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Studies on the role of polyamines associated with the ribosomes from Bacillus stearothermophilus.

ABSTRACT: 1. Spermine and spermidine were the main polyamines detectable in Bacillus stearothermophilus. 2. When grown at 65 degrees B. stearothermophilus contained lower concentrations of polyamines per mg. of RNA than when grown at 45 degrees or at 55 degrees . 3. Ribosomes isolated from B. stearothermophilus in 0.01m-tris-hydrochloric acid buffer (pH7.4)-0.01m-magnesium chloride contained sufficient polyamines to neutralize between 4% and 9% of their RNA phosphorus. 4. Removal of polyamines from the ribosomes by dialysis against m-potassium chloride did not appreciably alter the hypochromicity or thermal denaturation profiles of the ribosomes when measured in 0.01m-tris-hydrochloric acid buffer (pH7.4)-0.01m-magnesium chloride, though it did cause a loss of ribosome particles sedimenting at greater than 78s. 5. When ribosomes were dialysed against acridine orange solutions acridine orange bound to the ribosomes and did not displace spermine, but when a mixture of ribosomal RNA and spermine was dialysed against acridine orange the acridine orange displaced the spermine. It is concluded that polyamines in the ribosomes are less accessible for displacement by acridine orange than when polyamines are bound to ribosomal RNA.

SUBMITTER: Stevens L

PROVIDER: S-EPMC1198861 | biostudies-other | 1968 Jul

REPOSITORIES: biostudies-other

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Project description:1. Particulate enzyme preparations obtained from Bacillus stearothermophilus B65 by digestion with lysozyme were shown to catalyse teichoic acid synthesis. With CDP-glycerol as sole substrate the preparations synthesized 1,3-poly(glycerol phosphate). It was characterized by alkaline hydrolysis, by glucosylation to the alkali-stable 2-glucosyl-1,3-poly(glycerol phosphate) with excess of UDP-glucose and a Bacillus subtilis Marburg enzyme system, by degradation of this latter product with 60%HF and periodate oxidation of the resulting glucosylglycerol. The specificity of the B. subtilis system previously reported (Glaser & Burger, 1964), was confirmed in the present work. 2. Pulse-labelling experiments, followed by periodate oxidation of the product and isolation of formaldehyde from the glycerol terminus of the polymer, showed that the B. stearothermophilus enzyme system transferred glycerol phosphate units to the glycerol end of the chain. The transfer reaction was irreversible. It was not determined if these poly(glycerol phosphate) chains were synthesized de novo, but it was shown that the newly synthesized oligomers were bound to much larger molecules. 3. When the B. stearothermophilus enzyme system was supplied with both CDP-glycerol and UDP-glucose, 1-glucosyl-2,3-poly(glycerol phosphate) was synthesized in addition to the 1,3-isomer. The former polymer was characterized by acid and alkaline hydrolysis, degradation with HF and periodate oxidation of the resulting glucosylglycerol, and periodate oxidation of the intact polymer followed by mild acid hydrolysis. This latter procedure removed the glucose substituents without disrupting the poly(glycerol phosphate) chain. 4. The poly(glycerol phosphate) isomers were distinguished by glucosylation with the B. subtilis enzymes and alkaline hydrolysis, the 2,3-isomer remaining alkali-labile. The proportion of 2,3-poly(glycerol phosphate) in the product increased with increasing amounts of UDP-glucose in the incubation mixture, but the total glycerol phosphate incorporated into products remained constant. It is suggested that the synthetic pathways of the two poly(glycerol phosphate) species may share a rate-limiting step.

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Studies on the role of polyamines associated with the ribosomes from Bacillus stearothermophilus.

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