Plasma vitamin B6 concentrations in Nigerian adolescents.

Vitamin B6 status was assessed from dietary and plasma vitamin B6 concentration using Saccharomyces uvarum as test organism, and erythrocyte alanine amino transferase activity (E-ALAT). The subjects participating in the study were 72 males and 30 females (aged 10-18 years) who resided in a boarding institution. Mean daily dietary vitamin B6 and protein intakes were 1.56 +/- 0.42 mg and 63.0 +/- 9.6 g respectively. The corresponding mean plasma vitamin B6 concentration was 194 +/- 44.2 nmol/l. Neither age, sex nor menarche had significant effect (P less than 0.05) on plasma vitamin B6 concentration of these adolescents. Dietary vitamin B6 but not protein intake correlated with plasma vitamin B6 (r = 0.3076, P less than 0.002). However, low dietary vitamin B6/protein ratio (less than 0.02 mg/g) was not reflected in plasma vitamin B6 concentration, but low plasma vitamin B6 concentration (120-179 nmol/l) corresponded to low E-ALAT activity after in vitro addition of pyridoxal phosphate (E-ELAT 16 per cent). A stimulation above 25 per cent, 16-25 per cent and below 16 per cent was used as an indicator of poor, marginal and adequate vitamin B6 status, respectively. Based on these criteria 30.7 per cent, 17.8 per cent and 51.5 per cent of subjects, with corresponding mean plasma vitamin B6 of 150 +/- 28.4, 192 +/- 8.5 and 237 +/- 18.7 nmol/l are of deficient, marginal and adequate vitamin B6 status, respectively.

Examination of protein sequence homologies. VI. The evolution of Escherichia coli L7/L12 equivalent ribosomal proteins ('A' proteins), and the tertiary structure.

Sequence homologies among 23 complete and two partial sequences of ribosomal 'A' proteins from eukaryotes, metabacteria, eubacteria and chloroplasts, equivalent to Escherichia coli L7/L12, were examined using a correlation method that evaluates sequence similarity quantitatively. Examination of 325 comparison matrices prepared for possible combinations of the sequences indicates that 'A' protein sequences can be classified into two types: one is the "prototype" from eubacteria and chloroplasts, and the other is the "transposition type" from eukaryotes and metabacteria, which must have resulted from the internal transposition of the prototype sequence. The transposition type of eukaryotes can further be classified into P1 and P2 lines. Sequences of the P1 line are closer to those of metabacteria than to those of the P2 line. Eleven gaps, as deletion or insertion sites of amino acid residues, are necessary for an alignment of all the sequences. According to the crystallographic data for the C-terminal fragment (CTF) from E. coli L7, all the gaps involved in the CTF are located between segments that correspond to structural and functional elements such as alpha helix, beta strand, turning loop or hinge part. The existence of specific "preservation units" in these molecules is suggested. In contrast, the transposition site is located at the center of an alpha helix element that is involved in a folding domain, indicating that the transposition event was extremely drastic.

Fractionation and characterization of yeast proteins.

Fractional profile of proteins from baker's, brewer's, and alcoholic yeasts was studied, applying a consecutive treatment of commercial biomasses, with solutions with a gradient of ionic strength and pH. It was found that a large part (35-50%) of total proteins from investigated biomasses is extracted by means of 0.01 M KCl, pH 7.0, which together with proteins extracted by means of 0.65 M KCl, pH 9.5, makes the part of the easily extractable proteins (50-64% of the total protein amount). The remaining part of proteins is extracted by means of alkaline solutions only. The amino-acid composition of the isolated fractions indicated that they are suitable for usage as food product ingredients.

Peptide elongation factor 1 from yeasts: purification and biochemical characterization of peptide elongation factors 1 alpha and 1 beta (gamma) from Saccharomyces carlsbergensis and Schizosaccharomyces pombe.

Cytoplasmic elongation factor 1 alpha (EF-1 alpha) [corrected] was purified to homogeneity in high yield from the two different yeasts Saccharomyces carlsbergensis (S. carls.) and Schizosaccharomyces pombe (S. pombe). The purification was easily achieved by CM-Sephadex column chromatography of the breakthrough fractions from DEAE-Sephadex chromatography of cell-free extracts. The basic proteins have a molecular weight of 47,000 for the S. carls. factor and of 49,000 for the S. pombe factor. While the purified yeast EF-1 alpha s function analogously to other eukaryotic factors and the E. coli EF-Tu in Phe-tRNA binding and polyphenylalanine synthesis, the yeast factor unusually hydrolyzed GTP on yeast ribosomes upon addition of Phe-tRNA in the absence of poly(U) as mRNA. This novelty is probably owing to the yeast ribosomes, which are assumed to lack elongation factor 3-equivalent component(s). Trypsin and chymotrypsin selectively cleaved the two yeast factors to generate resistant fragments with the same molecular weight of 43,000 (by trypsin) and of 44,000 (by chymotrypsin), respectively. Those cleavage sites were characteristically protected by the presence of several ligands bound to EF-1 alpha such as GDP, GTP, and aminoacyl-tRNA. Based on the sequence analysis of the fragments generated by the two proteases, the partial amino acid sequence of the S. carls. EF-1 alpha was deduced to be in accordance with the N-terminal region covering positions (1) to 94 and two Lys residues at the C-terminal end of the predicted total sequence of the Saccharomyces cerevisiae (S. cerev.) factor derived from DNA analysis, except for a few N-terminal residues, confirming the predicted S. cerev. sequence at the protein level. EF-1 beta and EF-1 beta gamma were isolated and highly purified as biologically active entities from the two yeasts. EF-1 beta s from the two yeasts have the same molecular weight of 27,000, whereas component gamma of the S. carls. EF-1 beta gamma showed a higher molecular weight (47,000) than that of the S. pombe factor (40,000). It was also shown that a stoichiometric complex was formed between EF-1 alpha and EF-1 beta gamma from S. pombe. Furthermore, a considerable amount of Phe-tRNA binding activity was distributed in the EF-1H (probably EF-1 alpha beta gamma) fraction from freshly prepared cell-free extracts of yeast.

Cell-cell recognition in yeast. Molecular nature of the sexual agglutinin from Saccharomyces kluyveri 17-cells.

A previous study of Saccharomyces kluyveri 17-cell sexual agglutinin (alpha-agglutinin), solubilized by zymolyase (beta-glucanase) digestion of 17-cells and purified by affinity adsorption to immobilized 16-cell agglutinin (alpha-agglutinin), suggested that the major active component was a glycoprotein of 60,000 daltons and that a minor active component of 40,000 daltons was also present, possibly the result of proteolysis (Pierce, M., and Ballou, C. E. (1983) J. Biol. Chem. 258, 3576-3582). We now show that both of these active components are proteolytic fragments of a larger form with a molecular weight of 134,000, and that the latter is produced by proteolysis of a still larger species with a molecular weight of more than 200,000. Washed 17-cell wall fragments were labeled with 125I and digested with purified protease-free beta-1,3-glucanase, and the solubilized alpha-agglutinin was precipitated with antiserum raised against purified agglutinin containing a mixture of the 60,000- and 134,000-dalton forms. Gel electrophoresis in sodium dodecyl sulfate revealed a radioactive material with Mr greater than 200,000 that, on digestion with zymolyase containing an active protease, was converted sequentially to radioactive components with Mr = 134,000, 60,000, and 40,000.

Identification of yeast 60 S ribosomal proteins crosslinked to rRNA by 2-iminothiolane.

Saccharomyces carlsbergensis 60 S ribosomal subunits were treated with the hetero-bifunctional crosslinking agent 2-iminothiolane and then subjected to mild UV irradiation to introduce protein-rRNA crosslinks. The major crosslinked products were identified as proteins L2, L3, L5, L19 and L23 of which L5 was found to be crosslinked at a 3-5-fold higher efficiency than the other four. Several additional proteins were cross-linked to a detectable but much lower extent.

A note on the presence of novel DNA species in the spoilage yeasts Zygosaccharomyces bailii and Pichia membranaefaciens.

Two novel covalently closed circular DNA species of 5.4 and 6.0 kilobases were detected in strains of Zygosaccharomyces bailii with a rapid small scale isolation procedure. The 5.4 kb species was found in four strains and both species were found in three strains. A novel, covalently-closed circular DNA species of 6.9 kb was detected in four of 12 strains of Pichia membranaefaciens . Plasmid DNA (2 micron) (that is CCC DNA of approximately 6 kb in Saccharomyces cerevisiae) was detected in 38 of 40 strains of Sacch . cerevisiae confirming reports of the widespread distribution of this plasmid.

Detection of a yeast polyphosphate fraction localized outside the plasma membrane by the method of phosphorus-31 nuclear magnetic resonance.

Non-penetrating cations, like UO2+(2) and Eu3+, are bound to the outside of yeast cells in a reversible fashion. Binding of these ions was attended with a decrease of the 31P NMR polyphosphate signal. Subsequent addition of EDTA to the suspension restored the original spectrum. These experiments confirm the localization of a polyphosphate fraction outside the plasma membrane of yeast.

Thermal and Mg2+ dependent behavior of pseudouridines at 39th and 55th of yeast tRNAPhe.

Pseudouridine psi 55 alone and both psi 55 and psi 39 in yeast tRNAPhe are selectively modified with fluorescent reagent of 4-bromomethyl-7-methoxycoumarin (BMC). The change of fluorescence intensity was measured as a function of temperature and Mg2+ concentration. Fluorescent quenching shows the stacked and unstacked forms of Y base, dependent on Mg2+ concentration. In contrast, Mg2+ had no effect on psi 55-BMC in T psi C loop at 20 degrees C. Fluorescence on titrating Mg2+ exhibited a kind of Mg2+-induced structural collapse at the corner of L-structure. The melting of psi 55-BMC takes place at 70 degrees C in 10mM Mg2+. At very low Mg2+ concentration, melting takes place at 35 degrees C. The melting of psi 39-BMC, located near the anticodon loop, was observed before the unfolding of the whole structure of tRNAPhe. A conformational transition of the anticodon loop takes place at a lower temperature and it is also expected in the quenching experiment of Y base.

Localization of polyphosphates at the outside of the yeast cell plasma membrane.

Under appropriate experimental conditions toluidine blue is bound to the yeast cell surface, without penetrating into the cells. Based on experimental observations it is highly probable that the dye is bound to polyphosphates, localized outside the plasma membrane. The probable localization of polyphosphates outside the plasma membrane is important in the context of the proposed involvement of polyphosphates in glucose transport in yeast.

[Characterization of cell wall constituents involved in the flocculation of Saccharomyces uvarum yeast strains]. / Caractérisation des constituants pariétaux impliqués dans la floculation de levures Saccharomyces uvarum.

Cell walls of flocculent strains (0006) and non flocculent strains (0019) of Saccharomyces uvarum (Carlsbergensis), grown in different media and taken in both growth and stationary phases, were treated with water and with 2 per cent (W/V) potassium hydroxide. This treatment yielded four fractions (FI, FII, FIII and FR). The fractions FI isolated from the flocculent cell walls contained more mannose and less protein than the corresponding fractions FI isolated from the non flocculent cell walls. The amino-acid composition was also different between the two types of fractions. A radioactive labelling technique revealed that the FI and the walls from flocculent cells bound on average two to three times as much 45Ca as did the FI and the walls from non flocculent yeast. The substitution of carboxyl groups in FI and walls with glycine methyl ester led to a great drop of the 45Ca binding capacity. This result suggests that carboxyl groups of the cell walls are involved in the flocculation process. But flocculation seems to be a phenomenon more complex than the simple formation of a Ca2+ bridge, the involvement of "lectin like" components easily removed from the cell walls, should not be rejected.

[Relationship between aeration and sterol distribution in yeasts]. / Raspredelenie sterinov po fraktsiiam u drozhzhevykh organizmov v zavisimosti ot ikh aératsii.

The content of sterols in yeast organisms changes noticeably depending on the cultivation conditions. This content sharply decreases when the cells are transferred from aerobic to anaerobic conditions. The proportion between various sterol fractions also changes. The content of bound sterols under aerobic conditions reaches 85-90% of the total sterol content; that of free sterols is only 10-15%. Under anaerobic conditions, the quantity of bond sterols decreases to 70-75% while that of free sterols increases respectively to 25-30%. The fraction of free sterols increases whereas that of bound sterols decreases under aerobic conditions if oleic acid is added to the cultural broth. The content of all of the sterol fractions increases if oleic acid is added under anaerobic conditions. The same correlation is found when ergosterol is added to the medium under anaerobic conditions. Only the total sterol content and the content of sterol esters increase, rather than the content of free sterols, when cholesterol is added to the medium under anaerobic conditions.

Some yeast mitochondrial RNAs are circular.

11S and 18S fractions of yeast mitochondrial RNAs, isolated by electrophoresis through agarose gels, have been found by electron microscopy to contain approximately 50% circular molecules. Circles in the 11S fraction have a contour length of 0.36 +/- 0.02 micron, which is approximately equal to the length of the majority of linear molecules also present. Circles in the 18S fraction have an average length of 0.78 +/- 0.11 micron. The size distribution is broader than for the 11S fraction, and we cannot exclude the possibility that more than one size class may be present. The 11S circular RNA forms circular R loops and RNA-DNA hybrids with DNA fragments of the oxi 3 region of mtDNA, which contains the structural gene for subunit 1 of cytochrome oxidase. As judged from the electron micrographs, the complete RNA participates in hybrid formation and the sequences coding for it appear to be continuous. Both 11S and 18S circles withstand treatment with DNAase and pronase. They are not eliminated by treatment with 1 M glyoxal in 50% formamide for 1 hr at 50 degrees C. We conclude that they are covalently closed. The function of the circular RNAs is unknown. They may be active as mRNAs, storage forms, or arise in a cut-and-splice process which generates mRNAs from longer transcripts.