Changes in cell-wall polysaccharide composition of developingNitella internodes

Changes in the uronide, neutral-polysacharide, and cellulose composition of the cell wall ofNitella axillaris Braun were followed throughout development of the internodes and correlated with changes in growth rate. As the cells increased in length from 4 to 80 mm during development, the relative growth rate decreased. Cell wall thickness, as measured by wall density, increased in direct proportion to diameter, indicating that cell-wall stress did not change during elogation. Cell-wall analyses were adapted to allow determination of the composition of the wall of single cells. The total amounts of uronides, neutral sugars and cellulose all increased during development. However, as the growth rate decreased, the relative proportions of uronides and neutral sugars, expressed as percent of the dry weight of the wall, decreased, while the proportion of cellulose increased. The neutral sugars liberated upon hydrolysis ofNitella walls are qualitatively similar to those found in hydrolysates of higher plant cell walls: glucose, xylose, mannose, galactose, arabinose fucose and rhamnose. Only the percentage of galactose was found to increase in walls of mature cells, while the percentage of all other sugars decreased. The rate of apposition (g of wall material deposited per unit wall surface area per hour) of neutral polysaccharides decreased rapidly with decreasing growth rate during the early stages of development. The rate of apposition of uronides decreased more steadily throughout development, while that of cellulose, after an early decline, remained constant until dropping off at the end of the elongation period. These correlations between decreasing growth rate and decreasing rate of apposition of neutral sugars and uronides indicate that synthesis of these cell-wall components could be involved in the regulation of the rate of cell elongation inNitella.     

Analysis of Saccharomyces cerevisiae hexose carrier expression during wine fermentation: both low- and high-affinity Hxt transporters are expressed

The transport of glucose and fructose into yeast cells is a critical step in the utilization of sugars during wine fermentation. Hexose uptake can be carried out by various Hxt carriers, each possessing distinct regulatory and transport-kinetic properties capable of influencing yeast fermentation capacity. We investigated the expression pattern of the hexose transporters Hxt1 to 7 at the promoter and protein levels in Saccharomyces cerevisiae during wine fermentation. The Hxt1p carrier was expressed only at the beginning of fermentation, and had no role during stationary phase. The Hxt3p carrier was the only one to be expressed throughout fermentation, displaying maximal expression at growth arrest and slowly decreasing in abundance over the course of the stationary phase. The high-affinity carriers Hxt6p and Hxt7p displayed similar expression profiles, with expression induced at entry into stationary phase and persisting throughout the phase. The expression of these two carriers occurred despite the presence of high amounts of hexoses, and the proteins were stably expressed when the cells were starved for nitrogen. The Hxt2p transporter was only transiently expressed during lag phase, which suggests a role for the protein in growth initiation. Characterization of glucose transport kinetics indicated the presence of a shift in the low-affinity component that is consistent with a predominant expression of Hxt1p during growth phase and of Hxt3p during stationary phase. In addition, a high-affinity uptake component consistent with functional expression of Hxt6p/Hxt7p was identified during stationary phase.     

Metabolism of cell wall polysaccharides in cell suspension cultures of Populus alba in relation to cell growth

Changes in the composition of cell walls and extracellular polysaccharides (ECP) were studied during the growth of suspension-cultured Populus alba cells. Three growth phases, namely the cell division phase, cell elongation phase and stationary phase, were distinguished. The active deposition of polysaccharides in cell wall fractions (50 m M Na₂CO₃-, 1 M KOH-, 4 M KOH-soluble and 4 M KOH-insoluble) was observed during the elongation phase. A 50 m M Na₂CO₃-soluble pectic fraction mainly composed of 1,4-linked galactan and arabinan except acidic sugars. The 1,4-linked galactan decreased markedly during elongation. In 1 and 4 M KOH-soluble hemicellulosic fractions, non-cellulosic 1,4-glucan and xyloglucan were observed as major components, respectively. These polysaccharides also decreased during elongation. A large amount of polysaccharides was secreted into the medium as ECP. Neutral sugars were detected predominantly by sugar composition analysis. Acidic sugars, such as galacturonic acid, were less than 12% of total. In this study, active metabolism of pectic polysaccharides in addition to hemicellulosic polysaccharides, especially neutral side chains of pectin, during cell growth, was clarified.     

Carbohydrate Metabolism and Activity of Pyrophosphate: Fructose-6-Phosphate Phosphotransferase in Photosynthetic Soybean (Glycine max, Merr.) Suspension Cells

Activity of pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was investigated in relation to carbohydrate metabolism and physiological growth stage in mixotrophic soybean (Glycine max Merr.) suspension cells. In the presence of exogenous sugars, log phase growth occurred and the cells displayed mixotrophic metabolism. During this stage, photosynthetic oxygen evolution was depressed and sugars were assimilated from the medium. Upon depletion of medium sugar, oxygen evolution and chlorophyll content increased, and cells entered stationary phase. Activities of various enzymes of glycolysis and sucrose metabolism, including PFP, sucrose synthase, fructokinase, glucokinase, UDP-glucose pyrophosphorylase, and fructose-1,6-bisphosphatase, changed as the cells went from log to stationary phases of growth. The largest change occurred in the activity of PFP, which was three-fold higher in log phase cells. PFP activity increased in cells grown on media initially containing sucrose, glucose, or fructose and began to decline when sugar in the medium was depleted. Western blots probed with antibody specific to the -subunit of potato PFP revealed a single 56 kilodalton immunoreactive band that changed in intensity during the growth cycle in association with changes in total PFP activity. The level of fructose-2,6-bisphosphate, an activator of the soybean PFP, increased during the first 24 hours after cell transfer and returned to the stationary phase level prior to the increase in PFP activity. Throughout the growth cycle, the calculated in vivo cytosolic concentration of fructose-2,6-bisphosphate exceeded by more than two orders of magnitude the previously reported activation coefficient (K_a) for soybean PFP. These results indicate that metabolism of exogenously supplied sugars by these cells involves a PFP-dependent step that is not coupled directly to sucrose utilization. Activity of this pathway appears to be controlled by changes in the level of PFP, rather than changes in the total cytosolic level of fructose-2,6-bisphosphate.     

Long-term effects of growth regulators on growth and turnover of symplastic and apoplastic sugars in the suspension subculture of kidney bean

Suspension cells of kidney bean were grown for 42 d in MS medium supplemented with growth regulators (2.0 mgL^-1 2,4-D and 0.5 mgL^-1 kinetin) or without At the stationary growth phase (42 d), the sugars were fractionated into the symplastic (ethanol and starch) and apoplastic [low-molecular pectin (lm-pectin), high-molecular pectin (hm-pectin), hemicellulose, and cellulose] sugars. The neutral sugars (NS) of hm-pectin and hemicellulose fractions were analyzed by GLC. The growth of the suspension cells in the liquid MS media, in terms of settled cell volume (SCV), remained similar, to the end of the experiment, irrespective of the presence or absence of growth regulators, indicating the nonnecessity of the exogenous growth regulators for the subculture. Total sugar (TS) of the ethanol fraction and NS of the Im-pectin of the suspension cells grown in the medium with growth regulators were higher than in the medium without growth regulators. However, starch content in the starch fraction and uronic acid (UA) content of the Im-pectin fraction did not exhibit any differences. From these results, it was suggested that the growth regulators modulated the structure of the cell wall polysaccharide. Analysis of the NS composition of the hm-pectin fractions revealed that the Rha, Arb, and Gal contents in the presence of growth regulators were higher than in the absence, while the Xyl, Man, and Glc contents in the presence of growth regulators were higher than in the absence, indicating the turnovers of rhamnogalacturonan and/or arabinogalactan. On the other hand, analysis of NS composition of hemicellulose fractions revealed that the Ara and Glc contents in the presence of growth regulators was higher than in the absence, whereas Xyl and Glc contents were nearly consistent, indicating the turnovers of arabinaogalactan I or II. The cellulose contents remained similar, irrespective of the presence (19.1%) or absence (18.7%) of growth regulators.     

Metabolism and Solubilization of Cellulose by Clostridium cellulolyticum H10

When Clostridium cellulolyticum was grown with cellulose MN300 as the substrate, the rates of growth and metabolite production were found to be lower than those observed with soluble sugars as the substrate. At low cellulose concentrations, the growth yields were equal to those obtained with cellobiose. The main fermentation products from cellulose and soluble sugars were the same. Up to 15 mM of consumed hexose, a change in the metabolic pathway favoring lactate production similar to that observed with soluble sugars was found to occur concomitantly with a decrease in molar growth yield. With cellulose concentrations above 5 g/liter, accumulation of soluble sugars occurred once growth had ceased. Glucose accounted for 30% of these sugars. A kinetic analysis of cellulose solubilization revealed that cellulolysis by C. cellulolyticum involved three stages whatever cellulose concentration was used. Analysis of these kinetics showed three consecutive enzymatic activity levels having the same K_m (0.8 g of cellulose per liter, i.e., 5 mM hexose equivalent) but decreasing values of V_max. The hypothesis is suggested that each step corresponds to differences in cellulose structure.     

A possible role of cellulose-binding protein A (CBPA) in the adhesion of Eubacterium cellulosolvens 5 to cellulose

The cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5 is a modular enzyme comprised of a catalytic domain, a cellulose-binding domain and a cell wall-binding domain. Cellobiose-grown cells changed their adhesion ability to cellulose depending on the growth phase. On the other hand, carboxymethyl cellulose (CMC)-grown cells bound to cellulose regardless of their growth phase. The distribution of CBPA in the culture supernatant and cell fractions changed depending on the carbon source contained in the medium and growth phase. The cellobiose-grown cells harvested from the culture of the late stationary growth phase did not bind to cellulose, but their adhesion ability was recovered by treatment with recombinant CBPA. Moreover, cellobiose-grown cells harvested from the culture of an early exponential growth phase bound to cellulose, but their adhesion ability was inhibited by treatment with anti-CBPA antiserum. CBPA rapidly decreased the viscosity of CMC, indicating that CBPA was endoglucanase. The results obtained in this study indicate that CBPA plays an important role in the adhesion of E. cellulosolvens 5 cells to cellulose.     

Adhesive properties of five mesophilic, cellulolytic Clostridia isolated from the same biotope

Abstract Adhesion to cellulose of five strains of mesophilic, cellulolytic clostridia , isolated from a municipal waste digestor, was found to be a reversible phenomenon. The type of attachment for the five strains conformed to a multilayer adhesion. In a first step, attachment to the adhesion site occurred by cell-cellulose interaction. In a second step, cell-cell interactions were identified. The five strains adhered slightly better to magazine paper and Whatman No. 1 filter paper than to newspaper and cardboard. Two strains, C401 and A22, were studied in more detail. The two strains, harvested in stationary phase, presented a heterogeneous population which could be separated: (i) as 'unbound' cells, corresponding to cells remaining in suspension from cellulose-grown cultures; and (ii) as 'bound' cells, coming from two successive washes with 50 mM Tris HCl, pH 7.0, which released 'bound' cells. In adhesion measurements, eluted cells ('bound' cells) adhered better to the cellulose than the 'unbound' cells. Strain C401 adhered better than strain A22 to the cellulose: 1.9-fold for the 'bound' cells and 3.6-fold for the 'unbound' cells. Adhesion of the two isolates was enhanced by the presence of calcium (10 mM). Cellobiose and glucose had no effect on strain A22 adhesion. Conversely, adhesion of strain C401 to cellulose was enhanced by cellobiose at a concentration of 1.5 g I⁻¹, but 85% inhibited by a concentration of 5.0 g I⁻¹. The two strains adhered to the same site on Whatman filter paper and unspecific interactions between the two strains occur.     

Uptake of 13C-glucose by cell suspensions of carrot (Daucus carota) measured by in vivo NMR: Cycling of triose-, pentose- and hexose-phosphates

After a lag phase of 2 days, batch-grown cells of carrot ( Daucus carota L.) cv. Flakkese entered the exponential growth phase and started to accumulate sucrose and hexoses. Short-term feeding ¹³C-glucose in this period resulted in only minor labelling of sucrose or fructose. CO₂ production from [1-¹³C]- and [6-¹³C]-glucose revealed, that at least 40% of the added glucose passed through the oxidative pentose phosphate pathway (OPPP), up to 40% through glycolysis leaving only minor ¹³C-glucose for incorporation in various cell components in the exponential growth phase. After about 11 days of culture, the medium sugars were exhausted, cells entered the stationary growth phase and consumed stored sugar. Both neutral and acid invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13) increased 50% from day 0 to days 11–13; thereafter their levels decreased again. Labelling with ¹³C-glucose resulted in the accumulation of labelled sucrose and fructose during the stationary growth phase. Sucrose labelling was transient, i.e. after 6 h its level started to decrease again. Labelled fructose, however, evolved slower and increased even after 8 h. In sucrose and fructose up to 20% of the ¹³C-label was exchanged from C-1 to C-6 carbons, indicating intensive cycling of at least 40% of the carbon between hexoses and triose phosphates. In the stationary phase only 10% of the labelled glucose passed through the OPPP and about 30% passed through the respiratory pathway; the remaining 60% was incorporated in cell constituents and sugars. Comparing the various cycles revealed that the regulation of the OPPP operated relatively independently from the cytosolic cycling of hexose phosphates through sucrose and from the cycling between hexose phosphates and triose phosphates.     

Photoautotrophic potato cells: Transition from heterotrophic to autotrophic growth

Cells of potato (Solanum tuberosum L.) were obtained which were capable of photoautotrophic growth in liquid suspension culture under a photon flux density of 90–110 μmol m^?2 s^?1 PAR and in an atmosphere enriched with 2% CO₂. These photoautotrophic cells contained between 100 to 200 μg Chl (g fresh weight)^?1 and fixed CO₂ at a maximum rate of 16 μmol CO₂ (g fresh weight)^?1h^?1. In order to obtain cells capable of photoautotrophic growth it was necessary to adapt highly chlorophyllous heterotrophic cells (>50 μg Chl (g fresh weight)^?1) for growth in medium with 2.5 g sucrose 1^?1 (photomixotrophic cells). The photomixotropic cells had a Chl content of ca 100 μg Chl (g fresh weight)^?1 and were capable of photosynthetic activity which allowed them to survive after sugars had been depleted from the medium. It was from the photomixotrophic cells that cells capable of photoautotrophic growth were obtained. Heterotrophic cells initially established in liquid medium with 25 g sucrose I^?1 from chlorophyllous callus contained about 50 to 150 μg Chl (g fresh weight)^?1. However, after 5 to 10 passages the Chl content decreased to a maximum of 15 μg Chl (g fresh weight)^?1. These cells could not be adapted to photomixotrophic or photoautotrophic growth. These cells also were not able to regain Chl or initiate high rates of CO₂ fixation during the stationary phase of growth as did photomixotrophic cells or chlorophyllous heterotrophic cells. The loss of Chl exhibited by the cells during adaption to heterotrophic growth could be attributed at least in part to unbalanced growth (when cell division and growth exceeds Chl accumulation). Sucrose appeared to have an inhibitory effect directly on photosynthesis independent of Chl accumulation.     

Carbon assimilation in carrot cells in liquid culture

Assimilation of carbohydrates by carrot (Daucus carota L. cv Danvers) cells in liquid culture was studied to delineate the major metabolic pathways used in transformation of external carbohydrates to UDP-glucose. The cells grown on either sucrose or glucose for several years proved equally capable of utilizing each of these sugars. Sucrose was rapidly hydrolyzed extracellularly to glucose and fructose, and glucose was preferentially taken up. Uptake of fructose was slower and delayed until glucose was nearly depleted from the medium. Concentrations of cellular sugars, mainly glucose and sucrose, increased during late logarithmic phase of growth and decreased during the plateau phase. Continuous labeling of the cells with d-[¹⁴C]glucose resulted in rapid accumulation of radioactivity in glucose-6-phosphate and UDP-glucose. Because there was virtually no uptake of sucrose, UDP-glucose was likely derived from glucose-1-phosphate in a reaction catalyzed by UDP-glucose pyrophosphorylase and not directly from sucrose. Concentrations of major nucleotides and nucleotide sugars were maximal during the early logarithmic phase of growth and decreased several-fold in the stationary phase. A modified `energy charge' for adenylates calculated with the omission of AMP decreased steadily from 0.9 to 0.8 during the course of culture cycle. An analogous uracil nucleotide ratio was considerably lower (0.85) during early culture, decreased to about 0.7 for the entire logarithmic phase, and returned to initial values as cells entered stationary phase. The uracil nucleotide ratio may provide a useful index to assess the coupling between the energy available in phosphoanhydride bond in adenine nucleotides and the demand for sugar for polysaccharide synthesis through uridine diphosphate-sugar pools.     

Two penicillin binding proteins of Haemophilus influenzae are lost after cells enter stationary phase

Abstract The penicillin binding proteins (PBPs) of 4 representative isolates of Haemophilus influenzae were studied using crude membrane preparations and whole cells grown to the logarithmic and stationary phases of growth. Relative binding, % of total bound, and binding affinities were compared. The PBP patterns were similar for crude membranes and whole cells for all 4 strains tested at each phase of growth. However, PBP 2 was slightly reduced and PBP 4 was markedly reduced with whole-cell labelling in comparison to crude membranes. 8 PBPs were detected in cells labelled during the logarithmic phase of growth, while 6 were detected in stationary phase cells. The pBPs 'lost' in stationary phase (PBPs 4 and 6) with apparent M _r of 62 000 and 45 000, respectively, have a high affinity for ampicillin ( I ₅₀≃ 0.04 μ g/ml). This suggests that these proteins may have an important role in cell growth, and are targets for β-lactam substrates.     

Polysaccharides of the cellular slime mold II. Change of intra- and extracellular polysaccharides during growth phase of Dictyostelium discoideum NC-4

The three intra- and extracellular polysaccharide fractions were isolated during the growth phase of Dictyostelium discoideum NC-4, and the change in content of component sugars of four fractions during the culture period was examined. Myxamoebae most extensively contain a polysaccharide fraction extracted with phenol-water (polysaccharide fraction I) in a quantity of about 15–23% per dry cell. After 15 h the uronic acid formed in the polysaccharide fraction I, and the cell, could be aggregated. The glucosamine content in the polysaccharide fraction I reached a maximum as the myxamoebae entered the exponential phase, and a large amount of galactose was produced as the cell entered the stationary phase. The phenol-water extract from the cells of the stationary phase was reacted with concanavalin-A.     

Regulation of cell-wall polysaccharide components by CaCl2 in suspension cultures of kidney bean (Phaseolus vulgaris)

We cultured the suspension cells of kidney bean in MS media supplemented with one of five concentrations of CaCI₂ [0,22,44 (control), 88, or 176 mg/L], and harvested them at the logistic (15 d) and early-stationary (30 d) phases. Cells grown at concentrations higher than 22 mg/L showed better proliferation than those at 0 mg/L The rate of proliferation also increased with higher concentrations. We fractionated the individual sugars into symplastic (EtOH and starch) and apoplastic (low-molecular pectin, high-molecular pectin, hemicellulose, and cellulose) components. Cells treated at the highest concentration (176 mg/L) exhibited the greatest amount of sugar in the EtOH and starch fraction during the logistic phase. In contrast, cells in the early stationary phase had the highest level of sugar at treatment concentrations of less than 22 mg/L. For treatment concentrations higher than 22 mg/L on Day 15, more pectin and hemicellulose was detected at greater amounts compared with those cells treated with 0 mg/L. However, at Day 30, concentrations higher than 44 mg/L induced greater amounts of pectin and hemicellulose than from the other concentrations. Cellulose was more abundant with the 0 mg/L treatment, and contents ranged from 17.4 to 25.5% in the primary cell walls over all treatment concentrations. These results indicate that CaCI₂ modulates both symplastic and apoplastic sugar metabolism. Therefore, we suggest that the cell-wall structure may define the mode of polysaccharide biosynthesis during cell growth.     

Solid-State Fermentation with Trichoderma reesei for Cellulase Production

Cellulase yields of 250 to 430 IU/g of cellulose were recorded in a new approach to solid-state fermentation of wheat straw with Trichoderma reesei QMY-1. This is an increase of ca. 72% compared with the yields (160 to 250 IU/g of cellulose) in liquid-state fermentation reported in the literature. High cellulase activity (16 to 17 IU/ml) per unit volume of enzyme broth and high yields of cellulases were attributed to the growth of T. reesei on a hemicellulose fraction during its first phase and then on a cellulose fraction of wheat straw during its later phase for cellulase production, as well as to the close contact of hyphae with the substrate in solid-state fermentation. The cellulase system obtained by the solid-state fermentation of wheat straw contained cellulases (17.2 IU/ml), β-glucosidase (21.2 IU/ml), and xylanases (540 IU/ml). This cellulase system was capable of hydrolyzing 78 to 90% of delignified wheat straw (10% concentration) in 96 h, without the addition of complementary enzymes, β-glucosidase, and xylanases.     

Variation in melanosome numbers in cultured B-16 melanoma cells

Melanosomes from B-16 mouse melanoma cells in culture were isolated by treatment of pigmented cells with 2% SDS, sonication, and heating at 100°C. The total number of melanosomes in cultures of B-16 mouse melanoma cells increased exponentially during the rapid phase of sigmoid growth. The numbers of melanosomes per cell decreased during rapid phase of growth, and repigmentation was observed only when the cultures attained the stationary growth phase. BUdr at a minimum concentration of 0.5 μg/ml decreased both cell growth and numbers of melanosomes per cell, and completely inhibited repigmentation following a period of active growth. Cells cultured in 0.1 μg/ml BUdr grew at the same rate as untreated cells but contained fewer melanosomes/cell and lower total numbers of melanosomes during the late stages of the growth cycle.     

Photosynthesis and carbon metabolism in photoautotrophic cell suspensions of Chenopodium rubrum from different phases of batch growth

Rapidly dividing photoautotrophic cell suspensions from Chenopodium rubrum L. assimilated about 85 μmol CO₂ (mg chlorophyll)⁻¹ h⁻¹. During the late stationary phase of culture growth, CO₂ fixation rate was reduced to about 60 μmol CO₂ (mg chlorophyll)⁻¹ h⁻¹. Actively dividing cells characteristically incorporated a smaller proportion of ¹⁴C into starch than cells from non-dividing stationary phases. In rapidly dividing cells, [¹⁴C]-turnover from free sugars, sugar-phosphates, organic and amino acids was substantially higher compared to non-dividing cells from stationary growth phase. Higher proportions of photosynthetically fixed carbon were channelled into proteins, lipids and structural components in actively dividing cells than in non-dividing cells. In the latter. ¹⁴C was preferentially channeled into starch, and a striking increase in starch accumulation was observed. The transfer of non-dividing, stationary growth-phase cells into fresh culture medium resulted in an increase in the maximum extractable activities of some enzymes involved in the glycolytic and dark respiratory pathways and in the citric acid cycle. In contrast, the maximum extractable activities of the chloroplastic enzymes, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.38) and NADP⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) were highest after the cells had reached the stationary growth phase.     

A MODIFIED CELL WALL MUTANT OF THE RED MICROALGA RHODELLA RETICULATA RESISTANT TO THE HERBICIDE 2,6-DICHLOROBENZONITRILE1

The rigid component of the cell walls of red macroalgae, cellulose, is lacking in the red microalgae. Instead, the cells are encapsulated within an amorphous polysaccharide. These complex sul fated polysaccharides are composed of at least 10 different sugars, but their structure is not known, When the herbicide 2,6-dichlorobenzonitrile (DCB), a compound that specifically inhibits cellulose biosynthesis, was applied to cultures of the red microalga Rhodella reticulata upon inoculation, growth was inhibited. When added during the stationary phase of growth (after cell division had ceased), DCB did not affect cell number but it did inhibit polysaccharide production. A spontaneous mutant resistant to DCB was selected; it had physiological characteristics similar to those of the wild-type parent. The composition of the cell wall polysaccharide of the mutant was totally modified, being composed almost entirely (98% of its dry matter, as compared to 2.9% in the wild type) of methyl galactose, but retaining the same sulfate content. The molecular mass of the mutant polysaccharide was, however, similar to that of the wild-type parent (～6 × 10⁶ daltons), although its viscosity was significantly lower.     

Sensitivity of various yeasts to crude cellulolytic enzyme complexes fromTrichoderma reesei

Summary Twenty-six yeast strains, representative of different yeast genera, were tested for their sensitivity to crude extracellular cellulolytic enzyme complexes obtained from the fungusTrichoderma reesei QM 9414 and its mutants M 6 and MHC 22 (Microcrystalline cellulose was the sole carbon source.) Practically all the yeast strains tested were found to be sensitive, exhibiting signs of cellwall weakening and lysis during prolonged incubation with the emzymes fromTrichoderma. Under growth conditions, the effect of cellulolytic enzymes on yeast cells and their growth rates was much less pronounced. However, at increased cellulase concentrations (5 mg/ml) in the growth medium, lysis of stationary phase yeast cells was observed.     

Colchicine-binding activity of carrot tubulin at different culture age

Tubulin contents in the extract from cultured carrot cells at different growth phases were investigated by measuring colchicine-binding activity. The addition of vinblastine and dithiothreitol to the reaction mixture appreciably improved the stability of both free and colchicine-bound tubulins. Colchicine-binding activity in the cell extract obtained from stationary phase was more labile than that from log phase though the extract showed higher affinity to colchicine. After purification, however, tubulin from the cells at different growth phases showed the same affinity and its colchicine-binding activity was much more stable than in crude extract. The colchicine-binding activity in the crude extract was corrected for the decay during measurement and apparent difference in the affinity so that the activity in the cells containing different kind and amount of interefering substances could be compared. The corrected amount of colchicine that binds to the 100,000×g extract was 46 pmol/10⁵ cells at log phase. It decreased with the progression of culture age from linear to stationary phase. Combining the data with the morphological observation, it was suggested that the log phase cells contained larger free tubulin pool than the linear or stationary phase cells.