Stability Of Protoplasts Research Articles

Auxin‐Induced Expansion Growth of Cells and Protoplasts of Yeast

AbstractUsing an auxin‐responsive mutant of Sacchairomyces ellipsodeus, expansion growth of cells caused by auxin was studied especially in comparison with that of protoplasts. Indole‐3‐acetic acid induced detectable cell expansion growth in 3 hours in a buffered simple solution where no cell division occurred. The auxin‐induced expansion growth was inhibited by an antiauxin, trans‐cinnamic acid. Actinomycin D, chloramphenicol and cycloheximide inhibited the auxin‐induced cell expansion growth. Protoplasts did not expand in response to auxin under the condition where intact cells did. The stability of protoplasts was not changed by the low auxin concentration (20 mg/1) which induced cell expansion. High concentrations (100–1000 mg/1) of auxin caused protoplasts to burst even under an osmotically stable condition.

Steroid lysis of protoplasts and effects of stabilizers and steroid antagonists.

Six synthetic antimicrobial steroids were examined for indications of their mechanism of action. Dequadin acetate, cetyl pyridinium chloride (CPC), and sodium deoxycholate were studied for comparison. Aerated cells of Sarcina lutea were washed, suspended in 1.06 M sucrose, and converted to protoplasts with 20 mug/ml of lysozyme. Lysis was measured optically at 650 mmu as a decrease in optical density. Screening tests with 50 mug/ml of each compound showed five steroids and CPC to be lytic. Protoplasts were strongly protected from lysis by pretreatment with 0.001 to 0.004 M spermine tetrahydrochloride. Other polyamines, such as spermidine phosphate, were less protective, and putrescine was ineffective. Uranyl nitrate (5 x 10(-4) M) rapidly agglutinated protoplasts and protected them from rupture by the lytic agents. Similar studies with 0.001 to 0.004 M Mg(++) showed varying degrees of protection, which, in most cases, was only temporary. Steroidal lysis did not appear to be related to chelation, since ethylenediaminetetraacetate did not cause lysis alone and antagonized some lytic compounds. Lecithin, Tween 80, Tween 20, and Span 20 at 0.05% exhibited certain effects on protoplast stability. Span 20 strongly prevented lysis by steroids. Tween 20 alone quickly caused protoplast rupture. Lecithin and Tween 80, which also caused lysis alone, interfered with lytic steroids and CPC. The test compounds were both inhibitory and lethal to cells of Sarcina lutea. The results suggest that direct action on cell membranes may be chiefly responsible for the antimicrobial properties of the steroids.

Steroid Lysis of Protoplasts and Effects of Stabilizers and Steroid Antagonists

Six synthetic antimicrobial steroids were examined for indications of their mechanism of action. Dequadin acetate, cetyl pyridinium chloride (CPC), and sodium deoxycholate were studied for comparison. Aerated cells of Sarcina lutea were washed, suspended in 1.06 M sucrose, and converted to protoplasts with 20 mug/ml of lysozyme. Lysis was measured optically at 650 mmu as a decrease in optical density. Screening tests with 50 mug/ml of each compound showed five steroids and CPC to be lytic. Protoplasts were strongly protected from lysis by pretreatment with 0.001 to 0.004 M spermine tetrahydrochloride. Other polyamines, such as spermidine phosphate, were less protective, and putrescine was ineffective. Uranyl nitrate (5 x 10(-4) M) rapidly agglutinated protoplasts and protected them from rupture by the lytic agents. Similar studies with 0.001 to 0.004 M Mg(++) showed varying degrees of protection, which, in most cases, was only temporary. Steroidal lysis did not appear to be related to chelation, since ethylenediaminetetraacetate did not cause lysis alone and antagonized some lytic compounds. Lecithin, Tween 80, Tween 20, and Span 20 at 0.05% exhibited certain effects on protoplast stability. Span 20 strongly prevented lysis by steroids. Tween 20 alone quickly caused protoplast rupture. Lecithin and Tween 80, which also caused lysis alone, interfered with lytic steroids and CPC. The test compounds were both inhibitory and lethal to cells of Sarcina lutea. The results suggest that direct action on cell membranes may be chiefly responsible for the antimicrobial properties of the steroids.

STABILIZATION OF STREPTOCOCCUS FAECALIS PROTOPLASTS BY SPERMINE.

Harold, F. M. (National Jewish Hospital, Denver, Colo.). Stabilization of Streptococcus faecalis protoplasts by spermine. J. Bacteriol. 88:1416-1420. 1964.-Lysis of protoplasts of Streptococcus faecalis subjected to osmotic shock was prevented by the presence of 10(-3)m spermine and other divalent cations. Protein and nucleic acids were largely retained, but compounds of low molecular weight were discharged into the medium and the capacity for glycolysis was lost. Under these conditions, spermine was bound to the protoplasts. It could not be removed by washing with water or nonelectrolytes, but was displaced by salts, polyanions, and polycations. Removal of the spermine restored the osmotic fragility of the protoplasts, which could once again be protected from lysis by impermeant solutes. Protoplasts were also stabilized, in the absence of osmotic shock, by prolonged incubation with cations in 0.5 m sucrose. By either procedure, the protoplasts became resistant not only to osmotic lysis but also to sonic oscillation. It is concluded that the stabilization of protoplasts resulted from ionic binding of the cation to acidic sites on the external surface of the plasma membrane. This conferred upon the membrane additional mechanical strength, perhaps by the cross-linking of subunits, but did not alter its permeability to extracellular solutes.

STABILIZATION OF PROTOPLASTS AND SPHEROPLASTS BY SPERMINE AND OTHER POLYAMINES

Tabor, Celia W. (National Institute of Arthritis and Metabolic Diseases, Bethesda, Md.). Stabilization of protoplasts and spheroplasts by spermine and other polyamines. J. Bacteriol. 83:1101-1111. 1962.-Spermine (10(-3)m) or spermidine prevents lysis of lysozyme-produced protoplasts of Escherichia coli W, E. coli B, and Micrococcus lysodeikticus in hypotonic media. Spheroplasts prepared by the action of penicillin are also stabilized by these concentrations of spermine and spermidine, but the protection is not as complete. Streptomycin, polylysine, and Ca(++) are also effective or partially effective stabilizers, but 1,4-diaminobutane, 1,5-diaminopentane, ornithine, Mg(++), and monovalent cations have no protective action at 10(-3)m concentration, and only a slight effect at higher concentrations. The osmotic stability conferred on protoplasts by spermine is irreversible. However, the protective effect of polyamines against lysis is not accompanied by restoration of viability to lysozyme protoplasts. There is a marked reduction in the loss of ultra-violet-absorbing material from the protoplasts to the medium when 10(-3)m spermine is present.