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.     

Electrochemical sterilization of bacteria adsorbed on granular activated carbon

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.     

Development of Microbodies in Sunflower Cotyledons and Castor Bean Endosperm during Germination

In cotyledons of sunflower seedlings glyoxysomal and peroxisomal enzymes exhibit different rates of development during germination. The total activity of isocitrate lyase, a glyoxysomal marker enzyme, rapidly increased during the first 3 days, and then decreased 89% by day 9. Exposure to light accelerated this decrease only slightly. The specific activity of glyoxysomal enzymes (malate synthetase, isocitrate lyase, citrate synthetase, and aconitase) in the microbody fraction from sucrose density gradients increased between days 2 and 4 about 2- to 3-fold, and thereafter it remained about constant in light or darkness.     

The isolation of endosome-derived vesicles from rat hepatocytes.

Intracellular 5'-nucleotidase involved in membrane circulation in rat hepatocytes is latent, and is protected from inhibition when whole cells are incubated with inhibiting antiserum at 2 degrees C [Stanley, Edwards & Luzio (1980) Biochem. J. 186, 59-69]. These two criteria were used to identify intracellular membrane vesicles containing 5'-nucleotidase on Ficoll density gradients. A sharply defined turbid band containing intracellular 5'-nucleotidase isolated on density gradients was further fractionated by immunoadsorption of plasma-membrane fragments derived from the cell surface of surface-inhibited cells on to an anti-(immunoglobulin G) immunoadsorbent. The resulting non-adsorbed membrane fraction consisted of vesicles of uniform size (approx. 65 nm diam.), but was not identifiable as any known organelle. This fraction could account for approx. 5% of the total cell 5'-nucleotidase activity, and the enzyme activity measured was 55% latent. The fraction had a restricted polypeptide composition but similar phospholipid composition compared with plasma membrane. We suggest that the vesicles observed in this fraction were derived from the endocytic pathway.     

Mitochondrial and Peroxisomal β-Oxidation of Stearic and Lignoceric Acids by Rat Brain

Crude subcellular fractions were prepared from adult rat brains by differential centrifugation of brain homogenates. Greater than 98% of the cellular mitochondrial marker enzyme activity sedimented in the heavy and light mitochondrial pellets, and less than 1% of the activity sedimented in microsomal pellets. Lysosomal marker enzyme activities mainly (71-78% of cellular activity) sedimented in the heavy and light mitochondrial pellets. Significant amounts of the lysosomal marker enzyme activity also sedimented in the crude microsomal pellets (9-13% of total) and high-speed supernatants (14-16% of total). The specific activities of microsomal and peroxisomal marker enzyme activities were highest in the crude microsomal pellets. Fractionation of the crude microsomal pellets on Nycodenz gradients resulted in the separation of the bulk of the remaining mitochondrial, lysosomal, and microsomal enzyme activities from peroxisomes. Fatty acyl-CoA synthetase activities separated on Nycodenz gradients as two distinct peaks, and the minor peak of the activities was in the peroxisomal enriched fraction. Fatty acid beta-oxidation activities also separated as two distinct peaks, and the activities were highest in the peroxisomal enriched fractions. Mitochondria were purified from the heavy mitochondrial pellets by Percoll density gradients. Fatty acyl-CoA synthetase and fatty acid beta-oxidation activities were present in both the purified mitochondrial and peroxisomal enriched fractions. Stearoyl-CoA synthetase activities were severalfold greater compared to lignoceroyl-CoA synthetase, and stearic acid beta-oxidation was severalfold greater compared to lignoceric acid beta-oxidation in purified mitochondrial and peroxisomal enriched fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

(2'-5') oligoadenylate synthetase in chicken embryo erythrocytes and immature red blood cells

The appearance and induction of (2'-5')oligoadenylate synthetase (2-5A synthetase) in chicken embryo erythrocytes during development, and the activity and molecular size of this enzyme in immature red blood cells from anemic chickens were studied. Enzyme activity first appeared in the embryos on the 15th day of incubation, a marked increase being seen 1 or 2 days after hatching. In erythrocytes from early embryos without 2-5A synthetase activity, chicken interferon (5 IU/ml at most) induced the production of a large amount of the enzyme. In immature red blood cells from anemic chickens, only a small amount of 2-5A synthetase was detected in the nuclear fraction. The cytoplasmic fraction contained the smaller enzyme (about 45 kilodaltons), but the larger enzyme (85-120 kilodaltons) was scarcely detected in either fraction. The larger enzyme may be synthesized during the maturation of red blood cells.     

Chitin synthesis in Candida albicans: comparison of yeast and hyphal forms.

Chitin synthesis was studied in both yeast and hyphae of the dimorphic fungus Candida albicans. Incorporation of N-acetyl-d-[1-(3)H]glucosamine ([(3)H]GluNAc) into an acid-alkali-insoluble fraction was 10 times greater in hyphal-phase cells. A crude preparation of chitin synthetase was obtained from sonically treated protoplasts of both forms of Candida. Enzyme activity, which was determined by using [(14)C]UDP-GLuNAc as a substrate, was exclusively associated with the 80,000 x g pellet from sonically treated protoplasts of both forms. It was determined that enzyme activity (nanomoles of [(14)C]UDP-GluNAc incorporated per milligram of protein) was approximately 2 times greater in hyphae versus yeast cells. Enzyme activity in both yeast and hyphae increased six- to sevenfold when the enzyme preparations were preincubated with trypsin. A vacuolar fraction, obtained from yeast cells but not from hyphae, stimulated enzyme activity when incubated with either yeast or hyphal enzyme preparations. Membrane fractions from protoplasts coated with [(3)H]concanavalin A before disruption were isolated by Renografin density gradient centrifugation. Chitin synthetase activity was preferentially associated with the concanavalin A-labeled fraction, suggesting that the enzyme was located on the plasma membrane. In addition, enzyme activity in protoplasts treated with cold glutaraldehyde before disruption was significantly greater than in protoplasts that were sonically disrupted and then treated with cold glutaraldehyde, indicating that the enzyme resides on the inner side of the plasma membrane.     

Increased synthesis of phosphatidylserine decarboxylase in a strain of Escherichia coli bearing a hybrid plasmid. Altered association of enzyme with the membrane.

A strain of Escherichia coli bearing a hybrid plasmid containing the psd gene, starved for isoleucine by the addition of valine, produces amounts of phosphatidyl-serine decarboxylase, a membrane-bound enzyme, about 40-fold higher than wild type. At least 98% of the enzyme from cells with high levels of decarboxylase is isolated in the inner, cytoplasmic membrane fraction if the cells are broken by osmotic lysis of spheroplasts following treatment with lysozyme/EDTA. In contrast, if cells containing these large amounts of enzyme are disrupted by sonication, 40 to 45% of the activity is recovered in the 100,000 times g supernatant fraction, whereas with wild type cells, only 5 to 10% is recovered in this fraction. About half of the decarboxylase in membranes saturated with the enzyme is thus only loosely bound, and readily removed by sonication, but not by osmotic lysis. This apparent saturation of the membrane with decarboxylase seems specific, since two other membrane-bound enzymes, phosphatidyl-glycerophosphate synthetase, and CDP-diglyceride synthetase, are not displaced into the supernatant fraction upon sonication. Fractionation on columns of agarose and by centrifugation through gradients of sucrose revealed that the decarboxylase in the supernatant is associated with lipid, in a complex with an apparent molecular weight of at least 5 times 10(6).     

Chitosomes and chitin synthetase in the asexual life cycle of Mucor rouxii: spores, mycelium and yeast cells

To help understand the subcellular machinery responsible for cell wall formation in a fungus, we determined the abundance and subcellular distribution of chitin synthetase (chitin synthase, EC 2.4.1.16) and chitosomes in the asexual life cycle of Mucor rouxii. Cell-free extracts of ungerminated sporangiospores, hyphae/mycelium in exponential and stationary phase, and yeast cells were fractionated by isopycnic centrifugation in sucrose density gradients. The total amount of chitin synthetase per cell increased exponentially during aerobic germination of spores. In all developmental stages, the profile of chitin synthetase activity encompassed a broad range of sucrose density (d = 1.12-1.22) with two distinct zones: a low-density chitosome zone (d = approx. 1.12-1.16) and a high-density, mixed-membrane zone (d = approx. 1.16-1.22). Chitosomes were a major reservoir of chitin synthetase in all stages of the life cycle, including ungerminated spores. Two kinds of chitin synthetase profiles were recognized and correlated with the growth state. In nongrowing cells (ungerminated sporangiospores and stationary-phase mycelium), the profile was skewed toward lower densities with a sharp chitosome peak at d = 1.12-1.13. In actively growing cultures (aerobic mycelium or anaerobic yeast cells), the entire profile of chitin synthetase was displaced toward higher densities; the average buoyant density of chitosomes was higher (d = 1.14-1.16), and more chitin synthetase was associated with denser (d = 1.16-1.23) membrane fractions. In all life cycle stages, chitosomal chitin synthetase was almost completely zymogenic. In contrast to the enzyme from spores or from growing cells, samples of chitosomal chitin synthetase from stationary-phase mycelium were unstable and contained a high proportion of larger vesicles in addition to the typical microvesicles. The presence of chitosomes in ungerminated spores indicates that these cells are poised to begin synthesizing somatic (= vegetative) cell walls at the onset of germination. The increased buoyant density of chitosomes in actively growing cultures suggests that the composition of these microvesicles changes significantly as they mobilize chitin synthetase to the cell surface.     

Localization of phospholipid biosynthetic enzyme activities in cell-free fractions derived from Rhodopseudomonas sphaeroides

The phospholipid biosynthetic enzyme activities: CDP-diglyceride synthetase, phosphatidylglycerophosphate synthetase, PGP phosphatase, phosphatidylserine (PS) synthase, PS decarboxylase, and S-adenosyl-L-methionine:phosphatidylethanolamine (AdoMet:PE) N-methyltransferase were detected in crude cell-free extracts of Rhodopseudomonas sphaeroides. CDP-diglyceride synthetase and phosphatidylglycerophosphate synthetase co-enriched with penicillin-binding protein activity, a known cytoplasmic membrane marker, throughout fractionation of cell-free extracts of both chemoheterotrophically and photoheterotrophically grown cells. PS decarboxylase also co-enriched with the cytoplasmic membranes in fractions derived from chemoheterotrophically and photoheterotrophically grown cells, but substantially greater quantities of PS decarboxylase activity was found in the chromatophores derived from photoheterotrophically grown cells than could be accounted for by cytoplasmic membrane contamination of this sample. PS synthase (60% of the recovered activity) and S-adenosyl-L-methionine:phosphatidylethanolamine N-methyltransferase (90% of the recovered activity) were found in the supernatant fraction after high speed centrifugation of crude cell lysates, suggesting that these enzyme activities were not tightly membrane associated. The localization of phospholipid biosynthetic enzyme activity in R. sphaeroides is discussed in terms of the biosynthesis of the photosynthetic membranes.     

Cellular Localization of Acetyl-Coenzyme A Synthetase in Yeast

In cells of Saccharomyces cerevisiae grown with glucose in standing cultures, the microsomal fraction had the highest specific activity for acetyl-coenzyme A synthetase and contained the greatest fraction of the total activity regardless of when the cells were harvested during growth. The addition of acetate did not affect the distribution of the enzyme, nor did subsequent aeration of such cells in phosphate buffer even in the presence of glucose, acetate, or succinate. In cells grown aerobically, however, the microsomal fraction had the highest specific activity and the greatest fraction of the total activity only until the cells reached the stationary phase. After this time, most of the activity was associated with the mitochondrial fraction. Finally, 3 or 4 days after inoculation, this fraction appeared to lose most of the enzyme to the microsomal and soluble fractions. Chloramphenicol, at concentrations that interfered with respiration but not with fermentation, prevented the association of acetyl-coenzyme A synthetase with the mitochondrial fraction in aerated cells, but it did not appreciably affect the large increases in enzyme activity observed during aerobic incubation. Cells grown with glucose under strict anaerobic conditions contained barely detectable amounts of acetyl-coenzyme A synthetase.     

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     

Complete purification and immunochemical analysis of S-adenosylmethionine synthetase from bovine brain

We have purified S-adenosylmethionine (AdoMet) synthetase about 3000-fold from bovine brain extract. The Km values of the enzyme for L-methionine and ATP were 10 and 50 microM, respectively. An apparent molecular mass of the enzyme was estimated to be 160 kDa by gel filtration on a Sephacryl S-200 column. Sucrose density gradient centrifugation gave a sedimentation coefficient of 8 S. Polyacrylamide gel electrophoresis of the purified enzyme in native system revealed a single protein band, whereas two polypeptide bands with molecular masses of 48 kDa (p48) and 38 kDa (p38) were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme. Antibody against bovine brain AdoMet synthetase was prepared by injecting the purified enzyme into a rabbit. Immunoblot analysis revealed that the antibody recognized both p48 and p38 in the impure enzyme preparations from bovine brain as well as in the purified enzyme. Specific antibodies against p48 and p38 were separated from the immunoglobulin fraction by an affinity purification, both of which inhibited the enzyme activity. These results indicate that AdoMet synthetase from bovine brain consists of two different polypeptides, p48 and p38.     

Assembly and activation of the NADPH:O2 oxidoreductase in human neutrophils after stimulation with phorbol myristate acetate

Phagocytic leukocytes contain an activatable NADPH:O2 oxidoreductase. Components of this enzyme system include cytochrome b558, and three soluble oxidase components (SOC I, SOC II, and SOC III) found in the cytosol of resting cells. Previously, we found that SOC II copurifies with, and is probably identical to, a 47-kDa substrate of protein kinase C. In the present study we investigated the change in location of several of these oxidase components after activation of intact neutrophils with phorbol myristate acetate (PMA) and separation of subcellular fraction on sucrose density gradients. On Western blots with fractions of resting cells, the alpha subunit of cytochrome b558 was detected with a monoclonal antibody as a doublet of Mr 22,000 and 24,000 in the specific granules and as a single band of Mr 24,000 in the plasma membrane. PMA induced an increase of cytochrome b558 in the plasma membrane, including the Mr 22,000 band. PMA also induced translocation of the 47-kDa protein from the cytosol to the membrane fraction, as revealed by in vitro phosphorylation experiments. When NADPH oxidase activity was determined in a cell-free system in the presence of sodium dodecyl sulfate and GTP with plasma membranes from resting cells, cytosol from PMA-treated cells was deficient compared with cytosol from resting cells. This deficiency could be partially restored by the addition of SOC I. Concomitantly, SOC I activity appeared in the plasma membranes of PMA-treated cells. These studies support the hypothesis that PMA stimulation of neutrophils results in assembly of oxidase components from the cytosol and the specific granules in the plasma membrane with subsequent expression of NADPH oxidase activity.     

The site of cellulose synthesis. Hormone treatment alters the intracellular location of alkali-insoluble beta-1,4-glucan (cellulose) synthetase activities

Membrane preparations from growing regions of 8-day old Pisum sativum epicotyls contain multiple beta-1,4-glucan (cellulose) synthetase activities (UDP- or GDP-glucose: beta-1,4-glucan-glucosyl transferase), and the levels of some of these are influenced by treatments with the growth hormone, indoleacetic acid (IAA). When membranes from control epicotyl segments (zero time) are fractionated by isopycnic sedimentation in sucrose density gradients, all of the synthetase activities are associated mainly with Golgi membrane (density 1.55 g/cm3). After decapitation and treatment of epicotyls with IAA, synthetases also appear in a smooth vesicle fraction (density 1.11 g/cm3) which is rich in endoplasmic reticulum (ER) marker enzyme. Major fractions of these synthetases are not recovered in association with plasma membrane or washed cell walls. When [14-C]sucrose is supplied in vivo to segments +/- IAA, radioactive cellulose is deposited only in the wall. Cellulose or cellodextrin precursors do not accumulate in those membranes in which synthetase activities are recovered in vitro. In experiments where tissue slices containing intact cells are supplied with [14C]sugar nucleotide in vitro, alkali-insoluble beta-1,4-glucan is synthesized (presumably outside the protoplast) at rates which greatly exceeded (20-30 times) those obtained using isolated membrane preparations. Progressive disruption of cell structure results in increasing losses of this high activity. These results are consistent with the interpretation that Golgi and ER-associated synthetases are not themselves loci for cellulose synthesis in vivo, but represent enzymes in transit to sites of action at the wall:protoplast omterface. There they operate only if integrity of cellular organization is maintained.     

Application of enzymehistochemical methods to isolated subcellular fractions and to sucrose-ficoll density gradients

Summary To compare histochemical and biochemical determinations of enzyme activities, enzymehistochemical procedures are applied to sections of pellets of subcellular fractions. These investigations are of value to determine the subcellular localization of histochemically demonstrable enzyme activities and to test the homogeneity of an isolated fraction. In homogenating duckling liver a great part of the endothelial cells is not destructed and consequently is found in the nuclear fraction. Kupffer cell lysosomes land in the heavy mitochondrial fraction, whereas hepatocyte lysosomes are chiefly found in the light mitochondrial fraction. -Glucuronidase activity shows a preferentially microsomal localization. Application of enzymehistochemical staining reactions to discontinuous gradients and comparison with biochemical data provides additional information about the validity of an enzymehistochemical reaction. In rat liver the tetrazolium reductases show a distinctly dual localization: activity in the mitochondrial band and in microsomal bands. As to their localization in different bands of the gradients non-specific esterases demonstrate a clear pH-dependency.     

Pyrimidine reducing enzymes of rat liver.

Dihydrouracil dehydrogenase (NADP+) (EC 1.3.1.2) was partially purified from the cytosol fraction of rat liver and fractionated by disc gel electrophoresis. A major and minor band were visualized by staining for enzyme activity. The substrate specificity of these bands was investigated. It was found that both bands were two to three times more active with dihydrothymine as substrate than with dihydrouracil in the presence of NADP+ and the optimum pH of 7.4. Mitochondrial fractions containing most of the NADH-dependent uracil reductase of rat liver cells were fractionated by centrifugation in sucrose density gradients. Two procedures involving linear or discontinuous gradients were used. By both, good separation of NADH- and NADPH- dependent reductases was achieved. Marker enzyme studies supported the view that the NADH-dependent enzyme is located principally in mitochondria whereas the NADPH-dependent enzyme is mainly in plasma and endoplasmic reticulum membranes. For the NADH-dependent reductase the apparent Km for thymine at pH 7.4 was 1.39 times that found for uracil whereas for the NADPH-dependent enzyme the apparent Km values were similar for the two substrates at this pH. Dihydrouracil was the principal product isolated by paper chromatography from the reaction mixture containing a partially purified fraction of mitochondria, uracil and NADH at pH 7.4. This fraction also catalyzed the formation of radioactive carbon dioxide from [2-14C]uracil. The proportion of CO2 formed by the mitochondria was about 10% of that formed by the original homogenate.     

Particulate forms of phenylalanyl-tRNA synthetase from Ehrlich ascites cells

Over 80% of the phenylalanyl-tRNA synthetase activity in Ehrlich ascites cell homogenates was found to be associated with the high speed particulate fraction. This enzyme activity occurred in two principle forms: activity bound to the ribosomes, and activity as part of a complex sedimenting at approximately 25S in a sucrose density gradient. The ribosome-associated enzyme was shown to be bound to the 60S ribosomal subunit. Exposure of the ribosomes to RNA resulted in removal of synthetase activity from the ribosomes and the concomitant appearance of activity in a complex sedimenting at 25S.     

Temporal appearance of bacteriophage T4-modified valyl tRNA synthetase in Escherichia coli.

Bacteriophage T4-induced modification of Escherichia coli vlayl-tRNA synthetase (EC 6.1.1.9) requires: synthesis of a phage-gene specified tau factor, addition of the factor to host valyl-tRNA synthetase to produce a urea-stable enzyme, and interaction of the modified enzyme with tRNA to produce a more rapidly sedimenting valyl-tRNA synthetase activity on sucrose density gradients. This report demonstrates that the coincident, chloramphenicol-sensitive appearance of urea-stable and rapidly sedimenting valyl-tRNA synthetase activity are immediate early phage functions. It implies that once the tau factor is synthesized, further interactions are stoichiometric rather than catalytic. The potential for valyl-tRNA synthetase modification accumylates when E. coli is infected with T4 PHAGE IN THE PRESENCE OF CHLORAMPHINICOL AND IS EXPRESSED DURING THE RESUMPTION OF PROTEIN SYNTHESIS WHEREAS FURTHER RNA synthesis is inhibited by rifampicin. The modification phenomenon occurs similarly in several strains of E. coli and represents a novel virus-host interaction.     

The biosynthesis of ribonuclease and its accumulation in protein bodies in the cotyledons of mung bean seedlings

Germination and seedling growth of mung bean are accompanied by a 7- to 10-fold increase in the ribonuclease content of the cotyledons. The increase occurs during the first 4 days of seedling growth and precedes the senescence of the cotyledons. Separation of the RNases in the cotyledons by polyacrylamide gel electrophoresis indicates the presence of several minor bands in seeds imbibed for 24 hr. On the second day of seedling growth a new major band with an R_f of 0.76 is present. In 4- to 5-day old seedlings this major band accounts for nearly all the RNase activity in the tissue. The characteristics of this RNase show that it is a plant ribonuclease I (pH optimum of 5.0; MW 16,000; activity preferentially inhibited by purine nucleotides; no activity toward DNA; no phosphodiesterase activity). When the seedlings are grown in 66% D₂O the RNase activity undergoes a density shift of 0.61% indicating that the increase in enzyme activity is due to the de novo synthesis of the enzyme molecules. A method is described for the isolation of protein bodies from protoplasts of storage parenchyma cells. Fractionation of protoplast lysates on Ficoll gradients results in the recovery of a high proportion (75%) of intact protein bodies. On these gradients RNase activity comigrates with α-mannosidase, a protein body marker enzyme indicating that the newly synthesized RNase accumulates in the protein bodies. We suggest that the synthesis of RNase in the cotyledons and its accumulation in the protein bodies indicates that protein bodies may function in the degradation of cellular macromolecules other than the reserves stored within them.