Selectivity in cornified envelop binding of ceramides in human skin and the role of LXR inactivation on ceramide binding

The cornified lipid envelope (CLE) is a lipid monolayer covalently bound to the outside of corneocytes and is part of the stratum corneum (SC). The CLE is suggested to act as a scaffold for the unbound SC lipids. By profiling the bound CLE ceramides, a new subclass was discovered and identified as an omega-hydroxylated dihydrosphingosine (OdS) ceramide. Bound glucosylceramides were observed in superficial SC layers of healthy human skin. To investigate the relation between bound and unbound SC ceramides, the composition of both fractions was analyzed and compared. Selectivity in ceramide binding towards unsaturated ceramides and ceramides with a shorter chain length was observed. The selectivity in ceramide species bound to the cornified envelope is thought to have a physiological function in corneocyte flexibility. Next, it was examined if skin models exhibit an altered bound ceramide composition and if the composition was dependent on liver X-receptor (LXR) activation. The effects of an LXR agonist and antagonist on the bound ceramides composition of a full thickness model (FTM) were analyzed. In FTMs, a decreased amount of bound ceramides was observed compared to native human skin. Furthermore, FTMs had a bound ceramide fraction which consisted mostly of unsaturated and shorter ceramides. The LXR antagonist had a normalizing effect on the FTM bound ceramide composition. The agonist exhibited minimal effects. We show that ceramide binding is a selective process, yet, still is contingent on lipid synthesized.     

A new 6-hydroxy-4-sphingenine-containing ceramide in human skin.

A new ceramide consisting of 6-hydroxysphingosine linked to a non-hydroxyacid was found in human epidermal lipid. This ceramide was sought because its fatty acid and sphingoid moieties are present in other combinations in human epidermal ceramides. To isolate the new ceramide, the mixture of ceramides in human epidermal lipid was first separated into fractions by thin-layer chromatography (TLC), and then each fraction was further purified by TLC after acetylation of all hydroxyl groups. TLC after acetylation revealed that one of the fractions isolated in the first TLC step contained two components, namely, the ceramide consisting of sphingosine linked to an alpha-hydroxyacid and an unknown ceramide. The new ceramide constituted about 9% of the total ceramides, and was shown by NMR spectroscopy to be N-acyl-6-hydroxysphingosine.     

Characterization and direct quantitation of ceramide molecular species from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry

A rapid, simple, and reliable method has been developed for the characterization and quantitation of ceramide molecular species directly from chloroform extracts of biological samples by electrospray ionization tandem mass spectrometry (ESI/MS/MS). By exploiting the differential fragmentation patterns of deprotonated ceramide ions, individual 2-hydroxy and nonhydroxy ceramide molecular species were readily identified by ESI/MS/MS with the neutral loss of fragments of mass 256.2 and 327.3 which correspond to sphingosine derivatives. The ions generated from the neutral loss of 256.2 (i.e., [M - H - 256.2](-)) are unique for ceramides with N-acyl sphingosine with the 18-carbon homolog. However, the sensitivity for nonhydroxy ceramides in ESI/MS/MS with the neutral loss of 256.2 is approximately threefold higher than that for 2-hydroxy ceramides. The ions resulting from the neutral loss of 327.3 (i.e., [M - H - 327.3](-)) are specific for 2-hydroxy ceramides. Additionally, all ceramides including both 2-hydroxy and nonhydroxy forms can be confirmed and accurately quantitated by ESI/MS/MS with the neutral loss of 240.2 after correction for (13)C isotope factors. This methodology demonstrated a 1000-fold linear dynamic range and a detection limit at the subfemtomole range and was applied to directly quantitate ceramide molecular species in chloroform extracts of biological samples including brain tissues and cell cultures.     

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.     

Cellular distribution of the hamster liver specific nucleolar antigens

The immunochemical localization of hamster liver nucleolar antigens in subcellular fractions (nuclei, 10,000 x g pellet, 100,000 x g pellet and supernatant), nuclear substructures (chromatin, nuclear matrix, nuclear envelope, nucleoli, RNP particles and nucleosomes), and three classes of nonhistone chromosomal proteins with different affinities to DNA (NHCP1, NHCP2 and NHCP3) from nuclease-sensitive and nuclease-resistant chromatin fractions of hamster liver were studied. Six main nucleolar antigens with mol. wts 27,000; 29,000; 30,000; 36,000; 45,000; and 46,000 were found in subcellular fractions, nuclear substructures and classes of non-histone proteins of hamster liver. The antigens with mol.wts of approx. 27,000; 29,000; and 36,000 which were absent in hamster pancreas, spleen and Kirkman--Robbins hepatoma nuclei, seem specific for liver tissue.     

Many ceramides

Intensive research over the past 2 decades has implicated ceramide in the regulation of several cell responses. However, emerging evidence points to dramatic complexities in ceramide metabolism and structure that defy the prevailing unifying hypothesis on ceramide function that is based on the understanding of ceramide as a single entity. Here, we develop the concept that "ceramide" constitutes a family of closely related molecules, subject to metabolism by >28 enzymes and with >200 structurally distinct mammalian ceramides distinguished by specific structural modifications. These ceramides are synthesized in a combinatorial fashion with distinct enzymes responsible for the specific modifications. These multiple pathways of ceramide generation led to the hypothesis that individual ceramide molecular species are regulated by specific biochemical pathways in distinct subcellular compartments and execute distinct functions. In this minireview, we describe the "many ceramides" paradigm, along with the rationale, supporting evidence, and implications for our understanding of bioactive sphingolipids and approaches for unraveling these pathways.     

Enzymatic deoxyglucosylation of ceramides by microsomes of BHK-21 cells. The effect of deoxyglucose treatment and herpesvirus infection

The microsomal fractions of cultured hamster fibroblasts (BHK-21 cells) catalyze the incorporation of glucose from UDPglucose or of deoxyglucose from UDPdeoxyglucose into a reaction mixture with liposomes consisting of ceramide and phosphatidylcholine. The microsomal fractions also catalyze the transfer of glucose from UDPglucose to endogenous acceptors. The specific activity of ceramide deoxyglucoside or ceramide glucoside formation was significantly higher when microsomal preparations obtained from deoxyglucose-treated or herpesvirus-infected BHK-21 cells were used as the glucosyltransferase source. Deoxyglucose was incorporated from UDPdeoxyglucose into hydroxy- and nonhydroxy-fatty acid-containing ceramides at approximately the same rate. Competitive inhibition of deoxyglucosylation of ceramides by UDPglucose suggests that both reactions were catalyzed by the same enzyme, viz. UDPglucose:ceramide glucosyltransferase. This inhibition of glycosphingolipid synthesis may account, in part, for the inhibitory effect of deoxyglucose on lipid-containing viruses.     

Evidence for a high activity of sphingomyelin biosynthesis by phosphocholine transfer from phosphatidylcholine to ceramides in lung lamellar bodies

Biosynthesis of sphingomyelin from ceramides was investigated in lung subcellular fractions by incubating a lyophilized mixture of albumin and subcellular fraction (0.1-0.2 mg of protein) coated with [acyl-14C]-ceramide and phosphatidyl[methyl-3H]choline in Tris-buffer. The lamellar-body-rich fraction exhibited the highest specific activity for sphingomyelin biosynthesis measured by 14C incorporation into sphingomyelins or by [3H]phosphocholine transfer from phosphatidylcholines. Plasma membranes formed the next most active fraction, followed by the 'smooth' and, then, the 'rough' endoplasmic reticulum. Sphingomyelin biosynthesis by lamellar bodies was optimum at pH 7.4 and was inhibited by sphingomyelins formed. Slight inhibitory effects were also observed with Mn2+, Ca2+ and lysophosphatidylcholine. Phosphocholine transfer from CDPcholine was not observed under the reaction conditions employed. Ceramide conversion and phosphocholine transfer increased with ceramide concentration to reach a maximum at about 0.06 mM. The highest conversion rate was observed when 18:1 ceramide was used as an acceptor. When 1-palmitoyl-2-oleoylphosphatidylcholine was the phosphocholine donor, the overall biosynthesis of sphingomyelin was much higher than when using dipalmitoylphosphatidylcholine. These results suggest the possible involvement of the studied reaction in the control of the degree of saturation of the surfactant phosphatidylcholine.     

Ceramides and ceramide synthases in cancer: Focus on apoptosis and autophagy

Different studies corroborate a role for ceramide synthases and their downstream products, ceramides, in modulation of apoptosis and autophagy in the context of cancer. These mechanisms of regulation, however, appear to be context dependent in terms of ceramides’ fatty acid chain length, subcellular localization, and the presence or absence of their downstream targets. Our current understanding of the role of ceramide synthases and ceramides in regulation of apoptosis and autophagy could be harnessed to pioneer the development of new treatments to activate or inhibit a single type of ceramide synthase, thereby regulating the apoptosis induction or cross talk of apoptosis and autophagy in cancer cells. Moreover, the apoptotic function of ceramide suggests that ceramide analogues can pave the way for the development of novel cancer treatments. Therefore, in the current review paper we discuss the impact of ceramide synthases and ceramides in regulation of apoptosis and autophagy in context of different types of cancers. We also briefly introduce the latest information on ceramide synthase inhibitors, their application in diseases including cancer therapy, and discuss approaches for drug discovery in the field of ceramide synthase inhibitors. We finally discussed strategies for developing strategies to use lipids and ceramides analysis in biological fluids for developing early biomarkers for cancer.     

Mouse anti-ceramide antiserum: a specific tool for the detection of endogenous ceramide.

Ceramide is a pivotal molecule in signal transduction and an essential structural component of the epidermal permeability barrier. The epidermis is marked by a high concentration of ceramide and by a unique spectrum of ceramide species: Besides the two ceramide structures commonly found in mammalian tissue, N-acylsphingosine and N-2-hydroxyacyl-sphingosine, six additional ceramides differing in the grade of hydroxylation of either the sphingosine base or the fatty acid have been identified in the epidermis. Here we report on the characterization of an IgM-enriched polyclonal mouse serum against ceramide. In dot blot assays with purified epidermal lipids the antiserum bound to a similar extent to N-acyl-sphingosine (ceramide 2), N-acyl-4-hydroxysphinganine (ceramide 3), and N-(2-hydroxyacyl)-sphingosine (ceramide 5), whereas no specific reaction was detected with glycosylceramides, sphingomyelin, free sphingosine, phospholipids, or cholesterol. In contrast, a monoclonal IgM antibody, also claimed to be specific for ceramide, was shown to bind specifically to sphingomyelin and therefore was not further investigated. In thin-layer chromatography immunostaining with purified lipids a strong and highly reproducible reaction of the antiserum with ceramide 2 and ceramide 5 was observed, whereas the reaction with ceramide 1 and ceramide 3 was weaker and more variable. Ceramide 2 and ceramide 5 were detected in the nanomolar range at serum dilutions of up to 1:100 by dot blot and thin-layer immunostaining. In thin-layer chromatography immunostaining of crude lipid extracts from human epidermis, the antiserum also reacted with N-(2-hydroxyacyl)-4-hydroxysphinganine (ceramide 6) and N-(2-hydroxyacyl)-6-hydroxysphingosine (ceramide 7). Furthermore, the suitability of the antiserum for the detection of endogenous ceramide by immunolight microscopy was demonstrated on cryoprocessed human skin tissue. Double immunofluorescence labeling experiments with the anti-ceramide antiserum and the recently described anti-glucosylceramide antiserum (Brade et al., 2000, Glycobiology 10, 629) showed that both lipids are concentrated in separate epidermal sites. Whereas anti-ceramide stained the dermal and basal epidermal cells as well as the corneocytes, anti-glucosylceramide staining was concentrated in the stratum granulosum. In conclusion, the specificity and sensitivity of the reagent will enable studies on the subcellular distribution and biological functions of endogenous ceramide.     

Systematic analyses of free ceramide species and ceramide species comprising neutral glycosphingolipids by MALDI-TOF MS with high-energy CID

Free ceramides and glycosphingolipids (GSLs) are important components of the membrane microdomain and play significant roles in cell survival. Recent studies have revealed that both fatty acids and long-chain bases (LCBs) are more diverse than expected, in terms of i) alkyl chain length, ii) hydroxylation and iii) the presence or absence of double bonds. Electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been well utilized to characterize sphingolipids with high throughput, but reports to date have not fully characterized various types of ceramide species such as hydroxyl fatty acids and/or trihydroxy-LCBs of both free ceramides and the constituent ceramides in neutral GSLs. We performed a systematic analysis of both ceramide species, including LCBs with nona-octadeca lengths using MALDI-TOF MS with high-energy collision-induced dissociation (CID) at 20 keV. Using both protonated and sodiated ions, this technique enabled us to propose general rules to discriminate between isomeric and isobaric ceramide species, unrelated to the presence or absence of sugar chains. In addition, this high-energy CID generated ^3,5A ions, indicating Hex1-4Hex linkage in the sugar chains. Using this method, we demonstrated distinct differences among ceramide species, including free ceramides, sphingomyelins, and neutral GSLs of glucosylceramides, galactosylceramides, lactosylceramides, globotriaosylceramides and Forssman glycolipids in the equine kidneys.     

New Evidence for the Role of Ceramide in the Development of Hepatic Insulin Resistance

Aim

There are few and contradictory data on the role of excessive accumulation of intracellular sphingolipids, particularly ceramides, in the development of hepatic insulin resistance. In our study we assessed accumulated sphingolipid fractions and clarify the mechanisms of hepatic insulin resistance development as well as involvement of fatty acid and ceramide transporters in this process.

Methods

In culture of primary rat hepatocytes, exposed to high concentration of palmitic acid (0.75mM) during short and prolonged incubation, high performance liquid chromatography was used to assess intra- and extracellular sphingolipid fractions content. Degree of palmitate-induced insulin resistance was estimated by measuring changes in phosphorylation of insulin pathway proteins by western blotting as well as changes in expression of different type of transporters.

Results

In our study short and prolonged exposure of primary hepatocytes to palmitic acid resulted in increased intracellular accumulation of ceramide which inhibited insulin signaling pathway. We observed a significant increase in the expression of fatty-acid transport protein (FATP2) and ceramide transfer protein (CERT) what is consistent with enhanced intracellular ceramide content. The content of extracellular ceramide was increased nearly threefold after short and twofold after long incubation period. Expression of microsomal triglyceride transfer protein (MTP) and ATP-binding cassette transporter (ABCA1) was increased significantly mainly after short palmitate incubation.

Conclusion

Our data showed that increase in intarcellular ceramide content contributes to the development of hepatic insulin resistance. We suggest pivotal role of transporters in facilitating fatty acid influx (FATP2), accumulation of ceramides (CERT) and export to the media (MTP and ABCA1).     

Subcellular Distribution of UDP-Galactose:Ceramide Galactosyltransferase in Rat Brain Oligodendroglia

Oligodendrocytes isolated from 18-19-day-old rat brain were homogenized in 0.32 M sucrose. The homogenate was centrifuged at 100,000 g for 50 min in a gradient containing 0.8, 1.05, and 1.3 M sucrose. Three discrete bands were obtained at the interfaces 0.32-0.8 (F1), 0.8-1.05 (F2), and 1.05-1.3 M (F3). The distribution of UDP-galactose:ceramide galactosyltransferase (CgalT) activity in each fraction was measured using liposomes containing normal fatty acid-containing ceramides (NFA-CgalT activity) or 2-hydroxy fatty acid-containing ceramides (HFA-CgalT activity). Although detection of both CgalT activities was possible in all fractions, HFA-CgalT activity was enriched in F1 and F2 fractions, which also showed an enrichment of Golgi and endoplasmic reticulum markers, respectively. It is interesting that NFA-CgalT activity was significantly enriched in the F2 fraction. These results suggest that hydroxylated and nonhydroxylated galactocerebrosides may be synthesized at different intracellular locations.     

Regulation of mitochondrial ceramide distribution by members of the BCL-2 family

Apoptosis is an intricately regulated cellular process that proceeds through different cell type- and signal-dependent pathways. In the mitochondrial apoptotic program, mitochondrial outer membrane permeabilization by BCL-2 proteins leads to the release of apoptogenic factors, caspase activation, and cell death. In addition to protein components of the mitochondrial apoptotic machinery, an interesting role for lipids and lipid metabolism in BCL-2 family-regulated apoptosis is also emerging. We used a comparative lipidomics approach to uncover alterations in lipid profile in the absence of the proapoptotic proteins BAX and BAK in mouse embryonic fibroblasts (MEFs). We detected over 1,000 ions in these experiments and found changes in an ion with an m/z of 534.49. Structural elucidation of this ion through tandem mass spectrometry revealed that this molecule is a ceramide with a 16-carbon N-acyl chain and sphingadiene backbone (d18:2/16:0 ceramide). Targeted LC/MS analysis revealed elevated levels of additional sphingadiene-containing ceramides (d18:2-Cers) in BAX, BAK-double knockout MEFs. Elevated d18:2-Cers are also found in immortalized baby mouse kidney epithelial cells lacking BAX and BAK. These results support the existence of a distinct biochemical pathway for regulating ceramides with different backbone structures and suggest that sphingadiene-containing ceramides may have functions that are distinct from the more common sphingosine-containing species.     

Secondary ion mass spectrometry for sulfoglycolipids: application of negative ion detection

A series of underivatized sulfoglycolipids (SM4g, lyso-SM4g, SM4s, SM3, SM2, SB2, and SB1a) from various tissues were analyzed by both positive (POS-SI-MS) and negative (NEG-SI-MS) secondary ion mass spectrometry. By POS-SI-MS were detected the molecular ions of sulfoglycolipids in the form with sodium or potassium together with some fragment ions useful for the carbohydrate sequence determination. The analysis of monosulfogangliotriaosyl- or monosulfogangliotetraosylceramide and bis-sulfoglycolipid was difficult due to noise in the high mass region. On the other hand, NEG-SI-MS of sulfoglycolipids gave more intense signals from molecular ion of (M-H)- for monosulfoglycolipids and [M-H+Na)-H)- for bis-sulfoglycolipid. Many fragment ions useful for the elucidation of the carbohydrate sequences were also obtained with significant intensities. The fragmentation was assessed to occur at the glycosidic linkages to form ions of the oligosaccharides with or without ceramide. These ions were useful for sugar sequencing and also for distinguishing the differences in the position of the sulfate group. The intensities of saccharide ions without sulfate were lower than those with sulfates. In the case of SB2 and SB1a, containing 2 mol of sulfate ester groups, the molecular ion was detected as [M-H+Na)-H)-. Also, fragment ions with 2 mol of sulfate were detected as the sodium-additive form. It was concluded that NEG-SI-MS is a very useful technique for the structural elucidation of higher sulfoglycolipids.     

Accumulation of protein-bound epidermal glucosylceramides in beta-glucocerebrosidase deficient type 2 Gaucher mice

The epidermal permeability barrier for water is essentially maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the main components of these membranes, derive in large part from hydrolysis of glucosylceramides mediated by the lysosomal enzyme beta-glucocerebrosidase. As analyzed in this work, the beta-glucocerebrosidase deficiency in type 2 Gaucher mice (RecNci I) resulted in an accumulation of all epidermal glucosylceramide species accompanied with a decrease of the related ceramides. However, the levels of one ceramide subtype, which possesses an alpha-hydroxypalmitic acid, was not altered in RecNci I mice suggesting that the beta-glucocerebrosidase pathway is not required for targeting of this lipid to interstices of the stratum corneum. Most importantly, omega-hydroxylated glucosylceramides which are protein-bound to the epidermal cornified cell envelope of the transgenic mice accumulated up to 35-fold whereas levels of related protein-bound ceramides and fatty acids were decreased to 10% of normal control. These data support the hypothesis that in wild-type epidermis omega-hydroxylated glucosylceramides are first transferred enzymatically from their linoleic esters to proteins of the epidermal cornified cell envelope and then catabolized to protein-bound ceramides and fatty acids, thus contributing at least in part to the formation of the lipid-bound envelope.     

Apoptosis occurs via the ceramide recycling pathway in human HaCaT keratinocytes

Keratinocytes contain abundant ceramides compared to other cells. However, studies on these cells have mainly focused on the barrier function of ceramide, while their other roles, such as those in apoptosis or cell cycle arrest, have not been well addressed. In this study, we investigated the apoptosis-inducing effect of exogenously added cell-permeable ceramides in HaCaT keratinocytes. We found that N-hexanoyl sphingosine (C6-ceramide) induced apoptosis efficiently through the accumulation of long chain ceramides. On the other hand, N-acetyl sphingosine (C2-ceramide) induced neither apoptosis nor accumulation of long chain ceramides. We also found that exogenously added C6-ceramide was hydrolyzed to sphingosine and then reacylated in long chain ceramides (ceramide recycling pathway), but that C2-ceramide was not hydrolyzed and thus not recycled. We propose that this is the basis for the chain length-specific heterogeneity observed in ceramide-induced apoptosis in these cells. These results also imply that keratinocytes utilize exogenous sphingolipids or ceramides to coordinate their own ceramide compositions.     

Protein kinase activity in rat testis interstitial tissue. Effect of luteinizing hormone and other factors.

Protein kinase activity was determined in subcellular fractions of rat testis interstitial tissue after incubation of the intact tissue with LH (luteinizing hormone) in vitro. Various factors that might have changed the activity of this enzyme during preparation of the fractions before assay were also investigated. The following results were obtained. 1. LH and 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) added together during incubation of the interstitial tissue caused a twofold increase in the protein kinase activity in the total tissue homogenate and subcellular fractions (12000g X 5 min pellet and 105000g X 60 min supernatant and pellet). 2. A decrease of approx. 40% in the total amount of protein kinase recovered in the soluble fraction (105000g supernatant) occurred in tissue incubated with LH and 3-isobutyl-1-methylxanthine when compared with the controls. No change in total activity was found in the other fractions. 3. LH and 3-isobutyl-1-methylxanthine caused an increase in cyclic AMP concentration in the soluble fraction (from 30 +/- 6 to 450 +/- 40 pmol/mg of protein, means +/- S.E.M., n = 4), but there was little or no increase in the particulate fractions [from 9 +/- 1 to 13 +/- 3 pmol/mg of protein (n = 3) and from 6 +/- 2 to 23 +/- 11 pmol/mg of protein (n = 3) in the 12000g and 105000g pellets respectively]. 4 Addition of 3-isobutyl-1-methylxanthine alone had little effect on protein kinase activity or cyclic AMP concentrations. 5. Little or no protein kinase activity could be demonstrated in subcellular particulate fractions unless Triton X-100 was added; the effect of this detergent was shown to be at least partly due to the inhibition of adenosine triphosphatase activity. 6. In the presence of Triton X-100 approx. 57% of the total protein kinase activity in the homogenate was found in the 105000g supernatant compared with 11% in the 105000g pellet and 32% in the 12000g pellet. 7. In contrast with adipose-tissue protein kinase [Corbin et al. (1973) J. Biol. Chem. 248, 1813-1821] the relative amounts of cyclic AMP-dependent and -dependent enzyme were not affected by dilution of the interstitial-tissue fractions. NaCl (0.5 M) decreased the estimated total amount of protein kinase activity.     

Activation of guinea pig polymorphonuclear leukocytes with soluble stimulators leads to nonrandom distribution of NADPH oxidase in the plasma membrane

Guinea pig polymorphonuclear leukocytes (PMN) were briefly activated with soluble stimulators such as sodium myristate (SM) or phorbol myristate acetate (PMA) and then disrupted by the nitrogen cavitation method to study the subcellular distribution of NADPH oxidase, which is responsible for O2 - generation. Fc-receptor and 5'-nucleotidase activities were measured as plasma membrane markers. 1) The homogenate was first fractionated by differential centrifugation. The O2- -generating activity of PMN activated either by SM or PMA was recovered in a 2 X 10(4) g pellet which contained a large amount of granules and about 50% of the plasma membrane markers, but not in a 1 X 10(5) g pellet which consisted of plasma membranes and few granules. 2) Further separation of the 2 X 10(4) g pellet from PMA-activated PMN was attempted by an iso-osmotic Percoll density gradient centrifugation. The O2- -generating activity was recovered in light fractions in which plasma membrane markers were found, but neither in specific nor in azurophil granules. The 1 X 10(5) g pellet showed a similar distribution of the plasma membrane markers to that of the 2 X 10(4) g pellet, except that the peak of the O2- -generating activity was much smaller on an identical density gradient. The results showed that NADPH oxidase is located in the plasma membranes precipitated by centrifugation at 2 X 10(4) X g but not in the ones precipitated at 1 X 10(5) X g. The results suggest that the plasma membrane of activated PMN has a mosaic distribution of NADPH oxidase.     

Isolation of rat intestinal microsomes: partial characterization of mucosal cytochrome P-450

A procedure is presented for the isolation of subcellular fractions from small intestinal mucosal cells in the rat. The mucosal cells were detached by a scraping procedure resulting in an almost complete harvest of all types of cells as judged by light microscopy. Homogenization using a Potter-Elvehjem Teflon-glass device at high speed with ensuing sonication was found to be necessary for complete disruption of the cells. The subcellular fractions obtained after differential centrifugation--10,000g pellet, 105,000g pellet (microsomal fraction), and supernatant--were characterized with respect to different marker enzymes. The highest yield of 7-ethoxyresorufin-O-deethylase and NADPH-cytochrome c reductase activity in the microsomal fraction was achieved after resuspension and recentrifugation of the 10,000g pellet. Addition of anti-P-450 beta-naphthoflavone (BNF)-B2 antibodies to the incubation mixture resulted in almost complete inhibition of the O-deethylation of 7-ethoxyresorufin whereas addition of anti-P-450 phenobarbital (PB)-B2 had no effect. The presence of BNF-inducible isozymes was demonstrated by the Western blotting technique not only in intestinal microsomes from BNF-treated rats, but also in microsomes from untreated rats. Anti-P-450 BNF-B2 was also used in the peroxidase-antiperoxidase method for studies on the localization of cytochrome P-450. No BNF-inducible cytochrome P-450 could be detected in untreated rats, whereas BNF treatment resulted in a general staining of the whole villus.