Disrupted Yeast Cells Research Articles

Untersuchungen zur Separation aufgeschlossener Hefesuspensionen

AbstractBy the means of the electronic particle counting, characteristics of industrial contrifuges are found out in order to characterize the performance in the case of the separation of cell debris of mechanically disrupted yeast cells. The knowledge about the particle size distribution complements the usual determination of the dry matter content in the supernatant. Thus an optimization of the separation of tiny particles is facilitated.

Characteristics of mechanically disrupted bakers' yeast in relation to its separation in industrial centrifuges

AbstractThe viscosity, density, and sedimentation characteristics of suspensions of whole and mechanically disrupted yeast cells were measured. Mechanical disruption increases the suspension viscosity and its non‐Newtonian behavior. Experiments showed a good correlation between laboratory‐ and industrial‐scale centrifugation results.

A rapid method for the isolation of ribonuclease from yeast (Saccharomyces carlsbergensis).

A ribonuclease (RNAase; EC 3.1.14.1) from brewer's yeast was purified 90-fold. Crude RNAase was initially separated from other proteins by precipitation at pH 4.0 after incubation of the mechanically disrupted yeast cells at pH 6.0 and 52 degrees C for 30 min. The RNAase was purified from the supernatant by ultrafiltration with a PM-30 membrane and adsorption chromatography on hydroxyapatite. RNAase preparation was free of phosphatase, deoxyribonuclease and phosphodiesterase activities. It showed maximum activity at pH 6.0 and a temperature optimum of 52 degrees C with yeast RNA as substrate. This RNAase hydrolysed yeast RNA to nucleoside 3'-phosphates and showed no evidence of base specificity.

Biosynthesis of Ergosterol in Cell-Free System of Yeast<xref ref-type="fn" rid="fn1"><sup>1</sup></xref><xref ref-type="fn" rid="fn2"><sup>2</sup></xref>

We previously proposed the occurrence of multiple pathways in the ergosterol biosynthesis of yeast, based on the results of examination of 14C-incorporation into sterols from L-[methyl-14C]methionine which was given to the intact cells of yeast. This led us to investigate the validity of the pathways by experiments with the cell-free system. Highly active cell-free extracts could be prepared by disruption of yeast cells with a Vibrogen Cell Mill in the presence of 0.1 mM dithiothreitol. This preparation catalyzed 14C-incorporation from [14C]methionine into ergosterol with a high yield. This preparation was found to be favorable for elucidation of ergosterol synthesis, since only a small amount of radioactivity was incorporated into fatty acid ester form of sterols which were reported to be inactive as a substrate for sterol synthesis reaction. Time course experiment of 14C-incorporation from [14C]methionine into various sterols under aerobic conditions showed that ergosta-8,24(28)-dien-3 beta-ol was a precursor for ergosta-7,24(28)-dien-3 beta-ol and that radioactivities were converted through ergosta-5,7,24(28)-trien-3 beta-ol and ergosta-5,7,22,24(28)-tetraen-3 beta-ol into ergosterol with time. In contrast, similar experiments under anaerobic conditions showed that ergosta-7,24(28)-dien-3 beta-ol accumulated and very little conversion of radioactivity into ergosterol occurred. In addition, the results indicated that oxygen was required for the introduction of double bond into 22 position as well as into 5 position. The results obtained with the cell-free system supported the validity of the proposal of multiple pathways of ergosterol synthesis in the intact cells.

Yeast cell wall, membrane, and soluble marker polypeptides identified by comparative two-dimensional electrophoresis.

Yeast cell wall, plasma membrane, total spheroplast, and total soluble protein fractions were isolated from exponentially growing Saccharomyces cerevisiae batch cultures. The cell wall, plasma membrane, and soluble protein fractions were obtained by mechanical disruption of intact yeast cells under identical osmotic conditions. Electron micrographs of purified wall fractions appeared free of vesicular membrane contamination and micrographs of plasma membrane vesicles were free of cell wall contamination. Various stages of cell wall purification were monitored by electron microscopy and comparative two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This resulted in the identification of a glycopeptide designated 16w in the cell wall fraction, with an apparent isoelectric point of 5.0 and an apparent molecular weight of 25 000. Protein analyses of soluble and plasma membrane protein fractions failed to detect component 16w. Two-dimensional protein analyses of total cellular homogenates were capable of resolving the cell wall glycopeptide 16w. However, protein separations of spheroplasts formed by glusulase degradation of the cell wall complex did not detect 16w. These observations suggest that component 16w is unique to the cell wall fraction. In addition, comparison of two-dimensional gels of soluble and plasma membrane proteins, with a total cellular homogenate, tentatively identified several polypeptides unique to each of the soluble and plasma membrane fractions.

Biosynthesis of sterols by a yeast homogenate. Incorporation of mevalonic acid into cholesta-5,7,24-trien-3beta-ol and 5alpha-cholesta-7,24-dien-3beta-ol.

Incubation of (3RS,2R)-[2-14C,2-3H]mevalonic acid and (3RS, 2S)-[2-14C,2-3H]mevalonic acid with mechanically disrupted yeast cells resulted in C27-metabolites. Two (14C5, 3H4)-metabolites, cholesta-5,7,24-trien-3beta-ol and 5 alpha-cholesta-7,24-dien-3beta-ol, were isolated and characterized. The impairment of the 24-methyl transferase system was confirmed by the lack of incorporation of 14C into the sterol fraction on incubation of S-adenosyl-L-[methyl-14C]methionine with the yeast homogenate. The results indicate that interference with the (C-24)-alkylating system did not prevent the transformation of lanosterol to the cholesta-5,7,24-trien-3beta-ol and to 5 alpha-cholesta-7,24-dien-3beta-ol. It can therefore be inferred that transformations of the nucleus and of the side chain can function independently. However our results do not provide a definition of the actual sequence of the metabolic events between lanosterol and ergosterol.

The adaptive acid phosphatase of Tetrahymena pyriformis GL-9. Effect of endocytosis of various nutrients.

An adaptive increase in the cellular acid phosphatase activity ofTetrahymena pyriformis GL-9 was produced in response to ingestion of particulate materials such as yeast cells, bacteria or puff-ball spores. The particulate component of disrupted yeast cells also produced the response, whereas it was not initiated by the soluble cellular components of yeast. Macromolecules such as glycogen and ribonucleic acid evoked the response, whereas glucose, casein hydrolysate, albumin and latex particles failed to do so. It is suggested that this lysosomal enzyme increase is related to the quality of the material ingested and not to its particulate nature or to the endocytic uptake process itself; although a joint effect of endocytosis (apart from physically taking in the material) and quality of material cannot be excluded. The results also indicate that there is a relationship between ingestion of certain materials and cell division, even when taking place in a soluble growth medium which is entirely adequate nutritionally.

Chapter 4 Preparation of RNA and Ribosomes from Yeast

Publisher Summary This chapter discusses the preparation of RNA and ribosomes from yeast. Most methods used for the preparation of RNA and ribosomes from extracts of mammalian cells or bacteria can be directly applied either to yeast spheroplasts or to disrupted yeast cells. Yeast RNA can be radioactively labeled in vivo with purine and pyrimidine bases, phosphate- 32 P, and methyl-labeled methionine. Yeasts are able to utilize purines and pyrimidines added to the medium, although the free bases are not normal intermediates in the biosynthesis of nucleotides. Uracil and adenine are the bases most commonly used to label RNA. It is difficult to evaluate the usefulness for preparative purposes of the various labeling protocols, because the specific activities of the labeled RNAs are rarely determined. In the chapter, preparation of ribosomal subunits and polyribosomes and preparation of the high-molecular-weight rRNAs and low-molecular-weight rRNAs are discussed. An overview of polyacrylamide gel electrophoresis of low-molecular-weight RNAs and high-molecular-weight rRNA precursors is also presented in the chapter.

The influence of organophosphorus insecticides on yeast dehydrogenases activity

The interactions of organophosphorus insecticides (OPI) with yeast dehydrogenases in vitro were studied. A significant stimulation of dehydrogenase activity (up to 700%) occured with the intact cells. In contrast to this, however, dehydrogenases of disrupted yeast cells were inhibited. The mechanism stimulating the activity of yeast dehydrogenase by OPI is probably based on the formation of an unidentified complex of OPI with a lipophilic component of the cell membrane. The cell membrane modified in such a way is characterized by a higher permeability for the system substrate-acceptor H+.

Toxicity of Blastomyces dermatitidis.

Following injections of the insoluble portion of disrupted yeast cells ofBlastomyces dermatitidis animals exhibited a biphasic pyrogenic response, a decrease in total serum proteins, and there was no release of interferon.

Characterization of a new class of circular DNA molecules in yeast

This paper describes the isolation of a pure population of covalently closed circular twisted DNA molecules from yeast. These molecules are homogeneous in size, that is consist of monomers of 2.2μ and of multiple length oligomers of n x 2.2μ. While no data rule out the mitochondrial origin of this DNA, its actual intracellular localization remains unknown; it displays the same buoyant density as the main nuclear DNA and therefore is not the heavy nuclear satellite DNA (γ-DNA described by Moustacchi and Williamson (1966)); although circular molecules represent only 1 to 5 % of the total DNA, they can be prepared in sizable and reproducible amounts by a method based on the use of mechanical disruption of yeast cells rather than lysis by snail gut juice.

Antígenos del paracoccidioides brasiliensis para las Reacciones serológicas

The sera from normal subjects gave negative results with the following antigens used in the complement-fixation tests: 1) polysaccharide prepared according toFava Netto's technic; 2) a filtrate of shaked cultures followingAjello et al.'s technic; 3) an aqueous extract of mechanically disrupted yeast cells ofP. brasiliensis.

Intracellular distribution and characterization of yeast RNA

1. 1. Disrupted yeast cells were separated by centrifugation into two pellets, sedimenting at 15 000 × g and 40 000 × g, and a supernatant fraction. These fractions contained, respectively, 25, 15 and 60 % of the total cell RNA. 2. 2. In the presence of sodium deoxycholate or in buffers of low Mg 2+ concentration the fraction containing the 80-S ribosomes (40 000 × g supernantant) yielded 60-S and 50-S particles. Deoxycholate released only 50-S and smaller components from the 15 000 × g and 40 000 × g pellets. 3. 3. Sodium lauryl sulfate extraction of the 40 000 × g supernatant resulted in the liberation of 4 RNA components which sedimented at 26 S, 18 S, 13 S and 11 S. Similar treatment of the 15 000 × g and 40 000 × g pellets extracted only 13-S and 11-S components. 4. 4. RNA prepared by phenol extraction of the total disrupted cell mass or the 40 000 × g pellet yielded, in the presence of EDTA, two components which sedimented at 13 S and 11 S. 5. 5. It is concluded that RNA's with sedimentation coefficients of 13 S and 11 S are naturally occurring components of the cell and are present as subunits in ribosomal RNA. Less than 6 % of the RNA of the 15 000 × g and 40 000 × g pellets is present as 80-S ribosomes.