Lipoprotein enrichment in orange insoluble particulate matter reproducibly appearing in cryoprecipitate.

BACKGROUND AND OBJECTIVE: Cryoprecipitate prepared from two whole blood donations from the same donor contained insoluble orange particulate material (OPM). We sought to identify the OPM. MATERIALS AND METHODS: OPM was recovered from the blood product by centrifugation, dissolved in sodium dodecyl sulphate (SDS) and analysed by SDS-polyacrylamide gel electrophoresis and immunoblotting. RESULTS: Solubilized OPM was enriched in apolipoproteins B and E, but not apolipoprotein A1, immunoglobulin G or albumin, suggesting lipoprotein enrichment in OPM. Subsequent clinical laboratory blood tests confirmed low-density lipoprotein hyperlipidaemia with normal triglyceride levels. Further, cryoprecipitate production from this donor was prevented by implementation of national predominantly male plasma policies. CONCLUSION: Cryoprecipitate produced from hyperlipidaemic donors may contain insoluble particles that render it inappropriate for transfusion.

Scavenger receptor class B type I in high-density lipoprotein metabolism, atherosclerosis and heart disease: lessons from gene-targeted mice.

The scavenger receptor class B type I (SR-BI) is a multi-ligand receptor that can mediate the binding and bi-directional lipid transfer between high-density lipoproteins (HDLs) and cells. It is expressed in a variety of tissues, including the liver, and in macrophages in atherosclerotic plaques. The physiological role of SR-BI has been tested in vivo by the genetic manipulation of SR-BI levels in mice. Mice lacking SR-BI exhibit impaired hepatic-selective HDL cholesterol uptake and increased atherosclerosis, suggesting that SR-BI is required for hepatic reverse cholesterol transport and normally protects against atherosclerosis. Surprisingly, elimination of SR-BI in apolipoprotein E knockout mice results in rapid development of occlusive coronary artery disease, accompanied by spontaneous myocardial infarction, reduced heart function and early death, which points to a role for SR-BI in protection against coronary heart disease. The in vivo role of macrophage SR-BI has been less clear. We have used bone-marrow transplantation to demonstrate that bone-marrow-derived SR-BI also normally protects against atherosclerosis in low-density lipoprotein receptor knockout mice. These results suggest that SR-BI may have multiple protective effects against atherosclerosis in different tissues.

Hepatic cholesterol and bile acid metabolism and intestinal cholesterol absorption in scavenger receptor class B type I-deficient mice.

The scavenger receptor class B type I (SR-BI), which is expressed in the liver and intestine, plays a critical role in cholesterol metabolism in rodents. While hepatic SR-BI expression controls high density lipoprotein (HDL) cholesterol metabolism, intestinal SR-BI has been proposed to facilitate cholesterol absorption. To evaluate further the relevance of SR-BI in the enterohepatic circulation of cholesterol and bile salts, we studied biliary lipid secretion, hepatic sterol content and synthesis, bile acid metabolism, fecal neutral sterol excretion, and intestinal cholesterol absorption in SR-BI knockout mice. SR-BI deficiency selectively impaired biliary cholesterol secretion, without concomitant changes in either biliary bile acid or phospholipid secretion. Hepatic total and unesterified cholesterol contents were slightly increased in SR-BI-deficient mice, while sterol synthesis was not significantly changed. Bile acid pool size and composition, as well as fecal bile acid excretion, were not altered in SR-BI knockout mice. Intestinal cholesterol absorption was somewhat increased and fecal sterol excretion was slightly decreased in SR-BI knockout mice relative to controls. These findings establish the critical role of hepatic SR-BI expression in selectively controlling the utilization of HDL cholesterol for biliary secretion. In contrast, SR-BI expression is not essential for intestinal cholesterol absorption.

Cellular and physiological roles of SR-BI, a lipoprotein receptor which mediates selective lipid uptake.

High-density lipoproteins (HDL) play an important role in protection against atherosclerosis by mediating reverse cholesterol transport - the transport of excess cholesterol from peripheral tissues to the liver for disposal. SR-BI is a cell surface receptor for HDL and other lipoproteins (LDL and VLDL) and mediates the selective uptake of lipoprotein cholesterol by cells. Overexpression or genetic ablation of SR-BI in mice revealed that it plays an important role in HDL metabolism and reverse cholesterol transport and protects against atherosclerosis in mouse models of the disease. If it plays a similar role in humans then it may be an attractive target for therapeutic intervention. We will review some of the recent advances in the understanding of SR-BI's physiological role and cellular function in lipoprotein metabolism.

Scavenger receptor class B type I (SR-BI) and high-density lipoprotein metabolism: recent lessons from genetically manipulated mice.

The scavenger receptor BI is a cell surface lipoprotein receptor for selective high-density lipoprotein (HDL) cholesterol uptake in the liver and steroidogenic tissues. Studies of genetically manipulated strains of mice have revealed that SR-BI plays a key role in regulating HDL metabolism, cholesterol transport to steroidogenic tissues and bile cholesterol secretion. Furthermore, SR-BI protects against the development of atherosclerosis and is required for normal female fertility. If SR-BI has similar functions in lipoprotein metabolism and atherosclerosis in humans, it may represent a new target for the prevention and/or treatment of atherosclerotic cardiovascular disease.

The role of the high-density lipoprotein receptor SR-BI in cholesterol metabolism.

The HDL receptor scavenger receptor class B type I (SR-BI), which mediates selective HDL cholesterol uptake, plays a role in murine HDL metabolism, reverse cholesterol transport and whole-body cholesterol homeostasis. SR-BI is found in the liver, where its expression is regulated by estrogen, dietary cholesterol and fat, and controls murine plasma HDL cholesterol levels and bile cholesterol secretion. SR-BI is also highly expressed in rodent steroidogenic cells, where it facilitates cholesterol uptake for storage or steroid hormone synthesis and where its expression is regulated by trophic hormones. The detailed mechanism(s) underlying SR-BI-mediated selective cholesterol uptake have not yet been elucidated. Further analysis of the molecular and cellular bases of SR-BI regulation and function should provide new insights into the physiology and pathophysiology of cholesterol metabolism.

Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology.

The high density lipoprotein (HDL) receptor SR-BI (scavenger receptor class B type I) mediates the selective uptake of plasma HDL cholesterol by the liver and steroidogenic tissues. As a consequence, SR-BI can influence plasma HDL cholesterol levels, HDL structure, biliary cholesterol concentrations, and the uptake, storage, and utilization of cholesterol by steroid hormone-producing cells. Here we used homozygous null SR-BI knockout mice to show that SR-BI is required for maintaining normal biliary cholesterol levels, oocyte development, and female fertility. We also used SR-BI/apolipoprotein E double homozygous knockout mice to show that SR-BI can protect against early-onset atherosclerosis. Although the mechanisms underlying the effects of SR-BI loss on reproduction and atherosclerosis have not been established, potential causes include changes in (i) plasma lipoprotein levels and/or structure, (ii) cholesterol flux into or out of peripheral tissues (ovary, aortic wall), and (iii) reverse cholesterol transport, as indicated by the significant reduction of gallbladder bile cholesterol levels in SR-BI and SR-BI/apolipoprotein E double knockout mice relative to controls. If SR-BI has similar activities in humans, it may become an attractive target for therapeutic intervention in a variety of diseases.

Identification of caveolin-1 as a fatty acid binding protein.

In an attempt to identify high affinity, fatty acid binding proteins present in 3T3-L1 adipocytes plasma membranes, we labeled proteins in purified plasma membranes with the photoreactive fatty acid analogue, 11-m-diazirinophenoxy[11-3H]undecanoate. A single membrane protein of 22 kDa was covalently labeled after photolysis. This protein fractionated with caveolin-1 containing caveolae and was immunoprecipitated by an anti-caveolin-1 monoclonal antibody. Furthermore, 2D-PAGE analysis revealed that both the alpha and beta isoforms of caveolin-1 could be labeled by the photoreactive fatty acid upon photolysis, indicating that both bind fatty acids. The saturable binding of the photoreactive fatty acid suggests caveolin-1 has a lipid binding site that may either operate during intracellular lipid traffic or regulate caveolin-1 function.

The efficient cellular uptake of high density lipoprotein lipids via scavenger receptor class B type I requires not only receptor-mediated surface binding but also receptor-specific lipid transfer mediated by its extracellular domain.

The class B type I scavenger receptor, (SR-BI), is a member of the CD36 superfamily of proteins and is a physiologically relevant, high affinity cell surface high density lipoprotein (HDL) receptor that mediates selective lipid uptake. The mechanism of selective lipid uptake is fundamentally different from that of classic receptor-mediated uptake via coated pits and vesicles (e.g. the low density lipoprotein receptor pathway) in that it involves efficient transfer of the lipids, but not the outer shell proteins, from HDL to cells. The abilities of SR-BI and CD36, both of which are class B scavenger receptors, to bind HDL and mediate cellular uptake of HDL-associated lipid when transiently expressed in COS cells were examined. For these experiments, the binding of HDL to cells was assessed using either 125I- or Alexa (a fluorescent dye)-HDL in which the apolipoproteins on the surface of the HDL particles were covalently modified. Lipid transfer was measured using HDL noncovalently labeled by the fluorescent lipid 1,1'-dioctadecyl-3,3, 3',3'-tetramethylindocarbocyanine perchlorate. Although both mSR-BI and human CD36 (hCD36) could mediate the binding of HDL in a punctate pattern across the surfaces of cells, only mSR-BI efficiently mediated the transfer of lipid to the cells. Analysis of point mutants established that the major sites of fatty acylation of mSR-BI are Cys462 and Cys470 and that fatty acylation is not required for receptor clustering, HDL binding, or efficient lipid transfer. Generation of mSR-BI/hCD36 domain swap chimeras showed that the differences in lipid uptake activities between mSR-BI and hCD36 were not due to differences between their two sets of transmembrane and cytoplasmic domains but rather result from differences in their large extracellular loop domains. These results show that high affinity binding to a cell surface receptor is not sufficient to ensure efficient cellular lipid uptake from HDL. Thus, SR-BI-mediated binding combined with SR-BI-dependent facilitated transfer of lipid from the HDL particle to the cell appears to be the most likely mechanism for the bulk of the selective uptake of cholesteryl esters from HDL to the liver and steroidogenic tissues.

A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism.

Plasma high density lipoprotein (HDL), which protects against atherosclerosis, is thought to remove cholesterol from peripheral tissues and to deliver cholesteryl esters via a selective uptake pathway to the liver (reverse cholesterol transport) and steroidogenic tissues (e.g., adrenal gland for storage and hormone synthesis). Despite its physiologic and pathophysiologic importance, the cellular metabolism of HDL has not been well defined. The class B, type I scavenger receptor (SR-BI) has been proposed to play an important role in HDL metabolism because (i) it is a cell surface HDL receptor which mediates selective cholesterol uptake in cultured cells, (ii) its physiologically regulated expression is most abundant in the liver and steroidogenic tissues, and (iii) hepatic overexpression dramatically lowers plasma HDL. To test directly the normal role of SR-BI in HDL metabolism, we generated mice with a targeted null mutation in the SR-BI gene. In heterozygous and homozygous mutants relative to wild-type controls, plasma cholesterol concentrations were increased by approximately 31% and 125%, respectively, because of the formation of large, apolipoprotein A-I (apoA-I)-containing particles, and adrenal gland cholesterol content decreased by 42% and 72%, respectively. The plasma concentration of apoA-I, the major protein in HDL, was unchanged in the mutants. This, in conjunction with the increased lipoprotein size, suggests that the increased plasma cholesterol in the mutants was due to decreased selective cholesterol uptake. These results provide strong support for the proposal that in mice the gene encoding SR-BI plays a key role in determining the levels of plasma lipoprotein cholesterol (primarily HDL) and the accumulation of cholesterol stores in the adrenal gland. If it has a similar role in controlling plasma HDL in humans, SR-BI may influence the development and progression of atherosclerosis and may be an attractive candidate for therapeutic intervention in this disease.

Scavenger receptor BI--a cell surface receptor for high density lipoprotein.

The receptor-mediated transfer of lipids between cells and lipoproteins plays an important role in lipoprotein metabolism and cardiovascular disease. Although there have been many valuable studies of HDL binding to tissues, cells and membranes, and of the potential role of such binding in the transport of lipids between HDL and cells, much less is known about HDL receptors than about receptors for other lipoproteins (e.g. LDL, chylomicrons, vitellogenin). Here we review recent studies of the class B, type I scavenger receptor, which appears to be a physiologically relevant, cell surface HDL receptor that mediates the selective uptake of lipids by cells.

Murine SR-BI, a high density lipoprotein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae.

The class B, type I scavenger receptor, SR-BI, was the first molecularly well defined cell surface high density lipoprotein (HDL) receptor to be described. It mediates transfer of lipid from HDL to cells via selective lipid uptake, a mechanism distinct from receptor-mediated endocytosis via clathrin-coated pits and vesicles. SR-BI is expressed most abundantly in steroidogenic tissues (adrenal gland, ovary), where trophic hormones coordinately regulate its expression with steroidogenesis, and in the liver, where it may participate in reverse cholesterol transport. Here we have used immunochemical methods to study the structure and subcellular localization of murine SR-BI (mSR-BI) expressed either in transfected Chinese hamster ovary cells or in murine adrenocortical Y1-BS1 cells. mSR-BI, an approximately 82-kDa glycoprotein, was initially synthesized with multiple high mannose N-linked oligosaccharide chains, and some, but not all, of these were processed to complex forms during maturation of the protein in the Golgi apparatus. Metabolic labeling with [3H]palmitate and [3H]myristate demonstrated that mSR-BI was fatty acylated, a property shared with CD36, another class B scavenger receptor, and other proteins that concentrate in specialized, cholesterol- and glycolipid-rich plasma membrane microdomains called caveolae. OptiPrep density gradient fractionation of plasma membranes established that mSR-BI copurified with caveolin-1, a constituent of caveolae; and immunofluorescence microscopy demonstrated that mSR-BI colocalized with caveolin-1 in punctate microdomains across the surface of cells and on the edges of cells. Thus, mSR-BI colocalizes with caveolae, and this raises the possibility that the unique properties of these specialized cell surface domains may play a critical role in SR-BI-mediated transfer of lipids between lipoproteins and cells.

Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells.

The class B, type I scavenger receptor, SR-BI, binds high density lipoprotein (HDL) and mediates the selective uptake of HDL cholesteryl ester (CE) by cultured transfected cells. The high levels of SR-BI expression in steroidogenic cells in vivo and its regulation by tropic hormones provides support for the hypothesis that SR-BI is a physiologically relevant HDL receptor that supplies substrate cholesterol for steroid hormone synthesis. This hypothesis was tested by determining the ability of antibody directed against murine (m) SR-BI to inhibit the selective uptake of HDL CE in Y1-BS1 adrenocortical cells. Anti-mSR-BI IgG inhibited HDL CE-selective uptake by 70% and cell association of HDL particles by 50% in a dose-dependent manner. The secretion of [3H]steroids derived from HDL containing [3H]CE was inhibited by 78% by anti-mSR-BI IgG. These results establish mSR-BI as the major route for the selective uptake of HDL CE and the delivery of HDL cholesterol to the steroidogenic pathway in cultured mouse adrenal cells.

A single point mutation in epsilon-COP results in temperature-sensitive, lethal defects in membrane transport in a Chinese hamster ovary cell mutant.

At the nonpermissive temperature of 39.5 degrees C, the Chinese hamster ovary cell conditionally lethal, temperature-sensitive (ts) mutant ldlF exhibits the following defects: rapid degradation of low density lipoprotein receptors, disruption of ER-through Golgi transport, and disintegration of the Golgi apparatus. All of these are corrected by transfection with an expression vector for wild-type epsilon-COP, a subunit of coatomers (Guo, Q., Vasile, E., and Krieger, M. (1994) J. Cell Biol. 125, 1213-1224). We now report the identification in ldlF cells of a point mutation in the epsilon-COP gene, Glu251 to Lys251, which prevents the corresponding cDNA from correcting the defects in transfected ldlF cells and the immunochemical analysis of the synthesis, structure, and stability of epsilon-COP. At the permissive temperature (34 degrees C), the steady state level of ts-epsilon-COP in ldlF cells was about half that of epsilon-COP in wild-type Chinese hamster ovary cells and the isoelectric point of ts-epsilon-COP was 0.14 pH units higher than that of the wild-type protein. The stability but not the biosynthesis of ts-epsilon-COP was temperature-sensitive (t1/2 > 6 h at 34 degrees C and approximately 1-2 h at 39.5 degrees C), and this accounts for the virtual absence of detectable ts-epsilon-COP protein in ldlF cells after incubation at 39.5 degrees C for > 6h. The steady state levels in ldlF cells of another coatomer subunit, beta-COP, and the peripheral Golgi protein ldlCp were not temperature-sensitive. Thus, a mutation in epsilon-COP that causes instability at 39.5 degrees C is responsible for all of the temperature-sensitive defects in ldlF cells, and the stability of beta-COP is not linked directly to that of epsilon-COP. ldlF cells should be useful for the future analysis of the structure and function of epsilon-COP, the assembly of COPs into coatomers, and the participation of coatomers in intracellular membrane transport.

The effect of intracellular pH on long-chain fatty acid uptake in 3T3-L1 adipocytes: evidence that uptake involves the passive diffusion of protonated long-chain fatty acids across the plasma membrane.

To understand the mechanism of long-chain fatty acid permeation of the plasma membrane in mammalian cells, the effects of changes in the cytoplasmic pH on the internalization of physiologically relevant, submicromolar concentrations of uncomplexed long-chain fatty acids were investigated in 3T3-L1 adipocytes. The acidification of the cytoplasm upon NH4Cl prepulsing of intact cells was accompanied by a rapid reduction of cellular long-chain fatty acid uptake (measured as the total accumulation of [9,10-3H]oleate). This was followed by a slow recovery to normal levels of uptake as the cytoplasmic pH recovered. Conventional filtration assays do not distinguish between fatty acid movement across the plasma membrane and intracellular steps, such as binding to cytoplasmic fatty acid-binding proteins or metabolism. While the in vitro binding of a photoreactive fatty acid, 11-m-diazirinophenoxy[11-3H]undecanoate, to a cytoplasmic fatty acid-binding protein was insensitive to changes in pH from pH 7.5 to 5.5, the in vitro conversion of oleate into oleoyl-CoA by cellular acyl-CoA synthetase decreased dramatically. Therefore, the labelling of the 15 kDa cytoplasmic fatty acid-binding protein in intact cells by the photoreactive fatty acid was used as a more direct measure of the permeation of the probe across the plasma membrane. Acidification of the cytoplasm resulted in an immediate reduction in the labelling of this protein in intact adipocytes. Its photolabelling recovered, however, upon the recovery of the cytoplasmic pH to normal levels. This was due to effects of the cytoplasmic pH on the permeation of the photoreactive fatty acid across the plasma membrane rather than its binding to the 15 kDa protein or metabolism in vivo. This is the first demonstration that the movement of physiologically relevant, submicromolar concentrations of uncomplexed long-chain fatty acids across the plasma membrane of intact cells is coupled to the cytoplasmic pH and suggests that it occurs by the diffusion of the protonated long-chain fatty acid through the lipid bilayer.

A direct role for serum albumin in the cellular uptake of long-chain fatty acids.

The interaction of long-chain fatty acids with cells is important for their uptake and metabolism, as well as their involvement in signalling processes. The majority of long-chain fatty acids circulating in plasma exist as complexes with serum albumin. Thus an understanding of the involvement of serum albumin in these processes is vitally important. The effect of serum albumin on the uptake of long-chain fatty acids was studied in 3T3-L1 adipocytes. Serum albumin had a stimulatory effect on oleate uptake at all ratios of oleate: serum albumin tested. Furthermore, the rate of oleate uptake was saturable with increasing concentrations of serum albumin when the oleate: serum albumin ratio, and therefore the concentration of uncomplexed oleate, remained constant. This was not due to uptake being limited by dissociation of oleate from serum albumin, because oleate did not appear to be limiting. Furthermore, at very high ratios of oleate: serum albumin, when the concentration of uncomplexed oleate was predicted to be large relative to the amount of oleate taken up by cells, the rate of oleate uptake was still dependent on the albumin concentration. Serum albumin, covalently labelled with the photoreactive fatty acid 11-m-diazirinophenoxy[11-3H]undecanoate, bound to cells in a manner exhibiting both saturable (Kd 66.7 microM) and non-saturable processes. These results indicate that the stimulatory effect of serum albumin on the rate of oleate uptake is due to a direct interaction of serum albumin with the cells and point to an involvement of albumin binding sites in the cell surface in the cellular uptake of long-chain fatty acids.

Membrane permeation and intracellular trafficking of long chain fatty acids: insights from Escherichia coli and 3T3-L1 adipocytes.

Long chain fatty acids are important substrates for energy production and lipid synthesis in prokaryotes and eukaryotes. Their cellular uptake represents an important first step leading to metabolism. This step is induced in Escherichia coli by growth in medium containing long chain fatty acids and in murine 3T3-L1 cells during differentiation to adipocytes. Consequently, these have been used extensively as model systems to study the cellular uptake of long chain fatty acids. Here, we present an overview of our current understanding of long chain fatty acid uptake in these cells. It consists of several distinct steps, mediated by a combination of biochemical and physico-chemical processes, and is driven by conversion of long chain fatty acids to acyl-CoA by acyl-CoA synthetase. An understanding of long chain fatty acid uptake may provide valuable insights into the roles of fatty acids in the regulation of cell signalling cascades, in the regulation of a variety of metabolic and transport processes, and in a variety of mammalian pathogenic conditions such as obesity and diabetes.

Identification of high affinity membrane-bound fatty acid-binding proteins using a photoreactive fatty acid.

A photoaffinity labeling method was developed to identify and characterize high affinity fatty acid-binding proteins in membranes. The specific labeling of these sites requires the use of low concentrations (nanomolar) of the photoreactive fatty acid 11-m-diazirinophenoxy-[11-3H]undecanoate. It was delivered as a bovine serum albumin (BSA) complex which serves as a reservoir for fatty acid and thus allows precise control of unbound fatty acid concentrations. The fadL protein of E. coli, which is required for fatty acid permeation of its outer membrane, was labeled by the photoreactive fatty acid neither specifically nor saturably when the probe was added in the absence of BSA; however when a nanomolar concentration of the uncomplexed probe was maintained in the presence of BSA, the labeling of the fadL protein was highly specific and saturable. This photoaffinity labeling method was also used to characterize a 22 kDa, high affinity fatty acid-binding protein which we have recently identified in the plasma membrane of 3T3-L1 adipocytes. This protein bound the probe with a Kd of 216 nM. The approach described is easily capable of identifying membrane-bound fatty acid-binding proteins and can distinguish between those of high and low affinities for fatty acids. It represents a general method for the identification and characterization of fatty acid-binding proteins.

Fatty acid uptake in Candida tropicalis: induction of a saturable process.

The rates of oleate uptake by Candida tropicalis cells grown on a high oleate concentration (3.5 mM oleate in the presence of 0.50% Brij 58) were higher than those observed in cells grown on glucose; however, oleate uptake was not saturable with substrate concentration. Cells grown at a low oleate concentration (1.0 mM oleate and 2.5% Brij 58) grew to a lower density and at a slightly slower rate; these cells were found to take up oleate at a rate 43-fold higher than cells grown on high oleate concentration. Furthermore, oleate uptake by the cells grown in low oleate medium was a saturable process with Kt and Vmax values of 56 microM and 15 nmol/(min.mg cell protein), respectively. The growth of C. tropicalis under low fatty acid concentration thus clearly results in the induction of a saturable process for its uptake. The total level of acyl-CoA synthetase activity in cells grown on the low oleate concentrations was only twofold higher than in high oleate or glucose grown cells; the level of this enzyme thus does not account for the saturable process and suggests that either the enzyme is regulated in vivo or else a hitherto unidentified enzyme is induced by growth in low concentrations of oleate.

Photoaffinity labeling and fatty acid permeation in 3T3-L1 adipocytes.

Long chain fatty acid uptake was investigated in 3T3-L1 cells. Differentiation of these cells from fibroblasts to adipocytes was accompanied by an 8.5-fold increase in the rate of oleate uptake. This was saturable in adipocytes with apparent Kt and Vmax values of 78 nM and 16 nmol/min/mg cell protein, respectively. A number of proteins in various subcellular fractions of differentiated cells were labeled with the photoreactive fatty acid 11-m-diazirinophenoxy[11-3H]undecanoate. A 15-kDa cytoplasmic protein was induced upon differentiation to adipocytes. This protein was labeled with the photoreactive fatty acid in cytoplasm isolated from differentiated adipocytes, but not in cytoplasm from undifferentiated, fibroblastic cells. Furthermore, a high affinity fatty acid binding protein of 22 kDa was identified in plasma membranes of undifferentiated cells, and its level of labeling increased 2-fold upon differentiation. These results indicate the usefulness of the photoreactive fatty acid in identifying cellular fatty acid binding proteins, and its potential to elucidate the spatial and temporal distribution of fatty acids in intact cells.