Isolation of organelles from carrot cell suspension cultures

Summary Organelles isolated from carrot cell suspension cultures by density gradient centrifugation and identified by their specific marker enzymes were found at the following mean densities on the sucrose gradient: microbodies 1.25 g/cm³ (catalase), mitochondria 1.18 (fumarase), endoplasmic reticulum 1.09 g/cm³ (NADH-cytochrome c reductase). Further enzyme assays were done for characterization of microbodies from carrot cultures.This work was supported by the Deutsche Forschungsgemeinschaft.     

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.     

Basal-lateral membranes from rabbit renal cortex prepared on a large scale in a zonal rotor

Basal-lateral membranes from the renal cortex of the rabbit were isolated by sucrose gradient centrifugation in a zonal rotor which allows for a large-scale preparation of these membranes. A heterogeneous population of membranes (P4) which contained 29% of the (Na+ + K+)-ATPase found in the homogenate of renal cortex was prepared by differential centrifugation. When pellet P4 was subjected to centrifugation in a sucrose gradient the activity of (Na+ + K+)-ATPase, a marker for basal-lateral membranes, could be separated from enzymatic markers of other organelles. The specific activity of (Na+ + K+)-ATPase was enriched 12-fold at a density of 1.141 g/cm3. Membranes (P alpha) contained in the (Na+ + K+)-ATPase-rich fractions consisted primarily of closed vesicles which exhibited probenecid inhibitable transport of rho-aminohippurate. These membranes did not exhibit Na+-dependent, phlorizin-inhibitable D-glucose transport. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins from P alpha revealed at least six major protein bands with molecular weights of 91000, 81000, 73000, 65000, 47000 and 38000. A small fraction of total alkaline phosphatase found in the homogenate was found in pellet P4. Membranes containing this alkaline phosphatase activity were distributed widely over the gradient, with peak activity found at a density of 1.141 g/cm3. In contrast, when brush borders were subjected to gradient centrifugation under the same conditions as P4, alkaline phosphatase was found in a narrow distribution, with peak activity at a density of 1.158 g/cm3. The principle subcellular localization of the alkaline phosphatase found in P4 could not be determined unambiguously from the data, but the activity did not seem to be primarily associated with classical brush borders.     

Isolation of microbodies from, sweet potato root tissue and their development during aging of slices

Microbodies were isolated from, sweet potato root tissue bydifferential and linear sucrose density gradient centrifugation.When the tissue was homogenized in the presence of PolyclarAT, the microbodies sedimented together with the mitochondriathrough the sucrose gradients. The microbodies had a densityof 1.25 g/cm³, and contained catalase and urate oxidase, butnot malate dehydrogenase, isocitrate lyase, glycolate oxidase,hydroxypyruvate reductase and the cyanide-insensitive palmitoylCoA-oxidation system. A small amount of o-diphenol oxidase alsoseemed to be present. Catalase, but not urate oxidase, activity in the crude extractincreased during aging of the sliced tissue. A similar resultwas obtained with the microbody fraction after linear sucrosedensity gradient centrifugation. We propose that microbodiescontaining only catalase develop during aging of sliced sweetpotato root tissue. ¹ This work was supported in part by a Grant-in-Aid (No. 311908)for Scientific Research from the Ministry of Education, Scienceand Culture, Japan. (Received June 20, 1979; )     

Subcellular localization of glyoxylate cycle enzymes in Ascaris suum larvae

Evidence is presented on the particulate nature of glyoxylate cycle enzymes in metazoa with the use of 15-day old larvae of the nematode Ascaris suum. Homogenization procedures were developed to disrupt the resistant nematode cuticle. Malate synthase and isocitrate lyase, key enzymes of the glyoxylate cycle, consistently sedimented with mitochondrial enzymes in differential pellets while catalase, a major peroxisomal enzyme, was always soluble. Isopycnic sucrose gradient centrifugation of the differential pellet yielded two protein peaks: one at 1.18 g/cm3 (characteristic for mitochondria), and another at 1.23 g/cm3 (common for glyoxysomes and peroxisomes). Electron microscopy of these fractions revealed that the lighter peak consisted primarily of mitochondria, while the heavier band contained proteinaceous bodies termed "dense granules" morphologically resembling microbodies. SIgnificantly, both malate synthase and isocitrate lyase cosedimented with the mitochondrial marker enzymes in the lighter peak (1.18 g/cm3) and not with the dense granules. Further purification of mitochondria, accomplished by separating dense granules with a step gradient before isopycnic centrifugation, substantiated the evidence that microbodies (glyoxysomes) do not occur in these nematode larvae. Rough-surfaced membranes were alternatively considered as the subcellular site, but the evidence tends to favor localization of the glyoxylate bypass enzymes in the mitochondria.     

Sub-cellular fractionation of Trypanosoma brucei. Isolation and characterization of plasma membranes

A procedure is described for the isolation of sub-cellular fractions from bloodstream forms of Trypanosoma brucei. The method leaves intact most of the nuclei, mitochondria and microbodies. All the fractions have been chemically characterized and tested for 10 enzymatic markers. About 5% of total cell protein was isolated as a microsomal fraction containing mostly plasma membranes and endoplasmic reticulum vesicles. Plasma membranes were purified by high-speed centrifugation on magnesium-containing Dextran, and on linear sucrose-density gradients. The yield of membranes was approximately 0.3% of the total cell protein. The purified material had a sucrose density of 1.14 g/cm3 and consisted of smooth vesicles. Specific activity of the membrane markers Na+, K+, ouabain-sensitive ATPase and adenylate cyclase were 26- and 20-fold higher, respectively, than in total cells. Neither DNA nor RNA was detected. The sum of the cholesterol and phospholipid content was 0.99 mg/mg protein. The cholesterol/phospholipid molar ratio was 1:2.     

Microbodies of the alga Chara

Chara fragilis possesses microbodies with a remarkably large size of up to 2 µm in diameter . Many of the organelles contain huge nucleoids of amorphous material or paracrystalline inclusions. After isolation of the organelles by gradient centrifugation the specific density of the microbodies was determined to be 1.25 g cm⁻³. Catalase, glycolate oxidase and hydroxypyruvate reductase as well as enzymes of the fatty acid β -oxidation pathway were demonstrated to be constituents of the microbodies in Chara indicating that they are similar to those in green leaves. The data obtained are in agreement with the view that the Charophyceae and especially the algae in the subgroup of Charales are very closely related to the land plants.     

Physical and Biological Properties of Dengue-2 Virus and Associated Antigens

Dengue virus suspensions from mouse brain and cell culture were fractionated into three components by rate zonal centrifugation in sucrose gradients. Infectious virus sedimented in a single zone and possessed hemagglutinating (HA) and complement fixing (CF) activity. Electron micrographs showed the virion to be a spherical particle 48 to 50 nm in diameter with 7-nm spherical structures on its surface. Buoyant density in CsCl of virions from mouse brain was estimated at 1.22 g/cm(3) and from cell culture at 1.24 g/cm(3). During centrifugation of virions in CsCl, an additional HA component appeared with a buoyant density of 1.18 g/cm(3). It was shown in electron micrographs to consist of virion fragments. A noninfectious component with HA and CF activity sedimented in sucrose more slowly than intact virus, had a buoyant density of 1.23 g/cm(3) in CsCl, and appeared as "doughnut" forms measuring 13.8 to 14 nm in diameter. A third component, with CF activity and no HA activity, sedimented very little in sucrose gradients. Particles of the same size and shape as the spherical subunits on the surface of the virion were observed in electron micrographs.     

Isolation and Characterization of Microbodies from the Alga Chlorogonium elongatum

The alga Chlorogonium elongatum was grown autotrophically or heterotrophically on acetate. Cells harvested in the logarithmic phase of growth were disrupted, and the whole homogenates were fractionated on sucrose gradients. Protein and enzyme determinations carried out on the fractions led to the following conclusions. Chloroplast fragments which represent the major portion of particulate protein in autotrophic cells migrate to density 1.17 g/cm³. In heterotrophic cells, mitochondria comprise most of the particulate protein, and these particles accumulate at density 1.19 g/cm³, as shown by a peak of cytochrome oxidase in this region. Part of the catalase and uricase, two marker enzymes for microbodies, were found in the soluble fractions, but 60% or more of these activities were recovered at density 1.225 g/cm³ from autotrophic cells. Electron micrographs showed that in this region there were microbodies with a diameter of 0.4 micrometer. The isolated microbodies contained no isocitrate lyase, a marker enzyme of glyoxysomes. This enzyme was completely soluble and therefore seems not to be associated with organelles in this organism.     

Sialic acid binding activity of transmissible gastroenteritis coronavirus affects sedimentation behavior of virions and solubilized glycoproteins

The sedimentation behavior of transmissible gastroenteritis coronavirus (TGEV) was analyzed. Upon sucrose gradient centrifugation, the major virus band was found at a density of 1.20 to 1.22 g/cm(3). This high density was observed only when TGEV with a functional sialic acid binding activity was analyzed. Mutants of TGEV that lacked sialic acid binding activity due to a point mutation in the sialic acid binding site of the S protein were mainly recovered at a lower-density position on the sucrose gradient (1.18 to 1.19 g/cm(3)). Neuraminidase treatment of purified virions resulted in a shift of the sedimentation value from the higher to the lower density. These results suggest that binding of sialoglycoproteins to the virion surface is responsible for the sedimentation behavior of TGEV. When purified virions were treated with octylglucoside to solubilize viral glycoproteins, ultracentrifugation resulted in sedimentation of the S protein of TGEV. However, when neuraminidase-treated virions or mutants with a defective sialic acid binding activity were analyzed, the S protein remained in the supernatant rather than in the pellet fraction. These results indicate that the interaction of the surface protein S with sialoglycoconjugates is maintained after solubilization of this viral glycoprotein by detergent treatment.     

Inducible beta-oxidation pathway in Neurospora crassa.

An inducible beta-oxidation system was demonstrated in a particulate fraction from Neurospora crassa. The activities of all individual beta-oxidation enzymes were enhanced in cells after a shift from a sucrose to an acetate medium. The induction was even more pronounced in transfer to a medium containing oleate as sole carbon and energy source. Since an acyl-coenzyme A (CoA) dehydrogenase was detected instead of acyl-CoA oxidase, the former enzyme seems to catalyze the first step of the beta-oxidation sequence in N. crassa. After isopycnic centrifugation in a linear sucrose gradient, the intracellular organelles housing the fatty acid degradation pathway cosedimented (1.21 g/cm3) with the glyoxylate bypass enzymes isocitrate lyase and malate synthase and were clearly resolved from both mitochondrial marker enzymes (1.19 g/cm3) and catalase (1.26 g/cm3). On the basis of biochemical as well as morphological properties, these particles from N. crassa have recently been designated as glyoxysome-like particles (G. Wanner and T. Theimer, Ann. N.Y. Acad. Sci. 386:269-284, 1982). The failure to detect catalase, urate oxidase, and acyl-CoA oxidase indicate that these glyoxysome-like microbodies in N. crassa lack peroxisomal function and thus are clearly different from the various microbodies reported so far to contain a beta-oxidation pathway.     

Fractionation of the enzymes of the barley aleurone layer: Evidence for a soluble mode of enzyme release

Summary Aleurone cells of barley (Hordeum vulgare L.) contain microbodies as determined by histochemical localization with diaminobenzidine. These microbodies can be isolated from both water and gibberellic acid (GA₃) treated cells and identified on sucrose density gradients as glyoxysomes on the basis of their buoyant densities (1.25 g cm^-3) and their enzyme complement. Fractionation of aleurone layer homogenates by differential centrifugation after varying times of exposure to GA₃, however, does not indicate the presence of a discrete secretory vesicle containing either -amylase or -1,3-glucanase. Cytological evidence also suggests that at least -1,3-glucanase is not released from these cells by means of a discrete secretory vesicle.Supported by National Science Foundation Grant GB-27468. The assistance of Dr. William Breidenbach in the assay of glyoxysomal enzymes is gratefully acknowledged.     

Isolation and characterization of hydrophobic proteins (H proteins) in the membrane fraction of Bacillus subtilis. Involvement in membrane biosynthesis and the formation of biochemically active membrane vesicles by combining H proteins with lipid.

Cytoplasmic membranes of Bacillus subtilis, grown in complex medium containing glucose, were fractionated into three membrane subfractions [light band (1.155 - 1.158 g/cm3); medium band (1.181 - 1.183 g/cm3); heavy band (1.21 - 1.25 g/cm3)] by sucrose density gradient centrifugation. Among these subfractions, the light and medium bands consisted mainly of membranes but the heavy band consisted of an irregular arrangement or aggregate of small globular protein components of 5 - 8 nm in diameter. We named this H-protein. H-protein formed trilamellar unit membrane structure when combined with lipid. In pulse-labeling and pulse-chase experiments with radioactive leucine, it was found that H-protein consisted of the newest membrane protein synthesized in the cells and the label incorporated into H-protein was shifted into light and medium band of the membranes during the chase. Cytochromes were not found in H-protein. However, when H-protein was incubated with haem alpha and protohaem, these compounds were incorporated into the apoproteins of the cytochromes present in H-protein and form cytochromes a and b. Cytochromes were also formed in H-protein which were isolated from the cells grown in the presence of haemin (haemin-grown H protein). Succinate dehydrogenase activity was increased about 4-fold by combining H-protein or haemin-grown H protein with lipid. H-protein had no cytochrome oxidase activity; however, haemin-grown H protein was found to have some of the activity and this was increased about 4-fold by combining the protein with lipid. Haemin-grown H protein was also found to form succinate: cytochrome c oxidoreductase when combined with lipid and vitamin K2. On the other hand, succinate oxidase was required for the addition of lipid, vitamin K2 and cytochrome c. NADH oxidase was also found in haemin-grown H protein and was activated about 9-fold in constituted reaction systems. Vesicles formed by haemin-grown H protein and lipid, could accumulate alanine and proline by addition of NADH or reduced phenazine methosulfate. Alanine and proline was also accumulated into the vesicles when transport energy was supplied as a membrane potential introduced by K+-diffusion via valinomycin. These results would indicate that H-protein contains the apoprotein of cytochromes, and a carrier involved in the active transport of alanine and proline.     

Use of Zonal Ultracentrifuge Systems for Biophysical Studies of Rhinoviruses

This paper reports the use of zonal ultracentrifuge techniques to conduct biophysical studies of rhinoviruses grown with WI-38 cells. Good clean-out of infectivity from rhinovirus harvests was obtained with the continuous-flow B-V and B-IX rotors. Use of the B-V rotor resulted in the successful concentration of rhinovirus infectivity and antigenicity. Additional purification was achieved by the combined use of continuous-flow centrifugation and isopycnic banding procedures. Two particle sizes were found to be associated with the virus-infected cell harvests. The infectious 22-nm particle banded in density ranges of 1.38 to 1.40 g/cm(3) in CsCl and 1.26 to 1.27 g/cm(3) in potassium citrate. The 8.0 nm capsomere was composed of 2.0 nm subunits and banded with a density of protein at 1.28 g/cm(3) in CsCl. Equivalent sedimentation coefficients of 155 or 185, depending on particle density in sucrose, were calculated from rate zonal experiments by use of the B-IV zonal rotor.     

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.     

Intracellular distribution of hydroperoxide isomerase

Differential centrifugation of several plant extracts indicates that the majority of the hydroperoxide isomerase activity is present in the cytoplasm of the cell. However, lesser amounts of isomerase activity were found in the mitochondrial and microsomal fractions of sunflower seedlings. Sucrose density gradient centrifugation of extracts from sunflower, watermelon, and flax seedlings and from cauliflower buds showed that isomerase activity was associated with the mitochondria. There was no evidence for presence of hydroperoxide isomerase activity in the microbodies.     

Purification of vacuoles from Neurospora crassa.

The Neurospora crassa vacuole, defined by its content of basic amino acids, polyphosphate, protease, phosphatases, and alpha-mannosidase, was purified to near homogeneity. The procedure depends upon homogenization of snail gut enzyme-digested cells in a buffer osmotically stabilized with 1 M sorbitol, differential centrifugation of the extract, and sucrose density gradient centrifugation of the organellar pellet. Isopycnic centrifugation of vacuoles in 2.25 M sorbitol-Metrizamide density gradients yielded a peak (density, 1.31 g/cm3) of vacuolar markers coincident with 32P-phospholipids, trichloroacetate-insoluble 14C, and trichloroacetate-soluble 14C. A trail of macromolecular markers in the lighter portions of the gradient reflected, at least in part, heterogeneity of the vacuoles. Almost no contamination by mitochondria or glyoxysomes was detected. Vacuoles were very heterogeneous in size as estimated by velocity sedimentation, but most were larger than mitochondria. Variations of the osmotic strength of the medium were found to alter the equilibrium density of vacuole preparations from 1.06 g/cm3 to over 1.3 g/cm3. This explains the great variation in density reported previously for the "vacuole," the "vesicle," and the "protease particle" of N. crassa, all of which appear to be the same entity.     

The control of fruiting body formation in the ascomycete Sordaria macrospora Auersw. by arginine and biotin: a two-factor analysis

Summary The distribution of glyoxylate-cycle enzymes between microbodies and mitochondria was examined in ethanol-grown Aspergillus tamarii Kita. Particulate activities of catalase and the two glyoxylate by-pass enzymes, malate synthase and isocitrate lyase, were localized in the microbodies. The microbodies had a buoyant density of about 1.23 g cm^-3 after isopycnic centrifugation in linear sucrose gradients. Particulate activities of the other two glyoxycitrate synthase, together with that of succinate dehydrogenase were restricted to the mitochondria, which had a buoyant density of about 1.20 g cm^-3. Catalase also appeared to be localized in a second particle, perhaps the microbody inclusions or the Woronin bodies, having a buoyant density of about 1.26 g cm^-3.     

Interactions between different corneal proteoglycans.

Proteoglycans were extracted from bovine cornea with 4M-guanidinium chloride and purified by CsCl-density-gradient centrifugation. Under associative conditions two fractions were found: one capable of forming assemblies of high molecular weight and another lacking this property. The heavier fraction (density 1.59 g/ml) was eluted as a single retarded peak from Sepharose 2B, but on DEAE-Sephadex chromatography, gave two peaks: the first (eluted with 0.75 M-NaCl) contained mainly proteochondroitin sulphate and the second (eluted with 1.25 M-NaCl) mainly proteokeratan sulphate. Each of these proteoglycans was more retarded on Sepharose 2B than was the original sample from density-gradient centrifugation. Re-aggregation was obtained by recombination of the two fractions. The lighter fraction (density 1.44 g/ml), containing predominantly keratan sulphate chains, was eluted from DEAE-Sephadex as a single peak with 1.25 M-NaCl and was retarded on Sepharose 2B: this fraction was not able to form aggregates with proteochondroitin sulphate. Chemical analyses of the carbohydrate and protein moieties of the proteoglycans from DEAE-Sephadex confirmed that, in the cornea, different subunits are present with characteristic aggregation properties and hydrodynamic volumes.