Chitin synthetase activity is bound to the plasma membrane and to a cytoplasmic particulate fraction in Candida albicans germ tube cells

Abstract Subcellular distribution of chitin synthetase has been studied in germ tubes of Candida albicans . Two fractions with synthetase activity were separated from cell homogenates: (i) a mixed membrane fraction where the enzyme, partly in an active form, is associated with the plasma membrane (isopycnic centrifugation of mixed membrane fraction on linear sucrose gradients resolved a unique peak of activity matching with [³H]ConA-labelled membranes at a buoyant density of 1.195 g/ml); and (ii) a cytoplasmic fraction containing fully zymogenic enzyme associated with particles whose buoyant density (determined by isopycnic centrifugation on linear sucrose gradients) depended on the cell breakage conditions. The actual cytoplasmic fraction-enzyme may correspond to particles with buoyant density 1.135 g/ml (chitosomes), whereas the enzyme particles with other densities (1.085 and 1.165 g/ml) probably originated during cell disruption, as has been reported previously to occur during the preparation of yeast cell homogenates.     

Evidence that Blastocladiella emersonii zoospore chitin synthetase is located at the plasma membrane

Blastocladiella emersonii zoospores are not encased by a cell wall and do not detectably synthesize or contain chitin; accompanying de novo cell wall formation during zoospore encystment, chitin rapidly accumulates and is incorporated into the cell wall. Essential for understanding this abrupt change in chitin synthesis is the location of zoospore chitin synthetase. The enzyme has previously been reported to the sequestered with distinctive cytoplasmic organelles (gamma particles) characteristic for the zoospore cell type. Using similar differential and equilibrium density centrifugation procedures to those reported previously, we have observed the vast majority of zoospore homogenate chitin synthetase activity in fractions distinct from the gamma particle-enriched fractions. Over 90% of the homogenate enzyme activity could be recovered in a sucrose buoyant density region (1.14–1.18 g/ml) containing membranous elements and well separated from the region enriched for gamma particles (1.30–1.34 g/ml). When zoospores were surface-labelled with [³H]concanavalin A prior to homogenization, the buoyant density regions of radioactivity and of chitin synthetase activity exhibited nearly complete coincidence. At least the bulk of zoospore chitin synthetase appears to be located at the plasma membrane, rather than in gamma particles.     

Trypanosoma cruzi: subcellular distribution of glycolytic and some related enzymes of epimastigotes

The first six glycolytic enzymes in epimastigote Trypanosoma cruzi were shown to behave similarly during differential centrifugation, when maximum relative specific activity was found in the small granule fraction, and by isopycnic centrifugation, when the bulk of each activity coequilibrated on sucrose gradients with a modal density of 1.23 g/ml. All six showed substantial detergent latency in whole cell homogenates. Electron microscopic examination of fractions from a sucrose gradient with modal density 1.23 g/ml showed the presence of single membrane bound vesicles of diameter 0.2-0.8 micron. It was concluded that these six enzymes were contained in a microbodylike organelle, termed the "glycosome." Phosphoglucose isomerase (EC 5.3.1.9) also possessed substantial soluble activity. No microbody marker enzyme described in other sources could be detected. Peroxidase (EC 1.11.1.7) had an insignificant glycosomal component. Enzymes of amino acid and fatty acid metabolism were not detected in microbody fractions. Marker enzymes for the flagellar pocket and plasma membrane were suggested.     

beta-Glucosidase Activity in Corn Roots: Problems in Subcellular Fractionation

Preliminary results from differential centrifugation experiments, washing treatments, and enrichment in linear sucrose gradients at a density of 1.09 grams per cubic centimeter all indicated that β-glucosidase activity in corn root homogenates was associated with a membrane such as tonoplast. A subsequent sucrose density gradient centrifugation time course showed that the β-glucosidase was actually a soluble enzyme which moved into the gradients. The problem of soluble enzymes contaminating light density membranes in sucrose gradients and the question of centrifugation time necessary for membrane vesicles to reach isopycnic conditions are addressed.     

Mass isolation of cell surface membrane fragments from pigeon heart.

Cell surface membrane fragments were isolated and purified by successive rate zonal and isopycnic centrifugation of calcium oxalate-loaded pigeon heart microsomes in sucrose density gradients. The most highly purified cell membrane fraction sediments at a buoyant density of 1.105 g/ml. Some of the membrane pieces are present as open fragments and leaky vesicles, while others form tightly sealed vesicles of both inside-in and inside-out membrane orientation. The pigeon heart cell membrane preparation exhibits high (Na+ + K+ + Mg2+)-ATPase and adenylate cyclase activities. Additional activity of these enzymes is uncovered by sodium dodecyl sulfate and alamethicin, respectively. Electron microscopic inspection of the cell surface membrane preparation revealed (a) a predominance of thick-walled vesicles with smooth surfaces on negative staining and (b) binding of concanavalin A to the bulk of isolated membrane pieces following their incubation with the lectin.     

Chitin synthetase activity is bound to chitosomes and to the plasma membrane in protoplasts of Saccharomyces cerevisiae

The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacuolar proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40-110 nm; buoyant density (d) = 1.146 g/cm3). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density (d = 1.203 g/cm3) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with 3H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100-250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae.     

Nachweis und Lokalisation von Tryptophan-Synthetase in Samen von Juglans regia L.

Summary Cell-free extracts from seeds of Juglans regia synthesize tryptophan from L-serine and indole. Tryptophan synthetase has maximal activity in the range between pH 7 and 8. The enzyme is associated with a particulate fraction (density 1,210 g/ml) which is separated from the mitochondria (density 1,191 g/ml) after isopycnic density centrifugation on a continuous sucrose gradient.     

Sedimentation properties of chitosomes fromMucor rouxii

Summary This study was undertaken to assess the distribution and localization of chitin synthetase in a fungal cell and to evaluate the sedimentation behavior of chitosomes (microvesicular containers of chitin synthetase). Chitosomes were isolated from cell-free extracts of yeast cells ofMucor rouxii by rate-zonal and isopycnic sedimentation in sucrose density gradients. Because of their small size and low density, chitosomes were effectively separated from other subcellular particles. Rate-zonal sedimentation was a suitable final step for isolating chitosomes as long as ribosomes had been eliminated by enzymic digestion. By isopycnic centrifugation, chitosomes could be separated directly from a crude cell-free extract; they cosedimented with a sharp symmetrical peak of chitin synthetase at a buoyant density of d=1.14–1.15g/cm³; the only significant contaminants were particles of fatty acid synthetase complex. From such sedimentations, we estimated that 80–85% of the chitin synthetase activity in the cell-free extract was associated with chitosomes; the rest was found in two smaller peaks sedimenting at d=1.19–1.20 and d=1.21–1.22 (5–10%), and in the cell wall fraction (5–10%). By consecutive rate-zonal and isopycnic sedimentations, chitosome preparations with relatively few contaminating particles were obtained. Potassium/sodium phosphate buffer (pH 6.5)+MgCl₂ was the most effective isolation medium for chitosomes. Other buffers such as TRIS-MES+MgCl₂ led to massive aggregation of chitosomes and a change in sedimentation properties. This tendency of chitosomes to aggregate could explain why most of the chitin synthetase activity of a fungus is sometimes found associated with other subcellular structures,e.g., plasma membrane.     

Chitosome-like vesicles from gamma particles of Blastocladiella emersonii synthesize chitin

When gamma particles isolated from the aquatic fungus, Blastocladiella emersonii, were incubated in a supernatant derived from a homogenate of zoospores previously triggered to encyst with 50 mM KCl, they exhibited a three-fold increase in chitin synthetase activity and produced chitosome-like vesicles. Passage of such vesicles through a linear sucrose gradient resulted in a symmetrical distribution of chitin synthetase activity over a broad portion of the gradient, and the specific activity of the peak fraction was seven times greater than that of the gamma particles. After isopycnic sucrose gradient centrifugation, chitin synthetase activity occurred in a band of particles with a peak buoyant density of 1.18 g/cm³. Ultrastructural examination of negatively stained samples from the peak fraction revealed spheroidal, chitosome-like particles 70–120 nm in diameter. Suspension of these particles produced chitin microfibrils when incubated with uridine diphosphate N-acetylglucosamine, the substrate for chitin synthetase.Non Standard Abbreviations Used GlcNAc N-acetylglucosamine - UDP-GlcNAc uridine diphosphate N-acetylglucosamine - PYG agar 1.25 g of peptone, 1.25 g of yeast extract, 3 g of glucose, and 20 of agar per 1000 ml of water, the pH being adjusted to 6.8 with KOH after autoclaving - EGTA ethyleneglycol-bis (-aminoethylether)-N,N-tetraacetic acid     

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.     

Purification of cytoplasmic membranes and outer membranes from Proteus mirabilis

Summary A crude cell envelope suspension has been prepared from Proteus mirabilis after osmotic shock of penicillin-induced spheroplasts. Employing discontinuous sucrose gradients this cell envelope suspension can be fractionated into four fractions. Besides a pellet of remaining spheroplasts and an intermediate fraction with mixed composition a highly purified cytoplasmic membrane fraction and an outer membrane fraction have been obtained. The cytoplasmic membrane fraction is not contaminated with mucopeptide or outer membrane material. It has a buoyant density of 1.13 g/ml and a protein content of 38%. The specific activities of formate dehydrogenase and nitrate reductase and the content of cytochrome b₁ have increased sixfold in comparison with the crude cell envelope suspension. The outer membrane fraction contains only few contaminations with cytoplasmic membrane components and with mucopeptide.The gradient fractions have been characterized by electron microscopy and by polyacrylamide gel electrophoresis.     

An efficient preparative isopycnic flotation method for the isolation of chitosomes

Summary Chitin synthetase, a key enzyme in fungal cell wall biosynthesis, is located in chitosomes (microvesicles). To produce large quantities of chitosomes for immunochemical and biochemical characterization, we developed a two-step purification procedure in which isopycnic sucrose density gradients were centrifuged at ultra-high gravitational forces (fixed-angle rotor at 361,000×g R_av). Chitosomes from yeast cells ofMucor rouxii were separated from the soluble proteins and from the larger membranes by isopycnic centrifugation of the cell-free extract. The resulting crude chitosome sample was adjusted to a higher sucrose concentration, and a sucrose gradient was layered over the sample. Upon recentrifugation, the chitosomes moved up into the gradient and equilibrated at their buoyant density (1.15–1.16). This accelerated flotation separated contaminating particles of higher buoyant density (larger vesicles, ribosomes, and other miniorganelles) and yielded a large population of microvesicles with a mean diameter of 48.9±13.8 nm. This preparation contained vesicles essentially free of other particulate contaminants; more than 99% of the vesicles were smaller than 100 nm. When required, an additional velocity centrifugation step was added to remove the larger vesicles from the chitosome samples. This streamlined method for chitosome isolation was much simpler and faster than earlier isolation procedures, gave a high yield of functional chitosomes, and made the large scale isolation of these organelles possible.     

Utilization of enzyme markers to determine the location of plasma membrane from Pisum epicotyls on sucrose gradients

Using linear sucrose gradients, particulates derived from pea (Pisum sativum L. cv. Alaska) epicotyls have been fractionated and examined for marker enzyme activity. The coincidence of three reputed plasma-membrane markers [cellulase (EC 3.2.1.4), K⁺-stimulated Mg²⁺-ATPase, and glucan synthetase] at the same position on sucrose density gradients, in combination with electron microscopic evidence reported by G. Shore and G. Maclachlan (J. Cell Biol. 64, 557–571; 1975), indicates that plasma membrane of pea epicotyl has a buoyant density of about 1.13 g/cm³. This density disagrees with those usually reported for plant plasma membranes and also with recent reports for Pisum. It is, however, shown to be distinct from the equilibrium densities of enzymic markers for particulate components derived from Pisum endoplasmic reticulum (1.10–1.11 g/cm³), Golgi (1.12 g/cm³) and mitochondria (1.18 g/cm³). Furthermore, other recent literature indicates that the 1.13 g/cm³ buoyant density may be characteristic of the plasma membrane of many members of the Leguminosae. Our data indicate that the conditions of differential centrifugation (time, centrifugal force), coupled with the amount of protein utilized, affect the resolution and interpretation of profiles of marker enzymes on sucrose gradients (e.g. glucan synthetase and K⁺-stimulated Mg²⁺-ATPase were sometimes found to be associated not only with particles of 1.13 g/cm³ density, but with particles of higher densities as well). Particulate cellulase was found to be associated only with particles with equilibrium densities of about 1.13 g/cm³. Cellulase thus proved to be the most useful marker for establishing a differential centrifugation regime which would permit examination of the 1.13 g/cm³ particulate components with minimal contamination by particles of higher densities.     

Isolation and characterization of the Neurospora crassa endoplasmic reticulum

The endoplasmic reticulum from Neurospora crassa was identified by monitoring the activity of the putative enzyme marker phosphatidylcholine glyceride transferase. After differential centrifugation of a cell homogenate, phosphatidylcholine glyceride transferase activity initially copurified with plasma membrane H+-ATPase. However, isopycnic centrifugation of the whole-cell homogenate on a linear sucrose gradient separated the two enzyme activities into different fractions. The lighter membrane fraction exhibited characteristics that have been associated with the endoplasmic reticulum in other organisms: (i) the inclusion of magnesium caused this light membrane fraction to shift to a higher density on the gradient; (ii) it was highly enriched in cytochrome c reductase, an endoplasmic reticulum marker in other systems; and (iii) the morphology of the light fraction with and without added magnesium was clearly distinguishable from that of the plasma membrane fraction by electron microscopy. A reinvestigation of the location of chitin synthetase confirmed its association with the plasma membrane fraction even after separation of the lighter fractions.     

Chitin synthetase activity is bound to chitosomes anf to the plasma membrane in protoplasts of Saccharomyces cerevisiae

The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacoular proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40–110 nm; bouyant density (d) = 1.146 g/cm³). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density (d = 1.203 g/cm³) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with ³H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100–250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae.     

Analytical fractionation of cultured hepatoma cells (HTC cells)

Homogenates of HTC cells have been fractionated by differential centrifugation (in four particulate fractions: N, M, L, P, and a supernatant S) or isopycnic banding in linear sucrose gradients. On this basis, the following subcellular organelles may be characterized: (i) Mitochondria, detected by cytochrome oxidase and succinodehydrogenase, are collected in the M and L fractions, and equilibrate, as a narrow band, at a median buoyant density of 1.18 g/cm3. (ii) Lysosomes, detected by the latent hydrolases beta-glycerophosphatase and N-acetyl-beta-glucosaminidase, are largely sedimented in the M and L fractions, and display a broad density distribution pattern with a median value of 1.17 g/cm3. This density is decreased or increased after cultivation of the cells in presence of Triton WR-1339 or Dextran 500, respectively. The behavior of cathepsin D is somewhat at variance with that of the two other hydrolases. (iii) Plasma membrane is tentatively detected by alkaline phosphodiesterase I. Largely recovered in the P fraction, this enzyme equilibrates at a median density close to that of the lysosomal hydrolases; the bulk of cholesterol and about half of the leucyl-2-naphthylamidase are closely associated with alkaline phosphodiesterase I; HTC cells do not contain typical 5'-nucleotidase. (iv) Catalase-bearing particles, of high buoyant density (1.22 g/cm3) are present, but 30-40% of the catalase is also found readily soluble. NADPH- and NADH: cytochrome c reductase, and RNA show more complex distributions. It is suggested that the former enzyme is associated with the endoplasmic reticulum; as in liver, NADH reductase activity is shared between the endoplasmic reticulum and the mitochondria; half of the RNA is associated with free ribosomes of polysomes. True glucose-6-phosphatase could not be detected.     

Chitosomes from the wall-less “slime” mutant of Neurospora crassa

Cell-free extracts from the wall-less slime mutant of Neurospora crassa and the mycelium of wild type exhibit similar chitin synthetase properties in specific activity, zymogenicity and a preferential intracellular localization of chitosomes. The yield of chitosomal chitin synthetase from sline cells was essentially the same irrespective of cell breakage procedure (osmotic lysis or ballistic disruption) —an indication that chitosomes are not fragments of larger membranes produced by harsh (ballistic) disruption procedures. The plasma membrane fraction, isolated from slime cells treated with concanavalin A, contained only a minute portion of the total chitin synthetase of the fungus. Most of the activity was in the cytoplasmic fraction; isopycnic sedimentation of this fraction on a sucrose gradient yielded a sharp band of chitosomes with a buoyant density=1.125 g/ cm³. Approximately 76% of the total chitin synthetase activity of the slime mutant was recovered in the chitosome band. Because of their low density, chitosomes could be cleanly separated from the rest of the membranous organelles of the fungus. Apparently, the lack of a cell wall in the slime mutant is not due to the absence of either chitosomes or zymogenic chitin synthetase.Abbreviations Con A concanavalin A - d buoyant density in g/cm³ - GlcNAc N-acetyl-D-glucosamine - MES 2-[N-morpholino]ethanesulfonic acid - UDP-GlcNAc uridine diphosphate N-acetyl-D-glucosamine     

SEPARATION OF CATECHOLAMINE STORING SYNAPTOSOMES IN COLLOIDAL SILICA DENSITY GRADIENTS

Abstract— Catecholamine storing particles mainly from rat brain hypothalamus and corpus striatum have been isolated by isopycnic centrifugation in density gradients made of colloidal silica. As markers, tritium-labelled noradrenaline, endogenous noradrenaline and dopamine were measured. Cytochrome oxidase was determined as an indicator of mitochondria.
Two distinct populations of amine containing particles were recognized with densities of 1 , 03–1.04 g/ml and 1 , 045–1.065 g/ml in continuous isotonic gradients made of silica sol and a polymer. The light fraction was assumed to contain myelin fragments, light synaptosomes and possibly also catecholamine storage vesicles, while the other one was probably a heavy population of synaptosomes containing more mitochondria. Free mitochondria were found in a band at a density of 1 , 09–1.11.
The distribution pattern in isotonic gradients was compared with that in density gradients made of silica sol and sucrose or sucrose alone. The heavy population of the catecholamine particles was found to have a higher density in hypertonic gradients. Furthermore these synaptosomes seemed to lose more mitochondria and catecholamines than those in isotonic gradients probably due to the hypertonicity.
The present results confirm similar findings by other workers separating brain sub- cellular particles in isotonic gradients of Ficoll and sucrose.
Colloidal silica solutions might be of value for analytical centrifugation of brain sub-cellular particles, since it has a lower tonicity than sucrose, lower viscosity than Ficoll and furthermore it is very easy to handle. The silica sol is inexpensive and allows large scale work.     

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.     

Cytoplasmic and outer membranes separation in Rhodopseudomonas sphaeroides

A cell envelope fraction has been prepared after mechanical disruption of lysozyme-EDTA spheroplasts from depigmented Rhodopseudomonas sphaeroides (aerobically grown in the light). On linear sucrose gradients this fraction can be separated in a cytoplasmic membrane fraction and an outer membrane fraction. The cytoplasmic fraction (buoyant density: 1.18 g/cm³) has been characterized by its succinic dehydrogenase activity and by its composition. The outer membrane fraction (buoyant density: 1.21 g/cm³) does not contain any respiratory activity nor hemoproteins. The same fractionation has been done on cells repigmented in the dark by lowering the O₂ pressure. In that case the same two fractions have been detected in addition to the chromatophore fraction (buoyant density: 1.14 g/cm³). However both, and specially the outer membrane fraction, were contaminated by chromatophore material.