Isopycnic Density Centrifugation Research Articles

Proteomic profiling reveals key cancer progression modulators in shed microvesicles released from isogenic human primary and metastatic colorectal cancer cell lines

Extracellular vesicles comprise two main classes - exosomes and shed microvesicles (sMVs). Whilst much is known about exosome cargo content and functionality, sMVs are poorly understood. Here, we describe the large-scale purification of sMVs released from primary (SW480) and metastatic (SW620) human isogenic colorectal cancer (CRC) cell lines using a combination of differential ultracentrifugation and isopycnic iodixanol density centrifugation. The yield of SW480-sMVs and SW620-sMVs was 0.75 mg and 0.80 mg, respectively. Both SW480-/SW620-sMVs are heterogeneous in size (100–600 nm diameter) and exhibit identical buoyant densities (1.10 g/mL). In contrast to exosomes, sMVs are ALIX−, TSG101−, CD63− and CD9−. Quantitative mass spectrometry identified 1295 and 1300 proteins in SW480-sMVs and SW620-sMVs, respectively. Gene Ontology enrichment analysis identified ‘cell adhesion’ (CDH1, OCLN, CTN families), ‘signalling pathway’ (KRAS, NRAS, MAPK1, MAP2K1), and ‘translation/RNA related’ processes (EIF, RPL, HNRNP families) in both sMV types. Strikingly, SW480- and SW620-sMVs exhibit distinct protein signatures - SW480-sMVs being enriched in ITGA/B, ANXA1, CLDN7, CD44 and EGFR/NOTCH signalling networks, while SW620-sMVs are enriched in PRKCA, MACC1, FGFR4 and MTOR/MARCKS signalling networks. Both SW480- and SW620-sMVs are taken up by NIH3T3 fibroblasts resulting in similar cell invasion capability. This study provides, for the first time, molecular insights into sMVs and CRC biology.

Detection of novel non-M2-related antimitochondrial antibodies in patients with anti-M2 negative primary biliary cirrhosis

Objective:In 95% of patients with primary biliary cirrhosis (PBC) antimitochondrial antibodies (AMAs) can be detected reacting with at least one of the five components of the M2 antigen identified as...

Transgenic, Fluorescent Leishmania mexicana Allow Direct Analysis of the Proteome of Intracellular Amastigotes

Investigating the proteome of intracellular pathogens is often hampered by inadequate methodologies to purify the pathogen free of host cell material. This has also precluded direct proteome analysis of the intracellular, amastigote form of Leishmania spp., protozoan parasites that cause a spectrum of diseases that affect some 12 million patients worldwide. Here a method is presented that combines classic, isopycnic density centrifugation with fluorescent particle sorting for purification by exploiting transgenic, fluorescent parasites to allow direct proteome analysis of the purified organisms. By this approach the proteome of intracellular Leishmania mexicana amastigotes was compared with that of extracellular promastigotes that are transmitted by insect vectors. In total, 509 different proteins were identified by mass spectrometry and database search. This number corresponds to approximately 6% of gene products predicted from the reference genome of Leishmania major. Intracellular amastigotes synthesized significantly more proteins with basic pI and showed a greater abundance of enzymes of fatty acid catabolism, which may reflect their living in acidic habitats and metabolic adaptation to nutrient availability, respectively. Bioinformatics analyses of the genes corresponding to the protein data sets produced clear evidence for skewed codon usage and translational bias in these organisms. Moreover analysis of the subset of genes whose products were more abundant in amastigotes revealed characteristic sequence motifs in 3'-untranslated regions that have been linked to translational control elements. This suggests that proteome data sets may be used to identify regulatory elements in mRNAs. Last but not least, at 6% coverage the proteome identified all vaccine antigens tested to date. Thus, the present data set provides a valuable resource for selection of candidate vaccine antigens.

The Dynamin-like Protein Vps1p of the Yeast Saccharomyces cerevisiae Associates with Peroxisomes in a Pex19p-dependent Manner

Dynamins and dynamin-like proteins play important roles in organelle division. In Saccharomyces cerevisiae, the dynamin-like protein Vps1p (vacuolar protein sorting protein 1) is involved in peroxisome fission, as cells deleted for the VPS1 gene contain reduced numbers of enlarged peroxisomes. What relationship Vps1p has with peroxisomes remains unclear. Here we show that Vps1p interacts with Pex19p, a peroxin that acts as a shuttling receptor for peroxisomal membrane proteins or as a chaperone assisting the assembly/stabilization of proteins at the peroxisome membrane. Vps1p contains two putative Pex19p recognition sequences at amino acids 509-523 and 633-647. Deletion of the first (but not the second) sequence results in reduced numbers of enlarged peroxisomes in cells, as in vps1delta cells. Deletion of either sequence has no effect on vacuolar morphology or vacuolar protein sorting, suggesting that the peroxisome and vacuole biogenic functions of Vps1p are separate and separable. Substitution of proline for valine at position 516 of Vps1p abrogates Pex19p binding and gives the peroxisome phenotype of vps1delta cells. Microscopic analysis showed that overexpression of Pex19p or redirection of Pex19p to the nucleus does not affect the normal cellular distribution of Vps1p in the cytosol and in punctate structures that are not peroxisomes, suggesting that Pex19p does not function in targeting Vps1p to peroxisomes. Subcellular fractionation showed that a fraction of Vps1p is associated with peroxisomes and that deletion or mutation of the first Pex19p recognition sequence abrogates this association. Our results are consistent with Pex19p acting as a chaperone to stabilize the association of Vps1p with peroxisomes and not as a receptor involved in targeting Vps1p to peroxisomes.

Glycogen- and PP1c-targeting Subunit GM Is Phosphorylated at Ser48 by Sarcoplasmic Reticulum-bound Ca2+-Calmodulin Protein Kinase in Rabbit Fast Twitch Skeletal Muscle

Multifunctional Ca(2+)-calmodulin-dependent protein kinase (CaMKII) is a Ser/Thr protein kinase uniformly distributed within the sarcoplasmic reticulum (SR) of skeletal muscle. In fast twitch muscle, no specific substrates of CaMKII have yet been identified in nonjunctional SR. Previous electron microscopy data showed that glycogen particles containing glycogen synthase (GS) associate with SR at the I band level. Furthermore, recent evidence implicates CaMKII in regulation of glucose and glycogen metabolism. Here, we demonstrate that the glycogen- and protein phosphatase 1-targeting subunit, also known as G(M), selectively localizes to the SR membranes of rabbit skeletal muscle and that G(M) and GS co-localize at the level of the I band. We further show that G(M), GS, and PP1c assemble in a structural complex that selectively localizes to nonjunctional SR and that G(M) is phosphorylated by SR-bound CaMKII and dephosphorylated by PP1c. On the other hand, no evidence for a structural interaction between G(M) and CaMKII was obtained. Using His-tagged G(M) recombinant fragments and site-directed mutagenesis, we demonstrate that the target of CaMKII is Ser(48). Taken together, these data suggest that SR-bound CaMKII participates in the regulation of GS activity through changes in the phosphorylation state of G(M). Based on these findings, we propose that SR-bound CaMKII participates in the regulation of glycogen metabolism, under physiological conditions involving repetitive raises elevations of [Ca(2+)](i).

Association of the calpain/calpastatin network with subcellular organelles

The calcium-activated cysteine protease calpain is intimately involved in modulating cell adhesion and migration. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to both focal complexes/adhesions or the plasma membrane. Using confocal microscopy and isopycnic density centrifugation, the results demonstrate that calpain I and II, the 30 kDa regulatory subunit, and calpastatin associate with the endoplasmic reticulum and Golgi apparatus. Confocal microscopy reveals that calpain II colocalizes with the subcellular proteins calnexin and Rab6 in cells bound to laminin. To further verify this association, cell lysates prepared from laminin stimulated and unstimulated cells were subjected to isopycnic density centrifugation. The results reveal an increased association of calpain I, II, calpastatin, and the 30 kDa regulatory subunit with the endoplasmic reticulum and Golgi apparatus as evidenced by their position in the gradient relative to calnexin, Rab6, caveolin, and β1 integrin after laminin stimulation. This correlates with the accumulation of inducible calpain activity at the endoplasmic reticulum-Golgi apparatus interface. Further experiments established that calpain II colocalizes with phosphatidylinositol 4,5-bisphosphate. Finally, calpain II associates with membrane lipid rafts. These results provide new insights into how the calpain/calpastatin network is spatially and temporally regulated in cells binding to the extracellular matrix.

Molecular complexes that contain both c-Cbl and c-Src associate with Golgi membranes

Cbl is an adaptor protein that is phosphorylated and recruited to several receptor and non-receptor tyrosine kinases upon their activation. After binding to the activated receptor, Cbl plays a key role as a kinase inhibitor and as an E3 ubiquitin ligase, thereby contributing to receptor down-regulation and internalization. In addition, Cbl translocates to intracellular vesicular compartments following receptor activation. We report here that Cbl also associates with Golgi membranes. Confocal immunofluorescence staining of Cbl in a variety of unstimulated cells, including CHO cells, revealed a prominent perinuclear colocalization of Cbl and a Golgi marker. Both the prominent Cbl staining and the Golgi marker were dispersed by brefeldin A. Subcellular fractionation of CHO cells demonstrated that about 10% of Cbl is stably associated with membranes, and that Golgi-enriched membrane fractions produced by isopycnic density centrifugation and free-flow electrophoresis are also enriched in Cbl, relative to other membrane fractions. The membrane-bound Cbl was hyperphosphorylated and it co-immunoprecipitated with endogenous Src. By immunofluorescence, some Src colocalized with Cbl and Golgi markers, and Src, like Cbl, was present in the Golgi-enriched fraction prepared by sequential density centrifugation and free-flow electrophoresis. Transfection of an activated form of Src, but not wild-type Src, increased the amount of Src that co-immunoprecipitated with Cbl, and increased the intensity of Cbl staining on the Golgi. This result, together with the increased tyrosine phosphorylation of the membrane-associated Cbl, suggests that Golgi-associated Cbl could be part of a molecular complex that contains activated Src. The localization and interaction of Src and Cbl at the Golgi and the regulation of the interaction of Cbl with Golgi membrane suggest that this complex may contribute to the regulation of Golgi function.

Distribution of MUC1 in the normal human oral cavity is localized to the ducts of minor salivary glands

The change in expression of MUC1 from health to disease forms the basis of its use as a potential disease marker. Previous attempts at isolating MUC1 from normal, healthy human oral mucosa have, however, drawn conflicting conclusions as to its presence. Furthermore, when MUC1 was detected in the oral glycocalyx, it was not clear which cells were synthesising it. We examined human oral glycocalyx using pooled buccal smears from 50 normal individuals. Following isopycnic density centrifugation and membrane extraction with octyl glucoside and saponin, MUC1 was detected with the polyclonal antibody CT1. Immunohistochemistry using antibodies CT1 and BC2 was performed on sections from eight labial, seven palatal, four buccal, three retromolar pad, three dorsum of tongue and two ventral surface of tongue biopsies. In-situ hybridisation using MUC1 and cytoplasmic tail oligoprobes on sections from four palatal, seven labial and two retromolar pad biopsies was also carried out. MUC1 mRNA could only be detected in the minor salivary mucous glands. MUC1 has already been identified in the ducts of normal parotid and submandibular gland, and our findings demonstrate a similar distribution in minor salivary glands. We conclude that when present in the normal oral glycocalyx, the only oral source of MUC1 is from cell membranes of the minor salivary glands.

A Density Gradient Centrifugation Method for Separation of Peat

The conditions necessary for a laboratory—scale separation of the premaceral constituents of peat (i.e., the precursor to coal) are investigated. The method used is an isopycnic density centrifugation (DGC) technique aimed at isolating pure premacerals. This method, which is based on known density differences of various macerals, has been used successfully in coal separations but never with peats. The technique involved grinding the peats in a planetary ball mill to an approximate 10 μm average particle size and conducting dispersion tests using NaCl, Ca(NO3)2, CsCl, and TBE as solvents coupled with numerous surfactants. The density gradient centrifugation technique was run with both an aqueous and organic gradient using plain milled peat, demineralized peat, and demineralized methylated peat. The best separations were achieved in a single run if the peat was demineralized and dispersed with a wetting agent in an organic gradient. Analytical-scale separations were used to choose the most responsive peat to be used in a preparative-scale separation. A low and high density weight fraction, analyzed by flash pyrolysis, revealed distinctly different chromatograms, indicating that the peat had reached a reasonable degree of separation. Four peats of differing constitution are reviewed.

Characterization of Distinct Nuclear and Mitochondrial Forms of Human Deoxyuridine Triphosphate Nucleotidohydrolase

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase; EC 3.6.1.23) was purified from HeLa cells by immunoaffinity chromatography. Based on SDS-polyacrylamide gel electrophoresis, two distinct forms of dUTPase were evident in the purified preparation. These proteins were further characterized by a combination of NH2-terminal protein sequencing, mass spectrometry, and mass spectrometry-based protein sequencing. These analyses indicate that the two forms of dUTPase are largely identical, differing only in a short region of their amino-terminal sequences. Despite the structural difference, both forms of dUTPase exhibited identical binding characteristics for dUTP. Each form of dUTPase has a distinct cellular localization. Cellular fractionation and isopycnic density centrifugation indicate that the lower molecular weight form of dUTPase (DUT-N) is associated with the nucleus, while the higher molecular weight species (DUT-M) fractionates with the mitochondria. The DUT-N isoform is approximately 30-fold more abundant in HeLa cells than DUT-M as determined by densitometry. The NH2-terminal protein sequence of both DUT-N and DUT-M did not match previous reports of the predicted amino-terminal sequence for human dUTPase (McIntosh, E.M., Ager, D.D., Gadsden, M.H., and Haynes, R.H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8020-8024; Strahler, J.R., Zhu X., Hora, N., Wang, Y.K., Andrews, P.C., Roseman, N.A., Neel, J.V., Turka, L., and Hanash, S.M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4991-4995). A cDNA corresponding to the DUT-N isoform was isolated utilizing an oligonucleotide probe based on the determined NH2-terminal sequence. The cDNA contains a 164-amino acid open reading frame, encoding a protein of Mr 17,748. The DUT-N cDNA sequence matches the previously cloned cDNAs with the exception of a few discrepancies in the 5' end. Our data indicate a 69-base pair addition to the 5' end of the previously reported open reading frame.

Isolation and characterisation of human proximal tubular cells derived from kidney cortical segments

1. Human renal proximal tubular cells (HPTC) were isolated by collagenase digestion and purified following filtration and isopycnic Percoll density centrifugation. This method used cortical tissue obtained from surgical nephrectomies and was both rapid and simple, providing a preparation of cells with high viability (> 93 +/- 3%) and recovery (16 +/- 7 x 10(6) cells g-1 of cortical tissue). 2. Characterisation of the isolated cells showed that, in terms of morphology, enzyme profile, transport systems and hormonal responsiveness, they were > 95% proximal tubular. The transport systems obeyed Michaelis-Menten kinetics, with the kinetic parameters of the glucose transport system (Km = 2.5mM, Vmax = 7.7 nmol min-1 mg-1 protein) suggesting a higher proportion of PT cells originating from the S1-S2 segment of the nephron. Isolated HPTC also maintained levels of reduced glutathione (GSH) (11.9 +/- 3.2 nmol mg-1 protein) and exhibited cytochrome P450-dependent activity, levels of spectrally determined P450 being 0.22 +/- 0.07 nmol mg-1 protein. 3. These results demonstrate the isolation of a viable and functioning homogeneous preparation of HPTC from cortical tissue, with potential for use in short term pharmacological, physiological and toxicological studies.

Genetic and molecular analysis of DNA43 and DNA52: two new cell-cycle genes in Saccharomyces cerevisiae.

Two Saccharomyces cerevisiae genes previously unknown to be required for DNA synthesis have been identified by screening a collection of temperature-sensitive mutants. The effects of mutations in DNA43 and DNA52 on the rate of S phase DNA synthesis were detected by monitoring DNA synthesis in synchronous populations that were obtained by isopycnic density centrifugation. dna43-1 and dna52-1 cells undergo cell-cycle arrest at the restrictive temperature (37 degrees C), exhibiting a large-budded terminal phenotype; the nuclei of arrested cells are located at the neck of the bud and have failed to undergo DNA replication. These phenotypes suggest that DNA43 and DNA52 are required for entry into or completion of S phase. DNA43 and DNA52 were cloned by their abilities to suppress the temperature-sensitive lethal phenotypes of dna43-1 and dna52-1 cells, respectively. DNA sequence analysis suggested that DNA43 and DNA52 encode proteins of 59.6 and 80.6 kDa, respectively. Both DNA43 and DNA52 are essential for viability and genetic mapping experiments indicate that they represent previously unidentified genes: DNA43 is located on chromosome IX, 32 cM distal from his5 and DNA52 is located on chromosome IV, 0.9 cM from cdc34.

Isolation and characterization of rhoptries of Plasmodium falciparum

Rhoptries have been isolated from Plasmodium falciparum schizont-infected erythrocytes by isopycnic density centrifugation. Gradient fractions were analyzed by immunoblotting with antibodies against two polypeptides of 140 and 110 kDa, known to be components of the rhoptry. The proteins were present primarily in fractions with a density of 1.16 g ml −1. Electron microscopy of these fractions indicated they were enriched in rhoptries. For the most part, the isolated organelle retained in situ morphology, although some rhoptries were distorted, indicating the structure of some of the organelles is not rigid. Electrophoretic analysis of the rhoptry fractions indicated the presence of a number of proteins, many of which have not been identified to date. Properties of proteins in the isolated rhoptry were examined using the 140 and 110 kDa proteins as representative markers. Both proteins are present in a complex with a 130-kDa protein, as all three co-immunoprecipitate. At the late schizont stage, the rhoptry proteins are present in two distinct forms; a soluble form with an M r of 480 000 which would correspond to a single copy of the 140/130/110 kDa complex and a form that can be sedimented at 130 000 × g. Properties of the sedimentable form suggest that the proteins are included in structures that resemble membranes. Ionic detergents were required to solubilize the proteins while high concentrations of NaCl and Na 2CO 3 resulted in only partial solubilization. Furthermore, treatment of disrupted rhoptries with phospholipase A and C resulted in the release of proteins into the soluble form.

Ultrastructural characterization of human immunodeficiency virus type 1 Gag-containing particles assembled in a recombinant adenovirus vector system

The human immunodeficiency virus type 1 (HIV-1) Gag protein was expressed in A549 cells infected with recombinant adenovirus types 4 and 7, each carrying the HIV-1 gag and pro genes. The Gag protein was assembled into enveloped virus-like particles that budded from plasma and vacuolar membranes. The particles, isolated by precipitation and isopycnic density centrifugation, contained both processed and unprocessed Gag-associated proteins.

Two distinct steps in pullulanase secretion by Escherichia coli K12

Two distinct steps in the secretion of the extracellular, cell-surface-anchored lipoprotein pullulanase by Escherichia coli were uncoupled by allowing export of the enzyme to the cytoplasmic membrane via the signal peptide/sec-gene-dependent general export pathway, and then inducing the pulC-O operon of genes required for translocation to the cell surface. The secretion intermediate cofractionated mainly with intermediate-density vesicles when cells were gently lysed and the resulting vesicles were separated by isopycnic sucrose density centrifugation. Cytoplasmic forms of pullulanase (which are not exported because they lack a functional signal peptide) are more sensitive to heat inactivation, denaturation by sodium dodecyl sulphate and carboxymethylation than the intermediate and cell-surface forms. The latter are distinguished only by the fact that the secretion intermediate is less susceptible to proteinase K and trypsin, and is partially inaccessible to substrate or in an inactive conformation in sphaeroplasts. These and other results indicate that the secretion intermediate can acquire considerable higher-ordered structure, including disulphide bridges, before it is transported to the cell surface; this seems to rule out the possibility that it is threaded through this membrane as a locally unfolded polypeptide.

Selective extraction of outer-membrane proteins from membrane complexes of Pseudomonas maltophila by chloroform-methanol

An organic phase partitioning method is described for the selective purification of outer-membrane proteins (OMPs) from the total membrane complex of the opportunistic human and sheep pathogen, Pseudomonas maltophila. SDS-PAGE analysis confirmed that OMPs purified by chloroform-methanol treatment of the total membrane complex were not only identical to OMPs extracted from outer-membrane vesicles separated by sucrose gradient density centrifugation, but also possessed little or non-detectable levels of inner-membrane contaminants. Further analysis by enzyme linked immunosorbent assay (ELISA) and immunoblotting established that OMPs extracted by organic phase partitioning with chloroform-methanol retained antigenicity and serological activity indistinguishablee from OMPs that were present in outer-membrane vesicles resolved by isopycnic sucrose density centrifugation of sarkosyl-treated membrane complexes.

Characterization of reconstituted plasma membrane H+‐ATPase from maize roots

Corn (Zea mays L.) plasma membranes from KI‐washed microsomal fractions were further purified by isopycnic sucrose density centrifugation. An examination of separated fractions indicated that vesicles with nitrate‐insensitive proton transport copurified with fractions containing vanadate‐sensitive ATPase activity. The ATPase in purified plasma membrane was reconstituted into liposomes by a detergent dilution technique using deoxycholate. The reconstituted ATPase exhibited characteristics similar to those of the native enzyme. However, reconstituted preparations showed an enhanced sensitivity to vanadate, a diminished phosphatase activity and a high specific rate of ATP‐dependent H+‐transport. Apparent Ki values of reconstituted and native enzymes with respect to vanadate were 20 and 50 μM, respectively; the KJ value of the H+‐pumping of reconstituted ATPase was 30 μM. The proton pumping of reconstituted vesicles could be discharged rapidly by p‐trifluoromethoxyphenyl hydrazone (FCCP), hexokinase and vanadate. The hydrolysis of Mg‐ATP by both native and reconstituted ATPases obeyed simple Michaelis‐Menten plots with a Km between 0.5 and 0.6 mM. The reconstituted ATPase retained a pH profile similar to that of native enzyme with a maximum of pH 6.5.

Selective fractionation of hypoxic B16 melanoma cells by density gradient centrifugation

Cells of the mouse B16 melanoma growing in monolayer culture and as tumors were fractionated by isopycnic density centrifugation in a linear-density (1.02–1.20 g/ml) metrizamide gradient. Cultured cells concentrated into one or two distinct bands, with densities of 1.02–1.04 g/ml and 1.06–1.10 g/ml, depending on growth conditions. Cells subjected to extreme hypoxia (<0.02% O 2) banded predominantly at the lower density, and normally-oxygenated cells banded at the higher density. Fractionated tumor cells concentrated at both densities. Compared with cells at the higher density, lower-density cells incorporated more of the hypoxic cell radiosensitizer [ 14C]misonidazole and less [ 3H]thymidine in vivo, were less clonogenic but more resistant to X-irradiation in situ, and labeled to a lesser extent with intravenously-delivered Hoechst 33342 fluorochrome, a market for cells proximal to tumor blood vessels. Lower-density tumor cells were, therefore, enriched in non-proliferating radioresistant hypoxic cells from tumor regions remote from blood vessels.

Influence of growth phase, nutrition and hypoxia on heterogeneity of cellular buoyant densities in in vitro tumor model systems.

EMT6/Ro multicellular spheroids were sequentially dissociated into outer, middle and inner layers, and the buoyant densities of these subpopulations were compared to aerobic or exponential or plateau-phase monolayers by isopycnic density centrifugation using Percoll as the gradient medium. Spheroids were more heterogeneous in buoyant density distributions than monolayer cultures. The outer-layer cells of spheroids and fed plateau monolayers peaked at a similar density; the majority of middle-layer cells banded close to unfed plateau and exponential hypoxic monolayers, while most inner-layer cells banded where fed or unfed plateau hypoxic monolayer peaked in density. Our data suggest that metabolic heterogeneity due to growth phase, together with altered cellular microenvironment brought about by differences in oxygenation and external nutrient supplies between spheroids and monolayers, can be manifested in one biophysical parameter such as buoyant density.

Localization of 5-lipoxygenase within human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMN) stimulated with the Ca-ionophore A23187 or opsonized zymosan not only release marker enzymes of specific granules but secrete 5-lipoxygenase activity as well. In the presence of BSA cells incubated with [ 14C]AA were able to synthetize 5-HPETE but failed to produce 5-HETE, LTB 4, and its ω-oxidation metabolites. Subcellular fractionation studies by differential and isopycnic equilibrium density centrifugation demonstrated main lipoxygenase activity in particulate fractions consisting of specific granules, but not in cytosolic fractions. These results suggest the association of 5-lipoxygenase with specific granules. 5-lipoxygenase released from the cells upon appropriate stimulation reached its peak activity after 10 min and was then rapidly inactivated. It appears that the intermediate 5-HPETE may be generated extracellularly but has to re-enter the intracellular space for further metabolization.