Triosephosphate isomerase: energetics of the reaction catalyzed by the yeast enzyme expressed in Escherichia coli

Triosephosphate isomerase from bakers' yeast, expressed in Escherichia coli strain DF502(p12), has been purified to homogeneity. The kinetics of the reaction in each direction have been determined at pH 7.5 and 30 degrees C. Deuterium substitution at the C-2 position of substrate (R)-glyceraldehyde phosphate and at the 1-pro-R position of substrate dihydroxyacetone phosphate results in kinetic isotope effects on kcat of 1.6 and 3.4, respectively. The extent of transfer of tritium from [1(R)-3H]dihydroxyacetone phosphate to product (R)-glyceraldehyde phosphate during the catalyzed reaction is only 3% after 66% conversion to product, indicating that the enzymic base that mediates proton transfer is in rapid exchange with solvent protons. When the isomerase-catalyzed reaction is run in tritiated water in each direction, radioactivity is incorporated both into the remaining substrate and into the product. In the "exchange-conversion" experiment with dihydroxyacetone phosphate as substrate, the specific radioactivity of remaining dihydroxyacetone phosphate rises as a function of the extent of reaction with a slope of about 0.3, while the specific radioactivity of the products is 54% that of the solvent. In the reverse direction with (R)-glyceraldehyde phosphate as substrate, the specific radioactivity of the product formed is only 11% that of the solvent, while the radioactivity incorporated into the remaining substrate (R)-glyceraldehyde phosphate also rises as a function of the extent of reaction with a slope of 0.3. These results have been analyzed according to the protocol described earlier to yield the free energy profile of the reaction catalyzed by the yeast isomerase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dependence of inorganic polyphosphate chain length on the orthophosphate content in the culture medium of the yeast Saccharomyces cerevisiae

The content of inorganic linear polyphosphate (polyP) and the polymeric degree (n) of these compounds were determined in the process of growth of the yeast Saccharomyces cerevisiae VKM Y-1173 in a medium, which contained varying Pi amount with the constant level of all the necessary components. For this purpose, a combination of chemical methods of polyP extraction and 31P-NMR spectroscopy studies of their chain length were used. After 7 h of phosphate starvation, the yeast was shown to use almost completely the phosphate reserve in the form of polyP localized in various cell compartments to support their vitality. The polyP drop was followed by a considerable shortening of the polymer chain length of acid-soluble (polyP1) and two alkali-soluble (polyP3 and polyP4) fractions. Under the same conditions, the content of a salt-soluble fraction (polyP2) decreased almost 20-fold followed by a simultaneous increase of the chain length nearly 2-fold. As a result, fraction chain length ranged up to n = 40-45. Replacement of the yeast cells after phosphate starvation to a complete phosphate- and glucose-containing medium resulted in super-accumulation ("overcompensation") of polyP within 2 h mainly in polyP3 and, to a lesser degree, in polyP1, polyP2, and polyP5 fractions. In polyP4 fraction localized as polyP3 at the cell surface, the polyP super-accumulation was not detected. The increase of polyP amount in the fractions mentioned turned out not to be accompanied by simultaneous elongation of their chain length and occurred at the lowest level that is characteristic of a polymer level for each fraction. Further cultivation of the yeast on the complete medium during 2 h had little or no effect on polyP content in the cells but led to elongation of polyP chain length especially in the polyP3 and polyP4 fractions. A phenomenon of considerable elongation of polyP chain length against the background of their fixed content revealed in the yeast growing on the complete medium suggests that these organisms possess a previously unknown discrete way of polyP biosynthesis, which results first in the formation of comparatively low-molecular-mass chains followed by that of high-molecular-mass polymers.     

Large-scale preparation of the oligosaccharide phosphate fraction of Pichia holstii NRRL Y-2448 phosphomannan for use in the manufacture of PI-88.

Mild acid-catalysed hydrolysis of the extracellular phosphomannan of the yeast Pichia holstii NRRL Y-2448 produces a high-molecular-weight phosphomannan core, a low-molecular-weight oligosaccharide phosphate fraction, and a neutral oligosaccharide fraction. A method was developed for the large-scale preparation of the oligosaccharide phosphate fraction, consisting predominantly of the pentasaccharide phosphate, 6-O-PO3H2-alpha-D-Man-(1 --> 3)-alpha-D-Man-(1 --> 3)-alpha-D-Man-(1 C 3)-alpha-D-Man-(1 --> 2)-D-Man, for use in the manufacture of the promising new anti-cancer agent, PI-88. Further insights were also gained into the structure of the phosphomannan by HPLC analysis of the time course of the hydrolysis reaction.     

Stereochemical Control in Microbial Reduction. Part 10. Asymmetric Reduction of Alkyl 3-Methyl-2-Oxobutanoate with Immobilized Bakers' Yeast in Hexane

Esters of 3-methyl-2-oxobutanoic acid are reduced with bakers' yeast by three methods: free bakers' yeast in water, immobilized bakers' yeast in water, and immobilized bakers' yeast in hexane. Although (R)-hydroxy esters are obtained in all cases, the enantiomeric excess varies from 3% (reduction of the methyl ester with free bakers' yeast in water) to 93% (reduction of the butyl ester with immobilized bakers' yeast in hexane) depending on the structure of substrate and on the reaction conditions. The mechanism of the present stereochemical control is discussed.     

Characterization of phosphoprotein phosphatases and phosphorylase phosphatase from yeast

Three peaks of protein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) activity (fractions a, b and c) acting on muscle phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) were separated by DEAE-cellulose chromatography of yeast extracts. In contrast to fractions a and b, only fraction c was able to liberate phosphate from 32P-labelled inactivated yeast phosphorylase. The activity of fraction c on both substrates was totally dependent on the presence of bivalent metal ions (Mg2+, Mn2+), and was activated by Mg . ATP. Following freezing in the presence of mercaptoethanol, fractions a and b were also able to dephosphorylate yeast phosphorylase. Rabbit muscle phosphoprotein phosphatase inhibitors 1 and 2 showed that yeast phosphatases acting on muscle phosphorylase were inhibited by inhibitor 2 but not by inhibitor 1. The action of fraction c on yeast phosphorylase was not inhibited by either inhibitor. The native yeast phosphorylase phosphatase (EC 3.1.3.17) was purified 8000-fold by ion-exchange chromatography, casein-Sepharose chromatography and Sephadex G-200 gel filtration. The purified enzyme was unable to dephosphorylate rabbit muscle phosphorylase a, but acted on casein phosphate (Km 3.3 mg/ml). Molecular weight was estimated to be 78 000 and pH optimum 6.5-7.5. Activity of the enzyme was dependent on bivalent metal ions (Mg2+, Mn2+) and was inhibited by fluoride (Ki 20 mM) and succinate (Ki 10 mM).     

Inactivation of the thiamine transport system in Saccharomyces cerevisiae with O-bromoacetylthiamine

We have synthesized and characterized O-bromoacetylthiamine (BrAcThiamine), a new reagent for inactivating the thiamine transport system in Saccharomyces cerevisiae. A Lineweaver-Burk plot of data from the transport kinetic measurements showed that BrAcThiamine was a competitive inhibitor of thiamine transport in S. cerevisiae with a Ki value of 0.60 microM. Incubating BrAcThiamine with yeast cells at 40 degrees C in 0.05 M potassium phosphate buffer, pH 5.0, caused concentration- and time-dependently a remarkable loss of thiamine transport activity. The inactivating reaction of yeast thiamine transport by BrAcThiamine proceeded most effectively at pH 5.0, coinciding with the optimal pH of the transport activity. Thiamine and thiamine analogs (pyrithiamine and O-acetylthiamine) protected yeast thiamine transport activity against the inactivation by BrAcThiamine. In addition, it was found that a membrane fraction prepared from yeast cells treated with BrAcThiamine had a thiamine-binding activity only 20% of that from control cells without inactivating the binding activity of the soluble fraction. These results suggest that BrAcThiamine inactivates the uptake activity by irreversible binding to the binding site of carrier protein(s) in the thiamine transport system.     

Determination of the composition of the oligosaccharide phosphate fraction of Pichia (Hansenula) holstii NRRL Y-2448 phosphomannan by capillary electrophoresis and HPLC

The promising new anticancer agent, PI-88, is prepared by the sulfonation of the oligosaccharide phosphate fraction of the extracellular phosphomannan produced by the yeast Pichia (Hansenula) holstii NRRL Y-2448. The composition of the oligosaccharide phosphate fraction was determined by capillary electrophoresis (CE) with indirect UV detection using 6 mM potassium sorbate at pH 10.3 as the background electrolyte. Further confirmation of the composition was obtained by HPLC analysis of a sample dephosphorylated by treatment with alkaline phosphatase. The structure of the hexasaccharide component has been determined by isolation and NMR spectroscopic analysis of its dephosphorylated derivative. Additionally, the structure of a second, previously undetected tetrasaccharide component (a hexosamine) has been determined by isolation and NMR spectroscopic analysis of the acetate of its dephosphorylated derivative. It is demonstrated that CE is an ideal method for the quality control of the oligosaccharide phosphate fraction for use in the production of PI-88.     

Stereochemical Control in Microbial Reduction. Part 10. Asymmetric Reduction of Alkyl 3-Methyl-2-Oxobutanoate with Immobilized Bakers' Yeast in Hexane

Esters of 3-methyl-2-oxobutanoic acid are reduced with bakers' yeast by three methods: free bakers' yeast in water, immobilized bakers' yeast in water, and immobilized bakers' yeast in hexane. Although (R)-hydroxy esters are obtained in all cases, the enantiomeric excess varies from 3% (reduction of the methyl ester with free bakers' yeast in water) to 93% (reduction of the butyl ester with immobilized bakers' yeast in hexane) depending on the structure of substrate and on the reaction conditions. The mechanism of the present stereochemical control is discussed.     

CTP-dependent lipid kinases of yeast

Membrane fractions from yeast Saccharomyces cerevisiae catalyzed a transfer of gamma-phosphate from [gamma-32P]CTP into membranous lipids. Phosphorylated compounds were identified as phosphatidic acid and dolichyl phosphate (DolP). The membrane fraction also catalyzed phosphorylation of the exogenous dolichol. The activity of the phosphorylating enzymes could be modified by the yeast growing conditions; i.e., the enzyme from yeast grown aerobically favored the synthesis of phosphatidate over dolichyl phosphate in the ratio of 3:1, whereas the membrane fraction from anaerobically grown yeast synthesized PA and DolP in the ratio of 0.5:1. The activity of the phosphorylating enzymes could also be modified by divalent cations and the concentration of detergents. Phosphorylation of lipids does not occur in the presence of [gamma-32P]ATP and is not influenced by the presence of UTP or GTP. This result points to the specific role of CTP as a gamma-phosphate donor for the synthesis of phosphatidate and dolichyl phosphates in the yeast system.     

Alcohols dehydrate lipid membranes: an infrared study on hydrogen bonding.

The effects of alcohols (methanol, ethanol, and n-butanol) on the hydrogen bonding of dipalmitoylphosphatidylcholine (DPPC) were studied by Fourier-transform infrared spectroscopy (FTIR) in water-in-oil (carbon tetrachloride) reversed micelles. The bound O-H stretching mode of water, bonded to DPPC, appeared as a broad band at around 3400 cm-1. The O-H bending mode of this complex appeared as a weak broad band at 1644 cm-1. No free O-H signal was observed. When alcohols were added, a part of DPPC-bound water was replaced by the alcohols. The released 'free' water appeared at 3680 cm-1. This free O-H stretching band represents water-alcohol complex. A new broad band of O-H stretching appeared at 3235 cm-1, which represents the alcohol molecules bound to the phosphate moiety of DPPC. When the alcohol concentration was increased, the intensities of the free O-H stretching and bending bands increased. The P = O- antisymmetric stretching band at 1238 cm-1 became broader and shifted to lower frequencies. This means that alcohols interacted with the phosphate moiety and replaced the bound water. In the deconvoluted spectra of the C = O stretching mode, the ratio between the free sn-2 and the hydrogen-bonded sn-2 bands increased; a part of the bound water at the sn-2 carbon in the glycerol skeleton is also released and the free sn-2 signal increased. From the change in the intensity of the P = O- stretching band, the partition coefficients of alcohols between the phosphate region of DPPC and water were estimated: methanol 7.8, ethanol 16.7 at 22.0 degrees C in mole fraction bases. In molality, these values translates into methanol 0.21 and ethanol 0.45. These results indicate that short-chain alcohols interact with lipid membranes at the phosphate moiety at the hydrophilic head, weaken the membrane-water interaction, and destabilize membranes.     

Mechanisms for release of sediment-bound phosphate to water and the effects of agricultural land management on fluvial transport of particulate and dissolved phosphate

Sediment-bound phosphate includes organic and inorganic forms, but the inorganic fraction contains most of the P that can be released into water. The non-apatite inorganic P (NAIP) fraction of sediment-bound phosphate varies considerably with geochemistry and anthropogenic additions (fertilizer, livestock wastes, P adsorbed from municipal wastewater discharges). A small fraction of the NAIP is sufficiently labile to desorb into water, and this release can be described by dissolution or adsorption/ desorption mechanisms. Agricultural practices, such as phosphate fertility management and conservation tillage, which affect the levels of phosphate and sediment leaving the land, will determine the partition of sediment-bound P and dissolved P in water draining into lakes, with implications for the availability of that phosphate to phytoplankton.     

Studies on Microbial Metabolism of Sugar Nucleotides

The fermentative production of uridine diphosphate N-acetylglucosamine (UDPAG) from 5′-UMP and glucosamine by dried cells of baker’s yeast was studied. UDPAG was found to accumulate in a reaction system containing 5′-UMP, glucosamine, fructose, inorganic phosphate and magnesium ions with dried baker’s yeast as an enzyme source. UDPAG was separated from the reaction mixture by means of anion exchange column chromatography and was identified by several biochemical methods.

The reaction conditions for the fermentative production of UDPAG were examined. The yield of UDPAG was about 40~66% based on 5′-UMP added when fermentation conditions were optimized. The concentration of glucosamine and potassium phosphate buffer, and pH as well as the water content of dried cells greatly affected the formation of UDPAG.     

Characterization of the Repair of Injury Induced by Freezing Salmonella anatum

Fast freezing and slow thawing of Salmonella anatum cells suspended in water resulted in injury of more than 90% of the cells that survived the treatment. The injured cells failed to form colonies on the selective medium (xyloselysine-peptone-agar with 0.2% sodium deoxycholate) but did form colonies on a nonselective (xylose-lysine-peptone-agar) plating medium. In Tryptic soy plus 0.3% yeast extract broth or minimal broth, most of the injured cells repaired within 1 to 2 hr at 25 C. Tryptic soy plus yeast extract broth supported repair to a greater extent than minimal broth. Phosphate or citrate at concentrations found in minimal broth supported repair of some cells. MgSO(4), when present with inorganic phosphate or citrate or both, increased the extent of repair. The repair process in the presence of phosphate was not prevented by actinomycin D, chloramphenicol, and D-cycloserine, but was prevented by cyanide and 2,4-dinitrophenol (only at pH 6). This suggested that the repair process might involve energy metabolism in the form of adenosine triphosphate. The freeze-injured cells were highly sensitive to lysozyme, whereas unfrozen fresh cells were not. In the presence of phosphate or minimal broth this sensitivity was greatly reduced. This suggested that, at least in some of the cells, the injury involved the lipopolysaccharide of the cell wall and adenosine triphosphate synthesis was required for repair.     

Extracellular phosphomannan as a phosphate reserve in the yeast Kuraishia capsulata

We have found that extracellular phosphomannan is the main phosphate reserve in the yeast Kuraishia capsulata, in contrast to other yeast species effectively absorbing P_i. Under nitrogen starvation, K. capsulata absorbed essentially all P_i from the medium containing 240 mM glucose, 2.5 mM MgSO₄, and 11 mM KH₂PO₄. Inorganic polyphosphate level in the cells was about 14% of the P_i absorbed. Most of the P_i (～60%) was found in the fraction of extracellular phosphomannan that can be used as a carbon and phosphorus source by this yeast in deficient media.     

In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor

The in vitro synthesized precursor of the alpha-factor pheromone, prepro-alpha-factor, of Saccharomyces cerevisiae was translocated across yeast microsomal membranes in either a homologous or a wheat germ cell free system. Translocated prepro-alpha-factor was glycosylated, sedimented with yeast microsomal vesicles, and was protected from digestion by added protease, but was soluble after alkaline sodium carbonate treatment. Thus prepro-alpha-factor was properly sequestered within yeast microsomal vesicles, but was not integrated into the lipid bilayer. In marked contrast to protein translocation across mammalian microsomal membranes, translocation of prepro-alpha-factor across yeast microsomal membranes could occur posttranslationally. This reaction required protein components in the yeast microsomal fraction that could be inactivated by alkylation or proteolysis, was ATP-dependent, and was insensitive to the presence of a variety of uncouplers and ionophores.     

Effect of culture media on the composition of free amino acids in Saccharomyces cerevisiae yeast

The quantitative and qualitative compositions of free amino acids of the yeast Saccharomyces cerevisiae Y-503 cultivated in different nutrient media were studied by liquid chromatography. The yeast grown in the medium containing geothermal water was shown to accumulate more amino acids. During lyophilization, the stabilization of the physiological activity of the yeast in this nutrient medium was observed. The increased biological value of dry yeast was shown to depend on the content of free amino acids, including essential amino acids: arginine, histidine, leucine, isoleucine, lysine, threonine, serine and phenylalanine.     

Time-resolved magnetic resonance spectroscopic study of freeze-trapped yeast

Rapidly changing metabolic events in actively respiring yeast under strictly physiological conditions were approached by freeze-trapping analysis with 31P magnetic resonance spectroscopy. A 50% wet weight/volume suspension of Saccharomyces cerevisiae was freeze-trapped with 20% ethylene glycol and 5% methanol as an antifreezing agent, and with 3 mM creatine phosphate as an external standard. A phosphorus spectrum of the freeze-trapped yeast was measured for 20 min at -18 degrees C at 202.46 MHz, since energy-related phosphate compounds in yeast were stable at least for 20 min at the temperature. The time scale, defined as (time required for NMR measurement)/(time for trapping), was expanded by the freeze-trapping analysis by 67-times, since the time for trapping was 0.3 min. The use of creatine phosphate as an external standard enabled exact quantification of phosphorus resonances. 31P-NMR study of freeze-trapped yeast affords a well time-resolved and highly sensitive method to study phosphate metabolism.     

限磷对产甘油假丝酵母甘油合成与胞内磷积累的影响

研究了磷酸盐限量对产甘油假丝酵母甘油合成与胞内磷积累的影响。结果表明, 当酵母细胞从适磷或富磷培养基转接入低磷培养基时, 发酵过程中胞内积累的磷逐渐减少; 而当菌体从低磷培养基转接入适磷或富磷培养基时, 发酵过程中胞内聚磷酸盐的积累量迅速增加。当细胞在第14小时和第38小时从适磷培养基转接入低磷培养基时甘油得率分别高达60.9%和61.4%, 而甘油产率则分别为2.03 g/(L·h)和2.23 g/(L·h)。这些现象说明限制发酵培养基中的磷浓度是产甘油假丝酵母高产甘油的必要条件, 并为其反复分批发酵法生产甘油提供了重要依据。     

A rapid method for the purification of extrachromosomal DNA from eukaryotic cells.

A simple and efficient procedure to purify the low molecular weight extrachromosomal DNA from eukaryotic cells is described. Gentle lysis of cells with urea and sodium dodecyl sulfate in 0.24 M phosphate buffer (pH 6.8) is followed by the removal of high molecular weight bulk DNA by centrifugation. Protein and RNA are removed from the supernatant by hydroxyapatite chromatography in urea/phosphate buffer. Urea is then removed with 0.15 M phosphate buffer and the extrachromosomal DNA, virtually free from protein and RNA, is finally eluted in 0.5 M phosphate buffer. The procedure allows the recovery of about 99% simian virus 40 (SV40) DNA from infected monkey kidney cells in the extrachromosomal fraction. In normal mouse, monkey, andhuman cells, approximately 1% of total cell DNA appears to be extrachromosomal.     

Purification and properties of phosphorylase from baker's yeast

A rapid, reliable method for purification of phosphorylase, yielding 200-400 mg pure phosphorylase from 8 kg of pressed baker's yeast, is described. The enzyme is free of phosphorylase kinase activity but contains traces of phosphorylase phosphatase activity. Phosphorylase constitutes 0.5-0.8% of soluble protein in various strains of yeast assayed immunochemically. The subunit molecular weight (Mr) of yeast phosphorylase is around 100,000. The enzyme is composed of two subunits in various ratios, differing slightly in molecular weight and N-terminal sequence. Both are active. Only the enzyme species containing the larger subunit can form tetramers and higher oligomers. The activated enzyme is dimeric. Correlated with specific activity (1 to 110 U/mg), phosphorylase contained between less than 0.1 to 0.74 covalently bound phosphate per subunit. Inactive forms of phosphorylase could be activated by phosphorylase kinase and [gamma-32P]ATP with concomitant phosphorylation of a single threonine residue in the aminoterminal region of the large subunit. The small subunit was not labeled. The incorporated phosphate could be removed by yeast phosphorylase phosphatase, resulting in loss of activity of phosphorylase, which could be restored by ATP and phosphorylase kinase.