Polybase induced lysis of yeast spheroplasts

The polybasic macromolecules DEAE-dextran (diethylaminoethyl-dextran, molecular weight 500 000) and poly-dl-lysine (molecular weight 30 000–70 000) were adsorbed with a high affinity by spheroplasts of Candida utilis and, subsequently, induced lysis. The extent of lysis of spheroplasts and of the liberated vacuoles was studied under various conditions using α-glucosidase activity and soluble arginine as cytoplasmic and vacuolar markers, respectively. Adsorption of polybases was rapidly completed even at 0°C; however, with small doses, lysis was poor at 0–12°C and extensive at temperatures above 12°C. This permitted the completion of adsorption before initiating lysis. The purified vacuoles were also sensitive to polybases though less so than the spheroplasts; however, after lysis of spheroplasts the liberated vacuoles were well protected against the action of polybases. A treatment with polybases which disrupted more than 99% of the spheroplasts left at least 70% of the vacuoles intact. Potassium chloride in high concentrations and calcium chloride in low concentrations inhibited polybase induced lysis of spheroplasts by preventing or even reversing the polybase adsorption. A polyacidic macromolecule, dextran sulfate, could prevent but not reverse the adsorption of polybase and subsequent lysis. Metabolic inhibitors reduced the susceptibility of spheroplasts to polybase induced lysis. Vacuoles isolated from polybase lysed spheroplasts still contained large pools of soluble amino acids, and their ability to transport arginine specifically is a further indication of their functional integrity.     

Some properties of vacuoles isolated from Neurospora crassa slime variant

A method for the isolation of vacuoles based on polybase induced lysis of protoplasts of the cell wall deficient Neurospora crassa slime variant is described. Isolated vacuoles are characterized by 12 to 50 times increased specific activities of several hydrolases as compared with the total homogenate of protoplasts. Total -amino nitrogen, arginine, and polyphosphate are also greatly enriched in these vacuoles. Vacuoles are equipped with a permease for the transport of basic amino acids across the tonoplast.Non-Standard Abbreviation DEAE-dextran diethylaminoethyl-dextran     

Characterization of a specific transport system for arginine in isolated yeast vacuoles.

The transport of L-arginine was studied in isolated vacuoles of Saccharomyces cerevisiae. A centrifugation method allowed rapid separation of the fragile vacuoles from the incubation media so that initial uptake rates of [14C]arginine could be measured. Labelled arginine added to the medium was accumulated in the isolated vacuoles; it was found to exchange specifically with the arginine already present in the vacuoles. Such an exchange did not take place in intact spheroplasts. The pH dependence of the arginine transport in the vacuoles was tested. As the vacuoles are unstable in the pH range of optimal transport activity (pH above 7.0), the pH optimum of the transport reaction could not be determined. From the temperature dependence, the apparent energy of activation was calculated to be 9800 cal/mol. Arginine transport shows saturation kinetics with an apparent Km of 30 muM in the isolated vacuoles, and of 1.5 muM in the spheroplasts. Competition experiments with amino acids and arginine analogues demonstrated that the arginine transport in both vacuoles and spheroplasts, is highly specific. The two systems, however, were shown to have distinct specificities. The inhibition of vacuolar L-arginine transport by D-arginine, L-histidine, and L-canavanine was competitive with apparent Ki values of 60 muM, 400 muM and 600 muM respectively.     

高等植物液泡分离的研究进展

植物液泡是一种多功能细胞器。高等植物成熟液泡的分离近几十年来有了较大发展,目前已经形成了渗透裂解、多元碱化合物诱导裂解、机械破碎等几种较为成熟的方法。在初步工作的基础上,参考大量文献较详细地综述了国内外有关高等植物成熟液泡分离的研究进展。     

The transport of S-adenosyl-L-methionine in isolated yeast vacuoles and spheroplasts.

1. The properties of S-adenosyl-L-methionine accumulating system for both vacuoles and spheroplasts are described. Yeast vacuoles were obtained by a modified metabolic lysis procedure from spheroplasts of Saccharomyces cerevisiae. 2. Isolated vacuoles accumulate S-adenosyl-L-methionine by means of a highly specific transport system as indicated by competition experiments with structural analogs of S-adenosyl-L-methionine. The S-adenosyl-L-methionine transport system shows saturation kinetics with an apparent Km of 68 muM in vacuoles and 11 muM in spheroplasts. 3. S-Adenosyl-L-methionine accumulation into vacuoles does not require glucose, phosphoenolpyruvic acid, ATP, ADP nor any other tri- or di-phosphorylated nucleotides. It is insensitive to azide and 2,4-dinitrophenol which strongly inhibit the glucose-dependent accumulation of S-adenosyl-L-methionine in spheroplasts. 4. The transport of S-adenosyl-L-methionine into vacuoles is optimal at pH 7.4 and is insensitive to nystatin while the uptake of S-adenosyl-L-methionine into spheroplasts is optimal at pH 5.0 and is strongly sensitive to nystatin. On this basis it has thus been possible to measure both the intracytoplasmic and the intravacuolar pool of S-adenosyl-L-methionine. 5. Our results indicate the existence of a highly specific S-adenosyl-L-methionine transport system in the vacuolar membrane which is clearly different from the one present in the plasma membrane of yeast cells.     

Isolation and characterization of autolysis-defective mutants of Escherichia coli that are resistant to the lytic activity of seminalplasmin.

Two temperature-sensitive autolysis-defective mutants of Escherichia coli were isolated and shown to be resistant to lysis induced by seminalplasmin, an antimicrobial protein from bovine seminal plasma, as well as to lysis induced by ampicillin, D-cycloserine and nocardicin, at 37 or 42 degrees C but not at 30 degrees C. The mutants were, however, sensitive to inhibition of RNA synthesis by seminalplasmin even at the nonpermissive temperature. Temperature-resistant revertants of the mutants were sensitive to lysis induced by the various antibiotics at 37 or 42 degrees C. The mutations in both strains were mapped at 58 min on the E. coli linkage map. The lysis resistance of the mutants was phenotypically suppressed by the addition of NaCl. Partial suppression of the lysis-resistant phenotype was also observed in a relA genetic background.     

Purification of Streptococcus group C bacteriophage lysin.

A simple procedure for the purification of Streptococcus group C phage lysin to apparent homogeneity is described. The electrophoretically pure, enzymatically stable polypeptide of 98,000 molecular weight converted Streptococcus (groups A, F, and H) cells into spheroplasts within 5 min at 0 degrees C or within less than a minute at 37 degrees C.     

Requirement for a functional host cell autolytic enzyme system for lysis of Escherichia coli by bacteriophage phi X174

Escherichia coli VC30 is a temperature-sensitive mutant which is defective in autolysis. Strain VC30 lyses at 30 degrees C when treated with beta-lactam antibiotics or D-cycloserine or when deprived of diaminiopimelic acid. The same treatments inhibit growth of the mutant at 42 degrees C but do not cause lysis. Strain VC30 was used here to investigate the mechanism of host cell lysis induced by bacteriophage phi X 174. Strain VC30 was transformed with plasmid pUH12, which carries the cloned lysis gene (gene E) of phage phi X174 under the control of the lac operator-promoter, and with plasmid pMC7, which encodes the lac repressor to keep the E gene silent. Infection of strain VC30(pUH12)(pMC7) with phage phi X174 culminated in lysis at 30 degrees C. At 42 degrees C, intracellular phage development was normal, but lysis did not occur unless a temperature downshift to 30 degrees C was imposed. Similarly, induction of the cloned phi X174 gene E with isopropyl-beta-D-thiogalactoside resulted in lysis at 30 degrees C but not at 42 degrees C. Temperature downshift of the induced culture to 30 degrees C resulted in lysis even in the presence of chloramphenicol. These results indicate that host cell lysis by phage phi X174 is dependent on a functional cellular autolytic enzyme system.     

Lipoprotein synthesis in Escherichia coli spheroplasts: accumulation of lipoprotein in cytoplasmic membrane.

Synthesis of cell envelope proteins was studied in ethylenediaminetetraacetic acid-lysozyme spheroplasts of Escherichia coli ML30. The rate of incorporation of [3H]arginine into proteins in spheroplasts was about 30% of that of intact cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins synthesized in spheroplasts revealed the preferential synthesis of five polypeptides, one of which has been identified as the free form of murein lipoprotein. Lipoprotein synthesized in spheroplasts was found to be of same molecular size as that of mature lipoprotein. No prolipoprotein was observed even with a short pulse-labeling with [3H]arginine. On the other hand, significant accumulation of newly synthesized lipoprotein in the cytoplasmic membrane fraction of spheroplasts was observed. These results suggest that the processing of prolipoprotein occurs in the cytoplasmic membrane fraction of the cell envelope.     

Studies on purine transport and on purine content in vacuoles isolated from Saccharomyces cerevisiae.

The transport of purine derivatives into vacuoles isolated from Saccharomyces cerevisiae was studied. Vacuoles which conserved their ability to take up purine compounds were prepared by a modification of the method of polybase-induced lysis of spheroplasts. Guanosine greater than inosine = hypoxanthine greater than adenosine were taken up with decreasing initial velocities, respectively; adenine was not transported. Guanosine and adenosine transporting systems were saturable, with apparent Km values 0.63 mM and 0.15 mM respectively, while uptake rates of inosine and of hypoxanthine were linear functions of their concentrations. Adenosine transport in vacuoles appeared strongly dependent on the growth phase of the cell culture. The system transporting adenosine was further characterized by its pH dependency optimum of 7.1 and its sensitivity to inhibition by S-adenosyl-L-methionine. In the absence of adenosine in the external medium, [14C]adenosine did not flow out from preloaded vacuoles. However, in the presence of external adenosine, a very rapid efflux of radioactivity was observed, indicating an exchange mechanism for the observed adenosine transport in the vacuoles. In isolated vacuoles the only purine derivative accumulated was found to be S-adenosyl-L-homocysteine.     

Organization of the glycolytic enzymes in Escherichia coli

Differential centrifugation of osmotically lysed lysozyme-EDTA spheroplasts from Escherichia coli sedimented 50–70% of the glycolytic activities examined in a low speed pellet; the remaining activity, occurring in a high speed supernatant, contained the soluble enzymes of the cell. The distribution pattern of the enzymes could be altered by extrusion of the spheroplasts through the French Press or by lysis at different pH values. Electron micrographs of the pellet fraction revealed lysed spheroplasts mostly devoid of cellular constituents but consisting of cytoplasmic membranes surrounded by partially degraded cell wall fragments. Washing of the pellet showed that the enzymes were not all bound to the same degree to the membrane fraction. Throughput activity of the glycolytic pathway was demonstrated for the membrane fraction, but none was observed for the soluble fraction of the cell (i.e. for enzymes present in the supernatants) unless these were first concentrated by ultrafiltration. The supernatant from the lysed spheroplasts, together with a further supernatant obtained by washing the membrane pellet, was concentrated by ultrafiltration and chromatographed on a Bio-Gel column. The eluate contained glycolytic activities both in fractions corresponding to relatively high and relatively low molecular weight material The high molecular weight species, containing a proportion of all the enzymes studied, had a molecular weight of at least 1.2 × 10⁶. A multienzyme aggregate containing one each of the glycolytic enzymes would have a molecular weight of ～ 1.3 × 10⁶. The specific rate of pyruvate formation from glucose by the high molecular weight species was similar to that obtained from a preparation in which the fractions containing all the low molecular weight material enzyme activities were pooled and concentrated by ultrafiltration. Using the high molecular weight material, studies were made of the ability of added unlabelled glycolytic intermediates to compete for catalytic sites with intermediates produced endogenously from [¹⁴C₆] glucose. The relatively weak competition observed indicated a high degree of protection afforded the labelled intermediates derived from [¹⁴C₆] glucose.     

Endocytosis in Saccharomyces cerevisiae: internalization of enveloped viruses into spheroplasts.

When vesicular stomatitis virus was incubated with Saccharomyces cerevisiae spheroplasts at 37 degrees C, part of the virus was internalized by the spheroplasts as shown by the following criteria. (i) The spheroplast-associated virus was protected from proteinase K digestion, which releases surface-bound virus by degrading the envelope glycoproteins. (ii) The spheroplast-associated virus was resistant to mild Triton X-100 treatment, which readily solubilizes the virus. The same results were obtained with Semliki Forest virus. Internalization of the two viruses followed linear kinetics up to 90 min at 37 degrees C. Internalization was concentration- and temperature-dependent. At 11 degrees C no uptake could be detected for at least 2 h. Homogenization and organelle fractionation protocols were designed for the S. cerevisiae spheroplasts to study the compartments into which the virions were internalized. Three compartments containing both marker viruses could be separated in density gradients. One coincided with vacuole markers, one banded at a slightly higher and one at a similar density to the plasma membrane markers. Thus, S. cerevisiae spheroplasts appear to have the capability of endocytosing particulate markers like viruses. The companion paper describes internalization of two soluble macromolecules, alpha-amylase and fluorescent dextran, into intact cells.     

Haloalkaliphilic maltotriose-forming alpha-amylase from the archaebacterium Natronococcus sp. strain Ah-36.

A haloalkaliphilic archaebacterium, Natronococcus sp. strain Ah-36, produced extracellularly a maltotriose-forming amylase. The amylase was purified to homogeneity by ethanol precipitation, hydroxylapatite chromatography, hydrophobic chromatography, and gel filtration. The molecular weight of the enzyme was estimated to be 74,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibited maximal activity at pH 8.7 and 55 degrees C in the presence of 2.5 M NaCl. The activity was irreversibly lost at low ionic strength. KCl, RbCl, and CsCl could partially substitute for NaCl at higher concentrations. The amylase was stable in the range of pH 6.0 to 8.6 and up to 50 degrees C in the presence of 2.5 M NaCl. Stabilization of the enzyme by soluble starch was observed in all cases. The enzyme activity was inhibited by the addition of 1 mM ZnCl2 or 1 mM N-bromosuccinimide. The amylase hydrolyzed soluble starch, amylose, amylopectin, and, more slowly, glycogen to produce maltotriose with small amounts of maltose and glucose of an alpha-configuration. Malto-oligosaccharides ranging from maltotetraose to maltoheptaose were also hydrolyzed; however, maltotriose and maltose were not hydrolyzed even with a prolonged reaction time. Transferase activity was detected by using maltotetraose or maltopentaose as a substrate. The amylase hydrolyzed gamma-cyclodextrin. alpha-Cyclodextrin and beta-cyclodextrin, however, were not hydrolyzed, although these compounds acted as competitive inhibitors to the amylase activity. Amino acid analysis showed that the amylase was characteristically enriched in glutamic acid or glutamine and in glycine.     

Temperature-sensitive, colicin M-tolerant mutant of Escherichia coli.

A mutant sensitive to colicin M at 30 degrees C and tolerant at 42 degrees C to high concentrations of colicin M was isolated from Escherichia coli K-12. A temperature shift from 30 to 42 degrees C rescued all cells up to the time they started to lyse at 30 degrees C (25 min after addition of colicin M). The growth rate at 42 degrees C remained unaffected by colicin M. AT 42 degrees C the cell-bound colicin M was inactivated by trypsin, sodium dodecyl sulfate, and antiserum against colicin M. Ferrichrome competed with colicin M at 42 degrees C only during the initial adsorption to the common receptor protein in the outer membrane. Since cells lysed earlier at 30 degrees C when they had been preincubated with colicin M at 42 degrees C, we conclude that the process leading finally to cell lysis is initiated at 42 degrees C and stops at a later stage of colicin M trypsin, dodecyl sulfate, and antiserum when cells were transferred from 30 to 42 degrees C, we assume that colicin M is translocated from its target site towards the cell surface. The mutation conferring tolerance was mapped close to the rpsL gene.     

Ionic liquids as solvents for biopolymers: Acylation of starch and zein protein

Biopolymers such as starch and zein protein were found to be soluble at 80 °C in ionic liquids such as 1-butyl-3-methylimidazolium chloride (BMIMCl) and 1-butyl-3-methylimidazolium dicyanamide (BMIMdca) in concentrations up to 10% (w/w). Higher concentrations of biopolymers in these novel solvents resulted in solutions with too high viscosity to stir. Solutions of both starch and zein in BMIMCl were acylated with anhydrides in presence of pyridine to give acetyl starch and benzoyl zein with various degrees of substitution. Without pyridine the acylation reaction did not proceed. ¹H NMR and IR spectroscopies were used to determine the degree of substitution of starch. Viscosity studies indicated that the starch underwent slight reduction in molecular weight during the course of acylation. Starch was also soluble in other non-conventional solvents such as choline chloride/oxalic acid and choline chloride/ZnCl₂. However, zein was insoluble in these solvents.     

Enzymatic hydrolysis of cellulose in aqueous two-phase systems. I. Partition of cellulases from Trichoderma reesei

The partitioning of endo-beta-glucanase, exo-beta-glucanase, and beta-glucosidase from Trichoderma reesei QM 9414 in aqueous two-phase systems has been studied with the object of designing a phase system for continuous bioconversion of cellulose. The partitioning of the enzymes in two-phase systems composed of various water soluble polymeric compounds were studied. Systems based on dextran and polyethylene glycol (PEG) were optimal for one-sidedly partitioning the enzymes to the bottom phase. The influence of polymer molecular weights, polymer concentration, ionic composition of the medium, pH, temperature, and adsorption of the enzymes to cellulose on the enzyme partition coefficients (K) were studied. By combining the effects of polymer molecular weight and adsorption to cellulose, K values could be reduced for endo-beta-glucanase to 0.02 and for beta-glucosidase to 0.005 at 20 degrees C in a phase system of Dextran 40-PEG 40000 in the presence of excess cellulose, At 50 degrees C, K values were increased by a factor of two. In a phase system based on inexpensive crude dextran and PEG, the partition coefficient for endo-beta-glucanase was 0.16 and for beta-glucosidase was 0.14 at 20 degrees C with excess cellulose present.     

Osmotic Properties of Spheroplasts from Saccharomyces cerevisiae Grown at Different Temperatures

Spheroplasts were prepared from cells of Saccharomyces cerevisiae NCYC 366, grown at 30 or 15 C, by incubating cells with snail-gut juice after pretreatment with 2-mercaptoethanol. Walls of cells grown batchwise or in continuous culture at 15 C were more resistant to digestion with snail juice than walls on cells grown under the same conditions as 30 C. Spheroplasts lysed when suspended in hypotonic solutions of mannitol. The resistance of spheroplasts to osmotic lysis tended to increase when the test temperature was lowered below 30 C. The increased resistance was greater with spheroplasts from cells grown at 15 C. Cations, especially Ca²⁺, protected spheroplasts against osmotic lysis. In general, the protective effects, measured at 30 C, were smaller with spheroplasts from cells grown at 15 C compared with 30 C. Citrate and ethylenediaminetetraacetate (EDTA) decreased the resistance of spheroplasts to osmotic lysis. On the whole, the decrease was greater with spheroplasts from cells grown at 30 C rather than 15 C. In the presence of EDTA, spheroplasts from cells grown at 30 C were less resistant to osmotic lysis at 5 C than at 30 C; when spheroplasts from cells grown at 15 C were similarly examined, they were more resistant to lysis at 5 C than at 30 C. Spheroplast membranes from cells grown at 15 C had slightly but significantly greater contents of Mg²⁺, Ca²⁺, K⁺, and Na⁺ compared with spheroplast membranes from cells grown at 15 C. Mg²⁺ and Ca²⁺ were more easily extracted with EDTA from membranes of 30 C-grown cells than from 15 C-grown cells.     

Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein.

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.     

INDUCTION OF VACUOLATED SPHEROPLASTS AND ISOLATION OF VACUOLES IN CYANOBACTERIA1

There is still a great deal of debate about whether cyanobacteria contain vacuoles. This might in part reflect our limited ability to isolate vacuoles. We found and isolated vacuoles from different cyanobacteria during spheroplast preparation. Lysozyme treatment induced two kinds of spheroplasts: vacuolated spheroplasts and nonvacuolated spheroplasts. Upon breakage in distilled water, vacuolated spheroplasts released transparent, spherical, and colorless vacuoles with diameters ranging from 2.3 to 16 μm. Large vacuoles could be generated by fusion of two or three small vacuoles. Additionally, large vacuoles also could engulf small ones or other cellular bodies. The isolated vacuoles could tolerate hypotonic condition, and some could be drawn into a thread. Nonvacuolated spheroplasts released few vacuoles after breaking apart. This successful confirmation and isolation of vacuoles will allow studies of the origin and function of cyanobacterial vacuoles.     

Endocytosis in Saccharomyces cerevisiae: internalization of alpha-amylase and fluorescent dextran into cells.

In the preceding paper I reported that Saccharomyces cerevisiae spheroplasts were able to internalize particulate markers, enveloped viruses, into intracellular organelles. Here the internalization of soluble macromolecules into cells having an intact cell wall is described. alpha-Amylase was taken up into cells in a temperature- and concentration-dependent way. The kinetics of accumulation were linear for the first 20-40 min at 37 degrees C and then started to level off. Internalization of alpha-amylase into spheroplasts displayed similar characteristics, but the accumulation rate was about four times higher than into cells. Fluorescent dextran was used to mark morphologically the compartment into which internalization occurred. This marker was accumulated into the vacuole of the cells in a time-, temperature- and concentration-dependent way. A temperature-sensitive mutant deficient in exocytosis was found to be defective in intracellular accumulation of alpha-amylase and dextran. At the restrictive temperature, very little alpha-amylase accumulated into the cells and only faint staining of intracellular organelles with fluorescent dextran could be detected. At the permissive temperatures, accumulation of alpha-amylase and dextran into the mutant cells was comparable with accumulation into wild-type cells. I conclude that alpha-amylase and fluorescent dextran were internalized into S. cerevisiae cells and directed into the vacuoles.