The subcellular distribution of carotenoids in Neurospora crassa

The intracellular localization of carotenoids in the fungus Neurospora crassa was examined after completion of photoinduced biosynthesis of these pigments. Differential centrifugation of cell homogenates yielded subcellular fractions which were characterized by activities of several marker enzymes for cell constituents and in part purified by subsequent sucrose density gradient centrifugation. Most (ca 58%) of the carotenoids were found to be localized in lipid globules, but substantial amounts are also associated with two membrane fractions that were rich in membranes of the endoplasmic reticulum as indicated by high activities of NADPH- and NADH—cytochrome c reductase. These results, along with the coincidence in the distribution of both carotenoids and activities of specific marker enzymes in the sucrose density gradients, led to the conclusion that apart from lipid globules, carotenoids are also localized in membranes of the endoplasmic reticulum.     

Isolation of pig platelet membranes and granules distribution and validity of marker enzymes

A method for the subcellular fractionation of pig platelet homogenates by sucrose density gradient centrifugation is described. The procedure is simple, highly reproducible and yields two major particulate fractions and a soluble phase. One particulate fraction consists almost entirely of membrane fragments and is relatively free from granule contamination. The other particulate zone contains the platelet granules and mitochondria. The distribution on the gradients of the enzymes lactate dehydrogenase, succinate dehydrogenase, 5′-nucleotidase, leucyl β-naphthylamidase and cholinesterase has been studied and organelle localisation further substantiated by electron microscopy. The degree of solubilisation of certain marker enzymes during homogenisation has been investigated and the parallel release of these enzymes with the soluble phase marker enzyme lactate dehydrogenase, suggests they have a true biphasic location between the soluble and particulate components of the cell. No significant difference was found in the molar ratios of cholesterol to phospholipid in the subcellular fractions but the content of each lipid was twice as high in the membrane fraction as in the granule fraction.     

An electron microscopic study of the interaction in vitro of vimentin intermediate filaments with vesicles prepared from Ehrlich ascites tumor cell lipids

The interaction of intermediate filaments prepared from pure, delipidated vimentin with vesicles obtained from Ehrlich ascites tumor (EAT) cell lipids was studied employing sucrose density gradient centrifugation in combination with electron microscopy. In negative stain electron microscopy, preformed vimentin filaments were seen in lateral association with lipid vesicles; end-on contacts of filaments with liposomes were rarely detected. When the reaction of filaments with vesicles was carried out at 0 degree C, sucrose density gradient equilibrium centrifugation of the reaction products led to the banding of relatively light filament-vesicle meshworks in clear separation from free filaments and free vesicles. With certain vimentin and lipid preparations, occasionally partial breakdown of the filaments during centrifugation and banding of vesicle-free fragments in denser regions of the sucrose gradients was observed. However, when the reaction mixtures were incubated at 37 degrees C prior to sucrose gradient analysis, all filaments were released from vesicles and totally fragmented during centrifugation. Electron microscopy showed unraveling of the filament fragments into subfilament strands. Employing lipid vesicles labeled with [3H]cholesterol, a low but significant amount of radioactivity was found to be associated with the fragments in a non-vesicular form. Filament reconstitution experiments performed in the presence of EAT cell lipids revealed an inhibitory effect of vesicles on filament assembly, particularly at lower temperatures. The mechanical labilization of the filament structure by lipid vesicles might play a role in the redistribution of intermediate filaments in the course of certain cellular processes involving turnover and fragmentation of intracellular membrane systems.     

Glycosylation of pea cotyledon membranes

Pea cotyledons were injected with d-[(14)C]mannose or d-[(14)C]-glucosamine and incubated for 1 to 1.5 hours. Cotyledons were homogenized and subcellular fractions were isolated by differential centrifugation followed by linear sucrose density gradient centrifugation.Radioactivity that was precipitated by trichloroacetic acid was associated most extensively with rough endoplasmic reticulum, Golgi membranes, a membrane with a density of 1.14 grams per cubic centimeter (possibly plasma membrane) and an unidentified subcellular component with a density of 1.22 grams per cubic centimeter. Lower levels of incorporation were observed in protein bodies and mitochondria.Isolated membrane fractions were lipid-extracted to determine which components of the membrane contained the label. Rough endoplasmic reticulum contained the most extensively labeled lipids which had similar properties to the lipid intermediates thought to be involved in glycoprotein assembly. The lipid free residues of the various membrane fractions contained radioactivity that was released by protease treatment. Acid hydrolysis of the residues indicated that most of the radioactivity was associated with mannose or glucosamine. It appears that various subcellular components of the pea cotyledon possess glycoproteins that contain mannose and glucosamine.     

Ribulose 1,5-bisphosphate carboxylase and polyhedral bodies of Chlorogloeopsis fritschii

Ribulose 1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) activity was approximately equally distributed between supernatant and pellet fractions produced by differential centrifugation of disrupted cells of Chlorogloeopsis fritschii. Low ionic strength buffer favoured the recovery of particulate RuBP carboxylase. Density gradient centrifugation of resuspended cell-free particulate material produced a single band of RuBP carboxylase activity, which was associated with the polyhedral body fraction, rather than with the thylakoids or other observable particles. Isolated polyhedral body stability was improved by density gradient centrifugation through gradients of Percoll plus sucrose in buffer, which yielded apparently intact polyhedral bodies. These were 100 to 150 nm in diameter and contained ring-shaped, 12 nm diameter particles. It is inferred that the C. fritschii polyhedral bodies are carboxysomes. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of SDS-dissociated polyhedral bodies revealed 8 major polypeptides. The most abundant, with molecular weights of 52,000 and 13,000, correspond with the large and small subunits, respectively, of RuBP carboxylase.Abbreviations RuBP ribulose 1,5-bisphosphate - Ru5P ribulose 5-phosphate - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - EDTA ethylenediamine tetraacetic acid - Tris tris (hydroxymethyl) methylamine - IB isolation buffer - TCA trichloroacetic acid     

Plant glycoprotein biosynthesis. Uridine diphosphate N-acetyl-glucosaminyltransferase from horseradish root.

A particulate enzyme preparation from horseradish root tissue was shown to catalyze the transfer of 2-acetamido-2-deoxy-D-[14C1]glucose from uridine diphosphate 2-acetamido-2-deoxy-D-[14C1]glucose to an exogenous acceptor molecule derived from horseradish peroxidase. The acceptor was produced from purified peroxidase by the action of a mixture of glycoside hydrolases covalently bound to Sepharose. The membrane preparation containing the transferase was purified approximately 12-fold by aqueous two phase distribution and by discontinuous sucrose density gradient centrifugation. Hydrolysis of the reaction product yielded glucosamine as the only radio-labeled substance. Precipitation of the reaction product by antiserum against peroxidase showed that the label was incorporated into peroxidase. The transferase utilized the acceptor most efficiently when only 12% of the 2-acetamido-2-deoxy-D-glucose was removed from the acceptor. The acceptor lost no accepting capabilities when heated to 100 degrees C for 3 min prior to assay. Trypsin treatment caused a 14% decrease in label incorporated while pronase treatment caused a 93% decrease,     

Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: an anomalous effect of ficoll

An investigation was conducted into the isolation of plasma membrane vesicles from primary roots of corn (Zea mays L., WF9 × M14) by sucrose density gradient centrifugation. Identification of plasma membranes in cell fractions was by specific staining with the periodic-chromic-phosphotungstic acid procedure. Plasma membrane vesicles were rich in K⁺-stimulated ATPase activity at pH 6.5, and equilibrated in linear gradients of sucrose at a peak density of about 1.165 g/cc. It was necessary to remove mitochondria (equilibrium density of 1.18 g/cc) from the homogenate before density gradient centrifugation to minimize mitochondrial contamination of the plasma membrane fraction. Endoplasmic reticulum (NADH-cytochrome c reductase) and Golgi apparatus (latent IDPase) had equilibrium densities in sucrose of about 1.10 g/cc and 1.12 to 1.15 g/cc, respectively. A correlation (r = 0.975) was observed between K⁺-stimulated ATPase activity at pH 6.5 and the content of plasma membranes in various cell fractions. ATPase activity at pH 9 and cytochrome c oxidase activity were also correlated.     

Isolation and fractionation of the photosynthetic membranous organelles from Rhodopseudomonas spheroides

Molecular sieve chromatography and sucrose gradient centrifugation were used to prepare large quantities of purified chromatophores from Rhodopseudomonas spheroides. Electron micrographs of these chromatophores revealed that the final preparations were very homogeneous and free of non-chromatophore particulate material. As an additional check on purity, (14)C-l-phenylalanine-labeled aerobic cells, devoid of chromatophores, were mixed with unlabeled photosynthetic cells. The resulting preparation contained less than 1% of the radioactivity, originally located in non-chromatophore protein. The purified chromatophores were solubilized in 2-chloroethanol and separated into two fractions. Fraction P(1) contained 3 to 5% of the total chromatophore protein and could be resolved into 10 electrophoretic components. The second fraction, P(II), contained five electrophoretic components. One of these components had associated with it all of the pigment and phospholipid present in P(II). Preliminary immunochemical studies on these fractions are also reported.     

Raft composition at physiological temperature and pH in the absence of detergents

Biological rafts were identified and isolated at 37°C and neutral pH. The strategy for isolating rafts utilized membrane tension to generate large domains. For lipid compositions that led only to microscropically unresolvable rafts in lipid bilayers, membrane tension led to the appearance of large, observable rafts. The large rafts converted back to small ones when tension was relieved. Thus, tension reversibly controls raft enlargement. For cells, application of membrane tension resulted in several types of large domains; one class of the domains was identified as rafts. Tension was generated in several ways, and all yielded raft fractions that had essentially the same composition, validating the principle of tension as a means to merge small rafts into large rafts. It was demonstrated that sphingomyelin-rich vesicles do not rise during centrifugation in sucrose gradients because they resist lysis, necessitating that, contrary to current experimental practice, membrane material be placed toward the top of a gradient for raft fractionation. Isolated raft fractions were enriched in a GPI-linked protein, alkaline phosphatase, and were poor in Na⁺-K⁺ ATPase. Sphingomyelin and gangliosides were concentrated in rafts, the expected lipid raft composition. Cholesterol, however, was distributed equally between raft and nonraft fractions, contrary to the conventional view.     

Role of the cell wall in the ability of tobacco protoplasts to form callus

Cellular membranes from dark grown hypocotyls of Phaseolus aureus Roxb. were separated by centrifugation on a continuous sucrose gradient. Each gradient fraction was monitored for activity of inosine diphosphatase (EC 3.6.1.6) and the ability to transfer glucose from UDP-[¹⁴C]glucose to endogenous lipids in vitro. The highest incorporation of radioactivity into lipids occurred in a particulate fraction correlated with the Golgi apparatus, sedimenting at sucrose densities of 31.5–33% w/w. Three endogenous lipids were glucosylated in vitro. The two main lipids were characterized as steryl glucoside and acylated steryl glucoside; data from chromatography and hydrolysis of the third lipid suggests that it is dolichyl-monophosphate-glucoside. Steryl glucoside was found to be the main glucoside synthesized, but the proportion of the acylated form increased with time. The results are discussed in the context of the role of the Golgi apparatus as a centre of membrane modification within the plant cell.Abbreviations DMP-mannose dolichyl monophosphate mannose - ER endoplasmic reticulum - GA Golgi apparatus - ID-Pase inosine diphosphatase     

The subcellular distribution of endogenous and exogenous serotonin in brain tissue: comparison of synaptosomes storing serotonin, norepinephrine, and gamma-aminobutyric acid

Abstract— We have studied the subcellular distribution of exogenous and endogenous serotinin in slices from the hypothalamus and midbrain of several species. In a procedure which appears to label the endogenous pools, tissue slices were incubated with low concentrations of [³H]5-HT (5 × 10^-8 M), for 45 min, when there was apparent equilibrium between [³H]5-HT of tissue and medium. After the tissue slices were homogenized in 0-32 M-sucrose and subjected to differential centrifugation, the distribution of exogenous and endogenous 5-HT in pellets and supernatant fluid was similar. In some experiments, the crude mitochondrial pellets were resuspended in 0-32 M-sucrose, layered on linear, continuous density gradients of sucrose (1 -5-0-32 M), and centrifuged for short times (incomplete equilibrium centrifugation). The subcellular distribution of particulate tritium, total tritium, and particulate endogenous 5-HT was the same in portions of the gradients containing synaptosomes. The peak distribution of [³H]5-HT in sucrose gradients was separable from the peak for [¹⁴C]GABA by four to five fractions; potassium (a marker for cytoplasm occluded within synaptosomes) occurred in the regions of the gradients containing most of the labelled compounds. The distribution of monoamine oxidase activity (a mitochondrial marker) overlapped the distribution of [³H]5-HT after a 15 min centrifugation but appeared in denser regions of the gradient after centrifuging for 2 h. Particles containing [3H]5-HT and [I4C]NE were slightly but consistently separable in synaptosomal fractions isolated from the hypothalamus or midbrain of rat, guinea pig and hamster.     

Subcellular localization of a membrane-associated transglutaminase activity in rat liver

Fractionation of rat liver by homogenization and differential centrifugation revealed that only about 83% of the transglutaminase activity in the tissue is in a soluble form, and that the remainder is associated with the particulate fraction. This latter activity remained with the membranes even after they were extensively washed to remove 99% of such soluble enzymes as lactate dehydrogenase and aldolase. Subsequent fractionation of the membranes by isopycnic density gradient centrifugation in sucrose resulted in a single band of transglutaminase activity at a density of 1.194 g/cm3. This activity was coincident with the major band of plasma membranes, which was identified by its content of 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and leucine aminopeptidase activities. After treatment with digitonin and fractionation on sucrose gradients, the transglutaminase activity and the plasma membrane marker enzyme activities were found at a new density of 1.210 g/cm3, while the enzyme markers for the other membrane fractions remained unchanged. From these data, we conclude that approximately 17% of the transglutaminase activity in rat liver is specifically associated with the plasma membranes.     

Localization of the membrane-associated CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary cells with an altered membrane composition

The subcellular localization of the membrane-associated CTP:phosphocholine cytidylyltransferase was determined in Chinese hamster ovary cells in which the phospholipid composition had been altered by growth in the presence of N-methylethanolamine or treatment with phospholipase C. Cell homogenates were fractionated on Percoll density gradients, and marker enzyme activities were used to determine the location of the cellular membrane fractions. The peak of cytidylyltransferase activity occurred in the gradient at a density intermediate to that of the peaks of endoplasmic reticulum and plasma membrane markers. The profile of cytidylyltransferase activity most closely resembled that of the Golgi membrane marker; however, upon sucrose gradient centrifugation, the profile of the Golgi apparatus was very different from that of cytidylyltransferase. Differential centrifugation suggested a nuclear membrane association of the enzyme. Cytidylyltransferase was associated with a membrane fraction that sedimented when subjected to very low speed centrifugation (65 x g, 5 min). From Percoll gradient fractions, nuclei were identified by microscopy, and they migrated with cytidylyltransferase activity. The data are consistent with a localization of cytidylyltransferase in the nuclear membrane.     

Studies on the role of the oligomeric state and subunit III of cytochrome oxidase in proton translocation

Anion-exchange fast protein liquid chromatography in the presence of lauryldimethylamine N-oxide (LDAO) was introduced to separate cytochrome oxidase into different complexes that either did or did not contain subunit III. Both kinds of enzyme complex exhibited H+ translocation after reconstitution into phospholipid vesicles, but with a significantly (approx. 50-60%) reduced H+/e- ratio as compared with unchromatographed enzyme. The anion-exchange FPLC fractions of the enzyme (with or without subunit III) sedimented more slowly than the control enzyme upon sucrose gradient centrifugation in the presence of cholate and a high potassium phosphate concentration. When the control enzyme was subjected to the sucrose gradient centrifugation in the presence of LDAO or Triton X-100, instead of cholate, one band containing all subunits was observed, which sedimented slowly like the FPLC fractions. Transfer of this band to cholate medium, and reapplication on the sucrose gradient (with cholate), yielded both a slow- and a fast-migrating band after centrifugation. Enzyme complexes that sedimented slowly or rapidly in the sucrose gradients revealed longer and shorter elution times, respectively, in gel filtration FPLC. This suggests that these complexes corresponds to monomers and dimers of cytochrome oxidase. Solubilization of proteoliposomes and subsequent sucrose gradient centrifugation in cholate yielded one fast-migrating band for the untreated enzyme, but both a fast- and a slow-migrating band for the anion-exchange FPLC-treated enzyme, which was exclusively slow-migrating before reconstitution into liposomes. It is suggested that dimerisation of monomeric cytochrome oxidase may be favoured when the enzyme encounters a membranous milieu, and that the dimeric structure might be necessary for proton translocation.     

Characterization of a poly(A)+RNA-containing structural component directly associated with cytoplasmic membranes in dormant Artemia cysts

Poly(A)+RNA-containing material was extracted from the purified cytoplasmic membranes of dormant Artemia cysts by treatment with mild detergents. Sedimentation analysis of the extracts showed a predominant poly(A)-containing fraction at 40 S, associated with about 6% of the extracted proteins. Only limited amounts of poly(A)-containing material were found in the heavier fractions. Poly(A)+RNA extracted from the 40-S fraction sedimented around 14 S. The poly(A)-containing 40-S structures could be purified by treatment with non-ionic or zwitterionic detergents followed by resedimentation in sucrose gradients in the presence or absence of detergent. When the 40-S fraction was analyzed by isopycnic centrifugation in Cs2SO4 gradients, the main part of the poly(A)-containing material banded at a density of 1.27 g/ml. Electron-microscopic examination of this fraction revealed circular or slightly bullet-shaped profiles measuring 17-26 nm. When the 40-S fraction had been submitted to mild RNAase treatment prior to density gradient centrifugation, the material was displaced towards lower density and became less distinct. Purified 40-S particles showed a complex protein pattern not very similar to that of polyribosomal poly(A)+RNA-containing particles from developing embryos, but with components in common with unfractionated membranes. The particles also contained some lipids. The experiments indicate that a major part of the membrane-bound, latent poly(A)+RNA in dormant Artemia cysts occurs in the form of relatively uniform, detergent- and Cs2SO4-resistant structures, independent of ribosomes, but intimately associated with membrane components.     

Isolation and partial characterization of outer and inner membranes from encapsulated Haemophilus influenzae type b.

A method has been developed to separate the cell envelope of encapsulated (type b) Haemophilus influenzae into its outer and inner membrane components with procedures that avoided two problems encountered in fractionation of this envelope: (i) the tendency of the outer and inner membranes to hybridize and (ii) the tendency of the apparently fragile inner membrane to fragment into difficulty sedimentable units. Log phage cells, whose lipids were radioactively labeled, were lysed by passage through a French press. The lysate was applied to a discontinuous sucrose gradient, and envelope-rich material was collected by centrifugation onto a cushion of dense sucrose under carefully controlled conditions. This material was then further fractionated by isopycnic centrifugation in a sucrose gradient to yield four membrane fractions which were partially characterized. On the basis of their radioactivity, buoyant density, ultrastructure, polypeptide composition, and content of phospholipid, protein, lipopolysaccharide, and succinic dehydrogenase, these fractions were identified as follows: fraction 1, outer membrane vesicles with very little inner membrane contamination (less than 4%); fraction 2, outer membrane vesicles containing entrapped inner membrane; fraction 3, a protein-rich fraction of inner membrane; fraction 4, a protein-poor fraction of inner membrane. Fractions 3 and 4 contained about 25% outer membrane contamination.     

Cholesterol is required for the polarized secretion of erythropoietin in Madin-Darby canine kidney cells

It has already been reported that stably expressed exogenous human wild-type EPO (wtEPO) is preferentially secreted to the apical side and one of the three N-linked carbohydrate chains critically acts as an apical sorting determinant in Madin-Darby canine kidney (MDCK) cells. It has been suggested that lipid rafts are involved in the apical sorting of membrane and secretory proteins. To investigate the involvement of lipid rafts in the apical sorting of wtEPO, we examined the effect of cholesterol depletion with methyl-beta-cyclodextrin on the secretion polarity of EPO and analyzed Triton X-100 insoluble cell extracts by sucrose density gradients centrifugation in MDCK cells. We found that wtEPO was shifted in non-polarized direction by cholesterol depletion. Most of the wtEPO was not detectable in the raft fractions by sucrose density gradients centrifugation analysis. These results indicate that apical secretion of EPO involves a cholesterol-dependent mechanism probably not involving lipid rafts.     

CHARACTERIZATION OF MEMBRANES OBTAINED FROM ELECTRIC ORGAN OF THE ELECTRIC EEL BY SUCROSE GRADIENT FRACTIONATION AND BY MICRODISSECTION

Abstract— Two membrane fractions were obtained from electric organ tissue of the electric eel by sucrose gradient centrifugation of tissue homogenates. Electron microscopic examination showed that both fractions contained mainly vesicular structures (microsacs). Both the light and heavy fractions had a-bungarotoxin-binding capacity and Na⁺-K⁺ ATPase activity, while only the light fraction had AChE activity. The polypeptide patterns of vesicles derived from both the light and heavy fractions were examined by SDS-polyacrylamide gel electrophoresis and found to be very similar. The ratio of protein to phospholipid in the light vesicles was much lower than in the heavy vesicles, but the relative amounts of individual phospholipids in the two fractions were similar. A marked difference in the permeability of the light and heavy vesicles was observed by measuring efflux of both [¹⁴C]sucrose and ²²Na⁺, and also by monitoring volume changes induced by changing the osmotic strength of the medium. All three methods showed the heavy vesicles to be much more permeable than the light ones. Only the light vesicles displayed increased sodium efflux in the presence of carbamylcholine. The AChE in the light fraction does not appear to be membrane-bound, but is rather a soluble enzyme, detached from the membrane during homogenization, which migrates on the gradient similarly to that of the light vesicles. This is supported by the fact that the bulk of the AChE is readily removed by washing the vesicles. Moreover, under the conditions employed in our sucrose gradient separations,‘native’14 S + 18 S AChE exists in the form of aggregates which migrate very similarly to the major peak of AChE activity of tissue homogenates. Separated innervated and non-innervated surfaces of isolated electroplax were obtained by microdissection. α-Bungarotoxin-binding capacity was observed only in the innervated membrane. About 80% of the AChE was in the innervated membrane, and about 70% of the Na⁺-K⁺ ATPase in the non-innervated membrane. The data presented indicate that the light and heavy vesicle fractions separated by sucrose gradient centrifugation are not derived exclusively from the innervated and non-innervated membranes respectively, as previously suggested by others, but contain membrane fragments from both sides of the electroplax. The separation of two populations on sucrose gradients may be explained both by the differences in permeability and in protein to phospholipid ratios.     

Purification and partial characterization of plasma membranes from bovine spermatozoa

Bovine epididymal spermatozoa were subjected to nitrogen cavitation (600 psi for 10 min) to remove plasma membrane. Examination of the cavitated cells by electron microscopy revealed that the plasma membrane was preferentially removed from the periacrosomal and flagellar regions. Nuclear, mitochondrial and acrosomal membranes remained intact and attached to the spermatozoa, but the cytoplasmic droplets were frequently disrupted and their internal membrane-bound vesicles were released. Lower pressures (less than 200 psi) were relatively ineffective in removing the periacrosomal plasma membrane, while an intermediate pressure (400 psi) removed this membrane from about 70% of the spermatozoa. No apparent selectivity for removal of the periacrosomal and flagellar plasma membrane was observed as a function of cavitation pressure. The cavitated cells were separated from the plasma membranes by differential followed by linear sucrose density gradient centrifugation. Two distinct membrane populations were resolved on sucrose gradients and were designated Band I and Band II. Band I contained only spherical vesicles which arose from the plasma membrane. Surface labeling of intact cells confirmed the plasma membrane as the origin of Band I. The membranes of higher density comprising Band II were heterogeneous consisting of both spherical and flattened vesicles. When purified cytoplasmic droplets were cavitated and centrifuged on the sucrose gradient only Band II was obtained. These studies indicate that nitrogen cavitation of bovine epididymal spermatozoa can result in significant contamination of plasma membrane fractions by cytoplasmic droplet membranes unless appropriate differential centrifugation is used to separate the membrane fractions.     

The subsynaptosomal localization of histamine, histidine decarboxylase and histamine methyltransferase in rat hypothalamus

Abstract— We have examined the subcellular localization of histamine, histamine methyltransferase (EC 2.1.1.8) (HMT) and histidine decarboxylase (EC 4.1.1.22) in rat hypothalamus after osmotic lysis of synaptosome-containing primary particulate fractions. When crude mitochondrial fractions are subjected to osmotic lysis, histamine is retained within particulate structures, while HMT is released into the supernatant fluid. The majority of histidine decarboxylase activity is also recovered in the supernatant fluid, although more histidine decarboxylase than HMT is retained in particulate fractions. After sucrose gradient fractionation of osmotically lysed crude mitochondrial or microsomal pellets, histamine is also retained in particulate structures, with the greatest amount occurring in a fraction enriched in synaptic vesicles. In these sucrose gradients histidine decarboxylase activity shows a greater particulate localization than does HMT activity.