Partial purification, substrate specificity and regulation of alpha-L-glycerolphosphate dehydrogenase from Saccharomyces carlsbergensis.

alpha-L-Glycerolphosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) from Saccharomyces carlsbergensis was purified 400-fold. The enzyme preparation is free of interfering activities, such as glyceraldehyde phosphate dehydrogenase, alcohol dehydrogenase, triose phosphate isomerase and glycerolphosphatase. At pH 7.0 it is specific for NADH (Km = 0.027 mM with 0.8 mM dihydroxyacetone phosphate) and dihydroxyacetone phosphate (Km = 0.2 mM with 0.2 mM NADH). Between pH 5.0 and 6.0 the enzyme functions with NADPH, but only at 7% of the rate with NADH. Various anions (I- greater than SO42- greater than Br- greater than Cl-) act as inhibitors competing with the substrate dihydroxyacetone phosphate. Inorganic phosphate (Ki = 0.1 mM), pyrophosphate and arsenate are strong inhibitors. The nucleotides ATP and ADP are also inhibitory, but their action seems to be of the same type as the general anion competition (Ki = 0.73 mM for ATP). The results are consistent with the notion that the enzyme may regulate the redox potential of the NAD+/NADH couple during fermentation.     

Proteolytic enzymes of Saccharomyces carlsbergensis

1. Of four proteolytic enzymes isolated from autolysing Saccharomyces carlsbergensis, one is inactivated at about 45 degrees C, whereas the others are stable at 50 degrees C. pH optima for activity are from 3.0 to 8.0 but maximum stability is between pH6.0 and 6.5. All appear to be glycoproteins, the carbohydrate moiety containing glucose and mannose residues. 2. Lysed protoplasts of the same yeast release four proteolytic enzymes each of which have two pH optima at pH3.0 and 7.0 approximately. Compared with the enzymes from autolysed yeast, resistance to high temperature is much less, and they are not glycoprotein in nature. 3. The same yeast grown with N-acetyltyrosine ethyl ester as nitrogen source secretes into the medium four proteases believed to be glycoprotein in nature. Generally they resemble the enzymes from lysed protoplasts more than those from autolysing yeast.     

Physico-chemical properties of thiamine kinase from Saccharomyces carlsbergensis yeasts

The amino acid composition and intrinsic fluorescence were studied in thiamine kinase (ES 2.7.6.2) of brewer's yeast. The enzyme molecule is characterized by higher concentrations of amino acids which promote alpha-helix formation of the protein globule, the amount of residues (cysteine, proline) either binding or folding polypeptide chains being considerably high. Amino acids of middle and low hydrophobicities were the most frequent among the amino acid residues with nonpolar R-groups. The value for the protein isoelectric point was 6.21. The eigen pH value and isoionic point were in good agreement with the isoelectric point value and amounted to 6.28. The fluorescence spectrum has a maximum at 328 nm, half-width at 53 nm and a quantum yield at 0.14 nm. The tryptophane residues were located in hydrophobic surroundings, unexposed to anion quenchers and almost unexposed to cation ones. The fluorescence and phosphofluorescence parameters were sensitive to the conformational changes in the molecule. At pH of 5-9 the protein conformation remained unchanged. The temperature rise above 40 degrees C resulted in a disturbance in the nativity of the globule. The elevation of the enzyme concentration from 0.05 to 1 mg/ml increased the polarization degree from 0.115 to 0.194, the quantum yield and the spectrum position remaining unchanged. The results obtained develop knowledge of the equilibrium system of oligomerous forms of thiamine kinase with different catalytic properties.     

Analysis of the inducible MEL1 gene of Saccharomyces carlsbergensis and its secreted product, alpha-galactosidase (melibiase)

We have determined both the nucleotide sequence of the MEL1 gene of Saccharomyces carlsbergensis and the N-terminal amino acid (aa) sequence of its extracellular gene product, alpha-galactosidase (melibiase) (alpha-Gal). The predicted translation product of MEL1 is a pre-alpha-Gal protein containing an 18 aa N-terminal signal sequence for secretion. The purified enzyme is a dimer consisting of two 50-kDal polypeptides, each of which is glycosylated with no more than eight side chains. The 5'-flank of the MEL1 gene contains a region (UASm) having certain areas of sequence homology to similar sites found upstream of the structural genes GAL1, GAL7 and GAL10, which are also regulated by the action of the products of genes GAL4 and GAL80. There are three TATA boxes between UASm and the initiation codon of pre-alpha-Gal, as well as a typical yeast cleavage/polyadenylation sequence in the 3'-flank of the gene.     

Analysis of histones from the yeast Saccharomyces carlsbergensis.

Basic chromosomal proteins were isolated from the chromatin of the yeast Saccharomyces carlsbergensis by extraction with H2SO4 and were purified by ion-exchange chromatography. Electrophoresis of the purified fraction on acetic acid/urea gels revealed the presence of four main components. These four proteins were identified as histones H2A, H2B, H3 and H4 on the basis of their amino acid composition, molecular weight and solubility properties, all of which are very similar to the corresponding properties of the various histone proteins from other eukaryotic organisms. A fifth basic protein could be isolated from yeast chromatin by extraction with HClO4. The available evidence indicates this protein to be an H1-type histone. Yeast thus appears to contain a complete set of histone proteins which are strongly homologous to the histones occurring in higher eukaryotes.     

Isolation of a maltase structural gene from Saccharomyces carlsbergensis.

The maltase structural gene MAL6 of the yeast Saccharomyces carlsbergensis has been cloned by transformation of a maltose nonfermenting recipient strain with autonomously replicating chimeric recombinant plasmids. One recombinant plasmid, pMAL26, was shown by positive hybridization translation, as well as by Southern and Northern blot experiments, to carry the MAL6 structural gene.     

Purification and characterization of an alpha-glucosidase from Saccharomyces carlsbergensis.

alpha-Glucosidase (EC 3.2.1.20) was purified to homogeneity from logarithmically growing cells of Saccharomyces carlsbergensis. The purification involved the following steps: (a) ammonium sulfate fractionation; (b) Sephadex G-100 chromatography; (c) DEAE-cellulose chromatography; and (d) hydroxylapatite chromatography. This procedure gave a preparation judged to be greater than 98% pure by Na-DodSO4-polyacrylamide gel electrophoresis. The enzyme was shown to be a monomer of 63 000 daltons by gel filtration on Sephacryl S-200 under native conditions and by polyacrylamide gel electrophoresis under denaturing conditions. The Km values of the enzyme for the substrates maltose and p-nitrophenyl alpha-D-glucoside were found to be 1.66 X 10(-2) and 3.1 X 10(-4) M, respectively. The corresponding Vmax value for maltose was 44.8 X 10(-6) mol min(-1) mg(-1) and that for p-nitrophenyl alpha-D-glucoside was 134 X 10(-6) mol min-1 mg-1. The pH optimum for the purified enzyme was found to be between pH 6.7 and 6.8. The enzyme has an absolute anomeric specificity for alpha-glycosidic linkages and appears to recognize a glucosyl residue in alpha linkage on the nonreducing end of its substrate. For the strain used in this study, which carries the MAL 6 locus, only a single form of the enzyme was detected.     

Molecular analysis of alpha-galactosidase MEL genes from Saccharomyces sensu stricto

To infer the molecular evolution of yeast Saccharomyces sensu stricto from analysis of the alpha-galactosidase MEL gene family, two new genes were cloned and sequenced from S. bayanus var. bayanus and S. pastorianus. Nucleotide sequence homology of the MEL genes of S. bayanus var. bayanus (MELb), S. pastorianus (MELpt), S. bayanus var. uvarum (MELu), and S. carlsbergensis (MELx) was rather high (94.1-99.3%), comparable with interspecific homology (94.8-100%) of S. cerevisiae MEL1-MEL11. Homology of the MEL genes of sibling species S. cerevisiae (MEL1), S. bayanus (MELb), S. paradoxus (MELp), and S. mikatae (MELj) was 76.2-81.7%, suggesting certain species specificity. On this evidence, the alpha-galactosidase gene of hybrid yeast S. pastorianus (S. carlsbergensis) was assumed to originate from S. bayanus rather than from S. cerevisiae.     

Isolation of cloned ribosomal protein genes from the yeast Saccharomyces carlsbergensis

A colony bank of yeast dna obtained by cloning HindIII-generated fragments of total yeast nuclear DNA in Escherichia coli K-12 with the vector pBR322, was screened with a radioactive RNA probe enriched for a subset of ribosomal protein mRNAs. The selected recombinant DNA molecules were hybridized with poly(A)-containing mRNA under R-loop conditions. From the DNA-RNA hybrids the respective mRNAs were melted off and translated in vitro in a rabbit reticulocyte cell-free system. The translational products were analyzed by immunoprecipitation with antibodies raised against ribosomal proteins. The identity of the ribosomal protein gene products was further established by electrophoresis on two-dimensional gels. At least 15 recombinant DNA molecules were shown to contain ribosomal protein genes. Four of them, i.e. Y65, Y89, Y113 and Y138, have been characterized preliminarily.     

The cloning and expression of a gene encoding Old Yellow Enzyme from Saccharomyces carlsbergensis

We have identified a gene that encodes Old Yellow Enzyme in brewer's bottom yeast. The open reading frame encodes a polypeptide of 400 amino acids with Mr = 45,021. Using the T7 RNA polymerase system, recombinant enzyme was expressed in Escherichia coli. 17 mg of Old Yellow Enzyme was obtained from a 3-liter cell culture, and the recombinant enzyme had NADPH oxidase activity. On fast protein liquid chromatography separation, the recombinant enzyme showed a single large peak, while native enzyme from brewer's bottom yeast separated into five fractions on fast protein liquid chromatography. Southern blot analysis showed that there are at least two Old Yellow Enzyme genes in brewer's bottom yeast genomic DNA. These results suggest that the heterogeneity of Old Yellow Enzyme in Saccharomyces carlsbergensis is due to the presence of multiple genes.     

Letter: Electron microscopy of pyruvate kinase crystals from Saccharomyces carlsbergensis

Crystals of pyruvate kinase have been analysed by electron microscopy, optical diffraction and filtering, and the following parameters were obtained: $\text{2a = 93}\text{A}\text{?}$, $\text{2b = 126}\text{A}\text{?}$, $\text{l = 35·2}\text{A}\text{?}$. A comparison of these data with the parameters obtained from small-angle X-ray scattering measurements indicates that two molecules of the tetrameric enzyme are arranged in one packing unit.