has been measured by reversed-phase high-performance liquid chromatography (HPLC). The rate of incorporation of [3H]20:4 into the molecular species of glycerophospholipids was greatly influenced by their structures. The reversed-phase HPLC analysis allowed elucidation of the influence of structural differences, such as the nature of the polar head group, the fatty chain at the 1 position and the chemical form of the bond of the fatty chain attached at the 1 position on the uptake of [3H]20:4 by comparison of the specific radioactivities of arachidonoyl molecular species having the same structures, except that one of the three kinds of moiety was different. The specific radioactivities of the molecular species containing choline head groups were significantly higher than those containing ethanolamine and inositol moieties. The specific radioactivities of diacyl molecular species were considerably higher than those of ether-linked molecular species. The nature of the fatty chain attached at the 1 position also influenced the uptake of [3H]20:4 into glycerophospholipids. The arachidonoyl molecular species containing 18:1 at the 1 position were preferentially labelled with [3H]20:4 as compared to the corresponding 16:0-20:4 and 18:0-20:4 species either of diacyl or ether-linked glycerophospholipids. The present results suggest that the acyltransferase involved in the incorporation of 20:4 into glycerophospholipids has selectivity for the structures of glycerophospholipids and the order of selectivity of this enzyme for the arachidonoyl molecular species, deduced in the present experiments, was as follows: choline head group greater than ethanolamine and inositol groups, acyl bond greater than ether and vinyl ether bonds, 18:1 fatty chain greater than 16:0 and 18:0 fatty chains at the 1 position. Comparison of the metabolic activities of all major arachidonoyl molecular species of glycerophospholipids having a single structure is reported here for the first time.     

PHOSPHOLIPID METABOLISM IN LIGHT AND DARK ADAPTED EXCISED RETINA

Abstract— The phospholipid composition of, and the incorporation of labelled phosphorus into the different phospholipids of rat and calf retina have been studied. The influence of various conditions, such as dark and light adaptation, during the preparation of retina, lipid extraction and incubation of retina with radioactive phosphorus was investigated.
The phospholipid composition of rat retina did not differ significantly from that of calf retina and the different conditions of preparation and incubation did not modify the distributions.
The specific radioactivities of the different phospholipids of calf and rat retina, incubated in the presence of ³²P, distinguished in both species two groups of components characterized by the rate of labelling. Phosphatidic acid (PA) and inositol glycerophospholipids (PI) belonged to the first group and showed the highest uptake of labelled phosphorus; the second group, comprising choline glycerophospholipids (PC), serine glycerophospholipids (PS), sphingomyelin (SP), ethanolamine glycerophospholipids (PE) and cardiolipin (CL) showed low incorporation activities. Only SP was labelled differently in rat and calf retina. With the exception of PS, there was no evidence for the influence of light on the turnover of individual phospholipids. The finding that PS showed higher specific radioactivities when adaptation and incubation proceeded in the dark, seems to be of interest and needs further study.     

Turnover of phosphatidylcholine in cell membranes of adult rat brain

—Incorporation of radioactive choline into subcellular fractions of adult rat brains, after intracranial injection of the precursor, was studied at different times. Maximum labelling of phosphatidylcholine occurred at 5 h in all fractions, the highest specific radio-activity being found in mitochondria. The specific radioactivity diminished rapidly for a few hours after the peak of maximum incorporation and continued to decrease thereafter at a much slower rate. The type of decay curve found in all fractions except myelin, indicated the possible presence of two populations of phosphatidylcholine with quite different turnover rates. The significance of these findings is discussed.     

Different metabolic rates for arachidonoyl molecular species of ethanolamine glycerophospholipids in rat brain

The ethanolamine glycerophospholipids (EGP) contain most of the arachidonate (20:4, n-6) and adrenate (22:4, n-6), potential precursors of biologically potent prostaglandins and related compounds. Much better methods utilizing high performance liquid chromatography (HPLC) techniques are now available for the study of the molecular species of all three classes, namely diacyl, alkenylacyl (plasmalogen), and alkylacyl. Different molecular species may have different functions. This possibility was studied by examining the rates of incorporation of [3H]arachidonic acid into the three major molecular species of each of the three classes of ethanolamine glycerophospholipids. After the intracerebral injection of [3H]20:4 into rat brain, it was rapidly converted to 22:4(n-6). Of the total radioactivity, 10-20% was located in 22:4 in alkenylacyl and diacyl-GPE. In the alkylacyl-GPE, labeled 22:4 was preferentially incorporated and accounted for 50-60% of the total radioactivity. The primary arachidonoyl molecular species of alkenylacyl, alkylacyl, and diacyl-GPE were the 18:1-20:4, 16:0-20:4, and 18:0-20:4 species. The alkylacyl class contained almost equal proportions of these three molecular species. On the other hand, the 20:4 in alkenylacyl and diacyl classes was combined largely with 18:0 groups at the sn-1 position. In particular, the 18:0-20:4 species comprised about 80% of arachidonoyl molecular species of the diacyl class. In all three classes, the highest specific radioactivities were found in the 18:1-20:4 species, whereas the 18:0-20:4 species had the lowest specific radioactivity. Over the period 60 min-24 hr, the diacyl 18:0-20:4 and all three arachidonoyl molecular species of the alkenylacyl class increased in specific radioactivity more rapidly than the other arachidonoyl molecular species.     

Turnover of myelin lipids in aging brain

Turnover rates of myelin membrane components in mouse brains were determined by a method using stable isotope-labeling and mass spectrometry. The half-replacement times based on incorporation rates of newly synthesized molecules for young adult mice were 359 days for cholesterol, 20 days for phosphatidylcholine, 25 days for phosphatidylethanolamine, 94 days for cerebroside and 102 days for ganglioside GM1. The turnover rates of half-lives of myelin components were calculated from the decay curves of initially labeled molecules, and they were about the same as the half-replacement times. Individual components were thus revealed to be metabolized at different rates, and their turnover rates were differently affected by aging. As was observed with phospholipids, myelin pools appeared to be compartmentalized into rapidly and slowly exchanging pools. The turnover rates of cerebroside and GM1 decreased between the young and adult periods and slightly increased in senescence. The latter phenomenon may indicate an enhanced myelin turnover in senescence. The present study reveals the dynamic aspects of myelin membrane turnover during the life span of mouse.     

The molecular species of phospholipids of the cold-water soft coral <Emphasis Type="Italic">Gersemia rubiformis</Emphasis> (Ehrenberg, 1834) (Alcyonacea,Nephtheidae)

The chemical structures and contents of 68 molecular species of ethanolamine glycerophospholipids (PE), choline glycerophospholipids (PC), serine glycerophospholipids (PS), and inositol glycerophospholipids (PI) were determined in the asymbiotic soft coral Gercemia rubiformis (Ehrenberg, 1834) from the Sea of Okhotsk by the method of tandem high-resolution mass spectrometry. The major molecular species were 16:1e/20:4 PE, 16:0e/20:4 PC, 20:1/24:5 PS, and 16:0/24:5 PI. This study showed a significant similarity of the polar lipidomes of G. rubiformis and tropical species of alcyonarians with symbiotic microalgae and revealed the basic characteristic of the polar lipidome of these alcyonarians. It was found that the 24:5n-6 and 24:6n-3 acids (chemotaxonomic markers of soft corals) concentrate in the acyl groups of the molecular species of PS and PI, which can be used as molecular lipid markers in the study of the symbiotic and trophic relationships of soft corals.     

Selective changes to phosphatidylcholine and phosphatidylethanolamine molecular species in the developing fetal guinea pig liver and plasma

The molecular species composition of membrane phospholipids influences the activities of integral proteins and cell signalling pathways. We determined the effect of increasing gestational age on fetal guinea pig liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and plasma PC molecular species composition. The livers were collected from fetuses (n = 5/time point) at 5 day intervals between 40 and 65 days of gestation, and at term (68 days). Hepatic PC and PE molecular species composition was determined by electrospray ionisation mass spectrometry. An increasing gestational age was accompanied by selective changes in individual molecular species. The proportion of the sn-1 18:0 species increased relative to the sn-1 16:0 species in liver PC, but not PE, with an increasing gestational age. 1-O-alkyl-2-acyl PC species concentrations decreased significantly between 40 and 45 days of gestation (40%), and 65 and 68 days (54%). Total 1-O-alkenyl-2-acyl PE species concentration increased between days 60 and 65, due to a rise in 1-O-16:0 alkyl/20:4 content, and then decreased until term. Between day 40 and term, PC and PE sn-2 18:2n-6 species concentrations increased 3-fold. PC16:0/18:2 increased gradually throughout gestation, while PC18:0/18:2 content only increased after day 65. The overall increase in PE18:2n-6 content was due to PE18:0/18:2 alone. The composition of plasma PC essentially reflected hepatic PC. Overall, these data suggest differential regulation of hepatic PC and PE molecular species composition during development which is essentially independent of the maternal fatty acid supply.     

Metabolism of unique diarachidonoyl and linoleoylarachidonoyl species of ethanolamine and choline phosphoglycerides in rat testes

Selected molecular species of rat testicular 1,2-diradyl-sn-glycero-3-phosphocholines and 1,2-diradyl-sn-glycero-3-phosphoethanolamines were quantitated as their diradylglycerobenzoate derivatives, using a recently developed high-performance liquid chromatographic method. Increased amounts of docosapentaenoic acid were found in glycerophospholipids containing ether moieties compared with the diacyl phospholipids (e.g., docosapentaenoate-containing species comprised more than 80% of the alkylacyl subclass of the ethanolamine phosholipids as opposed to 29.3% of the diacyl subclass). Within 2 h after intratesticular injections of [5,6,8,9,11,12,14,15-3H]arachidonic acid, the 20:4-20:4 and 18:2-20:4 molecular species of the diacyl subclass of both the choline and ethanolamine glycerophosphatides had the highest specific radioactivities. These unique molecular species (20:4-20:4 and 18:2-20:4) also exhibited the largest percentage decrease in specific radioactivity 24 h after the intratesticular injections of [3H]arachidonic acid, which indicates these two species possess a high metabolic turnover. Two of the arachidonate-containing molecular species (18:1-20:4 and 18:0-20:4) in the ethanolamine plasmalogens showed only a small decrease in specific radioactivity, whereas a third species (16:0-20:4) actually had a 44% increase in specific radioactivity 24 h after the intratesticular injections of [3H]arachidonate. These data indicate that the 20:4-20:4, 18:2-20:4 and 18:1-20:4 species of phosphatidylcholine and/or phosphatidylethanolamine are most rapidly labeled after administration of [3H]arachidonic acid and that they appear to serve as the source of the [3H]arachidonate that is ultimately transferred to ethanolamine plasmalogens.     

Phosphatidylcholine molecular species involved in Γ-linolenic acid biosynthesis in microsomes from borage seeds

Boraginaceae seeds are particularly rich in Γ -linolenic acid (6,9,12-octadecatrienoic acid, Γ -18:3). In microsomes, the analysis of phosphatidylcholine (PC) molecular species by HPLC led to identification of 15 different molecular species; among them 4 contained Γ -18:3, mostly at position 2 of sn -glycerol. Time courses of acylation and desaturation in PC molecular species were examined when [¹⁴C]oleoyl-CoA or [¹⁴C]linoleoyl-CoA was provided as substrates to isolated microsomes. With [¹⁴C]oleoyl-CoA or [¹⁴C]linoleoyl-CoA and in the absence of NADH, 3 main labelled PC molecular species were found: 18:2/[¹⁴C]18:1, 16:0/[¹⁴C]18:1 and 18:1/[¹⁴C]18:1. When NADH was present in the incubation medium, the fatty acids were progressively desaturated by the Δ12- and Δ6-desaturases successively (with [¹⁴C]oleoyl-CoA as precursor) or by the Δ6-desaturase alone (with [¹⁴C]linoleoyl-CoA as precursor). In both types of experiments, 7 final desaturation products in microsomes were evidenced; among them, 3 contained radioactive Γ -18:3, i.e . 18:2/[¹⁴C] Γ -18:3, 18:1/[¹⁴C] Γ -18:3 and 16:0/[¹⁴C] Γ -18:3. While the Δ12-desaturase had no specificity for position on the glycerol backbone, labelled Γ -linolenic acid was recovered exclusively in the sn -2 position.     

Biosynthesis and degradation of gamma-glutamyltranspeptidase of rat kidney

gamma-Glutamyltranspeptidase (gamma GTP) of rat kidney is an intrinsic glycoprotein bound to the plasma membrane and composed of two nonidentical subunits and an amino-terminal portion of the heavy subunit anchors the enzyme to the membrane. The mechanisms of biosynthesis, post-translational processing and degradation of the enzyme were studied using mono-specific antibody raised to gamma-glutamyltranspeptidase purified from rat kidney. The following results were obtained. Double isotope labeling in vivo showed that gamma-glutamyltranspeptidase is synthesized as a precursor form with a single polypeptide chain of 78,000 daltons, and then processed post-translationally by limited proteolysis, resulting in two subunits of 50,000 and 23,000 daltons. Incorporation of [3H]leucine or [35S]methionine into the precursor form increased until 60 min after their intravenous injection, and a pulse-chase experiment showed that the half life of the precursor form was 53 min. [3H]Fucose and [3H]glucosamine could also be incorporated into the precursor form, showing that glycosylation of the enzyme occurs at the stage of the precursor form. Rat kidney labeled with [3H]fucose was subjected to subcellular fractionation. The Golgi fraction contained the glycosylated precursor form and a small amount of subunits, and the plasma membrane fraction contained mostly subunits with a significant amount of precursor, suggesting that post-translational processing of the precursor occurs on the plasma membrane. The apparent half lives of the native enzyme and the heavy and light subunits were all estimated as 4.3 +/- 0.5 days by labeling with [3H]leucine or [3H]fucose. gamma-Glutamyltranspeptidase has a different turnover rate from aminopeptidase M, which is located in the microvillus membrane close to gamma-glutamyltranspeptidase.     

Adaptation of biological membranes to temperature: molecular species compositions of phosphatidylcholine and phosphatidylethanolamine in mitochondrial and microsomal membranes of liver from thermally-acclimated rainbow trout

Summary Molecular species profiles were determined for both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of mitochondrial and microsomal membrane fractions from liver tissue of thermally-acclimated rainbow trout,Salmo gairdneri. The predominant molecular species of PC were 16:0/22:6, 16:0/18:1, 16:0/20:3 and 16:0/22:5, whereas predominant molecular species of PE were 18:1/20:4, 14:0/16:0, 18:0/22:6 and 18:1/22:6. PE possessed short chain saturates (primarily 14:0/16:0) and monoenes (primarily 14:0/16:1) not present in PC and larger proportions of polyunsaturated (18:0/22:6, 18:0/22:5 and 18:1/22:6. and diunsaturated molecular species than PC. Differences between membrane fractions were most evident in warm (20°C)-acclimated trout. Mitochondria contained higher proportions of long-chain, polyunsaturated molecular species of PE, but less of the corresponding species of PC than other membrane fractions. Rankings based on unsaturation index were accordingly: mitochondria heavy microsomes>light microsomes for PE, but heavy microsomes>light microsomes>-mitochondria for PC. Mitochondria were notable for high proportions of diunsaturated molecular species of both phosphatides. Growth at cold temperatures (5°C) was generally associated with a replacement of shorter chain mono- and dienoic molecular species (16:0/18:1, 16:1/18:1, 14:0/16:2 and 18:1/18:1 in the case of PC and 14:0/16:1, 14:0/16:2 and 16:1/18:1 for PE), and occasionally saturates, with long-chain, polyunsaturated molecular species (for PC, C_36–38: 16:0/22:6, 16:1/22:6, 16:0/20:3 and 16:0/20:5; for PE, C_38–40: 18:1/20:4, 16:1/22:6, 18:0/20:5, 18:2/20:4, 18:0/22:5 and 18:0/22:6). However, compositions of mitochondrial PE and PC from heavy microsomes were not significantly influenced by acclimation temperature. The role of phospholipase A₂, in addition to other metabolic processes, in mediating these changes is discussed.Abbreviations ACL average chain length - UI unsaturation index     

Mitochondrial turnover in liver is fast in vivo and is accelerated by dietary restriction: application of a simple dynamic model

'Mitochondrial dysfunction', which may result from an accumulation of damaged mitochondria in cells due to a slowed-down rate of mitochondrial turnover and inadequate removal of damaged mitochondria during aging, has been implicated as both cause and consequence of the aging process and a number of age-related pathologies. Despite growing interest in mitochondrial function during aging, published data on mitochondrial turnover are scarce, and differ from each other by up to one order of magnitude. Here we demonstrate that re-utilization of the radioactively labelled precursor in pulse-chase assays is the most likely cause of significant overestimation of mitochondrial turnover rates. We performed a classic radioactive label pulse-chase experiment using (14)C NaHCO(3), whose (14)C is incorporated into various amino acids, to measure mitochondrial turnover in mouse liver. In this system, the activity of the urea cycle greatly limited arginine dependent label re-utilization, but not that of other amino acids. We used information from tissues that do not have an active urea cycle (brain and muscle) to estimate the extent of label re-utilization with a dynamic mathematical model. We estimated the actual liver mitochondrial half life as only 1.83 days, and this decreased to 1.16 days following 3 months of dietary restriction, supporting the hypothesis that this intervention might promote mitochondrial turnover as a part of its beneficial effects.     

The polyadenylated RNA directing the synthesis of the rat myelin basic proteins is present in both free and membrane-bound forebrain polyribosomes.

Free and membrane-bound polyribosomes were isolated from the forebrain of actively myelinating 24-day-old rats. The poly(A)+ RNA (polyadenylated RNA) extracted from both fractions was translated in vitro in reticulocyte lysates [Hall & Lim (1981) Biochem. J. 196. 327-336] in the presence or absence of a heterologous microsomal membrane fraction from dog pancreas. The rat myelin basic proteins synthesized in vitro were isolated by CM-cellulose chromatography and by immunoprecipitation with purified anti-(myelin basic protein) antibody. The large (mol.wt. 18 500) and small (mol.wt. 16 000) myelin basic proteins were translational products of poly(A)+ RNA from both free and membrane-bound polyribosomes. The identity of the myelin basic proteins was verified by analysis of peptides generated by the cathepsin D digestion of the immunoprecipitated proteins synthesized in vitro, in comparison with authentic rat myelin basic proteins. Although several other translational products of membrane-bound polyribosomal poly(A)+ RNA were modified when microsomal membranes were present during translation, molecular weights of the myelin basic proteins themselves were unchanged. The myelin basic proteins synthesized in vitro also did not differ significantly in size from the authentic myelin basic proteins, indicating that these membrane proteins are unlikely to be synthesized as substantially larger precursor molecules. The presence of the specific mRNA species on both free and membrane-bound polyribosomes is compatible with the extrinsic location of the myelin basic proteins on the cytoplasmic surface of the myelin membrane.