Polybase induced lysis of yeast spheroplasts. A new gentle method for preparation of vacuoles

The polybasic macromolecules DEAE-dextran (diethylaminoethyl-dextran, molecular weight 500000) and poly-DL-lysine (molecular weight 30000-70000) were absorbed with a high affinity by spheroplasts of Candida utilis and subsequently, induced lysis. The extent of lysis of spheroplasts and of the libera...

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Veröffentlicht in:Archives of microbiology 1975-01, Vol.105 (3), p.319-327
Hauptverfasser: Durr, M, Boller, T, Wiemken, A
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container_title Archives of microbiology
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creator Durr, M
Boller, T
Wiemken, A
description The polybasic macromolecules DEAE-dextran (diethylaminoethyl-dextran, molecular weight 500000) and poly-DL-lysine (molecular weight 30000-70000) were absorbed 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 alpha-glucosidase activity and soluble arginine as cytoplasmic and vacuolar markers, respectively. Adsorption of polybases was rapidly completed even at 0 degrees C; however, with small doses, lysis was poor at 0-12 degrees C and extensive at temperatures above 12 degrees 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.
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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.</description><subject>Arginine - metabolism</subject><subject>Biological Transport - drug effects</subject><subject>Candida - cytology</subject><subject>Candida utilis</subject><subject>Cell Fractionation - methods</subject><subject>DEAE-Dextran - pharmacology</subject><subject>Hydrogen-Ion Concentration</subject><subject>Inclusion Bodies</subject><subject>Polylysine - pharmacology</subject><subject>Spheroplasts - cytology</subject><subject>Spheroplasts - drug effects</subject><subject>Temperature</subject><subject>Vacuoles - metabolism</subject><issn>0302-8933</issn><issn>1432-072X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1975</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpFkEtLw0AUhQfxVasb14KzciGk3nklk2UtVoWCghbcSJhmbmokzcSZROm_N6VFV_fC-c5ZfIScMxgxgOTmdgogZcIU3yMDJgWPIOFv-2QAAnikUyGOyUkInwCMa62PyCGXXAAbkPdnV60XJiAta9vlaGm1DmWgrqBrNKGloflA75qq_8OIjmmNP3SJdVshXWH74SwtnKeNx8Z405au3lS_Td65CsMpOShMFfBsd4dkPr17nTxEs6f7x8l4FuU8lm2kpGKAyiobx3GySBVaLaRKTBoXyHgMkGplbK5EmqAymCfI0liAVYZppqUYkqvtbuPdV4ehzVZlyLGqTI2uC5kWjKdJr2FIrrdg7l0IHous8eXK-HXGINuozP5V9vDFbrVbrND-oVt3fXy5jQvjMrP0ZcjmL7wPeulaCJDiFy8Zdpo</recordid><startdate>19750101</startdate><enddate>19750101</enddate><creator>Durr, M</creator><creator>Boller, T</creator><creator>Wiemken, A</creator><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>19750101</creationdate><title>Polybase induced lysis of yeast spheroplasts. 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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.</abstract><cop>Germany</cop><pmid>242301</pmid><doi>10.1007/BF00447152</doi><tpages>9</tpages></addata></record>
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subjects Arginine - metabolism
Biological Transport - drug effects
Candida - cytology
Candida utilis
Cell Fractionation - methods
DEAE-Dextran - pharmacology
Hydrogen-Ion Concentration
Inclusion Bodies
Polylysine - pharmacology
Spheroplasts - cytology
Spheroplasts - drug effects
Temperature
Vacuoles - metabolism
title Polybase induced lysis of yeast spheroplasts. A new gentle method for preparation of vacuoles
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