Hydrolytic enzymes in the central vacuole of plant cells

The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells, of tulip petals, and of pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast. (a) Purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation. (b) Hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation. (c) Vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated and had to be taken into account when the distribution of enzymes and of radioactivity was calculated.     

Subterminal polygalacturonase, a nonmacerating enzyme, attacks pectate from the reducing end

Nicotine was shown to be associated with mature vacuoles isolated from protoplasts of Nicotiana rustica. The vacuolar preparations also contained high levels of acid phosphatase, ATPase, and approximately 30% of the soluble protoplastic protein. The contamination of the vacuolar isolate by chlorophyll, succinate dehydrogenase, and NADPH cytochrome c reductase (markers for chloroplasts, mitochondria, and endoplasmic reticulum) was low. The enzymic activity associated with the vacuoles was not due to the exogenously supplied digestive enzymes used in the preparation of the protoplast. The relatively easy isolation of tobacco vacuoles makes this an excellent system for biochemical investigations of the vacuole.     

Intracellular Localization of Peptide Hydrolases in Wheat (Triticum aestivum L.) Leaves

Protoplasts from 8- to 9-day-old wheat (Triticum aestivum L.) leaves were used to isolate organelles which were examined for their contents of peptide hydrolase enzymes and, in the case of vacuoles, other acid hydrolases. High yields of intact chloroplasts were obtained using both equilibrium density gradient centrifugation and velocity sedimentation centrifugation on sucrose-sorbitol gradients. Aminopeptidase activity was found to be distributed, in approximately equal proportions, between the chloroplasts and cytoplasm. Leucyltyrosine dipeptidase was mainly found in the cytoplasm, although about 27% was associated with the chloroplasts. Vacuoles shown to be free from Cellulysin contamination contained all of the protoplast carboxypeptidase and hemoglobin-degrading activities. The acid hydrolases, phosphodiesterase, acid phosphatase, α-mannosidase, and β-N-acetylglucosamidase were found in the vacuole to varying degrees, but no β-glucosidase was localized in the vacuole.     

Identification of the leaf vacuole as a major nitrate storage pool

Highly purified vacuoles were isolated from protoplasts derived from green barley (Hordeum vulgare var. Numar) leaves, in order to determine their role as a NO₃⁻ storage sink. α-Mannosidase and acid phosphatase activities were used as markers to identify vacuoles, α-mannosidase being the more suitable. Nitrate and α-mannosidase, which were released from vacuoles destroyed during lysis of protoplasts, moved at unequal rates in the density gradient used for vacuole isolation. Purified vacuoles retained less NO₃⁻ than α-mannosidase during a single washing. Empirically determined corrections were used to account for NO₃⁻ movement in estimating the percentage of total cellular nitrate found in the vacuole. Vacuoles from plants grown in the presence of NO₃⁻ contained 58% of the total cellular NO₃⁻ and therefore represent a major NO₃⁻ storage pool.     

Determination of malate levels during the swelling of vacuoles isolated from guard-cell protoplasts

A method for the preparation of vacuoles from guard cells ofVicia faba L. is described. Vacuoles were released from guard-cell protoplasts by osmotic shock and purified on a Ficoll gradient. Contamination of the vacuoles was examined by assaying marker enzymes, such as fumarase, glucose-6-phosphate dehydrogenase, phosphofructokinase, acid phosphatase and mannosidase. Potassium ions in the incubation medium caused increases in the volume of the vacuoles by a factor of about 2.6, while the malate level remained unchanged. In contrast, malate synthesis was stimulated during the swelling phase when complete guard-cell protoplasts were exposed to K⁺. The possible role of K⁺ as an efficient osmotic effector is discussed.Abbreviations DEAE diethylaminoethyl - GCP guard-cell protoplast(s) - GCV guard-cell vacuoles(s) - MCP mesophyll cell protoplast(s) - MCV mesophyll cell vacuole(s)     

Vacuoles from Sugarcane Suspension Cultures : I. ISOLATION AND PARTIAL CHARACTERIZATION

Vacuoles were isolated from suspension cultures of sugarcane (Saccharum sp.) cells by centrifugation of protoplasts at high g force against a 12% (w/v) Ficoll solution. Distribution of marker enzymes and Concanavalin A binding showed an 11% contamination of the vacuole preparation by cytoplasmic components, mitochondria, and endoplasmic reticulum, and 18% contamination by plasma membrane. Acid phosphatase, carboxypeptidase, protease, peroxidase, and ribonuclease activities were enriched in isolated vacuoles. Carboxypeptidase was tonoplast-bound, whereas the other enzymes were soluble. Sucrose, reducing sugars, and free amino acids were measured in protoplasts and vacuoles during growth of cells in suspension culture. Sucrose and reducing sugar content of vacuoles increased as the culture aged, while free amino acids decreased sharply.     

Intracellular Localization of the Neurotoxin 2,4-Diaminobutyric Acid in Lathyrus sylvestris L. Leaf Tissue

The intracellular distribution of the neurotoxin 2,4-diaminobutyric acid (DABA) in mature leaves of the perennial legume Lathyrus sylvestris L. var `Lathco' (flatpea) was determined using subcellular fractions from mesophyll protoplasts. Chloroplasts contained about 15% of the cellular DABA. At least 75% of the DABA was vacuolar, based on the assumptions that each protoplast contained a single vacuole and that acid phosphatase occurred exclusively in the vacuole. DABA was not detectable in peroxisomal and mitochondrial fractions. Because the vacuole is not a major site of amino acid synthesis, this distribution implicates synthesis of DABA within chloroplasts with subsequent transport to and storage within the vacuoles of the mesophyll cells.     

Enzymic and protein character of tonoplast from hippeastrum vacuoles

The membrane of anthocyanin containing Hippeatrum petal vacuoles was examined for protein and enzyme content after purification by equilibrium density centrifugation. Light scattering, protein, and a Mg²⁺-dependent nucleotide specific ATPase were associated with membrane having a density of 1.08 to 1.12 grams per cubic centimeter. A small amount of acid phosphatase was also present in this region of the gradient, but this activity peaked at about 1.12 grams per cubic centimeter. A component of yeast tonoplast, α-mannosidase, was not significantly present. UDP-glucose, anthocyanidin-3-O-glucosyltransferase, thought to be a cytosol enzyme in Hippeastrum, was absent from tonoplast of vacuoles isolated by osmotic shock in 0.2 molar K₂HPO₄ or 0.35 molar mannitol. Vacuolar acid phosphatase was insensitive to ethylenediaminetetraacetate but was 80% inhibited by 10 millimolar KF, while ATPase was inactivated by 2 millimolar ethylenediaminetetraacetate and only 50% inhibited by 10 millimolar KF. Five major and about 9 minor polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane protein on 5 to 30 and 6 to 16% gradient gels.     

Sucrose phosphatase associated with vacuole preparations from red beet, sugar beet, and immature sugarcane stem

The specific phosphatase, sucrose phosphate phosphohydrolase (sucrose phosphatase, EC 3.1.3.24) was present in vacuole preparations from storage tissue of red beet (Beta vulgaris L.), sugar beet (Beta vulgaris L. cultivar Kawemono), and immature sugarcane (Saccharum spp. hybrid, cultivar NCO 310). In red beet vacuole preparations the specific activity of sucrose phosphatase, using the naturally occurring vacuole marker, betanin, as reference, was higher than the specific activity of cytoplasmic markers, phosphoenolpyruvate carboxylase and glucose 6-phosphate dehydrogenase, suggesting that sucrose phosphatase is associated with the vacuoles. High speed centrifugation of lysed vacuoles did not result in precipitation of the enzyme indicating that the enzyme is not tightly bound to the tonoplast. Sucrose phosphatase was more sensitive to inhibition by sodium vanadate and less sensitive to ammonium molybdate than was the nonspecific phosphatase which was also present in the extracts. Sucrose phosphatase might be part of the group translocator proposed recently to operate in the tonoplast of sugarcane and red beet.     

Isolation of protoplasts and vacuoles from cell suspension cultures of Coleus blumei Benth.

In order to study the accumulation and transport of rosmarinic acid in suspension cells of Coleus blumei we established an efficient method to isolate protoplasts and vacuoles. Protoplasts were disrupted by an osmotic shock in a medium with basic pH containing ethylenediamine tetraacetic acid. The resulting vacuoles were purified on a two-step Ficoll gradient. The comparison of the rosmarinic acid contents of cells, protoplasts and vacuoles showed that the depside is localized in the vacuole. Data concerning the yield and purity of the vacuoles are presented. In addition we show that at the physiological pH of the cytoplasm rosmarinic acid is present almost exclusively as an anion and cannot pass a membrane by simple diffusion. We therefore propose a carrier system for the transport of rosmarinic acid into the vacuole.Abbreviations EDTA ethylenediamine tetraacetic acid - HEPES 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethane sulfonic acid - HPLC high performance liquid chromatography - MES morpholinoethane sulfonic acid - NADP⁺ ß-nicotinamide adenine dinucleotide phosphate - PEG polyethylene glycol - RA rosmarinic acid - Tris Tris(hydroxymethyl)aminomethane     

Ultrastructure and cytochemical localization of acid phosphatase of laticifers in Euphorbia kansui Liou

Acid phosphatase (AcPase) activities are involved in the degeneration process of cytoplasm in plants. In this study, acid phosphatase was detected by the method of lead nitrate and cytochemical electron microscopy during the development of nonarticulated laticifers in Euphorbia kansui Liou. The most important feature in the differentiation of the laticifers in E. kansui is that the development of small vacuoles arises from endoplasmic reticulum (ER). The mature laticifers possess a thin layer of electron-dense peripheral cytoplasm in which the organelle cannot be distinguished and a large central vacuole filled with latex particles. AcPase cytochemistry studies show AcPase reaction products congregated into heaps are distributed along the tonoplast of central vacuole and around the mitochondria and plastids. Some small vacuoles which develop at later developmental stages of laticifers contain AcPase reaction products. As a result, the central vacuole is formed by cellular autophagy and fusion of small vacuoles which apparently arises from ER.     

Distribution of Sorbitol, Neutral Sugars, Free Amino Acids, Malic Acid and Some Hydrolytic Enzymes in Vacuoles of Apple Cotyledons

Vacuoles of apple cotyledons (Malus pumila Mill. var. domesticaSchneid.) were obtained by purification with Ficoll densitygradient centrifugation after the protoplasts were lysed byboth osmotic shock and the addition of EDTA. High levels ofacid protease and carboxypeptidase activity were detected inthe vacuoles along with acid phosphatase, phosphodiesteraseand ATPase. The distribution of sorbitol and other sugars inthe vacuoles, the protoplast and extracellular free space wasdetermined. About 45, 60 and 90% of the sorbitol, glucose andsucrose, respectively, contained in whole tissue were foundin the extracellular free space, and 54% of the sorbitol inthe protoplast was detected in the vacuole. The sorbitol inthe vacuoles and the extravacuole of the protoplast was about80 and 70%, respectively, of the total sugar content of thecell. Arginine was the most abundant free amino acid in theprotoplast, and about 90% of it was contained in the vacuole.More than 80% of the total amino acids and 50% of the proteinwere also located in the vacuole, as well as most of the malate.The amounts of the total sugars, total amino acids, proteinand malate in the vacuoles were to 250, 400, 48 and 9 µg/10⁶cells, respectively. The results suggest that the vacuoles ofapple cotyledons contain a large pool of amino acids and proteinsrather than sugars, and have a close connection with proteinbody degradation. ¹ This paper is contribution A-l37 of the Fruit Tree ResearchStation. (Received January 20, 1982; Accepted May 18, 1982)     

Acid hydrolases and their Release in Food Vacuole-Less Mutants of Tetrahymena thermophila*

SYNOPSIS. Mutants (NP1 and PSJ5) of Tetrahymena thermophila strains B and D 1968 exist that are unable to construct a functional oral apparatus and form food vacuoles at 37 C but which do so normally at 30 C. Food vacuole-less cells starved in dilute salt solution released similar amounts of acid phosphatase, β-N-acetyl-glucosaminidase and ±-glucosidase activity into the medium as wildtype cells during an 8-h period. Actively growing, food vacuole-less cells had ?50% less total protein, acid phosphatase, β-N-acetyl-glucosamin-idase, and ±-glucosidase per cell than wildtype cells after 72-h growth. During this time food vacuole-less cells released significant amounts of the 3 acid hydrolases into the growth medium. For each hydrolase, the total activity released from growing, food vacuole-less cells was less, on a per cell basis, than the amount released from food vacuole formers. The proportion of the total activity secreted by the mutant and the wildtype cells was the same for acid phosphatase and β-N-acetyl-glucosaminidase and somewhat lower for ±-glucosidase. It is concluded that the release of a significant amount of acid hydrolase activity from Tetrahymena is independent of food vacuole formation and may be analogous to the secretory activity of other nonphagocytic eukaryotic cells.     

Isolation and characterization of autophagic vacuoles from rat kidney cortex

The number of autophagic vacuoles in the proximal tubule cells of the rat kidney increased considerably after 3 h of vinblastine treatment. This increase was paralleled by stimulated proteolysis in an homogenate prepared from the cortex. We have taken advantage of this expansion in autophagic vacuoles in an effort to isolate these organelles from rat kidney cortex on a discontinuous Metrizamide gradient. Autophagic vacuoles have recently been purified from liver but not from other tissues. The purity of the isolated fraction was 95% of which 55% consisted of typical intact autophagic vacuoles containing sequestered organelles and 45% of other types of secondary lysosome. On plane section many of these displayed one or several intramatrical vesicles or flap like processes forming apparent vesicles at the pole of the organelles, which occasionally contained pinocytosed membranous material. These lysosomes were designated microautophagic vacuoles. It is suggested that the microautophagic vacuoles could be the morphological expression of uptake into lysosomes of small portions of cytosol. The isolated autophagic vacuole fraction was enriched in lysosomal enzymes (acid phosphatase and cathepsin D activities) and displayed high proteolytic rates, especially at acid pH.     

Presence of the cyanogenic glucoside dhurrin in isolated vacuoles from sorghum

Large numbers of vacuoles (10⁶-10⁷) have been isolated from Sorghum bicolor protoplasts and analyzed for the cyanogenic glucoside dhurrin. Leaves from light-grown seedlings were incubated for 4 hours in 1.5% cellulysin and 0.5% macerase to yield mesophyll protoplasts which then were recovered by centrifugation, quantitated by a hemocytometer, and assayed for cyanogenic glucosides. Mature vacuoles, released from the protoplasts by osmotic shock, were purified on a discontinuous Ficoll gradient and monitored for intactness by their ability to maintain a slightly acid interior while suspended in an alkaline buffer as indicated by neutral red stain. Cyanide analysis of the protoplasts and the vacuoles obtained there from yielded equivalent values of 11 μmoles of cyanogenic glucoside dhurrin per 10⁷ protoplasts or 10⁷ vacuoles. This work supports an earlier study from this laboratory which demonstrated that the vacuole is the site of accumulation of the cyanogenic glucoside in Sorghum.     

Association of calmodulin and an unconventional myosin with the contractile vacuole complex of Dictyostelium discoideum

mAbs specific for calmodulin were used to examine the distribution of calmodulin in vegetative Dictyostelium cells. Indirect immunofluorescence indicated that calmodulin was greatly enriched at the periphery of phase lucent vacuoles. The presence of these vacuoles in newly germinated (non-feeding) as well as growing cells, and the response of the vacuoles to changes in the osmotic environment, identified them as contractile vacuoles, osmoregulatory organelles. No evidence was found for an association of calmodulin with endosomes or lysosomes, nor was calmodulin enriched along cytoskeletal filaments. When membranes from Dictyostelium cells were fractionated on equilibrium sucrose density gradients, calmodulin cofractionated with alkaline phosphatase, a cytochemical marker for contractile vacuole membranes, at a density of 1.156 g/ml. Several high molecular weight calmodulin-binding proteins were enriched in the same region of the gradient. One of the calmodulin-binding polypeptides (molecular mass approximately 150 kD) cross-reacted with an antiserum specific for Acanthamoeba myosin IC. By indirect immunofluorescence, this protein was also enriched on contractile vacuole membranes. These results suggest that a calmodulin-binding unconventional myosin is associated with contractile vacuoles in Dictyostelium; similar proteins in yeast and mammalian cells have been implicated in vesicle movement.     

Transport of p-aminohippuric acid,uric acid and glucose in highly purified rabbit renal brush border membranes

A procedure for preparing highly purified brush border membranes from rabbit kidney cortex using differential and density gradient centrifugation is described. Brush border membranes prepared by this procedure were substantially free of basal-lateral membranes, mitochondria, endoplasmic reticulum and nuclear material as evidenced by an enrichment factor of less than 0.3 for (Na⁺ + K⁺)-ATPase, succinate dehydrogenase, NADPH-cytochrome c reductase and DNA. Alkaline phosphatase was enriched ten fold indicating that the membranes were enriched at least 30 fold with respect to other cellular organelles. The yield of brush border membranes was 20%.Transport of d-glucose by the membranes was identical to that previously reported except that the Arrhenius plot for temperature dependence of transport was curvilinear (E_A = 11.3–37.6 kcal/mol) rather than biphasic. Transport of p-aminohippuric acid and uric acid were increased by the presence of NaCl, either gradient or preequilibrated. However, no overshoot was obtained in the presence of a NaCl gradient, and KCl and LiCl also produced equivalent stimulation of transport suggesting a nonspecific ionic strength effect. Uptakes of p-aminohippuric acid and uric acid were not saturable, and were increased markedly by reducing the pH from 7.5 to 5.6. Probenecid (1 mM) reduced p-aminohippuric acid and uric acid (50 μM) uptake by 49% and 21%, respectively. We conclude that the uptake of uric acid and p-aminohippuric acid by renal brush border membranes of the rabbit occurs primarily by a simple solubility-diffusion mechanism.     

Isolation and characterization of plasma membrane from lactating bovine mammary gland

Plasma membranes were isolated from lactating bovine mammary gland. Two crude membrane fractions; medium/d 1.033 (light membrane) and 1.033/1.053 interfaces (heavy membrane), were obtained by Ficoll density gradient centrifugation of osmotically washed microsomal fraction. Two crude membranes were further purified separately by sucrose density gradient centrifugation. Both light and heavy membranes banded at a sucrose density of 1.14. The purified membranes appeared as heterogeneous smooth membrane vesicles on electron microscopy. The contaminating suborganelles were not detected. The yield of the purified membranes relative to the homogenate was 1.2%. The degree of purity of the membranes was shown by a great increase in the specific activity of 5′-nucleotidase over the homogenate of 20-fold for light membrane and of 16-fold for heavy membrane. The relative activities of Mg²⁺-ATPase, (Na⁺ + K⁺)-ATPase, γ-glutamyl transpeptidase, phosphodiesterase I, akaline phosphatase and xanthine oxidase were also high (12–18-times) and nearly 20% of these enzymes was recovered. The activity of marker enzyme for mitochondria, endoplasmic reticulum and Golgi apparatus was very low, while that of acid phosphatase for lysosome was relatively high (5-times). DNA and RNA contents were very low. The major polypeptides rich in other suborganelles were not detected profoundly in the membrane fraction and the polypeptide compositions in both light and heavy membranes were similar upon SDS-polyacrylamide gel electrophoresis.     

Isolation of Vacuoles from Root Storage Tissue of Beta vulgaris L

Morphologically intact and osmotically active vacuoles were isolated from root storage tissue of the red beet Beta vulgaris L., and the factors influencing both yield and stability of the vacuoles were determined. Successful isolation depended upon slicing the tissue in an apparatus specifically designed to cut open plant cells without the use of high shear forces and to liberate cellular organelles into an undisturbed reservoir of osmoticum. The resulting brei was centrifuged at 2,000g for 10 min to yield a pellet which contained many vacuoles but which also contained tissue fragments, nuclei, mitochondria, and plastids. The vacuoles were further purified by accelerated flotation through a Metrizamide step gradient. Biochemical assays, light microscopy, and electron microscopy confirmed that there was only trace contamination of the final vacuole preparation by other organelles. Isolated vacuoles were intact and retained their in vivo coloration.     

Vacuolar Localization of Endoproteinases EP(1) and EP(2) in Barley Mesophyll Cells

The localization of two previously characterized endoproteinases (EP₁ and EP₂) that comprise more than 95% of the protease activity in primary Hordeum vulgare L. var Numar leaves was determined. Intact vacuoles released from washed mesophyll protoplasts by gentle osmotic shock and increase in pH, were purified by flotation through a four-step Ficoll gradient. These vacuoles contained endoproteinases that rapidly degraded purified barley ribulose-1,5-bisphosphate carboxylase (RuBPCase) substrate. Breakdown products and extent of digestion of RuBPCase were determined using 12% polyacrylamide-sodium dodecyl sulfate gels. Coomassie brilliant blue- or silver-stained gels were scanned, and the peaks were integrated to provide quantitative information. The characteristics of the vacuolar endoproteinases (e.g. sensitivity to various inhibitors and activators, and the molecular weights of the breakdown products, i.e. peptide maps) closely resembled those of purified EP₁ and partially purified EP₂. It is therefore concluded that EP₁ and EP₂ are localized in the vacuoles of mesophyll cells.