Purification and properties of acid phosphatase from cultured tobacco cells

One component of acid phosphatase was purified from cultured tobacco cells. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate. The enzyme possesses high activity toward nucleoside di- and triphosphate, much less activity toward nucleoside monophosphates and sugar esters. The MWs of the phosphatase determined by Sephadex G-100 gel filtration and dodecyl sulfate gel electrophoresis were 74000 and 76000, respectively. The phosphatase showed high affinity for concanavalin A-Sepharose and single superimposed bands of protein and carbohydrate on gel electrophoresis, suggesting that it is a glycoprotein.     

Properties of acid phosphatase in the cell wall of tobacco cells cultured in vitro

A cell-wall fraction containing 10.0% dry weight of proteinwas isolated from tobacco (Nicotiana tabacum L.) XD-6 cellscultured in suspension. The fraction possessed high acid phosphataseactivity toward p-nitrophenyl phosphate, phenyl phosphate, inorganicpyrophosphate, adenosine diphosphate, nucleoside triphosphates,and bis- (p-nitrophenyl) phosphate; much less activity towardnaphthyl phosphate, ß-glycerophosphate, glucose-6-phosphate,fructose-6-phosphate, fructose-l,6-diphosphate, thiamine pyrophosphate;and no activity toward glucose-1-phosphate and inositol hexaphosphate.Metallic ions were not required for activation. The enzyme wasextracted from the wall with NaCl solution. The solubilizedenzyme resulted in a single peak on Sephadex G-150 chromatographywith activity toward p-nitrophenyl phosphate, pyrophosphate,and bis(p-nitrophenyl) phosphate. The activity of the solubilizedenzyme toward p-nitrophenyl phosphate was identical with thebound enzyme in its pH optimum, substrate specificity, ion inhibitionand Km value, but it was more sensitive to pH, substrate difference,inhibition, and heat treatment. ¹Present address: Department of Biology, Japan Women's University,Mejiro-dai, Bunkyo-ku, Tokyo, Japan (Received September 13, 1972; )     

Control of phosphatase release from cultured tobacco cells

The activity of cultured tobacco XD-6 cells to release phosphataseinto the medium was enhanced after a time lag of 2 to 3 daysfollowing a marked increase in intracellular level of the enzymeduring Pi-omitted culture. The enhanced release of phosphatasehad a Q₁₀ value of about 2.3, and was suppressed by 2,4-dinitrophenoland azide. Cycloheximide did not inhibit the enzyme release,but Pi caused a rapid and drastic decrease in the rate of release.The release is suppressed even by 1 µM Pi. These results indicate that the rate of enzyme release is notdirectly limited by synthesis of the enzyme, but limited bythe transport, which may be suppressed by Pi, of the enzymeto the outside of the cell membrane. ¹ Present address: Laboratory of Applied Microbiology, Facultyof Agriculture, Yamagata University, Tsuruoka, Yamagata-ken997, Japan. (Received June 28, 1977; )     

Analysis of extracellular macromolecules of cultured tobacco cells in comparison with cell-wall macromolecules

Rapidly growing, cultured tobacco cells secreted pectic substances, glycoprotein, and 50% ethanol-soluble polysaccharide into the medium. The extracellular pectic substances lacked rhamnose which was present in the cell-wall pectic substances. The protein moieties of the extracellular and cell-wall glycoproteins were hydroxy. proline-rich and similar in amino-acid composition. The sugar moiety of the cell-wall glycoprotein was similar in monosaccharide composition to that of the combined extracellular glycoprotein plus the 50% ethanol soluble polysaccharide.     

Purple acid phosphatase in the walls of tobacco cells

Purple acid phosphatase isolated from the walls of tobacco cells appears to be a 220 kDa homotetramer composed of 60 kDa subunits, which is purple in color and which contains iron as its only metal ion. Although the phosphatase did not require dithiothreitol for activity and was not inhibited by phenylarsine oxide, the enzyme showed a higher catalytic efficiency (k_cat/K_m) for phosphotyrosine-containing peptides than for other substrates including p-nitrophenyl-phosphate and ATP. The phosphatase formed as a 120 kDa dimer in the cytoplasm and as a 220 kDa tetramer in the walls, where Brefeldin A blocked its secretion during wall regeneration. According to our double-immunofluorescence labeling results, the enzyme might be translocated through the Golgi apparatus to the walls at the interphase and to the cell plate during cytokinesis.     

Regulation of phosphatase synthesis by orthophosphate in cultured tobacco cells

Phosphatase activity in cultured tobacco cells XD-6 increasedremarkably under phosphate-deficient culture conditions. Suchan increase did not occur in the activities of -amylase, ß-galactosidase,succinate dehydrogenase and catalase under the same cultureconditions. By replenishment with Pi, the increase in totalphosphatase activity was suppressed and the specific activitywas reduced to a low level. The suppression of increase in theactivity resulted form a repression of de novo synthesis ofthe enzyme. Added Pi also suppressed the release of phosphataseinto the culture medium. The effect of Pi was found to be greaterthan the effect on the increase in the intracellular activity. At least three phosphatases were extracted from XD-6 cells.One of the enzymes increased when the phosphatase synthesiswas increased by phosphate deficiency. ¹ Present address: Laboratory of Applied Microbiology, Facultyof Agriculture, Yamagata University, Tsuruoka, Yamagata 997,Japan. (Received May 18, 1977; )     

Chromatographic separation, of extracellular acid phosphatase of tobacco cells cultured under Pi-supplied and omitted conditions

An extracellular acid phosphatase preparation of tobacco XD-6cells cultured in suspension was resolved into three fractionsby sequential chromatography. Two of these were neutral pyrophosphatasewith diesterase activity, having optimum pH at 6.8. The otheris a nonspecific acid phosphatase having optimum pH at 5.8.The latter was concluded to be involved in the increase in extracellularactivity upon Pi-depletion. (Received August 31, 1976; )     

Effects of divalent cations on phosphatase secretion in cultured tobacco cells

Some differences were found between Mg²⁺- and Ca²⁺-stimulated phosphatase secretion in cultured tobacco cells. The effect of Mg²⁺ ions was greater than that of Ca²⁺ ions, and Ca²⁺ ions at below 1 mM rather depressed the secretion. Upon the addition of Mg²⁺ ions plus Ca²⁺ ions, a synergistic stimulation of the secretion occurred. Different influences on the effects of Mg²⁺ and Ca²⁺ ions on the secretion were exerted by treating cells with metabolic inhibitors that reduced the level of cellular metabolic energy. Phosphate (Pi) and arsenate did not depress the secretion in the presence of Mg²⁺ ions, but did depress it in the presence of Ca²⁺ ions. These results strongly suggested that the secretion of phosphatase involved at least two different steps affected by divalent cations.     

Stimulation of phosphatase release from cultured tobacco cells by divalent cations

Upon addition of divalent cations to the incubation medium ofcultured tobacco cells, the release of phosphatase into themedium increased and the time course of the release became biphasic.A rapid release (phase I release) occurred instantaneously afterthe addition and then a release at a constant rate (phase IIrelease) followed. Sodium and potassium ions did not affectthe enzyme release. Lanthanum ions caused the biphasic enzymerelease but inhibited the phase II release. The effects of temperature and metabolic inhibitors indicatedthat phase I release was limited by a diffusion process butphase II release was limited by an enzymatic reaction requiringmetabolic energy. From the results it was concluded that divalent cations enhancedthe enzyme release not only by stimulating the transport ofenzyme to the outside of the cell membrane, but also by liberatingthe enzyme retained on the exterior of the cells, e.g., thecell walls. The released phosphatase could be separated into two fractions,F-I and F-II. Only F-I was released by phase I release, whileboth F-I and F-II resulted from phase II release. This indicatedthat F-I was preferentially trapped on the exterior of the cells. ¹ These experiments were carried out at the Department of Botanyin the Faculty of Science of the University of Tokyo. (Received December 15, 1978; )     

Biosynthesis of cell-wall polysaccharides in cultured carrot cells

Biosynthesis of the cell wall in carrot cells (Daucus carota L.) cultured in a synthetic liquid medium was studied by measuring the incorporation of radioactive glucose and myo-inositol (MI). When the cells were fed with [¹⁴C]glucose in the presence of 0.01% MI, the label soon appeared in the neutral sugars in the cell wall but little radioactivity was found in the uronic-acid residues even after a prolonged incubation. On the other hand, radioactivity derived from [³H]MI was found to be distributed among uronic acids and pentoses but not in the hexose residues in the wall. The data indicate that MI is an important intermediate for the synthesis of acidic sugars in the wall of cultured carrot cells.Abbreviation MI myo-inositol     

Ultrastructural localization of acid phosphatase in cultured cells ofDaucus carota

Summary Acid phosphatase localization has been studied, using the lead salt method, in suspension-cultured cells of the wild carrot. Enzyme activity in most of the cells was restricted to the walls and vacuoles. However, in some senescent cells activity was also seen in the nucleus, at one face of the dictyosomes, and in nearby dictyosome-derived vesciles.The activity in the walls was closely associated with the central portion of the wall which ultimately disintegrates in auxin-containing media. However, the large vesicles which accumulate in this portion of the wall as it breaks down never showed acid phosphatase activity, nor did the multivesicular bodies which appear to transfer vesicular material into the wall space. Although multivesicular bodies in plant cells resemble the multivesicular lysosomes of animal cells, no evidence could be obtained in this study for the presence in such bodies of hydrolytic enzymes.     

Fatty acid composition in native and cultured human endothelial cells

Endothelial cells from human umbilical veins were isolated by collagenase treatment. Cells were cultured in the presence of either 20% fetal bovine serum (FBS) or 20% human serum (HS). At confluency, endothelial cell lipids were labeled with tracer concentrations of tritiated arachidonic acid, then extracted and separated into lipid subclasses by thin layer chromatography. The fatty acid composition of each lipid class was determined by glass capillary gas-liquid chromatography analysis and compared to that of cells freshly isolated from the cord (NC cells). The fatty acid compositions differed only in phospholipids. Polyunsaturated fatty acids (PFAs), arachidonic, and linoleic acids were depleted in FBS cell phospholipids and replaced by both stearic and oleic acids. No significant difference could be observed between NC cell and HS cell phospholipids. We conclude that PFAs might be decreased in FBS cells because of the relative paucity of PFAs in FBS as compared to HS. It seems therefore more convenient to cultivate endothelial cells in the presence of HS, especially in respect to their phospholipid content of arachidonic acid, which is the physiological reservoir for prostacyclin synthesis.     

Fatty acid composition in native and cultured human endothelial cells

Summary Endothelial cells from human umbilical veins were isolated by collagenase treatment. Cells were cultured in the presence of either 20% fetal bovine serum (FBS) or 20% human serum (HS). At confluency, endothelial cell lipids were labeled with tracer concentrations of tritiated arachidonic acid, then extracted and separated into lipid subclasses by thin layer chromatography. The fatty acid composition of each lipid class was determined by glass capillary gas-liquid chromatography analysis and compared to that of cells freshly isolated from the cord (NC cells). The fatty acid compositions differed only in phospholipids. Polyunsaturated fatty acids (PFAs), arachidonic, and linoleic acids were depleted in FBS cell phospholipids and replaced by both stearic and oleic acids. No significant difference could be observed between NC cell and HS cell phospholipids. We conclude that PFAs might be decreased in FBS cells because of the relative paucity of PFAs in FBS as compared to HS. It seems therefore more convenient to cultivate endothelial cells in the presence of HS, especially in respect to their phospholipid content of arachidonic acid, which is the physiological reservoir for prostacyclin synthesis. This work was supported by a grant from the Délégation Générale à la Recherche Scientifique et Technique, Paris, France (79.7.0091).     

Biosynthesis and processing of lysosomal acid phosphatase in cultured human cells

The biosynthesis of lysosomal acid phosphatase was studied in a normal human embryonic lung cell line, WI-38. Cells were labeled with radioactive leucine under a variety of conditions, the enzyme was immunoprecipitated using a monospecific antiserum raised against human liver lysosomal acid phosphatase, and the products were separated by electrophoresis and were visualized by fluorography. Lysosomal acid phosphatase constitutes 60% of the total tartrate-inhibitable acid phosphatase in WI-38. It is initially synthesized as a high-molecular-weight precursor polypeptide of 69 kDa. The precursor polypeptide is rapidly glycosylated and processed to a mature enzyme of 53-45 kDa via intermediates of 65 and 60 kDa in WI-38 cells. The 69-kDa precursor polypeptide is also converted to larger precursor polypeptides of 74 and 80 kDa. The multiplicity of precursor polypeptides is due at least in part to differences in the glycosylation and phosphorylation of the polypeptides. Sensitivity of phosphorylated oligosaccharide chains from precursor, mature and small polypeptides to endo-beta-hexosaminidase H-catalyzed cleavage suggests the presence of high-mannose phosphorylated oligosaccharide chains similar to those present on many other lysosomal enzymes. The effects of tunicamycin and ammonium chloride were also studied. In contrast to the effect of ammonium chloride on arylsulfatase A secretion, the lysosomal acid phosphatase in WI-38 cells was not secreted in the presence of NH4Cl. This is consistent with the existence of an alternate route for the transfer of lysosomal acid phosphatase into lysosomes. This alternate route may be the reason that I-cell fibroblasts contain a normal level of lysosomal acid phosphatase.     

Cell-wall lysing enzymes and products of cell-wall digestion elicit ethylene in citrus

Ethylene production was induced in Valencia oranges [ Citrus sinensis (L.) Osbcck] by injection of the fungal enzyme mixture Pectolyase ( Aspergillus japonicus ) which contains pectolytic enzymes into the peel. The mixture also stimulated production of 1-aminocyclopropane-1-carboxylic acid (ACC). Cycloheximide partially inhibited the Pectolyase-induced ethylene response. Pectin fragments, resulting from partial acid hydrolysis or Pectolyase digestion, caused an increase in ethylene production when injected into the peel of intact orange fruits. Pectic fragments produced by fungal enzymes are known to be elicitors of phytoalexins and in this study are shown to elicit ethylene in citurs.     

The demonstration of acid phosphatase in cultured 3T3 mouse cells

Summary Acid phosphatase has been demonstrated ultrastructurally in 3T3 and SV40-3T3 mouse cells using sodium -glycerophosphate and p-nitrophenyl phosphate as substrate. The former substrate only demonstrates the enzyme in lysosomes and elements of the Golgi apparatus while the latter demonstrates it in the cisternae of the endoplasmic reticulum and in the cell surface as well as at lysosomal sites. The significance of surface acid phosphatase activity is discussed in terms of sublethal autolysis.     

Molecular characterization of a novel yeast cell-wall acid phosphatase cloned from Kluyveromyces marxianus

A novel Kluyveromyces marxianus gene that encodes an acid phosphatase, Pho610, was cloned in Saccharomyces cerevisiae. The deduced amino acid sequence was distinct from S. cerevisiae phosphatases but similar to some fungal enzymes. A peculiar feature of the sequence is that it has hydrophobic stretches both at the N- and C-termini, which is a characteristic of the precursors of glycosylphosphatidylinositol(GPI)-anchored proteins. When the gene was expressed in S. cerevisiae, the active enzyme was recovered in the periplasmic fraction by glucanase digestion. The Pho610 polypeptide was highly glycosylated and a significant portion was covalently linked to the cell-wall glucan. The enzyme was secreted when the C-terminal region was truncated to remove the GPI signal. Therefore, Pho610 is a novel cell-wall protein having an enzyme activity.     

Metabolic engineering of cultured tobacco cells

Construction of a gene expression system in tobacco cultured cells (BY2) was studied. A 925 bp promoter fragment of a heat-shock protein gene (HSP18.2) of Arabidopsis thaliana showed clear heat-shock response of expression of the beta-glucuronidase (GUS) reporter gene in BY2 cells. Similar results were observed in a 500 mL flask and 3-L jar fermentor. Isolation of strong promoters in BY2 cells was tried. cDNA clones, in which the mRNA level is high in log-phase cells and the copy number in the genome is low, were isolated. These clones showed high homology with F1-ATPase (mitochondria type), elongation factor 1-alpha, and a gene with an unknown function of A. thaliana (clone 27), respectively. A 5'-flanking region of clone 27 showed 6.2 times the promoter activity of the CaMV35S promoter in BY2 cells. Three cDNA clones, which are expressed in the stationary growth phase of BY2 cells, were isolated by a differential screening. These clones showed high sequence homologies to alcohol dehydrogenase, pectin esterase, and extensin. Promoters of these genes will be useful in gene expression in high cell-density culture.     

The demonstration of acid phosphatase in cultured 3T3 mouse cells.

Acid phosphatase has been demonstrated ultrastructurally in 3T3 and SV40-3T3 mouse cells using sodium beta-glycerophosphate and p-nitrophenyl phosphate as substrate. The former substrate only demonstrates the enzyme in lysosomes and elements of the Golgi apparatus while the latter demonstrates it in the cisternae of the endoplasmic reticulum and in the cell surface as well as at lysosomal sites. The significance of surface acid phosphatase activity is discussed in terms of sublethal autolysis.     

Fatty acid hydroperoxide lyase in tobacco cells cultured in vitro

Fatty acid hydroperoxide lyase (HPO lyase) was found in green and non-green tobacco cells cultured in vitro. The HPO lyase activity in non-green cells was $\text{1}\text{3}$-$\text{1}\text{2}$ of that in green cells. When the cells were transferred from the light to dark conditions or vice versa, cells turned non-green or green according to the light conditions. The HPO lyase activity also changed according to the light conditions, but the changes in HPO lyase activities were not proportional to the changes in chlorophyll contents. These results suggest that at least two types of HPO lyases are present in the green cells. One type of HPO lyase is perhaps common both to the green and non-green cells; another one is chloroplastic. The fatty acid compositions of cells and substrate specificities of HPO lyase differed between green and non-green cells.