Enzymatic Maceration of Plant Tissue

Tissue cells of Vicia faba were separated from the leaf pieces by the pectinase treatment after which the chlorophyll determination method was employed. The separation proceeded at a constant rate for 3 hours', the maximum rate of separation was at pH 5.3. The optimal pH shifted to 5.5 in the presence of 0.001 M 2Na-ethylenediaminetetraacetic acid (EDTA), which accelerated the separation at pH levels higher than 5.5. The separation was enhanced by NaCl and MgCL₂ and delayed by CaCl₂ and hypertonicity produced by sugars. Indoleacetic acid (IAA) and naphthaleneacetic acid (NAA) accelerated the separation at physiological concentrations. The rate of separation differed markedly by the age of the leaf and the species which offered the substrate material.     

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; )     

Studies on the function of proplastids in the metabolism of in vitro-cultured tobacco cells III. Source of reducing power for amino acid synthesis from nitrite

Evidence to show the presence of glucose-6-phosphate dehydrogenase,6-phospho-gluconate dehydrogenase, and NADP-dependent malicenzyme in proplastids of in vitro-cultured tobacco cells wasobtained. Amino acid synthesis from nitrite and 2-oxoglutaratein the proplastids was stimulated by addition of 20 mM glucose-6-phosphate.6-Phosphogluconate, malate, and isocitrate did not affect thesynthesis. Nitrite reduction and glutamate synthesis in theproplastids are assumed to be supplied with NADPH₂ as the sourceof reducing power through the reactions catalyzed by glucose-6-phosphatedehyrdogenase and 6-phosphoglyconate dehydrogenase. (Received March 22, 1977; )     

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; )     

Studies on the function of proplastids in the metabolism of in vitro cultured tobacco cells I. Localization of nitrite reductase and NADP-dependent glutamate dehydrogenase

Proplastids were isolated from in vitro cultured tobacco cellsby brief centrifugation and gel filtration. The fraction containingintact proplastids was enriched 7.6-fold in carotenoid Contenton protein basis, 6.2-fold in NiR activity and 5.1-fold in NADPGDHactivity relative to the homogenate on a specific activity basis.We suggest that the proplastid is an organelle for nitrite reductionand reductive amination of -ketoglutarate. (Received August 17, 1976; )     

Studies on the function of proplastids in the matabolism of in vitro cultured tobacco cells II. Glutamine synthetase/glutamate synthetase pathway

Glutamate and glutamine were produced when intact proplastidsof in vitro cultured tobacco cells were incubated with nitriteand -ketoglutarate. ATP stimulated the production to a considerableextent. While glutamate was the main product of incubation inthe complete medium, glutamine production surpassed glutamateproduction when proplastids were incubated without -ketoglutarate.These facts suggests the operation of the glutamine synthetase/glutamatesynthetase pathway in the proplastids. This suggestion was substantiatedby the demonstration of both glutamine synthetase and glutamatesynthetase activities in the proplastid fraction. (Received December 24, 1976; )     

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.     

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; )     

Cell-wall column chromatographic identification of the native cell-wall acid phosphatase of cultured tobacco cells

Two fractions of acid phosphatase were eluted from a columnof isolated tobacco cell walls with a linearly-increasing gradientof NaCl. Although the isolated cell walls were found to havea large capacity to bind cytoplasmic acid phosphatase, the fractioneluted at 0.4 M NaCl was assumed to be the native cell-wallenzyme. (Received February 19, 1976; )     

Electrophoretic mobility of haploid cells, diploid cells, asci, and ascospores of Saccharomyces cerevisiae

The electrophoretic mobility of diploid cells, haploid cellsof different mating types, their cell walls, asci, and ascosporesof Saccharomyces cerevisiae was measured with a free-flow electrophoreticapparatus. Haploid -cells and ascospores exhibited higher mobilitythan diploid cells, haploid -cells, and asci. Similar differencesin mobility were found with isolated cell walls of the haploidand diploid cells. ² Present address: Lederle (Japan) Ltd., Kyobashi, Tokyo 104,Japan. (Received December 24, 1976; )