Advances in chemical proteomic evaluation of lipid kinases—DAG kinases as a case study

Advancements in chemical proteomics and mass spectrometry lipidomics are providing new opportunities to understand lipid kinase activity, specificity, and regulation on a global cellular scale. Here, we describe recent developments in chemical biology of lipid kinases with a focus on those members that phosphorylate diacylglycerols. We further discuss future implications of how these mass spectrometry–based approaches can be adapted for studies of additional lipid kinase members with the aim of bridging the gap between protein and lipid kinase–focused investigations.     

THE EFFECTS OF CARBAMYLCHOLINE ON INCORPORATION IN VIVO OF [1-14C]ARACHIDONIC ACID INTO GLYCEROLIPIDS OF MOUSE BRAIN

Abstract— The effects of carbamylcholine on incorporation of [1-¹⁴C]arachidonate into the glycerolipids in mouse brain synaptosome-rich and microsomal fractions were examined at 1, 3 and 10 min after intracerebral injection of the labeled precursor. When carbamylcholine was included with the labeled arachidonate, there was a decrease in the proportion of labeled fatty acid incorporated into the phospholipids. Among the phospholipids in the synaptosome-rich fraction, a decrease in incorporation of radioactivity into diacyl-glycerophosphoinositols and diacyl-glycerophosphocholines was observed at 1 and 3 min after injection. A decrease in labeling of diacyl-glycerophosphoethanolamines and diacyl-glycerophosphocholines in the microsomal fraction was observed at 3 and 10 min after injection. The decrease in phospholipid labeling was marked by an increase in labeling of diacylglycerols which was observed initially in the synaptosome-rich fraction, but also in the microsomal fraction at later time periods. Other lipid changes included an increase in triacylglycerol labeling which was found in the synaptosome-rich fraction and an increase in phosphatidic acid labeling which was found in the microsomal fraction. Results of the in vivo study have demonstrated changes in brain lipid metabolism during carbamylcholine stimulation. Furthermore, these changes appear to be initiated mainly in the synaptosome-rich fraction.     

Products of phospholipid metabolism in Bacillus stearothermophilus.

The products of phospholipid turnover in Bacillus stearothermophilus were determined in cultures labeled to equilibrium and with short pulses of [32P]phosphate and [2-3H]glycerol. Label lost from the cellular lipid pool was recovered in three fractions: low-molecular-weight extracellular products, extracellular lipid, and lipoteichoic acid (LTA). The low-molecular-weight turnover products were released from the cells during the first 10 to 20 min of a 60-min chase period and appeared to be derived primarily from phosphatidylglycerol turnover. Phosphatidylethanolamine, which appeared to be synthesized in part from the phosphatidyl group of phosphatidylglycerol, was released from the cell but was not degraded. The major product of phospholipid turnover was LTA. Essentially all of the label lost from the lipid pool during the final 40 min of the chase period was recovered as extracellular LTA. The LTA appeared to be derived primarily from the turnover of cardiolipin and the phosphatidyl group of phosphatidylglycerol. Three types of LTA were isolated; an extracellular LTA was recovered from the culture medium, and two types of LTA were extracted from membrane preparations or whole-cell lysates by the hot phenol-water procedure. Cells contained 1.5 to 2.5 mg of cellular LTA per g of cells (dry weight), over 50% of which remained associated with the membrane when cells were fractionated. Over 75% of the 3H label incorporated into the cellular LTA pool during a 90-min labeling period was released from the cells during the first cell doubling after the chase. Label lost from the lipid pool was incorporated into cellular LTA which was then modified and released into the culture medium.     

Subcellular distribution of protein kinase C/phorbol ester receptors in differentiating mouse keratinocytes

The activation of protein kinase C (PKC) by diacylglycerol or tumor promoters plays a pivotal role in signal transduction and subsequent activation of cellular processes. Since the activity of this enzyme is dependent on its immediate lipid domain, its relative distribution within the cell may be an important regulatory mechanism. We report here a relative decrease in PKC/phorbol ester receptor associated with the particulate fraction of mouse keratinocytes induced to differentiate by two separate systems. First, proliferating keratinocytes maintained in low Ca2+ (0.09 mM) serum-free medium were induced to differentiate rapidly by the addition of Ca2+ (1.8 mM). A 1.4-fold decrease in the percent of total phorbol receptor binding activity present in the particulate fraction and concomitant increase in binding in the cytosol fraction was evident 20 min after the Ca2+ addition. Second, in keratinocytes that differentiate over a 6 day cultivation period in serum-containing medium with Ca2+ concentration of 1.8 mM, a significant decrease in the percent of the phorbol receptor binding activity present in the particulate fraction was observed as the culture begins to differentiate on days 3 and 4. Maximal phorbol ester binding in the particulate fraction corresponded to the proliferative phase of the culture (day 2), while lower levels of PKC/phorbol ester binding to particulate fractions were noted during the early differentiative phase (days 3 and 4). Addition of the synthetic diacylglycerols 1-oleoyl-2-acetylglycerol or L-alpha-1,2 dioctanyl glycerol at 30 micrograms/ml to proliferating keratinocyte cultures induced a modest increase in two markers of terminal differentiation: cornified envelope formation and transglutaminase levels. These findings, taken together, support the hypothesis that PKC activation plays a role in the initial signalling events for keratinocyte differentiation.     

Sphingosine differentially inhibits activation of the Na+/H+ exchanger by phorbol esters and growth factors

The role of protein kinase C in activation of the plasma membrane Na+/H+ exchanger was studied in cultured vascular smooth muscle cells. The basic lipid, sphingosine, was used to block enzymatic activity of protein kinase C. Na+/H+ exchange was activated by phorbol 12-myristate 13-acetate (PMA), diacylglycerols, platelet-derived growth factor (PDGF), thrombin, or by osmotically-induced cell shrinkage. Intracellular pH and Na+/H+ exchange activity were measured using the intracellular pH indicator, 2',7'-bis(carboxyethyl)-5(6) carboxyfluorescein. Acting alone, both crude sphingosine and pure, synthetic C18 D-(+)-erythro-sphingosine raised pHi in a dose-dependent manner (from 6.95 +/- 0.02 to 7.19 +/- 0.09 over 10 min for 10 microM sphingosine). This alkalinization was not due to Na+/H+ exchange as it was not altered by t-butylamiloride (50 microM) nor by replacement of the assay medium with a Na(+)-free solution. Sphingosine-induced alkalinization did not require protein kinase C activity, since it was fully intact in protein kinase C-depleted cells. It was also not due to a detergent action of sphingosine on the cell membrane, since both ionic and non-ionic detergents caused cell acidification. Rather, alkalinization induced by sphingosine appeared to be due to cellular uptake of NH3 groups since N-acetylsphingosine showed no alkalinization. After the initial cell alkalinization, cellular uptake of [3H]sphingosine continued slowly for up to 24 h. The ability of PMA or dioctanoylglycerol to activate Na+/H+ exchange fell to 20% of control after 24 h of sphingosine exposure. At all times, C11 and N-acetylsphingosine failed to block PMA-induced activation of the exchanger. Activation of the Na+/H+ exchanger by sucrose, which does not depend on protein kinase C activity, was unaffected by sphingosine. Activation of Na+/H+ exchange by thrombin and PDGF was partially inhibited by 30 and 20%, respectively. These data indicate that both thrombin and PDGF activate Na+/H+ exchange by pathway(s) that are primarily independent of protein kinase C.     

sn-1,2-diacylglycerols and phorbol diesters: uptake, metabolism, and subsequent assimilation of the diacylglycerol metabolites into complex lipids of cultured cells

The cell-permeable diacylglycerol mediators have been shown to mimic partially the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured cells. In order to evaluate the metabolic stability of the lipid mediators, several radiolabeled diacylglycerols were synthesized and their uptake and intracellular fate in cultured HL-60 (human promyelocytic leukemia) cells was compared with TPA. In addition to whole cell assessment, the stability of diacyl lipids and TPA was evaluated in a buffer/water system and in the presence of serum and subcellular fractions. The compounds studied include 1,2-dioleoyl-sn-glycerol (DiOG), 1-oleoyl-2-acetyl-sn-glycerol (OaG), 1-palmitoyl-2-acetyl-sn-glycerol (PaG), the ether-linked analog 1-palmityl-2-acetyl-sn-glycerol (ePaG), and TPA. TPA was comparatively stable to lipase hydrolysis in all systems examined. First, the data show that within 5 min at pH 7.9, nearly 50% of the PaG (originally greater than 92% 1,2-isomer) had isomerized, and rapid formation of the 1,3-isomer also occurred with OaG and ePaG. The metabolism of OaG and PaG by serum hydrolases, using a reaction medium containing 10% serum, was chiefly by acetate hydrolysis; however, fatty acid was also liberated. After a 60-min incubation 68% of the [14C]OaG was converted, by serum enzymes, to monooleoylglycerol plus oleic acid. Heat-inactivation of serum reduced the enzymatic formation of fatty acid by 60-70%. ePaG was also metabolized by serum enzymes, but the ether-linked alkylglycerol product was stable. The results of cell-free studies (postmitochondrial supernatant) showed that cellular enzymes were present that could, like serum, convert the diacylglycerols to monoacylglycerols and free fatty acids. Studies using cultured cells showed that radiolabeled OaG, PaG, and ePaG were rapidly taken up by the cells and metabolized. Labeled metabolic products from the diacylglycerols appeared, in a time-dependent manner, in cellular phospholipids and triacylglycerols. The results from experiments employing 1-acyl-2-acetyl-sn-[3H]glycerol and [3H]acyl-2-acetyl-sn-glycerol indicate that the intracellular mode of mediator metabolism is via complete hydrolysis with subsequent incorporation of 3H-acyl groups into complex lipids. Data are also presented which show that a substantial amount of cellular lipid acyl group modification occurs and large amounts of glycerol are produced when cells are cultured with OaG. Collectively, these results demonstrate that the diacylglycerol mediators, when compared with TPA, are not stable and are metabolized by both serum and cellular enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transport and utilization of free fatty acids in Triatoma infestans

Triatoma infestans hemolymph has 0.31 mg/ml of free fatty acids and 2.8 mg/ml of diacylglycerols. Almost all the diacylglycerols are transported by lipophorin whereas free fatty acids are carried by lipophorin and a very high density lipoprotein. The binding of cis-parinaric acid to lipophorin was employed to specify the free fatty acid binding properties of lipophorin. Lipophorin has 10 binding sites of high affinity (3 x 10(7)) and approximately 45 binding sites of low affinity (1 x 10(6)). The relative rate of tissue incorporation of free fatty acids and diacylglycerols was measured by injecting insects with hemolymph previously labeled in both, free fatty acids and diacylglycerols. In this way, the half-life of the hemolymph free fatty acids was estimated to be about 4 min. Based on this result and taking into account the content of free fatty acids and diacylglycerols in hemolymph, the incorporation of free fatty acids, expressed in moles of fatty acids, seems to be 3.4 times higher than that of diacylglycerols. This finding can be applied to other insects.     

Distribution of dietary trans-octadecenoate among acyl-CoA and other lipid fractions of rat liver and heart

Groups of rats were fed diets containing 10% of either corn oil, partially hydrogenated soybean oil, or a mixture of the two. The partially hydrogenated oil contained a high level of trans-octadecenoate and a low level of linoleate, and all diets were adjusted to contain similar levels of cis-octadecenoate. The fatty acid compositions of five tissue lipid fractions from liver and heart (non-esterified fatty acids, acyl-CoA, diacylglycerols, triacylglycerols and phospholipids) were analyzed to measure the effect of the dietary supply on the accumulation of trans-octadecenoates and other fatty acids at different steps of glycerolipid synthesis. Although trans-octadecenoate was increased in all of the lipid fractions when the dietary supply was increased, the accumulation did not exceed 15% of the acyl chains in any of the lipid pools even when the dietary trans acid accounted for 46% of the fatty acids supplied in the diet. The trans-octadecenoate accumulated in a similar manner in the lipids of both liver and heart, and the amounts found in the acyl-CoA esters of both tissues were relatively low compared to the diet. A high dietary supply of trans-octadecenoate appeared to diminish the relative content of stearate in the acyl-CoA and phospholipid fractions. The level of cis-octadecenoate maintained in tissue phospholipids was similar to that in the acyl-CoA fractions, whereas the trans-octadecenoate content in phospholipids more closely resembled that in the diacylglycerols. Normal proportions of arachidonate were maintained in the tissue phospholipids during high intake of trans acids, even though lower levels were observed in the acyl-CoA and diacylglycerols of liver.     

Experiments towards quantification of saturated and polyunsaturated diacylglycerols by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry

As recently shown, different physiologically relevant lipid classes can easily be analyzed by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI–TOF MS). In the present study the first application of MALDI–TOF for the quantitative analysis of diacylglycerols is described. It is shown that the use of a suitable reference sample enables the quantification of diacylglycerols up to the picomolar range. The best reproducibility of quantitative results for diacylglycerols was obtained using a matrix of 2,5-dihydroxybenzoic acid in ethylacetate and incorporation of an internal standard of the same lipid class. A moderate laser power was used, resulting in a very low extent of fragmentation, allowing a quantification by using solely the highest signal arising from sodium adduct formation of diacylglycerols. A linear correlation between peak intensity and lipid concentration over one order of magnitude was found. The applicability of this new technique for the analysis of other lipids like phosphatidylcholines is also discussed.     

Changes of initiation mass and cell dimensions by the 'eclipse'

The minimum time (E) required for a new pair of replication origins (oriCs) produced upon initiating a round of replication to be ready to initiate the next round after one cell mass doubling, the 'eclipse', is explained in terms of a minimal distance (l(min)) that the replication forks must move away from oriC before oriCs can 'fire' again. In conditions demanding a scheduled initiation event before the relative distance l(min)/L(0.5) (L being the total chromosome length) is reached, initiation is presumably delayed. Under such circumstances, cell mass at the next initiation would be greater than the usual, constant Mi (cell mass per copy number of oriC) prevailing in steady state of exponential growth. This model can be tested experimentally by extending the replication time C using thymine limitation at short doubling times tau in rich media to reach a relative eclipse E/C < l(min)/L(0.5). It is consistent with results obtained in experiments in which the number of replication 'positions'n (= C/tau) is increased beyond the natural maximum, causing the mean cell size to rise continuously, first by widening, then by lengthening, and finally by splitting its poles. The consequent branching is associated with casting off a small proportion of normal-sized cells and lysing DNA-less cells. Whether or how these phenomena are related to peptidoglycan composition and synthesis are moot questions.     

Effect of fasting on the composition of the fat body lipid of Dipetalogaster maximus, Triatoma infestans and Panstrongylus megistus (Hemiptera: Reduviidae)

Modifications in content and lipid composition induced by fasting were examined in fat bodies from adults of Triatominae, Dipetalogaster maximus, Triatoma infestans and Panstrongylus megistus. With fasting, total lipid stores dropped approximately 50% for T. infestans and more than 70% for P. megistus. Total lipids analyzed by thin layer chromatography and fractionated by column chromatography on Unisil showed triacylglycerols as the main component in the three species, although P. megistus showed high levels of diacylglycerols (31–46%). Cholesterol amounted to 8–15%. In diacylglycerol fractions, C_16:0, C_18:1 and C_18:0 fatty acids were detected; their ratio varied with species but it was not dependent on nutritional status. In triacylglycerol fractions C_18:1 fatty acid was the major component at different times (48–68%); the ratio of monounsaturated to saturated in this fraction was 1.3, 2.6 and 1.2 for D. maximus, T. infestans and P. megistus respectively. The remarkable drop in lipid stores without noticeable changes in their relative composition would suggest that all types of lipid are used at similar rates. The higher content of diacylglycerols in P. megistus may be associated with the better flight performance of this species. Accepted: 4 August 1998     

Effect of cerulenin on cellular autolytic activity and lipid metabolism during inhibition of protein synthesis in Streptococcus faecalis.

Cellular autolytic activity as well as lipid and lipoteichoic acid metabolism have been studied in cultures of Streptococcus faecalis receiving various combinations of the following treatments: chloramphenicol addition, starvation for an essential amino acid (valine), and cerulenin treatment. Lipoteichoic acid initially accumulated in chloramphenicol-treated and amino acid-starved cells and decreased relative to the cellular mass in cerulenin-treated cells. The relative phosphatidylglycerol content of amino acid-starved cultures or of cultures treated with either antibiotic rapidly decreased upon initiation of each treatment. In all cases, cerulenin initially stimulated diphosphatidylglycerol synthesis. Pretreatment of cultures with cerulenin prevented the inhibition of cellular synthesis autolysis normally observed during chloramphenicol treatment, but did not affect amino acid starvation-induced inhibition of autolytic activity. Variations in the levels of the nonionic lipid fraction, predominantly diglycerides, correlated best with the patterns of autolytic activity observed during chloramphenicol treatment, whereas variations in the levels of diphosphatidylglycerol and lipoteichoic acid correlated best with the patterns of autolytic activity observed during amino acid starvation. Components of the nonionic lipid fraction were demonstrated to inhibit autolytic activity 50% in whole cell and in cell wall assays at 60 and 120 nmol/mg (dry weight), respectively.     

Axenic cultivation of Naegleria gruberi : Requirement for methionine

A simplified axenic medium for Naegleria gruberi strain NEG-M contains -methionine, dextrose, yeast extract, a macromolecular fraction of fetal calf serum, and phosphate buffer. Amoebae cultured in suspension in this medium grow with doubling times of 8–10 h (at 32 °C) to yield 2–4 × 10⁶ cells/ml. Amoebae from growing or early stationary phase cultures, transferred to nonnutrient buffer, differentiate synchronously into flagellates. Differentiation occurs reproducibly 80 min after initiation (time for 50% flagellates at 25 °C) if amoebae are taken from a culture maintained at pH 6.6.     

Effect of experimental diabetes and insulin on lipid metabolism in the isolated perfused rat lung.

The isolated perfused rat lung was used as a model to study the possible hormonal regulation of lipid metabolism in the mammalian adult lung. Experimental diabetes, whether induced by alloxan or streptozotocin, decreased the incorporation of [U-14C]glucose into neutral lipids and phospholipids of both the surfactant fraction and the residual fraction of the lung by 60-80%. Glucose incorporation into phosphatidylcholine and phosphatidylglycerol is decreased in experimental diabetes in both the surfactant and residual fractions to a comparable degree. Glucose incorporation is decreased in both the fatty acid and the glycerophosphocholine moieties of phosphatidylcholine isolated from the surfactant and residual fractions. Insulin treatment of normal animals 30 or 15 min prior to perfusion resulted in an approximate doubling of the incorporation of glucose into the phosphatidylcholine and phosphatidylglycerol isolated from the surfactant and residual fractions of the lung. The incorporation of glucose into palmitic acid isolated from phosphatidylcholine was also shown to increase similarly. The results of these investigations indicate that insulin may play a role in regulating the synthesis of the important lipid components of the mammalian pulmonary surfactant complex.     

Food reserves of scots pine (Pinus sylvestris L.)

Summary Starch, soluble sugars, triacylglycerols, diacylglycerols and free fatty acids were measured in 30-year-old Scots pine (Pinus sylvestris L.) trees during an annual cycle in the sapwood (youngest ten xylem rings). The radial distribution of carbohydrates and lipids was studied in the trunkwood of 90 -to 150-year-old Scots pine trees collected at the end of the growing season. Determination of the compounds was performed using specific enzymatic assays, capillary gas chromatography and thin layer chromatography. The amounts of glucose, fructose, sucrose, and galactose/arabinose in the sapwood were slightly higher in winter than in summer. Raffinose/stachyose increased up to 5-fold during the cold period. At the beginning of the growing season starch amounts rose, and then decreased in summer and autumn. No concentration changes were observed in the total amounts of diacylglycerols and fatty acids throughout the year. Triacylglycerol levels were slightly higher in February than in summer and autumn. Relative frequencies of individual fatty acids were similar in all lipid fractions. Glucose, fructose, sucrose, starch and triacylglycerols disappeared almost entirely at the transition zone from sapwood to heartwood. In contrast, free fatty acids and galactose/arabinose rose in centripetal direction, and diacylglycerols remained constant across trunk cross-sections. The relative amounts of individual fatty acids changed markedly in the free fatty acid fraction and in the triacylglycerols when crossing the sapwood-heartwood boundary. Concentration changes of reserve materials are discussed in relation to season, mobilization and translocation processes, dormancy, frost resistance, and heartwood formation. The results are compared to those found in needles.     

Role of membrane lipids and membrane fluidity in thermosensitivity and thermotolerance of mammalian cells

The role of membrane lipids and membrane fluidity in thermosensitivity of mammalian cells is not well understood. The limited experimental data in the literature have led to conflicting results. A detailed investigation of lipid composition and membrane fluidity of cellular membranes was undertaken to determine their relationship to cell survival after hyperthermia. Ehrlich ascites (EA) cells, mouse fibroblast LM cells, and HeLa S3 cells differed in thermosensitivity as expressed by a D0 of 3.1, 5.2, and 9.7 min, respectively, at 44 degrees C. No correlation with cellular thermosensitivity could be found with respect to the amount of cholesterol and to the cholesterol to phospholipid ratio in the particulate fraction of the cells. By growing the cells for some generations in different media, cholesterol and phospholipid content could be changed in the particulate fraction, but no difference in cell survival was observed. When mouse fibroblasts were grown for 24 hr in a serum-free medium supplemented with arachidonic acid (20:4), all subcellular membranes were about eight times richer in phospholipids containing polyunsaturated acyl (PUFA) chains and membrane fluidity was increased as measured by fluorescence polarization of diphenylhexatriene (DPH). The alterations resulted in a higher thermosensitivity. When mouse fibroblasts were made thermotolerant no change in cholesterol and phospholipid content could be found in the particulate fraction of the cells. The relative weights and the quality of the phospholipids as well as the fatty acid composition of the phospholipids appeared to be the same for normal and thermotolerant cells. Fluidity measurements in whole cells, isolated plasma membranes, and liposomes prepared from phospholipids extracted from the cells revealed no significant differences between normal and thermotolerant fibroblasts when assayed by fluorescence polarization (DPH) and electron spin resonance (5-nitroxystearate). It is concluded that the mechanism of thermal adaptation resulting in differences in lipid composition as reported in the literature differs from the mechanism of the acquisition of thermal tolerance. The lower heat sensitivity of thermotolerant cells, as initiated by a nonlethal triggering heat dose followed by an induction period at 37 degrees C, does not involve changes in lipid composition and membrane fluidity. However, a prompt and clear (also nonlethal) change in membrane fluidity by an increase in PUFA does result in an increased thermosensitivity, probably because of an indirect effect via the lipids in causing disfunctioning of proteins in the membrane and/or the cytoskeleton.     

Mice lacking phosphatidylinositol transfer protein-alpha exhibit spinocerebellar degeneration,intestinal and hepatic steatosis,and hypoglycemia

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and cellular functions. We now report that ablation of PITP alpha function leads to aponecrotic spinocerebellar disease, hypoglycemia, and intestinal and hepatic steatosis in mice. The data indicate that hypoglycemia is in part associated with reduced proglucagon gene expression and glycogenolysis that result from pancreatic islet cell defects. The intestinal and hepatic steatosis results from the intracellular accumulation of neutral lipid and free fatty acid mass in these organs and suggests defective trafficking of triglycerides and diacylglycerols from the endoplasmic reticulum. We propose that deranged intestinal and hepatic lipid metabolism and defective proglucagon gene expression contribute to hypoglycemia in PITP alpha-/- mice, and that hypoglycemia is a significant contributing factor in the onset of spinocerebellar disease. Taken together, the data suggest an unanticipated role for PITP alpha in with glucose homeostasis and in mammalian endoplasmic reticulum functions that interface with transport of specific luminal lipid cargoes.     

Formation of a complex between valine and intestinal mucosal lipid; its possible role in valine absorption

During intestinal absorption amino acids must traverse the lipid-rich epithelial cell membrane, possibly in a lipid-soluble form. In a search for such a form, we have determined the ability of lipid extracted from intestinal mucosa to bind valine. After incubation in a valine-containing medium this lipid (defined as the heptane-soluble fraction) contained, on the average, 3.63 micromoles of valine per 100 mg of lipid. Cyanide (0.002 m), 2,4-dinitrophenol (0.0002 m), and anaerobic conditions had little effect on this process. Valine uptake into the lipid fraction of mucosa was complete after 2.5 min. Of a number of sugars and amino acids tested, isoleucine, methionine, and leucine were the most potent inhibitors of valine uptake into lipid. The inhibition by leucine appeared to be competitive. A similar uptake of glucose into the mucosal lipid was not inhibited by leucine, methionine, or isoleucine but was inhibited by galactose. Various phosphoglycerides (but not sphingomyelin) from other sources, used in place of mucosal lipid, were able to carry 20-150 times as much valine into heptane-soluble fraction as were other lipid classes. Some characteristics of the complex are similar to those of the valine transport system.     

Study of mechanisms of photoreceptor membrane destabilization under modifying action of oxygen]

Induction of lipid peroxidation in the rod outer segments (ROS) of frog retina results in fragmentation of photoreceptor disc membranes and solubilization of lipoprotein rhodopsin complexes (sedimentation coefficient 2.7S). The substrates of lipid autooxidation are mainly docosahexa- and docosapentaenoyl residues of phosphatidyl ethanolamine. Large fragments (precipitation at 5000 gX30 min) and small vesicles (precipitation at 40 000 gX60 min; average diameter 1000 A) formed from lipoperoxidized ROS differ both in their chemical composition and structural organization. In small vesicles the content of O2-modified polyenoic acyls in the phospholipids is 3,7 times higher as compared to large fragments. Correspondingly, the capacity of hydrophobic areas in the small vesicle membranes evaluated by EPR spin-probing technique is lower than in the large fragments. A mechanism of the photoreceptor membrane destabilization under modification by molecular oxygen is proposed. It is based on a decrease in the value of surface tension upon accumulation of lipid peroxidation products in ROS, the former possessing the properties of non-ionic detergents.