Density distribution of guinea pig myenteric plexus nerve endings containing immunoreactive substance P

The present study was performed to investigate how myenteric plexus nerve endings containing substance P are distributed in sucrose density gradients in relation to nerve endings capable of taking up 3H-acetylcholine or 14C-noradrenaline. The peak of substance P-immunoreactivity (ISP) was found at a density of 1.157 +/- 0.001 g X ml-1, that of 3H-radioactivity at 1.160 +/- 0.002 and that of 14C-radioactivity at 1.162 +/- 0.002 g X ml-1 (mean +/- SEM, N = 6); there was considerable overlap. In a second set of experiments, the resuspended P2-pellet was layered upon a discontinuous density gradient consisting of 0.6, 1.0, 1.2 and 1.4 M sucrose. Nine fractions were recovered. There was a 2.5-3.4-fold increase in the relative specific activity of ISP in the 1.2 M fraction (density = 1.154 g X ml-1) and the adjoining interfaces. Conventional electron microscopy showed that synaptosomal elements were present in the transmitter-enriched fractions. It is concluded that the substance P-containing nerve endings of the guinea pig myenteric plexus co-distribute (and may be co-purified with) nerve endings utilizing noradrenaline or acetylcholine on sucrose density gradients.     

Extraction of plasma membranes from smooth muscle cells of the rabbit small intestine

A method is offered for isolation of subcellular fractions from small intestinal smooth muscle cells enriched by plasma membranes (PM). The method is based on differential centrifugation over sucrose density gradient. According to the localization of marker enzymes, the membrane fraction obtained with the use of 30% sucrose is considered to be optimal. The PM fraction is superior to the homogenate 10-fold on the average in the magnitude of Na, K-ATPase, 17-fold in Mg2+-ATPase, and 15-fold in that of 5'-nucleotidase activity. ATPase of PM is activated by Ca2+ in micro- and millimolar concentrations. It is suggested that Mg2+-dependent Ca-activated ATPase of PM is related to the Ca2+ content control in the cell.     

Fractionation of Isolated Rat CNS Myelinated Axons by Sucrose Density Gradient Centrifugation in a Zonal Rotor

Myelinated axons isolated from rat CNS brain stem by flotation in a buffered sucrose-salt medium were shocked by vigorous homogenization in hypotonie buffer and then fractionated on a 20-40% (wt/wt) linear sucrose gradient in a Beckman Ti-14 Zonal Rotor. After centrifu-gation to equilibrium, the gradient was fractionated on the basis of sucrose density into 13 individual fractions. The distributions of molecular markers related to myelin [(myelin basic protein, 2’3′-cyclic nucleotide 3′-phos-phodiesterase (EC 3.1.4.37), myelin-associated glycopro-tein (MAG)]; microsomes [CDP-choline:l,2 diglyceride cholinephosphotransferase (EC 2.7.8.2)]; mitochondria [cytochrome c oxidase (EC 1.9.3.1), monoamine oxidase (amine:oxygen oxidoreductase, deaminating, EC 1.4.3.4)], and axolemma [acetylcholinesterase (acetylcho-line hydrolase, EC 3.1.1.7), 5′-nucleotidase (5′-ribonu-cleotide phosphohydrolase, EC 3.1.3.5), Na⁺,K⁺-adeno-sine triphosphatase (EC 3.6.1.3), [³H]saxitoxin binding] were examined, as well as the protein composition and morphological appearance of the fractions. The myelin-related markers were most enriched in the 20-26% region of the gradient, although the MAG was broadly distributed throughout the entire gradient. The axolemma-related markers were most enriched in the 28-32% region of the gradient, whereas the microsomal and mitochondrial-related markers were enriched in the 35-40% region of the sucrose density gradient. Mixing experiments utilizing ¹²⁵I-labeled membrane preparations derived from cultured oligodendroglial and astroglial cells indicated that the constituents of the shocked myelinated axons were not significantly contaminated with glial membranes. The morphology of the fraction was consistent with the membrane molecular marker distribution: the light end of the gradient contained multilamellar myelin; fractions in the center of the gradient were enriched in un-ilamellar membrane fragments; the densest regions of the gradient were enriched in mitochondria. The myelin specific proteins were the prominent polypeptides in the 20-25% regions of the gradient, whereas polypeptides having a molecular weight of 50,000 or greater predominanted in the denser regions of the gradient. The significance of the distribution of these membrane markers and the utility of this fractionation procedure are discussed.     

LOCALIZATION OF ENZYMES WITHIN MICROBODIES

Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm³ which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50–60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [¹⁴C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21–1.22 g/cm³, whereas the original glyoxysomes appeared at density 1.24 g/cm³. Electron microscopy showed that the fraction at 1.21–1.22 g/cm³ was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance.     

Cerebroside Sulfotransferase in Golgi-Enriched Fractions from Rat Brain

Abstract: Golgi-enriched fractions were prepared from brainstems of 17-day-old rats by first floating off myelin, then fractionating the remaining pellet by a series of differential and density gradient centrifugations in sucrose. Fractions enriched in Golgi membranes were recovered at 0.46/0.76 m and 0.76/0.87 m interfaces on the final sucrose gradient as indicated by morphology and the biochemical markers thiamine pyrophosphatase and [³H]fucose-labeled glycoprotein. Morphology of the two fractions indicated very little contamination with myelin lamellae; however, the presence of significant levels of 2', 3'-cyclic nucleotidase in the lighter fraction suggested a contribution from oligodendroglial or myelin-related membranes. Cerebroside sulfotransferase was highly enriched in the lighter Golgi-enriched fraction relative to the denser fraction, the post-34, 880 x g microsomes, and the myelin-like fraction. In contrast, ceramide galactosyl transferase was more evenly distributed among the fractions. Our results show a more highly localized distribution of sulfatide synthesis than of galactocerebroside synthesis, probably in Golgi membranes or oligodendroglia-related membranes with similar properties.     

A novel method of chromosome distribution analysis in saccharose density gradient

The human chromosomes distribution in a sucrose density gradient was studied using a new computer method of the quantitative analysis of flow karyotypes. The dual-parameter flow distributions of human chromosomes fluorescence intensities of the sucrose density gradient fractions were analyzed to obtain the quantity of each chromosome. The chromosomes were found to distribute over sucrose density gradient as follows: 1) fractions with low sucrose density mostly contain chromosomes 1-7, and their quantity is increased between 1.4- to 3.2-fold in comparison with the control unfractionated suspension; 2) medium density fractions are enriched with chromosomes 8-20 up to 2.4-fold; 3) fractions with a high sucrose density mostly contain small chromosomes 21-22 and fragments of broken chromosomes. So the new method of quantitative analysis of flow karyotypes allows one to determine the efficiency of enrichment and the maximally enriched fraction for any chosen chromosome. Maximally enriched fractions maximize the rate of preparative flow sorting of individual chromosomes for research or biotechnology purposes.     

Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. I. Electrophoretic and immunochemical analyses

We have developed a fast and reliable method for the separation of two membrane fractions respectively enriched in outer and inner envelope membranes from isolated, intact, purified spinach chloroplasts kept in a hypertonic medium (0.6 M mannitol). This separation was achieved by osmotically shrinking the inner envelope membrane, thus widening the intermembrane space, and then subsequently removing the "loosened" outer envelope membrane by applying low pressure to the shrunken chloroplasts and slowly extruding them through the small aperture of a Yeda press under controlled conditions. By centrifugation of the mixture obtained through a discontinuous sucrose gradient, we were able to separate two membrane fractions having different densities (fraction 2 or light fraction, d = 1.08 g/cm3, and fraction 3 or heavy fraction, d = 1.13 g/cm3). The recent characterization of polypeptides localized on the outer envelope membrane from spinach chloroplasts, E10 and E24 (Joyard, J., Billecocq, A., Bartlett, S. G., Block, M. A., Chua, N.-H., and Douce, R. J. Biol. Chem., 258, 10000-10006) enabled us to characterize our two membrane fractions. Analyses of the polypeptides by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and immunoblotting have shown that fraction 2 (light fraction) was completely devoid of polypeptide E30, which is involved in the transport of phosphate across the inner envelope membrane, but was enriched in polypeptides E10 and E24. The reverse was true for fraction 3 (heavy fraction). Under these conditions, it is clear that fraction 2 is strongly enriched in outer envelope membrane whereas fraction 3 consisted mostly of inner envelope membrane. Indeed, by immunoelectrophoresis, we were able to demonstrate that, on a protein basis, fraction 2 contained about 90% of outer membrane, whereas fraction 3 contained about 80% of inner membrane. Further characterization of the outer envelope membrane was achieved by using thermolysin, a nonpenetrant protease.     

Characterization of three membrane fractions isolated from cells of Rhodopseudomonas capsulata adapting from chemotrophic to phototrophic conditions

By means of sucrose density centrifugation three membrane fractions, named light, medium and heavy have been isolated from cells of Rhodopseudomonas capsulata strain 37b4, adapting from chemotrophic to phototrophic growth conditions. Succinate dehydrogenase activity of aerobically grown cells was mainly confined to the heavy (chromatophore) fraction. Upon changing to phototrophic conditions the activity of the succinate dehydrogenase increased in the medium and light fraction. All fractions contain bacteriochlorophyll. NADH dehydrogenase of chemotrophically grown cells was enriched in the light and medium fraction but is increased in the heavy fraction under phototrophic growth conditions. The capacity of photophosphorylation is high in the light and heavy fraction. The results indicate a differentially incorporation of functional subunits into specific parts of the membrane system during membrane differentiation.Abbreviations Bchl bacteriochlorophyll - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCCD N,N-dicyclohexyl carbodiimide     

Subcellular Distribution of Aspartate Transaminase,Alanine Amino Transferase,Glutamate Dehydrogenases,and Lactate Dehydrogenase in Tetrahymena*

SYNOPSIS. Mitochondria and peroxisomes were isolated from homogenates of Tetrahymena pyriformis by sedimentation through a sucrose gradient. Succinate dehydrogenase was used as a mitochondrial marker; catalase and isocitrate lyase were used to mark the peroxisomal fraction. Lactate dehydrogenase, glutamate dehydrogenase, and alanine aminotransferase were found only in the mitochondrial fraction. Aspartate transaminase was found in both mitochondrial and peroxisomal fractions.     

Isolation of a highly enriched sarcolemma membrane fraction from canine heart.

A highly enriched sarcolemma preparation was isolated by differential centrifugation of a canine ventricular homogenate followed by centrifugation of a membrane fraction layered over 22% (w/v) sucrose. Ouabain binding, ouabain-sensitive potassium phosphatase activity and 5'-nucleotidase activity were enriched 19--27 fold over the homogenate whereas Ca2+-ATPase and succinate dehydrogenase activities were 0.75 and 0.36, respectively, of that for the homogenate. The isolation procedure was relatively rapid and yielded about 2.0 mg protein/100 g of ventricular muscle. The highest salt concentration used in the procedure was 0.6 M KCl and no detergents were employed. Initial characterization studies suggested that the sarcolemma-enriched fraction consists predominantly if not totally of freely permeable membrane vesicles and that the sarcolemma does not manifest a Ca2+-ATPase activity, at least within the limits of the assay procedures employed. This preparation was concluded to be about 1.5- to 4-fold more highly enriched with sarcolemmal markers than preparations obtained by previously published procedures. Accordingly, the preparation provides an improved basis for the probe of calcium movements that occur across the sarcolemma in association with the excitation-contraction-relaxation sequence of the mammalian myocardial cell.     

Evidence for a KCl-Stimulated, Mg-ATPase on the Golgi of Corn Coleoptiles

Membranes of corn (Zea mays, cv Trojan 929) coleoptiles were fractionated by sucrose density gradient centrifugation and the locations of organelles were determined using marker enzymes and electron microscopy. Latent IDPase (or UDPase) was selected as the Golgi marker and UDPG-sterol glucosyl transferase was selected as the plasma membrane (PM) marker, because they were clearly separable from markers for the other organelles. Golgi-rich and PM-rich fractions were studied in relation to their ATPase activities. The pH optimum of the KCl, Mg²⁺-ATPase of the PM-rich fraction from a step gradient was 6.0 to 6.5, while the Golgi-rich fraction had peaks at pH 6.0 to 6.5 and pH 7.5. It is hypothesized that the peak at pH 6.0 to 6.5 for the Golgi-rich fraction is due to PM-contamination, while the peak at pH 7.5 represents the activity of a Golgi ATPase. To reduce PM contamination, Golgi-rich fractions obtained from step or rate-zonal gradients were recentrifuged isopycnically on linear sucrose gradients. The distribution of KCl, Mg²⁺-ATPase activity was measured at pH 6.5 and 7.5. The pH 6.5 ATPase was coincident with UDPG-sterol glucosyl transferase, a PM marker, while the pH 7.5 ATPase overlapped with latent UDPase, a Golgi marker. These results provide strong evidence for a KCl, Mg²⁺-ATPase, active at pH 7.5, associated with the Golgi membranes of corn coleoptiles.     

Subcellular distribution of 2′,3′-cyclic nucleotide-3′-phosphodiesterase in cat peripheral nerve

Subcellular distribution of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) in desheathed, saline perfused cat sciatic nerve is reported. CNPase specific activity was enriched in the total particulate (P₂) fraction and was low in the soluble (S₂) fraction. “Light-myelin” floating above the 0.60 M sucrose phase had the highest CNPase activity, 2.5-fold over the crude homogenate (CH). By contrast, enzyme activity in “heavy myelin” floating above the 0.85 M sucrose interface was equal to that of the CH and accounted for only 12% of total activity. CNPase activity in the membranes floating above the 0.25 and 0.60 and 0.85 M sucrose phases comprised nearly 70% of the total CNPase activity. The “light myelin” fraction floating above the 0.60 M sucrose accounted for approx. 51% of the total CNPase activity. SDS-PAGE of membranes individually harvested from above the 0.25 and 0.60 and 0.85 M sucrose phases revealed the presence of myelin-specific proteins (P₀, P₁; and P₂). Electron microscope examination demonstrated the presence of myelin in each membrane fraction. The results of this study show that the majority of CNPase activity is associated with “light myelin” in cat peripheral nerve.     

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.     

Nucleases of cell fractions of rat brain tissues

Nucleases are found in different fractions of nerve cells in the rat brain. The nuclease denaturating preferably the one-chain DNA at pH 8.0 is located chiefly in the neuron nuclei. Fractions of cell nuclei containg mainly neuron nuclei or glial nuclei were obtained by the method of ultracentrifugation within the sucrose gradient.     

Isolation of transverse tubules by fractionation of sarcoplasmic reticulum preparations in ion-free sucrose density gradients

A new method for the preparation of transverse tubules (T-tubules) from rabbit skeletal muscles is reported. When crude sarcoplasmic reticulum (SR) preparations were centrifuged on sucrose density gradients containing buffering ions (buffered gradients) 70-80% of the material sedimented as a single heavy band in the region of 43% sucrose. When this fraction (or crude SR) was recentrifuged on sucrose gradients prepared free of buffer or other ions (ion-free gradients) the heavy band dissociated into three fractions of different densities. The lightest fraction sedimented at 28% sucrose and was identified as T-tubules on the basis of its nitrendipine and ouabain binding properties. The enzymatic properties, cholesterol contents, and protein compositions of the fractions obtained when SR is centrifuged on buffered and ion-free sucrose density gradients were measured. The T-tubules were enriched in cholesterol and in marker enzymes for surface membranes while the other fractions were shown to be terminal cisternae and longitudinal cisternae on the basis of their (Ca2+,Mg2+)-ATPase activities and characteristic protein profiles.     

A method for rapid isolation of rough and smooth microsomes and golgi apparatus from rat liver in the same sucrose gradient

A technique is presented for the rapid isolation, by centrifugation in the same sucrose gradient, of rough and smooth surfaced microsomes and a Golgi apparatus enriched fraction from rat liver. Ethanol treatment is not necessary. The purity and recovery of the isolated fractions, as judged from determinations of marker enzymes and morphological analyses, are similar to previous data obtained with more complicated procedures. The method is based on adding a 10000 g supernatant to a discontinuous sucrose gradient ranging from bottom to top (1.30 M–15 mM CsCl, 1.15 M–15 mM CsCl, 10000 g supernatant in 1.05 M–10 mM CsCl, 1.05 M sucrose and 0.3 M sucrose). The Golgi apparatus enriched fraction bands at the border line of 0.3/1.05 M sucrose. The smooth microsomes band at the bondary of 1.15/1.30 M sucrose and finally the rough microsomes are collected at the bottom of the gradient. All fractions are obtained within h.     

COMPARATIVE STUDIES ON MITOCHONDRIA ISOLATED FROM NEURON-ENRICHED AND GLIA-ENRICHED FRACTIONS OF RABBIT AND BEEF BRAIN

Fractions enriched in neuronal and glial cells were obtained from dispersions of whole beef brain and rabbit cerebral cortex by large-scale density gradient centrifugation procedures. The fractions were characterized by appropriate microscopic observation. Mitochondria were then isolated from these fractions by differential centrifugation of their homogenates. The two different types of mitochondria were characterized with respect to certain enzyme activities, respiratory rate, rate of protein synthesis, and their buoyant density in sucrose gradients. The mitochondria from the neuron-enriched fraction were distinguished by a higher rate of incorporation of amino acids into protein, higher cytochrome oxidase activity, and a higher buoyant density in sucrose density gradients. Mitochondria from the glia-enriched fraction showed relatively high monoamine oxidase and Na⁺- and K⁺-stimulated ATPase activities. The rates of oxidation of various substrates and the acceptor control ratios did not differ appreciably between the two types of mitochondria. The difference in the buoyant density of mitochondria isolated from the neuron-enriched and glia-enriched cell fractions was utilized in attempts to separate neuronal and glial mitochondria from the mixed mitochondria obtained from whole brain homogenates in shallow sucrose gradients. The appearance of two peaks of cytochrome oxidase, monoamine oxidase, and protein concentration in such gradients shows the potential feasibility of such an approach.     

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.     

Immunochemical studies on the putative plasmalemmal receptor for 1,25-dihydroxyvitamin D3 II. Chick kidney and brain.

Chick kidney and brain were analyzed for the subcellular distribution (if any) of a putative plasma membrane receptor for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Fractionation protocols were found to be based not only on differential centrifugation conditions, but also gentleness of resuspension procedures, and sufficiently dense Percoll gradients. The postnuclear pellets were resolved on 21.85% Percoll gradients overlayed on 2.4 M sucrose cushions. For both kidney and brain, fraction 1 (bottom of tube) was found to be enriched over whole homogenate 5.4- and 1.6-fold, respectively, in acid phosphatase activity, fractions 2 through 5 were enriched four- and eightfold, respectively, in succinate dehydrogenase activity, fraction 8 contained Golgi, as judged by a small peak of alpha-mannosidase activity, and fraction 9 was enriched sevenfold (for each tissue) in Na+,K+-ATPase activity. Western analyses, using a characterized antibody to the putative chick intestinal plasma membrane vitamin D receptor, revealed the highest levels of antigenicity in both chick kidney and brain in plasma membrane and Golgi fractions, followed by unidentified membranes in fractions 6 and 7 of Percoll gradients. Distribution of specific binding of [3H]1,25(OH)2D3 in Percoll gradient fractions paralleled that of antigenicity. Qualitatively, kidney plasma membrane contained more antigen than brain plasma membrane after Western blot analyses; these results were mirrored by differences in specific binding of the tritiated secosteroid (65 +/- 14.5 and 34 +/- 11.9 fmol/mg of protein, respectively).     

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.