Search PubMed⌕ Search

Biomedical subjects

K D Entian

Publications and source records attributed to K D Entian.

At least 91 records · Page 5Linked to original sources
PubMed1987

Purification procedure and N-terminal amino acid sequence of yeast malate dehydrogenase isoenzymes.

A method has been devised for the rapid isolation of malate dehydrogenase isoenzymes. First, anionic proteins were precipitated with polyethyleneimine, whilst hydrophobic malate dehydrogenase remained in the supernatant fluid. Secondly, the supernatant was 30% saturated with ammonium sulfate and the two isoenzymes were separated by hydrophobic phenyl-Sepharose CL-4B chromatography. For further purification the enzymes were chromatofocused, and polybuffer was removed by hydrophobic chromatography. Affinity chromatography with blue Sepharose CL-6B [1] was used as final purification step. The purified isoenzymes were homogeneous as shown by isoelectric focusing and could be used for N-terminal sequencing. 34 amino acid residues could be identified for the cytoplasmic isoenzyme and 56 amino acid residues for the mitochondrial isoenzyme. Although there are regions of strong homology between both isoenzymes, the sequence differences clearly showed support that both isoenzymes are coded by different genes. Sequence comparison clearly indicated that the N-terminus of the cytoplasmic enzyme extended that of the mitochondrial enzyme by 12 amino acid residues. The amino acid sequence of the extending sequence resembled that of leading sequences known for enzymes which are transported into the mitochondria. The assumed leading sequence is discussed with respect to its possible role in glucose inactivation.

Amino Acid Sequence↗
PubMed1987

Studies on the regulation of enolases and compartmentation of cytosolic enzymes in Saccharomyces cerevisiae.

Three enolase isoenzymes can be distinguished after electrophoresis of yeast crude extracts. After adding glucose to derepressed cells, there was a coordinated increase in the activity of enolase I and decrease in enolase II activity. Enolase I was found to be repressed and enolase II simultaneously induced by glucose. The third enolase activity remained unchanged and was identified as that of a hybrid enzyme. Enolase catalyses the first common step of glycolysis and gluconeogenesis. Gluconeogenic enolase I shows substrate inhibition for 2-phosphoglycerate (glycolytic substrate) and glycolytic enolase II is substrate-inhibited by phosphoenolpyruvate (gluconeogenic substrate). The gluconeogenic reaction was inhibited up to 45% by physiological concentrations of fructose 1,6-bisphosphate. To test for cytological compartmentation, a method was developed for isolating microsomes. Effective enrichment of rough and smooth endoplasmic reticulum was demonstrated by electron microscopy. No evidence was obtained for any compartmentation of either enolases or other glycolytic enzymes.

Carbon↗
PubMed1987

Isolation and expression analysis of two yeast regulatory genes involved in the derepression of glucose-repressible enzymes.

Yeast strains carrying one of the two regulatory mutations cat1 and cat3 are defective in derepression of several glucose-repressible enzymes that are necessary for utilizing non-fermentable carbon sources. Hence, these strains fail to grow on ethanol, glycerol or acetate. The synthesis of isocitrate lyase, malate synthase, malate dehydrogenase and fructose-1,6-bisphosphatase is strongly affected in cat1 and cat3 strains. Genes CAT1 and CAT3 have been isolated by complementation of the cognate mutations after transformation with an episomal plasmid gene library. The restriction map of CAT1 proved its allelism to the earlier isolated SNF1 gene. Both genes appear to exist as single-copy genes per haploid genome as indicated by Southern hybridization. Northern analysis has shown that the 1.35 kb CAT3 mRNA is constitutively expressed, independent of the carbon source in the medium. Derepression studies with CAT3 transformants using a multi-copy plasmid showed over-expression of glyoxylate cycle enzymes. This result would be consistent with a direct effector function for the CAT3 gene product.

Crosses, Genetic↗
PubMed1987

Cloning and sequence of the mdh structural gene of Escherichia coli coding for malate dehydrogenase.

The malate dehydrogenase gene of Escherichia coli, which is susceptible to catabolite and anaerobic repression, has been cloned using plasmic pLC32-38 of Clarke and Carbon (1976). The nucleotide sequence was determined of a 2.47 kbp fragment, containing the mdh structural gene. All information necessary for expression of the mdh structural gene was mapped within a 1.3 kbp SphI-BstEII fragment. Compared with the untransformed wild type, transformations with pUC19 vector, containing this fragment, gave up to 40-fold more malate dehydrogenase activity in both E. coli wild type and mdh mutant recipients. Catabolite repression was not affected in the transformants. A possible CRP binding site in the promotor region of the mdh gene provides evidence for a co-regulation with fumA gene, the structural gene of fumarase, which is also subject to catabolite repression. The structures for transcription initiation and termination were similar to those previously described for E. coli. Amino acid sequence homologies between pro- and eucaryotic malate dehydrogenases are discussed.

Amino Acid Sequence↗
PubMed1987

Isolation and characterization of the regulatory HEX2 gene necessary for glucose repression in yeast.

The HEX2 gene which is necessary for glucose repression and is involved in the regulation of hexokinase PII synthesis and maltose uptake, has been cloned by complementation of a hex2 mutant, and selection for restored growth on maltose. Glucose repression in the transformants was like that in the wild type. The HEX2 gene was localized within a 2.15 kb fragment. The restriction map was confirmed by Southern hybridization of genomic DNA. Based on 30 tetrads, the linkage between HEX2 and TRP1 was determined as 10 cM. Plasmid integration directed to the genomic site of the cloned gene also gave a similar linkage distance between the amino acid auxotroph plasmid marker and genomic TRP1. Gene disruption of HEX2 yielded nonrepressible transformants with elevated hexokinase PII activity showing inhibition by maltose; this provides clear evidence that the HEX2 gene has been isolated.

DNA Restriction Enzymes↗
PubMed1986

Glucose repression: a complex regulatory system in yeast.

Glucose repression is a complex regulatory system controlling numerous biochemical pathways. The triggering signal for glucose repression of many enzymes is given by hexokinase isoenzyme P11. In addition to glucose repression a glucose derepression system exists. Certain upstream activation sites have been identified for glucose derepression.

Allosteric Regulation↗
PubMed1985

Glucose repression and hexokinase isoenzymes in yeast. Isolation and characterization of a modified hexokinase PII isoenzyme.

Hexokinase PII, but not isoenzyme PI, has a unique role in glucose repression in yeasts [Entian, K.-D. (1980) Mol. Gen. Genet. 178, 633-637; Entian, K.-D. and Mecke, D. (1982) J. Biol. Chem. 257, 870-874; Entian, K.-D. and Fröhlich, K.-U. (1984) J. Bacteriol. 158, 29-35]. The number of hexokinase isoenzymes in crude extracts was re-examined by chromatofocusing. In addition to the known isoenzymes PI and PII, a third isoenzyme, PIIM, was detected. The activity of this enzyme was only about 5-10% of that of hexokinase PII and was independent of growth conditions. Experiments with hexokinase transformants and purified hexokinase isoenzymes clearly indicated that the PIIM form is also present in vivo. Fingerprint mapping of purified hexokinases showed that hexokinase PIIM is closely related to PII. Hybridization experiments between totally restricted yeast DNA and the previously isolated PII gene clearly indicated that PIIM is also coded by one of the two known hexokinase genes. No mRNA specific for hexokinase PIIM was detected after hybridization experiments with the previously cloned hexokinase PII gene [Fröhlich et al. (1984) Mol. Gen. Genet. 194, 144-148]. Hexokinase PIIM appears to be derived from hexokinase PII by a posttranslational event. The Km values of each of the purified isoenzymes, PII and PIIM, were identical for glucose, fructose and ATP. Both isoenzymes were strongly inhibited by high physiological concentrations for ATP; such inhibition has not been described previously. The possible role of hexokinase PIIM in glucose repression is discussed.

Chemical Phenomena↗
PubMed1985

The primary structure of the yeast hexokinase PII gene (HXK2) which is responsible for glucose repression.

The nucleotide sequence of the Saccharomyces cerevisiae gene encoding the glycolytic isoenzyme hexokinase PII (HXK2), which is responsible for triggering glucose repression, has been determined. The reading frame was identified by comparison with the N-terminal undecameric amino acid (aa) sequence, determined previously [Schmidt and Colowick, Arch. Biochem. Biophys. 158 (1973) 458-470]. The codon sequence was not random, with 82.1% of the aa specified by only 25 codons. The structural gene sequence corresponded to 1455 bp, coding for 485 aa residues, corresponding to the Mr of 53 800 for the HXK2 monomer. Five initiation regions spanning 162 bp and three termination sites spanning 29 bp were detected. Sequences with similarities to a 5'-TATAAA-3' sequence were located 24-39 bp upstream of each initiation region. The most pronounced initiation region corresponded to the 5'-TATAAA-3' sequence at position -152. Two of the minor initiation sites were inside the coding sequence in front of two ATG codons.

Amino Acid Sequence↗
PubMed1985

Complete nucleotide sequence of the hexokinase PI gene (HXK1) of Saccharomyces cerevisiae.

The nucleotide sequence of the yeast glycolytic hexokinase isoenzyme PI-gene, HXK1, has been determined by sequencing the yeast DNA insert of the previously isolated plasmid HXK1 clone [Entian et al., Mol. Gen. Genet. 198 (1984) 50-54]. The structural gene sequence included 1452 bp coding for 484 amino acid (aa) residues corresponding to the Mr of 153 605 for the HXK1 monomer. Several initiation regions and termination points were located using nuclease S1 mapping. The HXK1 sequence was 76% homologous with that of HXK2, which is responsible for triggering glucose repression in yeasts. Since HXK1 is not involved in this regulatory system, the regulatory function of HXK2 must correspond to one or more of the differences between both isoenzymes. Most changes in the amino acid sequence were statistically distributed; however, four clustered regions with more than five altered aa residues were identified.

Amino Acid Sequence↗
PubMed1985

Cloning of hexokinase structural genes from Saccharomyces cerevisiae mutants with regulatory mutations responsible for glucose repression.

The regulatory hexokinase PII mutants isolated previously (K.-D. Entian and K.-U. Fröhlich, J. Bacteriol. 158:29-35, 1984) were characterized further. These mutants were defective in glucose repression. The mutation was thought to be in the hexokinase PII structural gene, but it did not affect the catalytic activity of the enzyme. Hence, a regulatory domain for glucose repression was postulated. For further understanding of this regulatory system, the mutationally altered hexokinase PII proteins were isolated from five mutants obtained independently and characterized by their catalytic constants and bisubstrate kinetics. None of these characteristics differed from those of the wild type, so the catalytic center of the mutant enzymes remained unchanged. The only noticeable difference observed was that the in vivo modified form of hexokinase PII, PIIM, which has been described recently (K.-D. Entian and E. Kopetzki, Eur. J. Biochem. 146:657-662, 1985), was absent from one of these mutants. It is possible that the PIIM modification is directly connected with the triggering of glucose repression. To establish with certainty that the mutation is located in the hexokinase PII structural gene, the genes of these mutants were isolated after transforming a hexokinaseless mutant strain and selecting for concomitant complementation of the nuclear function. Unlike hexokinase PII wild-type transformants, glucose repression was not restored in the hexokinase PII mutant transformants. In addition mating experiments with these transformants followed by tetrad analysis of sporulated diploids gave clear evidence of allelism to the hexokinase PII structural gene.

Cloning, Molecular↗
PubMed1984

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae.

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cells. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ' Crabtree effect', was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate dehydrogenase was subject to glucose inactivation.

Fructose-Bisphosphatase↗
PubMed1984

Cloning of hexokinase isoenzyme PI from Saccharomyces cerevisiae: PI transformants confirm the unique role of hexokinase isoenzyme PII for glucose repression in yeasts.

Hexokinase isoenzyme PI was cloned using a gene pool obtained from a yeast strain having only one functional hexokinase, isoenzyme PI. The gene was characterized using 20 restriction enzymes and located within a region of 2.0 kbp. The PI plasmid strongly hybridized with the PII plasmids isolated previously (Fröhlich et al. 1984). Hence there was a close relationship between the two genes, one of which must have been derived from the other by gene duplication. In contrast, glucose repression was restored only in hexokinase PII transformants; PI transformants remained non-repressible. This observation provided additional evidence for the hypothesis of Entian (1980) that only hexokinase PII is necessary for glucose repression. Furthermore, glucose phosphorylating activity in PI transformants exceeded that of wild-type cells, giving clear evidence that the phosphorylating capacity is not important for glucose repression.

Cloning, Molecular↗
PubMed1984

Cloning and restriction analysis of the hexokinase PII gene of the yeast Saccharomyces cerevisiae.

Carbon catabolite repression in yeast depends on catalytic active hexokinase isoenzyme PII ( Entian 1980a ). A yeast strain lacking hexokinase isoenzymes PI and PII was transformed, using a recombinant pool with inserts of yeast nuclear DNA up to 10 kbp in length. One hundred transformants for hexokinase were obtained. All selected plasmids coded for hexokinase isoenzyme PII, none for hexokinase isoenzyme PI, and carbon catabolite repression was restored in the transformants. Thirty-five independently isolated stable plasmids were investigated further. Analysis with the restriction enzyme EcoRI showed that these plasmids fell into two classes with different restriction behaviour. One representative of each class was amplified in Escherichia coli and transferred back into the yeast hexokinase-deficient strain with concomitant complementation of the nuclear mutation. The two types of insert were analysed in detail with 16 restriction enzymes, having 0-3 cleavage sites on transformant vector YRp7 . The plasmids differed from each other by the orientation of the yeast insert in the vector. After yeast transformation with fragments of one plasmid the hexokinase PII gene was localised within a region of 1.65 kbp.

Cloning, Molecular↗
PubMed1984

Saccharomyces cerevisiae mutants provide evidence of hexokinase PII as a bifunctional enzyme with catalytic and regulatory domains for triggering carbon catabolite repression.

A selection system has been devised for isolating hexokinase PII structural gene mutants that cause defects in carbon catabolite repression, but retain normal catalytic activity. We used diploid parental strains with homozygotic defects in the hexokinase PI structural gene and with only one functional hexokinase PII allele. Of 3,000 colonies tested, 35 mutants (hex1r) did not repress the synthesis of invertase, maltase, malate dehydrogenase, and respiratory enzymes. These mutants had additional hexokinase PII activity. In contrast to hex1 mutants (Entian et al., Mol. Gen. Genet. 156:99-105, 1977; F.K. Zimmermann and I. Scheel, Mol. Gen. Genet. 154:75-82, 1977), which were allelic to structural gene mutants of hexokinase PII and had no catalytic activity (K.-D. Entian, Mol. Gen. Gent. 178:633-637, 1980), the hex1r mutants sporulated hardly at all or formed aberrant cells. Those ascospores obtained were mostly inviable. As the few viable hex1r segregants were sterile, triploid cells were constructed to demonstrate allelism between hex1r mutants and hexokinase PII structural gene mutants. Metabolite concentrations, growth rate, and ethanol production were the same in hex1r mutants and their corresponding wild-type strains. Recombination of hexokinase and glucokinase alleles gave strains with different specific activities. The defect in carbon catabolite repression was strongly associated with the defect in hexokinase PII and was independent of the glucose phosphorylating capacity. Hence, a secondary effect caused by reduced hexose phosphorylation was not responsible for the repression defect in hex1 mutants. These results, and those with the hex1r mutants isolated, strongly supported our earlier hypothesis that hexokinase PII is a bifunctional enzyme with (i) catalytic activity and (ii) a regulatory component triggering carbon catabolite repression (Entian, Mol. Gen. Genet. 178:633-637, 1980; K.-D. Entian and D. Mecke, J. Biol. Chem. 257:870-874, 1982).

Alleles↗
PubMed1983

Purification of three distinct enolase isoenzymes from yeast.

A method for the rapid isolation of yeast enolases, yielding three distinct isoenzymes, has been devised. In the first step anionic proteins were precipitated with polyethyleneimine, whereas hydrophobic enolase isoenzymes remained in the supernatant. Secondly, the supernatant was 45% saturated with ammonium sulfate and bound to phenyl-Sepharose CL-4B. Decreasing ammonium sulfate and simultaneously increasing ethylene glycol concentrations were used for elution. Finally, enolase isoenzymes were separated by chromatofocusing. The purified isoenzymes gave single bands after isoelectric focusing.

Ammonium Sulfate↗
PubMed1983

Studies on rapid reversible and non-reversible inactivation of fructose-1,6-bisphosphatase and malate dehydrogenase in wild-type and glycolytic block mutants of Saccharomyces cerevisiae.

Experimental conditions have been elaborated to test for reversibility of the malate dehydrogenase inactivation (E.C.1.1.1.37) after addition of glucose to derepressed yeast cells. Malate dehydrogenase inactivation was shown to be irreversible at all stages of inactivation. In contrast fructose-1,6-bisphosphatase inactivation (E.C.3.1.11) remained reversible for at least 30 min after addition of glucose. Rapid reversible inactivation of fructose-1,6-bisphosphatase and irreversible inactivation of malate dehydrogenase were additionally investigated in glycolytic block mutants. Normal inactivation kinetics were observed in mutants without catalytic activity of phosphoglucose isomerase (E.C.5.3.1.9), phosphofructokinase (E.C.2.7.1.11), triosephosphate isomerase (E.C.5.3.1.1) and phosphoglycerate kinase (E.C.2.7.2.3). Hence, neither type of inactivation depended on the accumulation of any glucose metabolite beyond glucose-6-phosphate. Under anaerobic conditions irreversible inactivation was completely abolished in glycolytic block mutants. In contrast rapid reversible inactivation was independent of energy provided by respiration or fermentation. Reversibility of fructose-1,6-bisphosphatase inactivation was tested under conditions which prevented irreversible malate dehydrogenase inactivation. In these experiments, fructose-1,6-bisphosphatase inactivation remained reversible for at least 120 min, whereas reversibility was normally restricted to about 30 min. This indicated a common mechanism between the irreversible part of fructose-1,6-bisphosphatase inactivation and irreversible malate dehydrogenase inactivation.

Adenosine Triphosphate↗