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PubMed1998

Use of fructose 1,6-diphosphate aldolase to detect tumour necrosis after transcatheter arterial embolization of hepatocellular carcinoma.

Transcatheter arterial embolization (TAE) is a popular and well-established devascularization treatment modality for hepatocellular carcinoma (HCC). The persistent retention of lipiodol on follow-up computed tomography (CT) scan and time-dependent decrease in size of the lipiodol-stained area of tumour after TAE does not reveal the biological death of tumour cells. Moreover, it is difficult to clinically evaluate the effective necrosis of tumour cells by TAE in cases of HCC that do not produce alpha-fetoprotein (AFP). We therefore studied the release of a relatively tumour-specific protein by the necrotic hepatoma cells to evaluate the effectiveness of TAE. Transcatheter arterial embolization was performed in 17 patients with the imaging diagnosis of HCC; either superselective (n = 6) or non-superselective (n = 11) techniques were used. We measured serum levels of relatively tumour-specific fructose 1,6-diphosphate (FDP) aldolase and non-tumour-specific fructose 1-phosphate (F1P) aldolase by substrate-specific enzymatic methods. Enzyme activities were performed before and after TAE. The time-dependent decrease in size of the lipiodol-stained areas was studied on follow-up CT scans after TAE. Pre- and post-treatment serum AFP levels were determined by radio-immunoassay. The six cases of superselective TAE underwent marked tumour regression by CT compared with the 11 cases of non-superselective TAE. Fructase 1,6-diphosphate aldolase output correlated well with post-necrotic tumour regression after TAE (r = 0.87, P= 0.001). The elevation of serum FDP aldolase was also significantly associated with a decrease in serum AFP (r = 0.72, P < 0.01). In contrast, serum F1P aldolase output was inversely correlated with either tumour regression or serum AFP concentrations after TAE. The serum levels of the tumour-specific enzyme FDP aldolase correlated significantly with effective tumour necrosis and consequent tumour regression after TAE. We suggest that measurement of FDP aldolase activity in serum after TAE can be used clinically to detect the degree of tumour necrosis by TAE.

Adult↗
PubMed1997

How thiamine diphosphate is activated in enzymes.

The controversial question of how thiamine diphosphate, the biologically active form of vitamin B1, is activated in different enzymes has been addressed. Activation of the coenzyme was studied by measuring thermodynamics and kinetics of deprotonation at the carbon in the 2-position (C2) of thiamine diphosphate in the enzymes pyruvate decarboxylase and transketolase by use of nuclear magnetic resonance spectroscopy, proton/deuterium exchange, coenzyme analogs, and site-specific mutant enzymes. Interaction of a glutamate with the nitrogen in the 1'-position in the pyrimidine ring activated the 4'-amino group to act as an efficient proton acceptor for the C2 proton. The protein component accelerated the deprotonation of the C2 atom by several orders of magnitude, beyond the rate of the overall enzyme reaction. Therefore, the earlier proposed concerted mechanism or stabilization of a C2 carbanion can be excluded.

Allosteric Regulation↗
PubMed1990

Cloning and expression of the gene encoding the fructose-1,6-diphosphate-dependent L-(+)-lactate dehydrogenase of Streptococcus mutans.

The fructose-1,6-diphosphate-dependent lactate dehydrogenase from Streptococcus mutans JH1000 was purified by a modification of published methods. The sequence of 27 amino-terminal amino acids was determined, which allowed us to construct a 17-base DNA probe that had 32-fold degeneracy. The probe was used to screen a genomic library in pBR322. Of 18 reactive clones, 1 was found that expressed lactate dehydrogenase (LDH) activity identical to that of S. mutans with regard to dependence on fructose-1,6-diphosphate, thermal inactivation profile, and inhibition by sodium oxamate. Extracts of this clone possessed a protein band that comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with purified LDH from JH1000. Compared with controls, the clone was shown to produce elevated amounts of L-(+)-lactic acid during growth in the presence of glucose, thereby indicating that the activity was expressed in vivo. This result was substantiated by demonstrating that the activity could complement a mutation in the fermentative D-(-)-LDH of Escherichia coli. Subcloning showed that the S. mutans LDH subunit is encoded by a 1.2-kilobase gene. Our ability to clone this gene is expected to have great practical significance in the construction of an effector strain for use in the replacement therapy of dental caries.

Amino Acid Sequence↗
PubMed1970

Depression of guanosine diphosphate-mannose pyrophosphorylase by mutations in two different regulator genes involved in capsular polysaccharide synthesis in Escherichia coli K-12.

Mutations in a regulator gene (capR) that causes increased synthesis of capsular polysaccharide and derepressed synthesis of several enzymes involved in polysaccharide synthesis also derepress synthesis of guanosine diphosphate (GDP)-mannose pyrophosphorylase. In addition, a second mucoid mutation (capS, which maps separately from capR) also results in the derepression of GDP-mannose pyrophosphorylase. New conditions for assaying GDP-mannose hydrolyase and GDP-l-fucose synthetase permitted us to show that these enzymes are also derepressed in the capS mucoid strain. Although phosphomannose isomerase and uridine diphosphate-galactose-4-epimerase are derepressed in capR mucoid strains, they are not derepressed in capS mucoid strains. A nonmucoid mutant of a strain containing the capR9 (mucoid) allele was deficient in GDP-mannose pyrophosphorylase.

Cell Wall↗
PubMed1973

Properties of ribonucleoside diphosphate reductase in nucleotide-permeable cells.

Ribonucleoside diphosphate (RDP) reductase activity can be readily assayed in ether-treated Escherichia coli cells. The rate of cytidine 5'-diphosphate (CDP) reduction observed in ether-treated cells by using saturating substrate concentrations is about 25% of the rate of de novo deoxyribonucleotide synthesis required to account for in vivo deoxyribonucleic acid synthesis. Optimal activity is observed in the presence of magnesium ions and a positive effector. Adenosine 5'-triphosphate (ATP), deoxy ATP (dATP), and deoxythimidine triphosphate serve as positive effectors, and dATP also serves as a negative effector. These effects on the activity in ether-treated cells resemble those observed in vitro with highly purified enzyme. When the RDP reductase activity in these cells is assayed by using high specific activity (3)H-CDP as substrate, even at nonsaturating substrate concentrations, the sensitivity of the assay is sufficient to make it useful for the assay of the low levels of reductase activity in cells not derepressed by thymine starvation or in cells containing mutationally altered RDP reductase. This assay is much easier to perform than the usual in vitro assay, since thioredoxin, thioredoxin reductase, and enzyme subunits B1 or B2 need not be first purified and added to the reaction mixtures.

Adenosine Triphosphate↗
PubMed1976

Hydrogenase and ribulose diphosphate carboxylase during autotrophic, heterotrophic, and mixotrophic growth of scotochromogenic mycobacteria.

Two key autotrophic enzyme systems, hydrogenase and ribulose diphosphate carboxylase, were examined in Mycobacterium gordonae and two other chemolithotrophic, scotochromogenic mycobacteria under different cultural conditions. In all three organisms both enzymes were inducible and were produced in significant levels only in the presence of the specific substrate, hydrogen or carbon dioxide. M. gordonae exhibited increased growth rates and yields, indicating mixotrophic growth, in the presence of a number of single organic substrates, including acetate, pyruvate, glucose, fructose, and glycerol. In contrast to other aerobic hydrogen autotrophs, the presence of either acetate or pyruvate did not repress ribulose diphosphate carboxylase, and mixotrophic growth was rapid with these substrates. In the absence of carbon dioxide, growth in glycerol medium under an atmosphere of hydrogen and oxygen was severely inhibited, even with cells preadapted to heterotrophic growth on glycerol. Cyclic adenosine monophosphate was not effective in inducing hydrogenase or carboxylase in heterotrophic, mixotrophic, or hydrogen-inhibited cultures.

Acetates↗
PubMed1989

Isolation and characterization of an Escherichia coli mutant having temperature-sensitive farnesyl diphosphate synthase.

The screening of a collection of highly mutagenized strains of Escherichia coli for defects in isoprenoid synthesis led to the isolation of a mutant that had temperature-sensitive farnesyl diphosphate synthase. The defective gene, named ispA, was mapped at about min 10 on the E. coli chromosome, and the gene order was shown to be tsx-ispA-lon. The mutant ispA gene was transferred to the E. coli strain with a defined genetic background by P1 transduction for investigation of its function. The in vitro activity of farnesyl diphosphate synthase of the mutant was 21% of that of the wild-type strain at 30 degrees C and 5% of that at 40 degrees C. At 42 degrees C the ubiquinone level was lower (66% of normal) in the mutant than in the wild-type strain, whereas at 30 degrees C the level in the mutant was almost equal to that in the wild-type strain. The polyprenyl phosphate level was slightly higher in the mutant than in the wild-type strain at 30 degrees C and almost the same in both strains at 42 degrees C. The mutant had no obvious phenotype regarding its growth properties.

Alkyl and Aryl Transferases↗
PubMed1996

Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase.

Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic interaction chromatography, catalyzed the interconversion of IPP and dimethylallyl diphosphate. Thus, the photosynthesis gene cluster encodes all of the enzymes required to incorporate IPP into the ultimate carotenoid and bacteriochlorophyll metabolites in R. capsulatus. More recent searches uncovered additional putative open reading frames for IPP isomerase in seed-bearing plants (Oryza sativa, Arabidopsis thaliana, and Clarkia breweri), a worm (Caenorhabditis elegans), and another eubacterium (Escherichia coli). The R. capsulatus enzyme is the smallest of the IPP isomerases to be identified thus far and may consist mostly of a fundamental catalytic core for the enzyme.

Amino Acid Sequence↗
PubMed1996

Phosphoribosyl diphosphate synthetase-independent NAD de novo synthesis in Escherichia coli: a new phenotype of phosphate regulon mutants.

Phosphoribosyl diphosphate-lacking (delta prs) mutant strains of Escherichia coli require NAD, guanosine, uridine, histidine, and tryptophan for growth. NAD is required by phosphoribosyl diphosphate-lacking mutants because of lack of one of the substrates for the quinolinate phosphoribosyltransferase reaction, an enzyme of the NAD de novo pathway. Several NAD-independent mutants of a host from which prs had been deleted were isolated; all of them were shown to have lesions in the pstSCAB-phoU operon, in which mutations lead to derepression of the Pho regulon. In addition NAD-independent growth was dependent on a functional quinolinate phosphoribosyltransferase. The prs suppressor mutations led to the synthesis of a new phosphoryl compound that may act as a precursor for a new NAD biosynthetic pathway. This compound may be synthesized by the product of an unknown phosphate starvation-inducible gene of the Pho regulon because the ability of pst or phoU mutations to suppress the NAD requirement requires PhoB, the transcriptional activator of the Pho regulon.

Escherichia coli↗
PubMed2000

Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci.

The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)-pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.

Acetyl-CoA C-Acetyltransferase↗
PubMed2001

Cloning, expression, and characterization of cis-polyprenyl diphosphate synthase from the thermoacidophilic archaeon Sulfolobus acidocaldarius.

cis-polyprenyl diphosphate synthases are involved in the biosynthesis of the glycosyl carrier lipid in most organisms. However, only little is known about this enzyme of archaea. In this report, we isolated the gene of cis-polyprenyl diphosphate synthase from a thermoacidophilic archaeon, Sulfolobus acidocaldarius, and characterized the recombinant enzyme.

Alkyl and Aryl Transferases↗
PubMed1963

EPIMERIZATION OF THYMIDINE DIPHOSPHATE GLUCOSE IN BACTERIAL EXTRACTS.

Tinelli, Regina (Washington University School of Medicine, St. Louis, Mo.), A. M. Michelson, and Jack L. Strominger. Epimerization of thymidine diphosphate glucose in bacterial extracts. J. Bacteriol. 86:246-251. 1963.-Extracts of Pasteurella pseudotuberculosis type IV and several other bacteria catalyzed the reversible interconversion of thymidine diphosphate (TDP)-d-glucose and TDP-d-galactose. Moreover, extracts of P. pseudotuberculosis catalyzed the synthesis of TDP-d-glucose from thymidine triphosphate and alpha-d-glucose-1-phosphate. The limitations of these observations and their possible significance are discussed.

Galactose↗
PubMed1966

Isolation of a Mutant of Escherichia coli with a Temperature-sensitive Fructose-1,6-Diphosphate Aldolase Activity.

Böck, August (Purdue University, Lafayette, Ind.), and Frederick C. Neidhardt. Isolation of a mutant of Escherichia coli with a temperature-sensitive fructose-1,6-diphosphate aldolase activity. J. Bacteriol. 92:464-469. 1966.-A mutant of Escherichia coli was isolated which was able to grow in rich medium at 30 C but not at 40 C. Upon exposure to 40 C, the cells immediately stopped ribonucleic acid (RNA) and deoxyribonucleic acid synthesis, but protein synthesis continued at a diminished rate for a short time. Addition of chloramphenicol did not release RNA synthesis from inhibition at 40 C. Synthesis of beta-galactosidase could be induced at high temperature despite the presence of glucose in the medium, indicating a lesion in glucose catabolism. Of many catabolic enzymes tested in cell-free extracts, only fructose-1,6-diphosphate aldolase activity appeared to be altered in the mutant cells. No activity was demonstrable in extracts of mutant cells grown at either 30 or 40 C, but determination of glucose-oxidation patterns revealed that the enzyme is probably active in vivo at 30 C. Temperature-resistant secondary mutants were found to have partially or fully restored aldolase activity, and temperature-resistant recombinants had normal aldolase activity, indicating that the growth pattern and the altered aldolase had a common genetic basis. Linkage data permitted the assignment of an approximate map location for the mutated aldolase gene.

Journal Article↗
PubMed1967

Effect of adenosine monophosphate, adenosine diphosphate, and reduced nicotinamide adenine dinucleotide on adenosine triphosphate-dependent carbon dioxide fixation in the autotroph Thiobacillus neapolitanus.

The observation that adenosine triphosphate (ATP)-dependent CO(2) fixation in extracts of chemosynthetic and photosynthetic autotrophs may be regulated in part by adenosine monophosphate (AMP) was extended to the strict autotroph Thiobacillus neapolitanus (X). In addition, this report presents data which include adenosine diphosphate (ADP) in the regulatory role. When the primary CO(2) acceptor, ribose-5-phosphate, was replaced by ribulose-1,5-diphosphate, no inhibition of CO(2) fixation occurred unless the Mg(++) concentration was limiting. A molar ratio of 5:1 AMP or ADP to ATP reduced the specific activity (micromoles of CO(2) fixed per milligram of protein per minute) of the extracts from 0.22 to 0.12 and 0.11, respectively. The reported stimulation of the carboxylative phase of ATP-dependent CO(2) fixation by reduced nicotinamide adenine dinucleotide (NADH(2)) was investigated. Adding NADH(2) to the extracts did not stimulate CO(2) fixation, even at carbonate levels from 0.05 to 30 mumoles, except in the absence of ribose-5-phosphate. Slight increases in CO(2) fixation were noted when the assay system was incubated in air instead of the usual helium atmosphere.

Adenine Nucleotides↗
PubMed1981

Galactosaemia: a new severe variant due to uridine diphosphate galactose-4-epimerase deficiency.

A baby presented on day 5 with symptoms of classical galactosaemia which are believed to be owing to a lack of uridine diphosphate-4-epimerase, rather than to the usual galactose-1-phosphate uridyl transferase defect. Apart from galactosaemia the condition was characterised biochemically by a red cell accumulation of galactose-1-phosphate and uridine diphosphate galactose. Galactose restriction modified the acute clinical and biochemical abnormality, but it appears essential to include some galactose in the diet in this condition to allow synthesis of galactosides, including the brain gangliosides.

Carbohydrate Epimerases↗
PubMed1988

Hydrogen peroxide induced adenosine diphosphate ribosyl transferase (ADPRT) response in patients with inflammatory bowel disease.

The sample population in this initial case control study of the adenosine diphosphate ribosyl transferase (ADPRT) response of inflammatory bowel disease patients included: 23 patients with ulcerative colitis (UC)-active and inactive, 13 patients with Crohn's disease (CD)-active and inactive, 14 first degree relatives of UC and CD patients, and 19 age-matched controls. Adenosine diphosphate ribosyl transferase activity was determined after one hour incubation with 1% plasma (the constitutive value) or with 1% plasma and 100 microM H2O2 (the activated value) with the resulting difference designated as the induced value. Statistically significant decrease in ADPRT activity was found for the constitutive, activated and induced values in human mononuclear leucocytes of UC and CD patients, compared with controls. The values in the first degree relatives of UC and CD patients were not significantly different from either the control or disease populations, indicating an intermediate ADPRT response. These results may be related to the nature of the immunological response of IBD patients and comparable with similar findings in other diseases with known DNA repair deficiencies--for example, colon cancer.

Adult↗
PubMed1963

Further observations on platelet aggregation induced by adenosine diphosphate thrombin and tri-ethyl tin.

Aggregation induced by adenosine diphosphate is inhibited by adenosine as well as by adenosine mono- and triphosphate. Thrombin-induced aggregation is also inhibited by adenosine and the monophosphate, but the triphosphate at a similar concentration is not inhibitory. Aggregation induced by tri-ethyl tin is accelerated by a critical concentration of triphosphate and delayed by a higher concentration. The tri-ethyl tin effect is inhibited by 5-hydroxytryptamine and by tryptamine. Platelets from patients treated with reserpine respond normally to these last two substances. Incubated platelets lose first their ability to respond normally to tri-ethyl tin, then to thrombin, and lastly to adenosine diphosphate. These results are briefly discussed.

Journal Article↗
PubMed1979

A new gene involved in expression of fructose-1,6-diphosphate aldolase activity in Escherichia coli.

A new gene, fdaB, has been mapped by transduction and partial diploid analyses and located adjacent to argA at 59.9 min on the Escherichia coli recalibrated linkage map. This gene is involved in expression of fructose-1,6-diphosphate aldolase activity and indirectly in ribosomal RNA synthesis. The temperature-sensitive mutant strain AA-157, containing the defective gene product of of fdaB, accumulates high concentrations of fructose 1,6-diphosphate at the nonpermissive temperature.

Chromosome Mapping↗