New arrangement of proteins and lipids in the stratum corneum cornified envelope

A new arrangement of proteins and lipids of stratum corneum (SC) cornified envelope (CE) is proposed. The chemical analysis of CE revealed the presence of free fatty acids (FFA), ceramides (Cer), and important percentages of glutamic acid/glutamine (Glx) and serine (Ser) residues. The molecular structure of these components suggests the existence of covalent links not only between Cer and Glx but also between FFA and Ser. The protein distribution of extracellular surface of CE, i.e., the proteins that could be involved in the bonds with lipids, was studied using post- and pre-embedding immunolabeling electron microscopy. Some loricrin (protein rich in Ser) was detected in the outermost part of the CE protein layer. The external arrangement of some domains of this protein may give rise to form linkages with FFA, yielding further insight into the CE arrangement in which Cer-Glx bonds and FFA-Ser bonds would be involved. Although the importance of fatty acids in the cohesion and barrier function of SC has been widely demonstrated, their role could be associated not only to the presence of these lipids in the intercellular lamellae but also in the CE, in the same way that Cer.     

Lipids and fatty acids of Leptospira interrogans serovar copenhageni virulent strain Shibaura.

Lipids and fatty acids of Leptospira interrogans serovar copenhageni virulent strain Shibaura were analyzed by thin-layer chromatography, gas-liquid chromatography, gas-mass spectrometry and infrared absorption spectrometry. The virulent cells possessed a characteristic lipid pattern consisting of free fatty acid (FFA) (41.8%), one major unidentified phospholipid (14.8%), phosphatidylethanolamine (PE) (12.9%), cholesteryl ester (CE) (9.3%), lysophosphatidylethanolamine (LPE) (4.9%) and diphosphatidyl-glycerol (DPG) (1.1%). Various fatty acids such as hexadecanoic (26.9%), hexadecenoic (15.4%), octadecenoic (26.5%) and octadecadienoic (27.4%) acids were detected in the FFA. The fatty acid composition of the major unidentified phospholipid distinctly differed from those of other lipids including PE, LPE, DPG and CE, and comprised mainly tetradecadienoic (53.6%), tetradecatrienoic (14.0%) and octadecanoic (13.8%) acids. This phospholipid with a large amount of polyunsaturated fatty acids with chain lengths of 14 carbon atoms was detected only in the lipids of the virulent cells.     

Combined LC/MS-platform for analysis of all major stratum corneum lipids,and the profiling of skin substitutes

Ceramides (CERs), cholesterol, and free fatty acids (FFAs) are the main lipid classes in human stratum corneum (SC, outermost skin layer), but no studies report on the detailed analysis of these classes in a single platform. The primary aims of this study were to 1) develop an LC/MS method for (semi-)quantitative analysis of all main lipid classes present in human SC; and 2) use this method to study in detail the lipid profiles of human skin substitutes and compare them to human SC lipids. By applying two injections of 10 μl, the developed method detects all major SC lipids using RPLC and negative ion mode APCI-MS for detection of FFAs, and NPLC using positive ion mode APCI-MS to analyze CERs and cholesterol. Validation showed this lipid platform to be robust, reproducible, sensitive, and fast. The method was successfully applied on ex vivo human SC, human SC obtained from tape strips and human skin substitutes (porcine SC and human skin equivalents). In conjunction with FFA profiles, clear differences in CER profiles were observed between these different SC sources. Human skin equivalents more closely mimic the lipid composition of human stratum corneum than porcine skin does, although noticeable differences are still present. These differences gave biologically relevant information on some of the enzymes that are probably involved in SC lipid processing. For future research, this provides an excellent method for (semi-)quantitative, ‘high-throughput’ profiling of SC lipids and can be used to advance the understanding of skin lipids and the biological processes involved.     

Changes induced by sarcolysine in vivo in the composition of DNA-bound lipids in sarcoma 37

The two DNA fractions were isolated from sarcoma 37 by the use of the phenol method: supramolecular complex of DNA (SC DNA, 60%) and "phenol" nuclear matrix DNA (PNM DNA, 40%). The lipids in SC DNA represented of light and tightly bound components, the latter was similar to the lipid composition of PNM DNA. SC DNA contains 20 micrograms of neutral lipids (NL) and 6.5 micrograms of phospholipids (PL), while PNM DNA contains 9.8 micrograms of NL and 3.5 micrograms of PL per mg DNA. SC DNA-bound lipids of sarcoma 37 are deficient in free cholesterol (FC, 13%), but rich in cholesterol esters (CE, 39%) and free fatty acids (FFA, 23%); very rich in cardiolipin (CL, 43%) and phosphatidylethanolamine (PE, 28%), but deficient in phosphatidylcholine (PC, 12%). The tumor contains triglycerides (TG) that is absent in DNA of the normal cells. The injection of sarcolysine (10 micrograms/kg) markedly increased (1.5-3 times) the content of all LN and PL fractions in SC DNA, which was accompanied by both the accumulation of FC, TG, PC and the reduction of the remaining lipid fractions in PNM DNA. It is supposed, that DNA-bound lipids may be the target for the action of sarcolysine.     

Effects of sphingomyelin/ceramide ratio on the permeability and microstructure of model stratum corneum lipid membranes

The conversion of sphingomyelin (SM) to a ceramide (Cer) by acid sphingomyelinase (aSMase) is an important event in skin barrier development. A deficiency in aSMase in diseases such as Niemann–Pick disease and atopic dermatitis coincides with impaired skin barrier recovery after disruption. We studied how an increased SM/Cer ratio influences the barrier function and microstructure of model stratum corneum (SC) lipid membranes. In the membranes composed of isolated human SC Cer (hCer)/cholesterol/free fatty acids/cholesteryl sulfate, partial or full replacement of hCer by SM increased water loss. Partial replacement of 25% and 50% of hCer by SM also increased the membrane permeability to theophylline and alternating electric current, while a higher SM content either did not alter or even decreased the membrane permeability. In contrast, in a simple membrane model with only one type of Cer (nonhydroxyacyl sphingosine, CerNS), an increased SM/Cer ratio provided a similar or better barrier against the permeation of various markers. X-ray powder diffraction revealed that the replacement of hCer by SM interferes with the formation of the long periodicity lamellar phase with a repeat distance of d = 12.7 nm. Our results suggest that SM-to-Cer processing in the human epidermis is essential for preventing excessive water loss, while the permeability barrier to exogenous compounds is less sensitive to the presence of sphingomyelin.     

Long-term biopermanence of ceramides,cholesteryl esters,and ether-linked triglycerides with very-long-chain PUFA in the cadmium-damaged testis

Cadmium is known to harm rat testis by causing the dose-dependent apoptotic or necrotic death of seminiferous epithelium cells. Here we investigated how this affects the lipids with long-chain (C₁₈–C₂₂) and very-long-chain (C₂₄–C₃₂) polyunsaturated fatty acids (VLCPUFA) typical of spermatogenic and Sertoli cells. A severe acute inflammatory reaction resulted from the massive necrotic death of these cells two days after a single high (4 mg/kg) dose of CdCl₂. This led to the conversion of most testicular glycerophospholipids to diradylglycerols (DRG) and free fatty acids (FFA) and of most sphingomyelins to ceramides (Cer). By day 30 the testis weight had decreased three-fold. The DRG and FFA had been metabolized but, unexpectedly, ceramides persisted. Also slow to disappear were VLCPUFA-containing triacylglycerols from former germ cells and ether-linked triglycerides and cholesteryl esters (CE) from former Sertoli cells. Similar results were observed 30 and 45 days after administering repeated small non pro-inflammatory CdCl₂ doses (1 mg/kg). At day 30 after both treatments, an amorphous material replaced the original seminiferous tubules and the interstitium was populated by macrophages. Species of CE and ether-linked triglycerides containing fatty acids other than VLCPUFA steadily accumulated in the irreversibly damaged testis, a manifestation of the activity of these cells. The long-term permanence of original VLCPUFA-containing neutral lipids, especially ceramides, indicates that these phagocytes were slow to clear out the acellular material contained in seminiferous tubules, pointing to a form of silent chronic inflammation as an additional outcome of the multifactorial commotion caused in the testis by experimentally administered cadmium.     

Negative regulation by Ser/Thr phosphorylation of HadAB and HadBC dehydratases from Mycobacterium tuberculosis type II fatty acid synthase system

The type II fatty acid synthase system of mycobacteria is involved in the biosynthesis of major and essential lipids, mycolic acids, key-factors of Mycobacterium tuberculosis pathogenicity. One reason of the remarkable survival ability of M. tuberculosis in infected hosts is partly related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate synthesis of these lipids in response to environmental changes are unknown. We demonstrate here that HadAB and HadBC dehydratases of this system are phosphorylated by Ser/Thr protein kinases, which negatively affects their enzymatic activity. The phosphorylation of HadAB/BC is growth phase-dependent, suggesting that it represents a mechanism by which mycobacteria might tightly control mycolic acid biosynthesis under non-replicating condition.     

Ceramide composition of the psoriatic scale

This paper investigates the ceramide composition of the psoriatic scale compared with that of normal human SC. A method was optimalized, based on TLC separation followed by densitometry, allowing the provision of good resolution and quantification of ceramide fractions from both normal and pathological specimens. Seven ceramide fractions were isolated and submitted to compositional analysis. The obtained results suggested a revisitation of previous ceramide designation. Therefore a simple classification is suggested, based on grouping ceramides carrying structural similarities under common codes. According to these rules, ceramides were grouped into five classes designated as: (1) Cer[EOS], which contains ester-linked fatty acids, ω-OH fatty acids and sphingosines; (2) Cer[NS], which contains non-OH fatty acids and sphingosines; (3) Cer[NP], which contains non-OH fatty acids and phytosphingosines; (4) Cer[AS], which contains α-OH fatty acids and sphingosines; (5) Cer[AP], which contains α-OH fatty acids and phytosphingosines. Analysis of ceramides from the psoriatic scale, compared to those from normal human SC, resulted in an impairment of the Cer[EOS] content as well as of the ceramides containing phytosphingosine, with concurrent increase in ceramides containing sphingosine, being the total amount maintained identical. Since one of the suggested pathways for phytosphingosine biosynthesis involves the water addition to the corresponding sphingosine double bond, we can speculate that the observed alterarion is due to a deranged water bioavailability, associated with psoriaris.     

Lipid organization in human and porcine stratum corneum differs widely, while lipid mixtures with porcine ceramides model human stratum corneum lipid organization very closely

The conformational disordering and lateral packing of lipids in porcine and human isolated stratum corneum (SC) was compared using Fourier transform infrared spectroscopy (FTIR). It was shown that SC of both species differ markedly, porcine SC lipids being arranged predominantly in a hexagonal lattice while lipids in human SC are predominantly packed in the denser orthorhombic lattice. However, the lipid organization of equimolar ceramide:cholesterol:free fatty acid (CER:CHOL:FFA) mixtures prepared with isolated porcine CER or human CER is very similar, only the transition temperatures differed being slightly lower in mixtures with porcine CER. Therefore, the difference in lateral packing between human and porcine stratum corneum is not due to the difference in CER composition. Furthermore, it is possible to use more readily available porcine CER in model lipid mixtures to mimic lipid organization in human SC. As the equimolar porcine CER:CHOL:FFA mixtures closely mimic the lipid organization in human SC, both human SC and this mixture were selected to examine the effect of glycerol on the lipid phase behaviour. It was found that high concentrations of glycerol change the lamellar organization slightly, while domains with an orthorhombic lateral packing are still observed.     

Fatty acid transporters in skin development,function and disease

Fatty acids in the epidermis can be incorporated into complex lipids or exist in a free form, and they are crucial to proper functions of the epidermis and its appendages, such as sebaceous glands. Epidermal fatty acids can be synthesized de novo by keratinocytes or taken up from extracutaneous sources in a process that likely involves protein transporters. Several proteins that are expressed in the epidermis have been proposed to facilitate the uptake of long-chain fatty acids (LCFA) in mammalian cells, including fatty acid translocase/CD36, fatty acid binding protein, and fatty acid transport protein (FATP)/very long-chain acyl-CoA synthetase. In this review, we will discuss the mechanisms by which these candidate transporters facilitate the uptake of fatty acids. We will then discuss the clinical implications of defects in these transporters and relevant animal models, including the FATP4 animal models and ichthyosis prematurity syndrome, a congenital ichthyosis caused by FATP4 deficiency. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Influence of different ceramides on the structure of in vitro model lipid systems of the stratum corneum lipid matrix

Human stratum corneum (SC) consists of several layers of keratinized corneocytes embedded in a lipid matrix of ordered lamellar structure which is considered to constitute the major barrier to percutaneous penetration. Artificial mixtures of SC lipids are often used as model systems to mimic the skin barrier or to investigate the effects of substances on the phase behaviour of the models. In the present study a SC lipid model composed of cholesterol, fatty acids and ceramides was used to investigate the effect of three different commercially available ceramide types on the microstructure and the physicochemical behaviour of the lipids. Polarized light microscopy, transmission electron microscopy, small-angle X-ray diffraction, wide-angle X-ray diffraction and differential scanning calorimetry (DSC) were used for physicochemical characterization. The results revealed a lamellar structure for all models but showed differences with regard to the thermal and optical behaviour depending obviously on the composition of the ceramide mixtures. A model containing a mixture of Cer[AS] was comparable to human SC lipids.     

Investigation of stratum corneum lipid model membranes with free fatty acid composition by neutron diffraction

The outermost epidermal layer, the stratum corneum (SC), is the main skin barrier. Studies of SC model systems enable characterization of the influence of individual lipids on the organization of the SC lipid matrix, which is the main pathway of water through the skin. This work presents a neutron diffraction study of the SC model membranes based on short-chain ceramide 6 with nearly realistic composition of free fatty acids (FFA) at physiological temperature of the SC. The influence of FFA and the effect of cholesterol–cholesterol sulfate substitution on the structure and hydration of the SC model membranes are described. The structure of the SC membrane with FFA is close to the structure of the earlier studied SC membrane based on short-chain palmitic acid (PA) and does not vary significantly under changes of the ratio of the main membrane components. FFA accelerates membrane swelling at the same low level of hydration of both PA- and FFA-containing membranes. The substitution of cholesterol sulfate by cholesterol in the membrane composition decreases membrane swelling and leads to phase separation in the model system.     

Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2

During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747–1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion.     

Changes in fatty acids of leaf polar lipids during chilling and post-chilling rewarming of Zea mays genotypes differing in response to chilling

Changes in fatty acids of leaf polar lipids: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) in maize seedlings of chiling-sensitive (CS) CM 7 and Co 151 lines and chilling-tolerant (CT) S 215 and EP 1 lines upon chilling for either 4 or 6 days in the dark and after rewarming for 4 days at original growth conditions were studied. The content of free fatty acids (FFA) in control leaves as well as alterations in the proportion of major fatty acids, unsaturation ratio (UR), double bond index (DBI) and changes in the proportion of heigh-temperature melting of both phosphatidylglycerol (htm-PG) and sulfoquinovosylglycerol (htm-SQDG) after chilling and rewarming of seedlings were estimated. FFA content in intact leaves was 2–3-fold higher in the chilling susceptible CM 7 line than in the other three inbreeds studied. After chilling for 6 days the level of FFA increased only in CM 7 and S 215 lines by about 30 %. Upon rewarming seedlings chilled for 6 days the level of FFA increased about two-fold in CS Co 151 line and CT EP 1 line and decreased in CS CM 7 line. Limited accumulation of FFAs during chilling and post-chilling rewarming of maize seedlings, did not correspond to the extent of polar lipid breakdown (Kaniuga et al. 1999b) probably due to the contribution of active oxidative systems to the peroxidation of fatty acids under these conditions. During rewarming seedlings chilled for 6 days major changes were observed in decrease of 18:3 and an increase of 16:0 in all four polar lipids studied with more pronounced changes in CS than CT lines. Similarly, in CS inbreeds a decrease in UR of fatty acids in MGDG, DGDG and SQDG after post-chilling rewarming was greater than in CT lines. Proportion of htm-fraction in both PG and SQDG increased after post-chilling rewarming in all four inbreeds, however to a lesser extent in CT than CS lines. A similar pattern of changes in DBI in CS and CT maize seedlings was observed in glycolipid and combine lipid classes. More extensive degradation of polar lipids in CS than CT maize inbreeds following galactolipase action in chloroplasts (Kaniuga et al. 1998) provides FFAs for initiation of peroxidation by LOX which is manifested by decrease of UR and DBI. This sequence of reactions during chilling and post-chilling rewarming appears to be a main route of fatty acids peroxidation responsible for secondary events involved in chilling injury. In addition, the extent of these changes differentiates CS and CT inbreeds. Contribution of esterified fatty acids in thylakoid lipids to direct peroxidation, may be of minor importance.     

Basic Nanostructure of Stratum Corneum Lipid Matrices Based on Ceramides [EOS] and [AP]: A Neutron Diffraction Study

The goal of this study was to investigate the nanostructure of SC lipid model membranes comprising the most relevant SC lipids such as the unique-structured ω-acylceramide [EOS] in a near natural ratio with neutron diffraction. In models proposed recently the presence of ceramide [EOS] and FFA are necessary for the formation of one of the two existent crystalline lamellar phases of the SC lipids, the long-periodicity phase as well as for the normal barrier function of the SC. The focus of this study was placed on the influence of the FFA BA on the membrane structure and its localization within the membrane based on the ceramides [EOS] and [AP]. The internal nanostructure of such membranes was obtained by Fourier synthesis from the experimental diffraction patterns. The resulting neutron scattering length density profiles showed that the exceptionally long ceramide [EOS] is arranged in a short-periodicity phase created by ceramide [AP] by spanning through the whole bilayer and extending even further into the adjacent bilayer. Specifically deuterated BA allowed us to determine the exact position of this FFA inside this SC lipid model membrane. Furthermore, hydration experiments showed that the presented SC mimic system shows an extremely small intermembrane hydration of ∼1 Å, consequently the headgroups of the neighboring leaflets are positioned close to each other.     

Ceramides and sphingomyelins with high proportions of very long-chain polyunsaturated fatty acids in mammalian germ cells

Very long-chain polyunsaturated fatty acids (VLCPUFA) have previously been shown to be components of sphingomyelin (SM) of mammalian testis and spermatozoa. Here we examined the fatty acids of testicular ceramide (Cer) in comparison with those of SM in some mammals with a special focus on the rat testis. In bull, cat, dog, rabbit, mouse, and rat, VLCPUFA were found in both testicular lipids, Cer having a higher percentage of VLCPUFA than SM. Rat testis had the highest percentage of VLCPUFA in both lipids, the major ones being 28:4n-6 and 30:5n-6. VLCPUFA-containing SM and Cer occurred in cells located in the seminiferous tubules, where germ cells had a higher percentage of these species than Sertoli cells. Seminiferous tubule fractionation showed that SM and Cer of mitochondria and lysosomes had mostly saturates and negligible VLCPUFA, the latter being important in the SM and Cer of microsomes and other membrane fractions. VLCPUFA were absent from the SM and Cer of rat prepuberal testis, increased with the onset of spermatogenesis to account for nearly 15 and 40% of the total fatty acids of testicular SM and Cer, respectively, remained at those levels throughout the adult life of fertile rats and tended to decrease at advanced ages. Four conditions that lead to selective death of germ cells in vivo, namely experimental cryptorchidism, post-ischemic reperfusion, focalized x-ray irradiation and treatments with the antineoplasic drug doxorubicin, caused the VLCPUFA to disappear from the testicular SM and Cer of adult fertile rats, showing that these lipids are specific traits of spermatogenic cells.     

Unique effects of different fatty acid species on the physical properties of the torpedo acetylcholine receptor membrane.

To study the effects produced by free fatty acids (FFA) on the biophysical properties of Torpedo marmorata nicotinic acetylcholine receptor-rich native membranes and to investigate the topology of their binding site(s), fluorescence measurements were carried out using the fluorescent probe Laurdan (6-dodecanoyl-2-(dimethylamino) naphthalene) and ADIFAB, an Acrylodan-derivatized intestinal fatty acid-binding protein. The generalized polarization (GP) of the former probe was used to learn about the physical state of the membrane upon FFA binding. Saturated FFA induced a slight increase in GP, whereas cis-unsaturated fatty acids decreased GP. Double bond isomerism could also be distinguished; oleic acid (18:1cis) induced a net disordering effect, whereas elaidic acid (18:1trans) produced no changes in GP. The changes in the efficiency of the F?rster energy transfer from the protein to Laurdan brought about by addition of FFA, together with the distances involved in this process, indicate that all FFA studied share a common site at the lipid-protein interface. However, despite being located at the same site, each class of FFA differs in its effect on the physical properties of the membrane. These data lead us to suggest that it is the direct action of FFA at the lipid-protein interface, displacing essential lipids from their sites rather than changes in bulk properties such as membrane fluidity that accounts for the effect of FFA on the acetylcholine receptor membrane.     

Arrangement of ceramide [EOS] in a stratum corneum lipid model matrix: new aspects revealed by neutron diffraction studies

The lipid matrix in stratum corneum (SC) plays a key role in the barrier function of the mammalian skin. The major lipids are ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA). Especially the unique-structured omega-acylceramide CER[EOS] is regarded to be essential for skin barrier properties by inducing the formation of a long-periodicity phase of 130 angstroms (LPP). In the present study, the arrangement of CER[EOS], either mixed with CER[AP] and CHOL or with CER[AP], CHOL and palmitic acid (PA), inside a SC lipid model membrane has been studied for the first time by neutron diffraction. For a mixed CER[EOS]/CER[AP]/CHOL membrane in a partly dehydrated state, the internal membrane nanostructure, i.e. the neutron scattering length density profile in the direction normal to the surface, was obtained by Fourier synthesis from the experimental diffraction patterns. The membrane repeat distance is equal to that of the formerly used SC lipid model system composed of CER[AP]/CHOL/PA/ChS. By comparing both the neutron scattering length density profiles, a possible arrangement of synthetic long-chain CER[EOS] molecules inside a SC lipid model matrix is suggested. The analysis of the internal membrane nanostructure implies that one CER[EOS] molecule penetrates from one membrane layer into an adjacent layer. A 130 angstroms periodicity phase could not be observed under experimental conditions, either in CER/CHOL mixtures or in CER/CHOL/FFA mixture. CER[EOS] can be arranged inside a phase with a repeat unit of 45.2 angstroms which is predominately formed by short-chain CER[AP] with distinct polarity.     

Identification of hydrophobic interactions between proteins and lipids: free fatty acids activate phospholipase C delta1 via allosterism

Lipids are well recognized ligands that bind to proteins in a specific manner and regulate their function. Most attention has been placed on the headgroup of phospholipids, and little is known about the role of the acyl chains in mediating any effects of lipids on proteins. In this report, free fatty acids (FFA) were found to bind and activate phospholipase C delta1(PLC delta1). The unsaturated FFA arachidonic acid (AA) and oleic acid were able to stimulate PLC delta1 up to 30-fold in a dose-dependent manner. The saturated FFA stearic acid and palmitic acid were less efficacious than unsaturated FFA, activating the enzyme up to 8-fold. The mechanism of activation appears to be due to a change in K(m) for substrate; 50 microM arachidonate reduced the K(m) for the soluble PLC substrate diC(4)PI from 1.7 +/- 0.6 mM to 0.24 +/- 0.04 mM (7-fold reduction). V(max) was not significantly altered. PLC delta1 bound to sucrose-loaded vesicles that contained AA in a concentration-dependent manner. A fragment of PLC delta1 that encompasses the EF-hand domain also bound to micelles containing AA using nondenaturing PAGE. This same fragment also inhibited AA activation of PLC delta1 in a competition assay. These results suggest that the function of the EF-hand domain of PLC delta1 is to bind lipid and to allosterically regulate catalysis. These results also suggest that esterified and nonesterified fatty acids can bind to and regulate protein function, identifying a functional role for hydrophobic interactions between lipids and proteins.