Biomedical subjects
Publications and source records attributed to L W Parks.
Rates of cell division and deoxyribonucleic acid synthesis after shift-up with grande and mitchondrial deoxyribonucleic-acid-less petite yeasts were studied. The results indicate that simple eukaryotes behave as prokaryotes.
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The transmethylation of methyl-(14)C-methionine and methyl-(14)C-adenosylmethionine into the nonsaponifiable lipids of anaerobically grown yeast during adaptation to aerobic conditions was investigated. The rate and extent of methyl transfer increased with aeration time and was dependent upon the presence of a fermentable carbon source and O(2). Methionine and adenosylmethionine uptake rates increased in adaptation buffer but did not seem to be the rate-limiting factor for transmethylation under the conditions studied. Thinlayer chromatography of the nonsaponifiable fraction after exposure to label showed the labeled product to be ergosterol. Samples taken after short-term exposure to label were composed of two labeled steroidal products, one with kinetics of an ergosterol precursor.
Yeast cells accumulate S-adenosyl-l-methionine (S-AM) when cultivated in the presence of l-methionine. Cell growth is inhibited by the addition of high concentrations of l-methionine. A number of investigators have attributed this to the depletion of adenosine triphosphate (ATP) as a consequence of the utilization of that mucleotide for S-AM formation. The cellular ATP pool of Saccharomyces cerevisiae was measured during growth inhibition caused by addition of excess l-methionine. Polyethylenimine thin-layer chromatography and subsequent autoradiography were used to quantitate the extracted ATP. Addition of l-methionine to a level of 5 mg/ml in a culture during exponential growth caused an increase in the doubling time of 40 to 50%. During this period, the cellular ATP level continued increasing normally and, as the cells entered stationary growth, receded to a level characteristic of an uninhibited stationary culture growth. After the addition of methionine, there was never an observed depletion of the ATP pool other than the normal fluctuation which occurs in an uninhibited culture. We have concluded that growth inhibition by excessive methionine does not result from limiting availability of ATP.
Whereas wild-type strains of Saccharomyces cerevisiae can synthesize up to 7% dry weight of ergosterol, a polyene-resistant mutant has been obtained which produces no ergosterol. Instead, a C-28 methyl sterol is produced, and it has been identified as Delta(8(9),22)-ergostadiene-3beta-ol. This sterol is converted to ergosterol by wild-type yeasts and is observed transiently in cells during aerobic adaption of anaerobically grown wild-type yeasts. The new sterol is proposed as an intermediate in ergosterol biosynthesis.
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The effects of inhibitors of bacterial deoxyribonucleic acid (DNA) synthesis upon logarithmically growing cultures of Saccharomyces cerevisiae were investigated. Cell division, ribonucleic acid (RNA) synthesis, and DNA synthesis were measured after addition of nalidixic acid, fluorodeoxyuridine, or phenethyl alcohol to cultures of yeast growing in defined and complex media. Both nalidixic acid and fluorodeoxyuridine had only temporary effects on nucleic acid synthesis in cultures growing in defined medium, and little or no observable effect on cultures growing in complex medium. Neither compound inhibited colony formation on complex solid medium, although growth was slow on defined solid medium. Phenethyl alcohol caused complete inhibition of DNA synthesis, RNA synthesis, and cell division in cultures growing in defined medium. In cultures growing in complex medium, RNA synthesis and cell division were inhibited to a lesser extent. A slight increase in DNA was observed in the presence of the inhibitor.
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The effect of different respiratory inhibitors on the ergosterol content of microaerobically grown non-proliferating yeast cultures was monitored during adaptation to oxygen. It was found that dinitrophenol, azide, and cyanide, which act on the mechanism of the respiratory chain, cause a marked stimulation of sterol production. Acriflavine and chloramphenicol, which affect the synthesis of the respiratory apparatus, caused a delay in the onset of ergosterol synthesis or a marked decrease in sterol content. The data obtained provide presumptive evidence that a component of sterol formation is synthesized on the 70S ribosomal system of the mitochondrion and induced in the presence of oxygen.
Serine transhydroxymethylase appears to be the first enzyme in the synthesis of the methyl group of methionine. Properties of serine transhydroxymethylase activity as assayed by the production of formaldehyde were correlated with properties of cell-free extracts for the methylation of homocysteine deriving the methyl group from the beta-carbon of serine. The reaction required pyridoxal phosphate and tetrahydrofolic acid, and was characterized in cell-free extracts with respect to Michaelis constant, pH optimum, incubation time, and optimal enzyme concentration. The activity was sensitive to inhibition by methionine, and to a much greater extent by S-adenosylmethionine. Serine transhydroxymethylase and the methylation of homocysteine reactions were not repressed by methionine and were stimulated by glycine. The activities of cell-free extracts for these reactions were significantly higher in cells in exponential than in stationary growth. When cells were grown in 10 mm glycine, the activities remained high throughout the culture cycle. The data indicated that glycine rather than methionine is involved in the control of the formation of the enzyme.
Synthesis of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein was determined in Saccharomyces cerevisiae during amino acid and pyrimidine starvation and during shift-up and shift-down conditions. During amino acid starvation, cell mass, cell number, and RNA continued to increase for varying periods. During amino acid and pyrimidine starvation, cell mass and RNA showed little increase, whereas total DNA increased 11 to 17%. After a shift from broth medium to a minimal defined medium, increase in RNA and protein remained at the preshift rate before assuming a lower rate. DNA increase remained at an intermediate rate during shift-down, and then dropped to a low rate. During shift-up from minimal to broth medium, increase in cell number, protein, and DNA showed varying lag periods before increasing to the new rate characteristic of broth medium; each of these quantities exhibited a step sometime in the first 2 hr after transfer to rich medium, suggesting a partial synchronous division. Immediately after shift-up, RNA synthesis assumed a high rate, and then dropped to a rate characteristic of growth in the rich medium after about 1 hr.
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The role of cystathionine in methionine biosynthesis in wild-type and auxotrophic strains of Saccharomyces cerevisiae was studied. Homocysteine and cysteinerequiring mutants were selected for detailed study. Exogenously supplied cystathionine, although actively transported by all strains tested, could not satisfy the organic sulfur requirements of the mutants. Cell-free extracts of the wild-type, homocysteine, and cysteine auxotrophs were shown to cleave cystathionine. Pyruvic acid and homocysteine were identified as teh products of this cleavage. A mutant containing an enzyme which could cleave cystathionine to homocysteine in cell-free experiments was unable to use cystathionine as a methionine precursor in the intact organisms. The significance of this finding is discussed.
A water-soluble complex containing ergosterol together with a component of yeast has been isolated. The complex can be isolated from commercial yeast extract to which ergosterol has been added or directly from whole yeast cells. The complexing component has the properties of a large polysaccharide and the binding between the sterol and the polysaccharide appears to be noncovalent. The complex is easily prepared and is stable in aqueous solution; ergosterol in this solution is metabolically available to yeast cells to which it is added.
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Extracts of Saccharomyces cerevisiae were used to develop a cell-free system capable of converting the beta-carbon of serine into the methyl group of methionine. No requirement for either S-adenosylmethionine or S-adenosylhomocysteine could be demonstrated for net methionine biosynthesis. Growth of the cells in B(12) did not affect the reaction. The mechanism for the methylation of homocysteine in yeast appears to be similar to the non-B(12) system in Escherichia coli.