Biomedical subjects
Publications and source records attributed to I Goldberg.
The nonpolar lipids of methanol-grown bacteria which utilize one-carbon (C1) compounds via the RMP pathway (Pseudomonas C, Pseudomonas methylotropha, and Methylomonas methanolica) were found to contain squalene in concentrations between 0.1 to 1.16 mg/g of cell (dry weight). Squalene could not be detected in lipid extracts of methanol-grown bacteria which utilize C1 compounds via the serine pathway.
Extracts of Pseudomonas C grown on methanol as a sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD+) and formate dehydrogenase (NAD+) were also detected in the extracts. The addition of D-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD+ or NADP+. In addition, the oxidation of [14C]formaldehyde to CO2, by extracts of Pseudomonas C, increased when D-ribulose 5-phosphate was present in the assay mixtures. The amount of radioactivity found in CO2, was 6;8-times higher when extracts of methanol-grown Pseudomonas C were incubated for a short period of time with [1-14C]glucose 6-phosphate than with [U-14C]glucose 6-phosphate. These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO2 both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant.
A sensitive and rapid bioassay for the determination of the antilipogenic compounds cerulenin and CM-55 is described. The bioassay is based on the inhibitory effect of cerulenin and CM-55 on the in vivo luminescence of an aldehyde-requiring mutant of the marine bacterium Beneckea harveyi. A total quantity as low as 0.1 mug of cerulenin can be determined within 15 min with an error of +/-2%. The bioassay, as presented, is specific for compounds that are known to inhibit fatty acid biosynthesis and, as such, it might be used as a general screening method for the detection of antilipogenic compounds.
The viability of Streptococcus lactis and Lactobacillus sp. A-12 after freezing at -17 degrees C for 48 h was better preserved when the cells were grown in medium supplemented with oleic acid or Tween 80 (polyoxyethylene sorbitan monooleate). A pronounced change in the cellular fatty acid composition was noted when the bacteria were grown in the presence of Tween 80. In S. lactis the ratio of unsaturated to saturated fatty acids increased from 1.18 to 2.55 and in Lactobacillus sp. A-12 it increased from 0.85 to 1.67 when Tween 80 was added to the growth medium. The antibiotic cerulenin markedly inhibited the growth of lactic acid bacteria in tomato juice (TJ) medium but had almost no effect on the growth of the bacteria in TJ medium containing Tween 80 (or oleic acid). The antibiotic inhibited markedly the incorporation of [1-C]acetate but had no inhibitory effect on the incorporation of exogenous [1-C]oleate (or [1-C]palmitate) into the lipid fractions of lactic acid bacteria. Thus, the fatty acid composition of lactic acid bacteria, inhibited by the antibiotic cerulenin, can be modulated by exogenously added oleic acid (or Tween 80) without the concurrent endogenous fatty acid synthesis from acetate. The data obtained suggest that cerulenin inhibits neither cyclopropane fatty acid synthesis nor elongation of fatty acid acyl intermediates. The radioactivity of cells grown in the presence of [1-C]oleate and cerulenin was associated mainly with cyclopropane Delta19:0, 20:0 + 20:1, and 21:0 acids. As a consequence, cerulenin caused a decrease in the ratio of unsaturated to saturated fatty acids in lactic acid bacteria as compared with cells grown in TJ medium plus Tween 80 but without cerulenin. Cerulenin caused a decrease in the viability of S. lactis and Lactobacillus sp. A-12 after freezing at -17 degrees C for 48 h only when Tween 80 was present in the growth medium. We conclude that the sensitivity of lactic acid bacteria to damage from freezing can be correlated with specific alterations in the cellular fatty acids.
Differences in phospholipid and fatty acid composition were found among bacteria using the serine or the ribulose monophosphate pathway for the assimilation of C1 compounds. These differences might be used as an additional criterion for the classification of methanol-utilizing bacteria.
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A methanol-oxidizing enzyme has been purified from Pseudomonas C, grown on methanol as a sole source for carbon and energy. The purification procedure involved ammonium sulphate precipitation, ion-exchange chromatography and gel filtration and resulted in a yield of 35.4%. Enzyme activity can be coupled to phenazine methosulfate and requires the presence of ammonium ions in the assay mixtures. The enzymes possesses a broad specificity for primary alcohols. Formaldehyde is also oxidized by the purified enzyme. The Km value for methanol is 15 muM. The optimum pH for the oxidation of both methanol and formaldehyde is about 10.4. The enzyme has a molecular weight of about 128000 and consists of two subunits each having a molecular weight of 60000.
Several bacteria utilizing C1-compounds as sole carbon sources were grown on these substrates in continuous culture. The molar yield values (g of cell dry wt/mol of substrate utilized) of bacteria which utilize C1-compounds via the ribulose monophosphate pathway were between 15.7 to 17.3 when grown on methanol; while the molar yield values of bacteria which use the serine pathway for the assimilation of C1-compounds varied between 9.8 and 13.1. The molar yield values of different bacteria which use the serine pathway decreased as the oxidation levels of the C1-growth substrates increased. On formaldehyde the values were between 7.2 to 9.6, whereas on formate the values varied from 3.3 to 6.9. It appears that bacteria utilize C1-compounds more efficiently via the ribulose monophosphate pathway than via the serine pathway. The oxidation step from methanol to formaldehyde (and from methylamine to formaldehyde) in the bacteria studied may be energy yielding. A comparison has been made between the experimental yield values obtained and theoretical values.
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The light-induced formation of the photosynthetic membranes (greening) in y-1 mutant of Chlamydomonas reinhardi requires synthesis of new proteins which become incorporated into the growing membranes. It has been shown previously (Eytan and Ohad, 1970) that proteins synthesized by both chloroplast and cytoplasmic ribosomes concur in the formation of functional photosynthetic membranes, indicating the presence of a mechanism permitting the specific transfer of membrane proteins synthesized in the cytoplasm into the chloroplast. Transfer of such proteins cannot yet be identified by the usual biochemical techniques unless they become part of the growing photosynthetic membranes. However, it is possible to follow their synthesis and translocation between the different cellular compartments by use of quantitative electron microscopic radioautography. In the present work, the radioautographic grain distribution among chloroplast, chloroplast membrane, nucleus, mitochondria, and the remainder of the cytoplasm (cytosol) was carried out following short radioactive pulse-labeling and chase during greening of dark-grown mutants in the presence or absence of protein synthesis inhibitors. The results indicate that transport of some of the proteins of cytoplasmic origin to their final location within the chloroplast is at least partially controlled by concomitant synthesis of proteins by the chloroplast ribosomes.
Pseudomonas C can grow on methanol, formaldehyde, or formate as sole carbon source. It is proposed that the assimilation of carbon by Pseudomonas C grown on different C1 growth substrates proceeds via one of two metabolic pathways, the serine pathway or the allulose pathway (the ribose phosphate cycle of formaldehyde fixation). This contention is based on the distribution of two key enzymes, each of which appears to be specifically involved in one of the assimilation pathways, glycerate dehydrogenase (serine pathway) and hexose phosphate synthetase (allulose pathway). The assimilation of methanol in Pseudomonas C cells appears to occur via the allulose pathway, whereas the utilization of formaldehyde or formate in cells grown on formaldehyde or formate as sole carbon sources appears by the serine pathway. When methanol is present together with formaldehyde or formate in the growth medium, the formaldehyde or formate is utilized by the allulose pathway.
On reexamination Pseudomonas C was found to be incapable of growth on formaldehyde or formate as a sole carbon source and to contain a hexose phosphate synthase activity when grown on methanol.
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Pseudomonas C was grown in continuous culture on methanol, formaldehyde, or formate as sole carbon source. On methanol mu(max) = 0.49/h and yield constant (Y) = 0.54; on formaldehyde and on unsupplemented media, mu(max) was about 0.2/h and Y was 0.15, whereas addition of p-aminobenzoic acid, folic acid, serine, or glycine to the medium raised Y to about 0.26 to 0.29, and addition of p-aminobenzoic acid, folic acid, serine, nicotinamide adenine dinucleotide, and Tween 80 raised the yield to 0.35. On formate and on unsupplemented media, mu(max) = 0.2/h and Y = 0.02, whereas addition of 0.1 mM p-aminobenzoic acid increased mu(max) to about 0.47 and Y to about 0.23. At low cell concentrations or growth rates a beneficial effect of CO(2) was observed. Formaldehyde or formate, when added together with methanol, were utilized simultaneously with the methanol.
Microsomal enzyme systems from mouse brain that catalyze, respectively, the elongation of palmitoyl-coenzyme A (palmitoyl-CoA), stearoyl-CoA, or arachidyl-CoA appear and reach maximal activity at different times after birth of the animal. A specific C(20)-CoA elongating system exists in mouse brain in addition to the previously recognized C(16)-CoA and C(l8)-CoA elongating enzymes. The C(20)-CoA elongation system is severely reduced in the mutant quaking mouse.
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The antibiotic cerulenin markedly inhibits the growth of Escherichia coli. The effects of the antibiotic on cellular syntheses were studied by measuring the incorporation of labeled precursors into lipids and macromolecules. During the first 40 min after the addition of cerulenin to a culture of growing cells, lipid synthesis was inhibited more than 90% and ribonucleic acid and deoxyribonucleic acid synthesis about 25%, whereas protein synthesis was not affected. At later periods after cerulenin addition (1 to 2 h), the inhibition of cell growth and of lipid and protein synthesis was complete. Upon removal of cerulenin from the culture, growth was restored and lipid synthesis resumed more rapidly than did the synthesis of protein. Addition of both palmitate and oleate, but not of either fatty acid alone, reversed the inhibition of growth by cerulenin. These findings support the conclusion that the antibiotic effects of cerulenin are due to a specific inhibition of fatty acid synthesis.
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