Effect of reactive oxygen species on the erythrocyte calcium-pump function in protein-energy malnutrition

The presence of detectagle amounts of non-heme iron in erythrocyte ghost membranes have been postulated to lead to the initiation of membrane lipid peroxidation and the attendant perturbation of membrane functions. We have investigated the presence of non-heme iron and endogenous products of lipid peroxidation in erythrocyte membranes of normal and kwashiorkor (KWA) subjects and assessed the susceptibility of the membranes to exogenously generated reactive oxygen species. The modulation of the basal and calmodulin-stimulated calcium-pumping activity of these membranes by reactive oxygen species was also assessed. The results show the presence of significant amounts of non-heme iron and endogenous free radical reaction products in the red cell membranes of KWA subjects compared with that of normal children. Estimation of the extent of lipid peroxidation in the presence of exogenously generated reactive oxygen species further revealed that erythrocyte ghost membranes of KWA subjects are more susceptible to oxidative stress than those of normal individuals. Although both the basal and calmodulin-stimulated activities of the membrane-bound Ca²⁺-pump enzyme in normal and KWA subjects were inhibited by oxygen-free radicals, the erythrocyte enzyme in KWA subjects showed higher susceptibility to inhibition by oxygen free radicals than that of normal individuals. We propose that the reduced erythrocyte calcium-pump function in KWA is not unconnected with excessive generation of reactive oxygen species.Abbreviations PMSF phenylmethylsulfonylfuloride - TLCK N--p-tosyl-l-lysine chloromethylketone - EGTA ethyleneglycol-bis (B-aminoethylether) N,N-tetraacetic acid - EDTA ethylene diamine tetraacetic acid - ATP Adenosine 5-triphosphate - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - Tris-HCl Tris (hydroxymethyl) aminomethane-hydrochloride - SDS Sodium dodecyl sulphate - TBAR thiobarbituric acid-reactive products TBA, thiobarbituric acid - TCA trichloroacetic acid     

Comparative Analysis of the Fatty Acid Composition of Microalgae Obtained by Different Oil Extraction Methods and Direct Biomass Transesterification

One of the main challenges for the successful production and use of microalgae for biodiesel production is to obtain a satisfactory level of fatty acid methyl esters (FAME). The aims of this study are to identify the best method of lipid extraction and provide high FAME levels and to evaluate their fatty acid profiles. Six lipid extraction methodologies in three microalgae species were tested in comparison with the direct transesterification (DT) of microalgal biomass method. The choice of extraction method affected both the oily extract yield and the FAME composition of the microalgae and consequently may affect the properties of biodiesel. The efficiency of different lipid extraction methods is affected by the solvent polarity, which extracts different target compounds from lipid matrix. Dichloromethane/methanol extraction and Folch extraction produced the largest oil extract yields, but extraction with hexane/ethanol resulted in the best ester profile and levels. Performing DT reduces the volume of extractor solvent, the time and cost of FA composition analysis, as well as, presents less steps for fatty acid quantification. DT provided biomass FAME levels of 50.2, 636.4, and 258.2 mg.g^?1 in Nannochlorophisis oculata, Chaetoceros muelleri, and Chlorella sp., respectively. On the basis of an analysis of the fatty acids profiles of different species, C. muelleri is a promising microalga for biodiesel production. Depending on the extraction method, Chlorella sp. and N. oculata can be considered as an alternative in obtaining arachidonic (Aa) and eicosapentaenoic (EPA) acids.     

Electron paramagnetic resonance studies of spectrin-phospholipid associations

The interaction of spectrin, a peripheral cytoplasmic protein of the erythrocyte membrane, with synthetic phospholipids was characterized by density gradient centrifugation, electron microscopy, and the paramagnetic resonance of nitroxide spin labels. The organic solvent 2-chloroethanol, which favors the stability of hydrophobic surfaces on proteins, was utilized in the formation of the protein-lipid systems. Spectrin, upon dialysis to remove 2-chloroethanol, was found to associate into extensive network-like aggregates and in the presence of dipalmitoylphosphatidylcholine, the spectrin aggregates were found to associate with liposomes formed during dialysis. This interaction, which was significantly enhanced by the presence of dipalmitoylphosphatidylethanolamine, was found to reduce the mobility of fatty acid spin labels incorporated into the lipid regions of the lipid-protein associations. Evidence was found which suggests that spectrin tends to stabilize the phospholipid vesicles against fusion and decrease lipid mobility, particularly near the polar bilayer surfaces.     

Impact of high dietary lipid intake and related metabolic disorders on the abundance and acyl composition of the unique mitochondrial phospholipid,cardiolipin

Excessive dietary lipid intake, coupled with lack of exercise, are the major causes of the development and progression of metabolic syndrome features e. g. obesity, hepatic steatosis, insulin resistance, type 2 diabetes and cardiovascular diseases. These metabolic diseases are associated with both structural and functional alterations of mitochondria. Cardiolipin (CL) is a unique phospholipid that is almost exclusively localized in the mitochondrial inner membrane. Cardiolipin is at the heart of mitochondrial metabolism playing a key role in several processes of mitochondrial bioenergetics as well as in mitochondrial membrane stability and dynamics, and in many of the mitochondrial-dependent steps of apoptosis. Indeed, alterations to CL content and acyl chain profile have been associated with mitochondrial dysfunction in multiple tissues in Barth syndrome and in many other physio-pathological conditions. After a brief overview of the biological roles of CL, we highlight the consequences of lipid overload-related nutritional manipulations as well as related metabolic disorders on both CL content and its fatty acid composition in the major metabolic tissues, the heart, muscle and liver. The goal of this review is to fill a void in the CL literature concerning the effects of CL abundance and form that arise following high lipid supplementation and the related metabolic disorders.     

Design and synthesis of mono and bicyclic tetrapeptides thioester as potent inhibitor of histone deacetylases

Inhibitors of histone deacetylases (HDACs) are a promising class of anticancer agents that have an effect on gene regulation. The naturally occurring cyclic depsipeptide FK228 containing disulfide and Largazole possessing thioester functionalities act as pro-drugs and share the same HDAC inhibition mechanism in cell. Inspired from these facts, we have reported bicyclic tetrapeptide disulfide HDAC inhibitors resembling FK228 with potent activity and enhanced selectivity. In the present study, we report the design and synthesis of several mono and bicyclic tetrapeptide thioester HDAC inhibitors that share the inhibition mechanism similar to Largazole. Most of the compounds showed HDAC1 and HDAC4 inhibition and p21 promoting activity in nanomolar ranges. Among these the monocyclic peptides 1, 2 and bicyclic peptide, 4 are notable demanding more advanced research to be promising anticancer drug candidates.     

Role ofβ-oxidation in inhibitingLactobacillus leichmanii by fatty acids

The effect of saturated fatty acids from 6∶0 to 16∶0 and oleic acid onLactobacillus leichmanii ATCC 4797 growing in non-skim-milk media was determined. The inhibition by lauric acid was higher than that obtained with any other fatty acid. A mutant (MC12) resistant to the fatty acid inhibition with high β-oxidation activity was also studied. A positive correlation between the ability ofL. leichmanii ATCC 4797 and its derivative MC12 to degrade fatty acids and their resistance to the fatty acid inhibition is shown in this report.     

Reactive oxygen species generation and antioxidant defense system in hydroponically grown wheat (Triticum aestivum) upon β-pinene exposure: an early time course assessment

We investigated the effect of β-pinene on reactive oxygen species (ROS: lipid peroxidation, membrane integrity, hydrogen peroxide and superoxide ions) generation and activity of antioxidant defense system during early hours of treatment (4, 8, 16 and 24 h) in hydroponically grown Triticum aestivum (wheat). β-Pinene reduced the root and shoot growth of the hydroponically grown wheat. However, the reduction was more pronounced in root length than in shoot length. β-Pinene enhanced ROS generation as indicated by increased levels of malondialdehyde (20–87 %), hydrogen peroxide (9–45 %) and superoxide ion (23–179 %) content, thereby suggesting lipid peroxidation and induction of oxidative stress in a time- and concentration-dependent manner. The oxidative damage was more pronounced at ≥10 µM β-pinene and at ≥8 h after exposure. β-Pinene caused a severe electrolyte leakage from wheat roots indicating membrane disruption and loss of integrity. Enhanced lipid peroxidation and loss of membrane integrity were confirmed by in situ histochemical studies. β-Pinene provoked increase in the activity of lipoxygenase and upregulation in the activities of antioxidant enzymes: catalases, superoxide dismutases, ascorbate peroxidases, guaiacol peroxidases and glutathione reductases. The enhanced activity of lipoxygenases evoked by β-pinene paralleled higher accumulation of MDA, thereby suggesting that antioxidant defense mechanism was not able to prevent β-pinene-induced lipid peroxidation.     

Fatty acids profile and temperature in the cultured marine diatom Odontella aurita

The diatom Odontella aurita has now been industrially cultured and commercialized as a dietary supplement rich in omega-3 fatty acids for several years. In this study, we investigated the effect of three temperatures (8, 16, and 24 °C) on the growth and fatty acid composition of cells harvested during the exponential and stationary growth phases. These temperatures were selected on the basis of photosynthesis responses previously obtained at different temperatures using a modulated fluorometer. Our results confirm that both growth and lipid composition were sensitive to culture temperature. Growth was reduced when O. aurita was cultured at low temperature (8 °C) compared to when it was cultured at high temperatures (16 and 24 °C), but the proportion of polyunsaturated fatty acids (PUFAs, 20:5 n-3 and 22:6 n-3) increased while the level of saturated fatty acids (SFAs, 14:0 and 16:0) decreased in the cells harvested during both the exponential and stationary growth phases. On the other hand, the cells grown at 24 °C displayed a marked decrease in PUFA and an increase in SFA levels. Harvesting time is also a critical parameter in achieving optimum n-3 PUFA productivity during batch cultivation. Indeed, changes in fatty acid composition with growth phase seem to be dependent on the culture temperature, with the most marked effects being observed at 24 °C. PUFA levels (i.e., levels of 20:5 n-3 and 22:6 n-3) increased during the stationary growth phase, while the proportion of SFAs and monounsaturated fatty acids (MUFAs) fell with time. As this species is currently grown in outdoor ponds with seasonal temperature variations (minimal and maximal average temperatures in winter and summer, from 3 to 9 °C and from 13 to 26 °C, respectively), this factor can be expected to have a strong influence on the fatty acid content and composition of the algal biomass harvested and commercialized.     

On the mechanism of respiratory complex I

The energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy and X-ray crystallography revealed the two-part structure of the enzyme complex. A peripheral arm extending into the aqueous phase catalyzes the electron transfer reaction. Accordingly, this arm contains the redox-active cofactors, namely one flavin mononucleotide (FMN) and up to ten iron-sulfur (Fe/S) clusters. A membrane arm embedded in the lipid bilayer catalyzes proton translocation by a yet unknown mechanism. The binding site of the substrate (ubi) quinone is located at the interface of the two arms. The oxidation of one NADH is coupled with the translocation of four protons across the membrane. In this review, the binding of the substrates, the intramolecular electron transfer, the role of individual Fe/S clusters and the mechanism of proton translocation are discussed in the light of recent data obtained from our laboratory.     

Biogenesis of mitochondria 20 the effects of altered membrane lipid composition on mitochondrial oxidative phosphorylation inSaccharomyces cerevisiae

1. The lipid composition of mitochondria isolated from a fatty acid desaturase mutant ofSaccharomyces cerevisiae may be extensively manipulated by growing the organism on defined supplements of unsaturated fatty acid (UFA).
2. The fatty acid composition of the mitochondrial lipids closely follows that of the whole cells from which the mitochondria are isolated. UFA-depleted mitochondria contain normal levels of sterols, neutral lipids and total phospholipids, but have much lower levels of phosphatidyl inositides.
3. UFA-depleted mitochondria possess a full complement of cytochromes, oxidase both NAD-linked and flavoprotein-linked substrates at normal rates, and have levels of succinate and malate dehydrogenases similar to those of UFA-supplemented mitochondria. However, UFA-depletion has a marked effect on the ability of cytochromec to reactivate the NADH oxidase activity of cytochromec-depleted mitochondria.
4. The efficiency of oxidative phosphorylation decreases progressively with the UFA content of the mitochondria, and oxidative phosphorylation is completely lost in mitochondria containing approximately 20% UFA.
5. The incorporation of UFA into the lipids of UFA-depleted mitochondriain vivo results in a recoupling of oxidative phosphorylation. Recoupling is insensitive to both chloramphenicol and cycloheximide, indicating that all the proteins necessary for oxidative phosphorylation are present in UFA-depleted mitochondria, and that the less of oxidative phosphorylation is a purely lipid lesion.
6. ATPase activity is apparently unaffected by UFA-depletion, but³²P_i-ATP exchange activity is lost in mitochondria which have been extensively depleted in UFA.
7. Valinomycin stimulates the respiration of UFA-supplemented mitochondria in media containing potassium, but has no effect on the respiration of UFA-depleted mitochondria, suggesting that active transport of potassium is lost as a result of UFA-depletion.

     

Profiling of fatty acid methyl esters from the oleaginous diatom Fistulifera sp. strain JPCC DA0580 under nutrition-sufficient and -deficient conditions

The marine oleaginous diatom, Fistulifera sp. strain JPCC DA0580, is a promising candidate for biodiesel production due to its high lipid content. In order to truly evaluate the potential of this strain as biodiesel feedstock as well as the impact of nutrition-deficiency to this strain, the proportion of the lipid fractions and fatty acid methyl ester (FAME) derived from Fistulifera sp. cultured under nutrition-sufficient or -deficient conditions were analyzed. The nutrition deficiency led to the increase of the total lipid content in the form of neutral lipids (NLs) accumulation and the decline of polar lipids compared with nutrition-sufficiency. Meanwhile, the total lipid productivity was not significantly changed under two nutrition conditions while the NL productivity under nutrition-deficient condition was much higher than nutrition-sufficient condition. The major FAME components, C14:0, C16:0, C16:1, and C20:5, contribute to over 90 % of total FAMEs under both nutrition conditions. A lower polyunsaturated FAME level were observed in the nutrition-deficient condition (9.9?±?0.2 %) compared with the nutrition-sufficient condition (19.8?±?1.2 %), suggesting the availability of the nutrition stress on the strain JPCC DA0580 for improvement of fuel quality as well as productivity. The lipid quality estimation based on the FAME profile revealed that the nutrition-deficiency could further improve the lipid quality of both total lipids and NL fraction. In addition, direct infusion ESI-Q-TRAP-MS/MS was carried out for the fractionated NL in order to estimate triacylglycerol (TAG) composition, suggesting a crucial role of the chloroplast in TAG synthesis.     

Gender-dimorphic regulation of muscular proteins in response to high fat diet and sex steroid hormones

To investigate the roles of gender-dependent obesity, we investigated the effects of high fat diet (HFD) and sex steroid hormones on the fiber-type dependent expression of contractile and metabolic regulatory pathway proteins in gastrocnemius (G) and soleus (S) muscle tissue of male and female rats. The results revealed that estrogen (E2) negatively influences body weight gain, whereas testosterone (DHT) has positive effects. Additionally, E2 appeared to play an essential role in initiating muscle contraction and mediating glucose and lipid metabolism events via AMPK and AKT pathways. The elevated expression of ERα contributed to the expression of muscular proteins in a fiber-type and gender-dependent manner. E2 treatment increased the protein levels of AMPK, thereby activating downstream lipid metabolic proteins such as PPARγ, ACSL1, LPL, and A-FABP. Such cooperatively activating proteins enhanced fatty acid oxidation, attenuating TG accumulation. E2 stimulated AKT and AMPK activation suggests that these proteins enhanced GLUT4 expression. More importantly, the S muscle of HFD-fed control females showed higher expressions of MYH, TPM1α, and TnI, while only MYH and TnI were upregulated in males treated with E2, indicating that females may be more resistant to HFD-increased metabolic complications. Similarly, E2 treatment enhanced metabolic regulatory proteins in males, indicating that they are more susceptible to metabolic dysregulation than females. To the best our knowledge, this is the first report to distinguish the fatty acid uptake and oxidation between two types of muscle in both genders.     

Structure of a protein catalyzing the formation of 11cis-retinal in the visual cycle of invertebrate eyes

A pigment made up of a protein able to bind retinal as well as retinol is described. The molecule consists of a dimer with a molecular weight of 50,000 which binds one molecule of retinal. The binding site for retinal is a Schiff base buried in the interior of the protein. Retinol is probably bound to the protein in the same site as for retinal, although not covalently, as suggested by the absorbance spectra. The protein, extracted from honeybee retina, is involved in visual pigment metabolism (1), and its structure may elucidate the mechanism of the stereospecific photoisomerization of alltrans-retinal to 11-cis-retinal.     

Structural and kinetic evidence that catalytic reaction of human UDP-glucose 6-dehydrogenase involves covalent thiohemiacetal and thioester enzyme intermediates

Biosynthesis of UDP-glucuronic acid by UDP-glucose 6-dehydrogenase (UGDH) occurs through the four-electron oxidation of the UDP-glucose C6 primary alcohol in two NAD⁺-dependent steps. The catalytic reaction of UGDH is thought to involve a Cys nucleophile that promotes formation of a thiohemiacetal enzyme intermediate in the course of the first oxidation step. The thiohemiacetal undergoes further oxidation into a thioester, and hydrolysis of the thioester completes the catalytic cycle. Herein we present crystallographic and kinetic evidence for the human form of UGDH that clarifies participation of covalent catalysis in the enzymatic mechanism. Substitution of the putative catalytic base for water attack on the thioester (Glu¹⁶¹) by an incompetent analog (Gln¹⁶¹) gave a UGDH variant (E161Q) in which the hydrolysis step had become completely rate-limiting so that a thioester enzyme intermediate accumulated at steady state. By crystallizing E161Q in the presence of 5 mm UDP-glucose and 2 mm NAD⁺, we succeeded in trapping a thiohemiacetal enzyme intermediate and determined its structure at 2.3 Å resolution. Cys²⁷⁶ was covalently modified in the structure, establishing its role as catalytic nucleophile of the reaction. The thiohemiacetal reactive C6 was in a position suitable to become further oxidized by hydride transfer to NAD⁺. The proposed catalytic mechanism of human UGDH involves Lys²²⁰ as general base for UDP-glucose alcohol oxidation and for oxyanion stabilization during formation and breakdown of the thiohemiacetal and thioester enzyme intermediates. Water coordinated to Asp²⁸⁰ deprotonates Cys²⁷⁶ to function as an aldehyde trap and also provides oxyanion stabilization. Glu¹⁶¹ is the Brønsted base catalytically promoting the thioester hydrolysis.     

Nonesterified fatty acids and lipid peroxidation

Oxygen free radicals damage cells through peroxidation of membrane lipids. Gastrointestinal mucosal membranes were found to be resistant to in vitro lipid peroxidation as judged by malonaldehyde and conjugated diene production and arachidonic acid depletion. The factor responsible for this in this membrane was isolated and chemically characterised as the nonesterified fatty acids (NEFA), specifically monounsaturated fatty acid, oleic acid. Authentic fatty acids when tested in vitro using liver microsomes showed similar inhibition. The possible mechanism by which NEFA inhibit peroxidation is through iron chelation and iron-fatty acid complex is incapable of inducing peroxidation. Free radicals generated independent of iron was found to induce peroxidaton of mucosal membranes. Gastrointestinal mucosal membranes were found to contain unusually large amount of NEFA. Circulating albumin is known to contain NEFA which was found to inhibit iron induced peroxidation whereas fatty acid free albumin did not have any effect. Addition of individual fatty acids to this albumin restored its inhibitory capacity among which monounsaturated fatty acids were more effective. These studies have shown that iron induced lipid peroxidation damage is prevented by the presence of nonesterified fatty acids.     

Iron content and degree of lipid peroxidation in liver mitochondria isolated from iron-loaded rats

1.The content of non-heme iron and the degree of lipid peroxidation were measured in liver mitochondria isolated from rats injected with either Jectofer (an iron-sorbitol-citric acid complex) or iron-nitrilotriacetate. 2. The sedimentation profiles of the mitochondria from controls and iron-treated rats as revealed by analytical differential centrifugation, indicated single population of mitochondria with $\text{s}{}_{\mathrm{4,B}}$ values of 13200± 560 S and 14200±590 S for controls and iron-loaded animals, respectively. In contrast, the sedimentation profiles of the acid phosphatase activity and the non-heme iron revealed marked polydispersities with at least three populations of particles for both controls and iron-loaded animals. 3. The mitochondria and iron-rich lysosomes were separated by density-gradient centrifugation in an isotonic medium of Percoll and sucrose. With this technique, the amount of non-heme iron in a mitochondrial fraction by differential centrifugation decreased from 69±28 nmol/mg protein to 5.6±1.1 nmol/mg protein and from 19.3±5.6 nmol/mg protein to 3.3±0.6 nmol/mg protein for Jectofer and iron-nitrilotriacetate injected rats, respectively. For control rats the amount of mitochondrial non-heme iron was about 2.7 nmol/mg protein both before and following density gradient centrifugation. The extra amount of non-heme iron still present in the purified mitochondrial fraction from iron-loaded rats, as compared to controls, was further characterized by the reactivity towards bathophenanthroline sulfonate. The results suggest that the extra iron was due to a small amount of either ferritin or hemosiderin still contaminaning the mitochondrial fraction. The amount of mitochondrial heme iron was the same in iron-loaded rats and controls. 4. The degree of lipid peroxidation in the mitochondria was estimated from the amount of malondialdehyde. The thiobarbituric acid method used for the quantitation of malondialdehyde was modified so that it was insensitive to variable amounts of iron present in the samples. No difference in the degree of lipid peroxidation was observed between the mitochondria from iron-loaded rats and controls. 5. In contrast to recent proposals (Hanstein, E.G. et al. (1981) Biochim. Biophys. Acta 678, 293–299), the present study showed that the amounts of non-heme iron and the degrees of lipid peroxidation are the same in mitochondria isolated from iron-loaded and control animals.     

Lipid Rafts in Neurodegeneration and Neuroprotection

The collective properties of the lipids that form biological membranes give rise to a very high level of lateral organization within the membranes. Lipid-driven membrane organization allows the segregation of membrane-associated components into specific lipid rafts, which function as dynamic platforms for signal transduction, protein processing, and membrane turnover. A number of events essential for the functional integrity of the nervous system occur in lipid rafts and depend on lipid raft organization. Alterations of lipid composition that lead to abnormal lipid raft organization and consequent deregulation of lipid raft-dependent signaling are often associated with neurodegenerative diseases. The amyloidogenic processing of proteins involved in the pathogenesis of major nervous system diseases, including Alzheimer’s disease and Parkinson’s disease, requires lipid raft-dependent compartmentalization at the membrane level. Improved understanding of the forces that control lipid raft organization will facilitate the development of novel strategies for the effective prevention and treatment of neurodegenerative and age-related brain diseases.     

Analysis of expressed sequence tags from a normalized cDNA library of perilla (Perilla frutescens)

Perilla (Perilla frutescens), an herbal plant belonging to the Lamiaceae family, has long been cultivated in Asia. Perilla is notable as an aroma-rich leaf vegetable and as the oilseed crop richest in omega-3 fatty acids. However, molecular analysis of this herbal plant is lacking due to insufficient genetic resources. Here, we constructed a normalized cDNA library from whole young perilla plants and analyzed expressed sequence tags (ESTs). A total of 4,582 uniESTs were generated from analysis of 5,435 ESTs. Among these, 307 uniESTs (6.7%) were identified as unique in perilla and 3,625 uniESTs were assigned at least one GO term through similarity searches in public databases. The most frequent GO terms were related to abiotic and biotic stress responses. In addition, we identified 141 uniESTs involved in lipid metabolism, of which four genes encoded fatty acid desaturases. We found one new candidate omega-3 fatty acid desaturase gene, in addition to the four that were previously reported. This analysis of uniESTs from perilla provides a valuable genetic resource to elucidate the molecular underpinnings of lipid metabolism and for molecular breeding of perilla species.     

Chemical composition of a purified membrane fraction from Sarcina aurantiaca in relation to growth phase

The effect of the age of a culture of Sarcina aurantiaca on the chemical composition of the total membrane fraction has been investigated. Whereas the protein content is constant and the carbohydrate increases with age, the lipid content decreases which may be a reflection of increased binding of the lipid to protein. The carbohydrates present in the membrane are mannose, galactose, glucose and ribose and their relative concentrations change from the exponential to stationary phase. As the cell goes into stationary phase, the amino acid composition of the protein changes with a large increase in alanine and smaller increases in lysine and methionine. The increase in alanine and lysine may be associated with an accumulation of incomplete wall precursors in the membrane during stationary phase since the murein peptide of S. aurantiaca is known to be of the l-lysine-l-alanine ₃ type.