Products Of Lipid Hydrolysis Research Articles

Microparticle fabricated from a series of symmetrical lipids based on dihydroxyacetone form textured architectures

We report the synthesis of a series of symmetrical lipids composed of dihydroxyacetone and even‑carbon fatty acids (eight to sixteen carbons), both components of the human metabolome, and characterize their formulation into porous microparticles through spontaneous emulsification without the use of additional porogens. Lipid hydrolysis products were identified by 1H NMR to validate lipid degradation into the parent metabolic synthons. Microparticle architecture, as determined by scanning electron microscopy, was lipid-length dependent, with shorter alkyl chains forming tight structures and longer alkyl chains forming larger pores with plate-like lipid architectures. In all cases, the lipids formed organized patterns, not irregular shapes. As a demonstration of the potential use of these solid lipid-based microparticles, the release kinetics of a model drug (piroxicam) was quantified showing that release was more greatly influenced by microparticle porosity, and hence surface area, than by hydrophobicity of the lipids.

THP-1 macrophage cholesterol efflux is impaired by palmitoleate through Akt activation.

Lipoprotein lipase (LPL) is upregulated in atherosclerotic lesions and it may promote the progression of atherosclerosis, but the mechanisms behind this process are not completely understood. We previously showed that the phosphorylation of Akt within THP-1 macrophages is increased in response to the lipid hydrolysis products generated by LPL from total lipoproteins. Notably, the free fatty acid (FFA) component was responsible for this effect. In the present study, we aimed to reveal more detail as to how the FFA component may affect Akt signalling. We show that the phosphorylation of Akt within THP-1 macrophages increases with total FFA concentration and that phosphorylation is elevated up to 18 hours. We further show that specifically the palmitoleate component of the total FFA affects Akt phosphorylation. This is tied with changes to the levels of select molecular species of phosphoinositides. We further show that the total FFA component, and specifically palmitoleate, reduces apolipoprotein A-I-mediated cholesterol efflux, and that the reduction can be reversed in the presence of the Akt inhibitor MK-2206. Overall, our data support a negative role for the FFA component of lipoprotein hydrolysis products generated by LPL, by impairing macrophage cholesterol efflux via Akt activation.

Fatty acid composition and phospholipid types used in infant formulas modifies the establishment of human gut bacteria in germ-free mice

Human milk fat contains high concentrations of medium-chained fatty acids (MCFA) and triacylglycerols emulsified by a sphingomyelin-rich phospholipid membrane (milk phospholipids, MPL). Infant formula comprises mainly long-chained fatty acids (LCFA) emulsified with dairy proteins and soy lecithin (SL) lacking sphingomyelin. Sphingomyelin content and saturation level of phospholipids affect the gut lipase activity, which alters the concentrations of lipid hydrolysis products in ileum and colon, and hereby putatively affects the competitive advantage of specific gut bacteria. Thus, differences in phospholipid and FA composition may modulate the establishment of the gut microbiota. We investigated effects of fatty acid (FA) composition and emulsification (MPL vs SL) ingested during establishment of human gut microbiota in germ-free mice, and found that cecal microbiotas from mice given MCFA-rich emulsions were characterized by high relative abundances of Bacteroidaceae and Desulfovibrionaceae, while LCFA-rich emulsions caused higher abundances of Enterobacteriaceae, Erysipelotrichaceae, Coriobacteriaceae and Enterococcaceae. Consumption of SL-emulsified lipids skewed the community towards more Enterococcaceae and Enterobacteriaceae, while MPL increased Bacteroidaceae, Desulfovibrionaceae, Rikkenellaceae and Porphyromonadaceae. Intake of SL increased cecal concentrations of iso-valeric and iso-butyric acids. This suggests that fat-type and emulsifiers applied in infant formula may have distinct effects on the establishment of the gut microbiota in formula-fed infants.

Diverse activity of human secretory phospholipases A2 on the migration of human vascular smooth muscle cells.

Investigation of the diversity of human secretory phospholipases A2 (sPLA2) on the migration of human vascular smooth muscle cells (VSMC). We investigated the impact of sPLA2 IIA, V, and X and of oleic acid, linoleic acid and lysophosphatidylcholine on the migration of human VSMC. Recombinant human sPLA2's and Boyden's chamber method were applied. sPLA2, IIA but not V or X enhanced migration of VSMC in a dose/time dependent manner. Oleic and linoleic acids, and lysophosphatidylcholine markedly enhanced migration. These results imply that sPLA2 IIA, which is known to be present in the arterial wall in the vicinity of VSMC, as well as products of lipid hydrolysis induced by sPLA2, enhance the migration of VSMC, and thus may contribute to atherogenic process.

Lipid hydrolysis products affect the composition of infant gut microbial communities in vitro.

Some lipid hydrolysis products such as medium-chained NEFA (MC-NEFA), sphingosine and monoacylglycerols (MAG) possess antibacterial activity, while others, including oleic acid, are essential for the optimal growth of Lactobacillus species. Thus, changes in the concentrations of NEFA and MAG in the distal ileum and colon can potentially selectively modulate the composition of the gut microbiota, especially in early life when lipid absorption efficacy is reduced. As medium-chained fatty acids are enriched in mothers' milk, such effects may be highly relevant during gut colonisation. In the present study, we examined the effect of selected NEFA, MAG and sphingosine on the composition of faecal microbial communities derived from infants aged 2-5 months during a 24h anaerobic in vitro fermentation. We tested lipid mixtures in the concentration range of 0-200μm, either based on MC-NEFA (10:0 to 14:0 and MAG 12:0) or long-chained NEFA (LC-NEFA; 16:0 to 18:1 and MAG 16:0) with and without sphingosine, representing lipid hydrolysis products characteristic for intestinal hydrolysis of breast milk lipids. Ion Torrent sequencing of the bacterial 16S ribosomal RNA gene revealed that the relative abundance of lactic acid-producing genera, including Lactobacillus and Bifidobacterium, was generally increased in the presence of 50μm or higher concentrations of MC-NEFA. For Bifidobacterium, the same effect was also observed in the presence of a mixture containing LC-NEFA with sphingosine. On the contrary, the relative abundance of Enterobacteriaceae was significantly decreased in the presence of both lipid mixtures. Our findings suggest that the high concentration of medium-chained fatty acids in breast milk might have functional effects on the establishment of the gut microbiota in early life.

Cold acclimation and low temperature resistance in cotton: Gossypium hirsutum phospholipase Dα isoforms are differentially regulated by temperature and light

Phospholipase Dalpha (PLDalpha) was isolated from cultivated cotton (Gossypium hirsutum) and characterized. Two PLDalpha genes were identified in the allotetraploid genome of G. hirsutum, derived from its diploid progenitors, G. raimondii and G. arboreum. The genes contained three exons and two introns. The translated products shared a 98.6% homology and were designated as GrPLDalpha and GaPLDalpha. Their ORFs encoded a polypeptide of 807 amino acids with a predicted molecular mass of 91.6 kDa sharing an 81-82% homology with PLDalpha1 and PLDalpha2 from A. thaliana. A possible alternative splicing event was detected at the 5' untranslated region which, however, did not result in alternative ORFs. Cold stress (10 degrees C or less) resulted in gene induction which was suppressed below control levels (25 degrees C or 22 degrees C growth temperature) when plants were acclimated at 17 degrees C before applying the cold treatment. Differences in the expression levels of the isoforms were recorded under cold acclimation, and cold stress temperatures. Expression was light regulated under growth, acclimation, and cold stress temperatures. Characterization of the products of lipid hydrolysis by the endogenous PLDalpha indicated alterations in lipid species and a variation in levels of the signalling molecule phosphatidic acid (PA) following acclimation or cold stress.

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A(2) treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.

Effect of Phospholipase A2 Hydrolysis Products on Calcium Oxalate Precipitation at Lipid Interfaces

Urinary stones are commonly composed of an inorganic component, calcium oxalate, or calcium phosphate and an organic matrix of lipids, carbohydrates, and proteinaceous matter. Of interest is the role that the organic matrix elements may play as catalysts for the heterogeneous nucleation of the calcium salts, and a number of studies have examined the role of lipids in calcium oxalate monohydrate (COM) formation. In this study, products of lipid hydrolysis from phospholipase A(2) (PLA(2)) are examined for their effect on COM formation using Langmuir monolayers as model lipid membrane assemblies. The enzyme PLA(2) hydrolyzes DPPC monolayers in the presence of a supersaturated calcium oxalate subphase, inducing the rapid and plentiful nucleation of calcium oxalate at the lipid interface. To investigate the cause of increased crystal formation in the presence of the enzyme, Langmuir monolayers modeling the hydrolysis products were investigated. Calcium oxalate crystal growth at a ternary monolayer of dipalmitoylphosphatidylcholine (DPPC), palmitic acid (PA), and a 22-carbon chain lysophospholipid (22:0 Lyso PC) dramatically increases relative to monolayers of just DPPC. Binary monolayers of DPPC with either PA or the 22:0 Lyso PC and single-component monolayers of PA were also studied. It is demonstrated that the fatty acid generated during lipid hydrolysis causes a significant increase in the extent of heterogeneous nucleation of calcium oxalate from supersaturated solutions. The results imply a possible link between increased phospholipase activity, which is associated with hyperoxaluria, and calcium oxalate precipitation.

Interfacial Mechanism of Phospholipase A2: pH-Dependent Inhibition and Me-β-cyclodextrin Activation

The pH-dependent activity of phospholipase A(2) (PLA(2)) from Naja mossambica mossambica venom and the membrane-water partitioning of the lipid hydrolysis products were investigated in solid-supported palmitoyl-oleyl-phosphatidylcholine-d(31) (POPC-d(31)) membranes using neutron reflection. At pH 5, PLA(2) interacts only weakly with the substrate membrane and leads to no observable membrane breakdown, which is consistent with protonation of the catalytic histidine (His48, pK(a) approximately 6.2). The rate of the lyso-lipid partitioning into the solution phase is the same at pH 9 as at pH 7.4, and the relative membrane-water partitioning of the products is essentially the same; that is, the fatty acid accumulates in the membrane, and only the lyso-lipid is solubilized. However, Me-beta-cyclodextrin (Me-beta-CD) activates PLA(2) irrespective of pH by facilitating the solubilization of the lyso-lipid product, but not the fatty acid, of which only 22% is encapsulated at pH 9. Since no product solubilization is observed at pH 5 in the absence of Me-beta-CD, this suggests that the hydrolytic mechanism of PLA(2) is not fully disabled at pH 5 but is inhibited by a mechanism, which is counteracted by Me-beta-CD-mediated release of the lyso-lipid. Me-beta-CD does not interact with the substrate membrane, which indicates that at low pH the product extraction occurs directly from the enzyme active site outside the immediate membrane-water interface, whereas at pH 7-9, direct solubilization of the lyso-lipid from the membrane can also contribute to activation of PLA(2).

Intestinal lipid absorption

Our knowledge of the uptake and transport of dietary fat and fat-soluble vitamins has advanced considerably. Researchers have identified several new mechanisms by which lipids are taken up by enterocytes and packaged as chylomicrons for export into the lymphatic system or clarified the actions of mechanisms previously known to participate in these processes. Fatty acids are taken up by enterocytes involving protein-mediated as well as protein-independent processes. Net cholesterol uptake depends on the competing activities of NPC1L1, ABCG5, and ABCG8 present in the apical membrane. We have considerably more detailed information about the uptake of products of lipid hydrolysis, the active transport systems by which they reach the endoplasmic reticulum, the mechanisms by which they are resynthesized into neutral lipids and utilized within the endoplasmic reticulum to form lipoproteins, and the mechanisms by which lipoproteins are secreted from the basolateral side of the enterocyte. apoB and MTP are known to be central to the efficient assembly and secretion of lipoproteins. In recent studies, investigators found that cholesterol, phospholipids, and vitamin E can also be secreted from enterocytes as components of high-density apoB-free/apoAI-containing lipoproteins. Several of these advances will probably be investigated further for their potential as targets for the development of drugs that can suppress cholesterol absorption, thereby reducing the risk of hypercholesterolemia and cardiovascular disease.

Purification of the used palm oil by adsorption

Purification of the used palm oil by adsorption The components of fresh vegetable oils are mainly non-polar. During frying of food, complex and multistage reactions occur in the oil, which produce various polar compounds. Active carbons are characterized by small contents of polar functional groups, which are responsible for adsorbing such polar compounds. Effective purification of the used frying oils must involve the removal of the polar substances. To improve the quality of the used palm oil, an active carbon oxidized by a hydrogen peroxide treatment was used. Such a carbonaceous adsorbent improves the quality of the oil used for frying food by purifying it from colour substances, lipid hydrolysis products, oxidation products and total polar compounds.

Accelerated Lipid Absorption in Mice Overexpressing Intestinal SR-BI

Dietary cholesterol absorption contributes to a large part of the circulating cholesterol. However, the mechanism of sterol intestinal uptake is not clearly elucidated. Scavenger receptor class B type I (SR-BI), major component in the control of cholesterol homeostasis, is expressed in the intestine, but its role in this organ remains unclear. We have generated transgenic mice overexpressing SR-BI primarily in the intestine by using the mouse SR-BI gene under the control of intestinal specific "apoC-III enhancer coupled with apoA-IV promoter." We found SR-BI overexpression with respect to the natural protein along the intestine and at the top of the villosities. After a meal containing [(14)C]cholesterol and [(3)H]triolein, SR-BI transgenic mice presented a rise in intestinal absorption of both lipids that was not due to a defect in chylomicron clearance nor to a change in the bile flow or the bile acid content. Nevertheless, SR-BI transgenic mice showed a decrease of total cholesterol but an increase of triglyceride content in plasma without any change in the high density lipoprotein apoA-I level. Thus, we described for the first time a functional role in vivo for SR-BI in cholesterol but also in triglyceride intestinal absorption.

Effect of the lipid hydrolysis products on the phospholipase A 2 action towards lipid monolayer

The effect of lauric acid (LA) and lysolauroyllecithin (LLL) on the hydrolysis of lipid in monolayer by phospholipase A 2 from Bee venom was studied. It was found that LLL inhibits phospholipase action under both high (39 mN/m) and low (25 mN/m) surface pressure. On the other hand, LA inhibits phospholipase action under the low surface pressure (15 mN/m or 25 mN/m), but increases enzyme activity under high surface pressure (39 mN/m). This activating effect can be suppressed by high ionic strength of the aqueous subphase. It is suggested that an increase of the negative surface charge of the lipid monolayer, followed by an increase of the local concentrations of the positively charged enzyme and calcium near the monolayer is a coupling factor between fatty acid accumulation and phospholipase activation. Such an autocatalytic process can only occur when the substrate is organised into monolayer, bilayer or micelles, therefore it can be considered as a reason for the substrate activation and induction time before lipid hydrolysis.

Characteristics of Mackerel Mince Lipid Hydrolysis

ABSTRACTThe lipid hydrolysis products in minced mackerel stored under vacuum at 2–3°C for 15 days were free fatty acids (FFA) and 1,2‐diglycerides (DG); no 1,3‐DG br monoglycerides were obsrved, Lysophosphati‐dylcholine fLPC and lvsoohosohatidvlethanolamine (LPE) increased and then ddcreased with time: Lysiphosphatidylserie was not observed. More FFA, 1,2‐DG, LPC, and LPE were detected in dark muscle than in light muscle. The initial fatty acid composition, by GC, showed no significant differences (α‐0.05) among whole, light and dark muscles. When the lipid extracts were separated into three fractions (1: mostly triglycerides; 2: mostly FFA, DG, and cholesterol; and 3: mostly phospholipid), the degree of unsaturation of the lipids in fraction 2 was between those in fraction 1 and 3 and increased with time. These findings support the possibility that fish lipid hydrolysis may affect fish wholesomeness.

Modulation of lipid derived mediators by polyunsaturated fatty acids

Cell stimulation by a number of agonists triggers the formation of products of lipid hydrolysis, which act either as intracellular mediators of signal transduction or as modulators of cell-cell interactions. This process is mediated by the activation of hydrolytic enzymes, the phospholipases (PLase), especially the A 2 and C, acting on cell phospholipids (PL). Among the major products being formed, the following: a) the inositol phosphates (IP), especially IP 3, and diacylglycerols (DAG) generated intracellularly from phosphoinositides through PLase C, b) the eicosanoids, the arachidonic acid (AA) metabolites produced through combined PLase A 2 and (cyclo- and lip-) oxygenase activities, and released from cells, c) the ether lipid PAF, derived from alkylacyl phosphatidylcholine through PLase A 2, have attracted the attention of investigators for their important biological roles. Interest has also been recently developed towards products of sphingolipid hydrolysis, sphingosine and ceramide, which are generated by various cell types after stimulation, and exert biological activities. Cell glycerophospholipids are rich in long-chain polyunsaturated fatty acids (PUFA) of the n-6, namely AA 20:4 n-6, and n-3, mainly docosahexaenoic acid (DHA) 22:6, series. These compounds are differentially distributed among the various PL classes and their levels in cells are modulated through the intake with the diet of either the 18-C fatty acids (FA), precursors, linoleic 18:2 n-6, and, alpha-linolenic 18:3 n-3, respectively-followed by conversion to their long-chain PUFA derivatives, or through the intake of the preformed compounds. Levels of the long-chain PUFA in membranes can also be modulated in cultured cells, through manipulation of the supply with the growth medium. The incorporation of exogenous FA in cell lipids, in this type of study, however, follows patterns which are different from those observed in vivo after dietary manipulations. The levels of long chain n-6 and n-3 PUFA in cell phosphoglycerides modulate the generation of lipid derived mediators, in various types of cells, after in vitro stimulation. These effects can be ascribed to various mechanisms: 1. 1. Changes in eicosanoid production as a consequence of: a) changes in the levels of AA, the major precursor of these compounds, b) accumulation of long-chain PUFA of the n-3 series, which act as precursors (e.g. eicosapentaenoic acid (EPA), 20:5) of eicosanoids with biological activities different from those of the products derived from AA, or inhibitors (EPA) and/or modulators (DHA) of the AA oxygenases. 2. 2. Changes in the stimulated formation of other products of PL hydrolysis, such as the IP and PAF, possibly as a consequence of a modified micro-environment of the PLases, or as a consequence of modified eicosanoid production. The fomation of other mediators, such as the cytokines, in specialized cells, is also affected by changes of cell PUFA, possibly as an additional consequence of altered eicosanoid formation. In this presentation, examples of changes of cell n-6 and n-3 PUFA, obtained in circulating cells (e.g. platelets, leukocytes) through feeding studies in animals and humans, or induced in cultured cells after FA supplementation, will be presented and discussed, in relation to the effects on the production of eicosanoids and other lipid derived mediators.

Sphingosine inhibits angiotensin-stimulated aldosterone synthesis

Sphingosine and other protein kinase C inhibitors were tested for their ability to inhibit aldosterone synthesis by bovine adrenal glomerulosa cells. Sphingosine inhibited angiotensin (AII)-stimulated aldosterone synthesis (IC 5 pf 5 μM). At doses that totally blocked steroidogenesis, sphingosine did not affect protein synthesis or [ 125I]AII binding to cells. Sphingosine also inhibited dibutyryl cyclic AMP (dbcAMP)-stimulated aldosterone synthesis. Sphingosine inhibited pregnenolone synthesis from cholesterol, but not the conversion of progesterone or 20α-hydroxycholesterol to aldosterone. These results suggest that sphingosine inhibits steroidogenesis at a locus close to that where stimulation occurs by AII and dbcAMP. Other protein kinase C inhibitors were tested. Retinal, 1-(5-isoquinolinesulfonyl)-2-methyl- piperazine dihydrochloride (H-7), and staurosporine inhibited aldosterone synthesis stimulated by AII and dbcAMP. Retinal and H-7 also inhibited progesterone conversion to aldosterone, and retinal blocked [ 125I]AII binding. Staurosporine was more specific, inhibiting AII-stimulated aldosteronogenesis at concentrations which had little effect on conversion of progesterone to aldosterone. Because they inhibited dbcAMP stimulation, none of the inhibitors was sufficiently specific to use as a probe of the role of protein kinase C. The IC 50 of sphingosine suggests that this or related products of lipid hydrolysis could act as endogenous regulators of adrenal cell function.

Human milk kills Giardia lamblia by generating toxic lipolytic products.

This study supports our previous hypotheses that normal human milk kills Giardia lamblia trophozoites in vitro and that this killing is due to the release of free fatty acids (FFAs) from milk triglycerides by action of the bile salt-stimulated lipase (BSL) of human milk. Heat-stable killing of G. lamblia was generated when normal human milk was preincubated with sodium cholate, which activates BSL. Moreover, both the skim-milk (containing BSL) and cream (containing mainly triglycerides) fractions were required to kill G. lamblia. We measured the toxicity of FFA and other products of lipolysis to G. lamblia. cis-Unsaturated FFAs (LD50 less than 12 microM), three of four monoglycerides, and four of five lysophosphatidylcholines were toxic to the parasites (LD50 less than 100 microM). In contrast, the parasites were not harmed by the corresponding diglycerides, phosphatidylcholines, triolein, or glycerol. Thus, products of lipid hydrolysis in the normal digestive tract are toxic to G. lamblia. We also demonstrated that albumin and conjugated bile salts, which bind FFA, partially protected trophozoites from killing by oleic acid.

Structural alterations of the inner mitochondrial membrane in ischemic liver cell injury

Mitoplasts were prepared from 3-h ischemic livers in an attempt to define the structural alterations in the inner membrane that may account for the functional deficiencies of ischemic mitochondria. Mitoplasts from both control and ischemic livers had similar specific activities of cytochrome oxidase and succinate-cytochrome c reductase. With both preparations, the specific activity of rotenone-insensitive NADH-cytochrome c reductase was 10-fold lower than in the mitochondria from which they were prepared. Ischemic mitoplasts had no respiratory control with ADP, and had a slightly reduced phospholipid to protein ratio and an increased cholesterol to protein ratio. As a result, the cholesterol to phospholipid molar ratio was increased from the control of 0.04 to 0.08. There were also differences in the content of individual phospholipid species. Phosphatidylcholine increased by 15%, while cardiolipin decreased by 60%. There were increases in sphingomyelin and in the lysophospholipids of phosphatidylcholine, ethanolamine, and cardiolipin. Pretreatment with chlorpromazine did not prevent these changes. Linoleic acid was decreased by 35% in ischemic phospholipids, and the content of free fatty acids was increased 4-fold. Electron spin resonance spectroscopy of mitoplasts spin labeled with either 5- or 12-doxyl stearic acid revealed an increased molecular order (decreased fluidity) of ischemic inner mitochondrial membranes consistent with the increased cholesterol to phospholipid ratio. The data indicate activation of a phospholipase A in ischemic mitochondria with the resulting accumulation of products of lipid hydrolysis. This conclusion further emphasizes the close similarity between the structural and functional consequences of ischemia in the intact animal and the effect on isolated mitochondria of the activation of the endogenous phospholipase A. In both cases the major functional alterations are attributable to changes in the permeability of the inner mitochondrial membrane induced by the accumulation of lysophospholipids.

Mobilisation of the major stored reserves in the embryo of fenugreek (Trigonella foenum-graecum L., Leguminosae), and correlated enzyme activities

Changes in total nitrogen, soluble amino nitrogen, lipid and phytate contents, and in the activities of proteinase (pH 7.0), isocitrate lyase and phytase were followed in the endosperm, cotyledons, and axis during germination of fenugreek seeds and subsequent growth of the seedlings. The endosperm is comprised largely of cell-wall galactomannans: the majority of the seed total nitrogen, lipid and phytate (5%, 8%, 0.44% of seed dry weight respectively) is localised within the cotyledons as stored reserves. Germination is completed after 10-14 h from the start of imbibition, but the major reserves are not mobilised during the first 24 h. Then the total nitrogen content of the cotyledons starts to decrease and that of the axis increases; there is a concomitant accumulation of soluble amino nitrogen in both cotyledons and axis. An increase in proteinase activity in the cotyledons correlates well with the depletion of total nitrogen therein. Depletion of lipid and phytate reserves in the different seed tissues constitutes a late event, occurring after 50 h from the start of imbibition, and is coincident with the final disintegration of the endosperm tissue. The depletion of phytate and stored lipids is accompanied by an increase in phytase and isocitrate lyase activity. It appears that the products of lipid hydrolysis are converted by gluconeogenesis to serve as the major source of sugars for the growing axis after the endosperm galactomannan has been completely mobilised.

Effects of lipids and their related compounds on the chloroplast functions.

Effects of lipids and their related compounds on the chloroplast functions were investigated. Whole lipids, i.e. methyl linolenate, monogalactosyidiglyceride, and lecithin induced only a slight effect on the photochemical activities; electron transport, H+ uptake, and cyclic photophosphorylation. Lecithin alone stimulated significantly O2 evolution. On the contrary, the lipid-hydrolysis products, i.e. linolenic acid, monogalactosylmonoglyceride, and lysolecithin caused a strong inhibition of all the photoactivities tested. They showed two different modes of inhibition, which arise probably from a situation whether the lipids affect coupling factor or not besides the membrane part in thylakoid. Peroxidized methyl linolenate and a-tocopherol were also examined and the effect of lipid oxidation was discussed.