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R Eliasson

Publications and source records attributed to R Eliasson.

At least 37 records · Page 2Linked to original sources
PubMed1993

Flavodoxin is required for the activation of the anaerobic ribonucleotide reductase.

The inactive anaerobic ribonucleotide reductase from Escherichia coli is transformed by a multienzyme system and S-adenosylmethionine + NADPH into a radical protein that is enzymatically active. One of the activating enzyme components was earlier shown to be ferredoxin (flavodoxin):NADP+ reductase. Here we present evidence that flavodoxin, but not ferredoxin, also is a component of the system. Light reduced deazaflavin can substitute for the flavodoxin system. An additional unidentified low-molecular weight component further stimulates the reaction.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1993

Escherichia coli ferredoxin NADP+ reductase: activation of E. coli anaerobic ribonucleotide reduction, cloning of the gene (fpr), and overexpression of the protein.

A specific ribonucleoside triphosphate reductase is induced in anaerobic Escherichia coli. This enzyme, as isolated, lacks activity in the test tube and can be activated anaerobically with S-adenosylmethionine, NADPH, and two previously uncharacterized E. coli fractions. The gene for one of these, previously named dA1, was cloned and sequenced. We found an open reading frame coding for a polypeptide of 248 amino acid residues, with a molecular weight of 27,645 and with an N-terminal segment identical to that determined by direct Edman degradation. In a Kohara library, the gene hybridized between positions 3590 and 3600 on the physical map of E. coli. The deduced amino acid sequence shows a high extent of sequence identity with that of various ferredoxin (flavodoxin) NADP+ reductases. We therefore conclude that dA1 is identical with E. coli ferredoxin (flavodoxin) NADP+ reductase. Biochemical evidence from a bacterial strain, now constructed and overproducing dA1 activity up to 100-fold, strongly supports this conclusion. The sequence of the gene shows an apparent overlap with the reported sequence of mvrA, previously suggested to be involved in the protection against superoxide (M. Morimyo, J. Bacteriol. 170:2136-2142, 1988). We suggest that a frameshift introduced during isolation or sequencing of mvrA caused an error in the determination of its sequence.

Amino Acid Sequence↗
PubMed1992

Characterization of components of the anaerobic ribonucleotide reductase system from Escherichia coli.

Anaerobic growth of Escherichia coli induces an oxygen-sensitive ribonucleoside triphosphate reductase system, different from the aerobic ribonucleoside diphosphate reductase (EC 1.17.4.1) of aerobic E. coli and higher organisms (Fontecave, M., Eliasson, R., and Reichard, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2147-2151). We have now purified and characterized two proteins from the anaerobic system, provisionally named dA1 and dA3. dA3 is the actual ribonucleoside triphosphate reductase; dA1 has an auxiliary function. From gel filtration, dA1 and dA3 have apparent molecular masses of 27 and 145 kDa, respectively. In denaturing gel electrophoresis, dA3 gives two bands of closely related polypeptides with apparent molecular masses of 77 (beta 1) and 74 (beta 2) kDa. Immunological and structural evidence suggests that beta 2 is a degradation product of beta 1 and that the active enzyme is a dimer of beta 1. dA1 activity coincides on denaturing gels with a band of 29 kDa and thus appears to be a monomer. The reaction requires, in addition, an extract from E. coli heated for 30 min at 100 degrees C. Potassium is one required component, but one or several others remain unidentified and are provisionally designated fraction RT. With dA3, dA1, RT, and potassium ions, CTP reduction shows absolute requirements for S-adenosylmethionine, NADPH (with NADH as a less active substitute), dithiothreitol, and magnesium ions, and is strongly stimulated by ATP, probably acting as an allosteric effector. Micromolar concentrations of several chelators inhibit CTP reduction completely, suggesting the involvement of (a) transition metal(s).

Aerobiosis↗
PubMed1992

Activation of the anaerobic ribonucleotide reductase from Escherichia coli by S-adenosylmethionine.

The anaerobic ribonucleoside triphosphate reductase from Escherichia coli reduces CTP to dCTP in the presence of a second protein, named dA1, and a Chelex-treated boiled extract of the bacteria, named RT. The reaction requires S-adenosylmethionine, NADPH, dithiothreitol, ATP, and Mg2+ and K+ ions. It occurs only under anaerobic conditions. We now show that the overall reaction occurs in two steps. The first is an activation of the reductase by dA1 and RT and requires S-adenosylmethionine, NADPH, dithiothreitol, and possibly K+ ions. In the second step, the activated reductase reduces CTP to dCTP with ATP acting as an allosteric effector. During activation, S-adenosylmethionine is cleaved reductively to methionine + 5'-deoxyadenosine. This step is inhibited strongly by S-adenosylhomocysteine and various chelators. The activation of the anaerobic reductase shows a considerable similarity to that of pyruvate formate-lyase (Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., and Gänzler, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1332-1335).

Anaerobiosis↗
PubMed1990

The anaerobic ribonucleoside triphosphate reductase from Escherichia coli requires S-adenosylmethionine as a cofactor.

Extracts from anaerobically grown Escherichia coli contain an oxygen-sensitive activity that reduces CTP to dCTP in the presence of NADPH, dithiothreitol, Mg2+ ions, and ATP, different from the aerobic ribonucleoside diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) present in aerobically grown E. coli. After fractionation, the activity required at least five components, two heat-labile protein fractions and several low molecular weight fractions. One protein fraction, suggested to represent the actual ribonucleoside triphosphate reductase was purified extensively and on denaturing gel electrophoresis gave rise to several defined protein bands, all of which were stained by a polyclonal antibody against one of the two subunits (protein B1) of the aerobic reductase but not by monoclonal anti-B1 antibodies. Peptide mapping and sequence analyses revealed partly common structures between two types of protein bands but also suggested the presence of an additional component. Obviously, the preparations are heterogeneous and the structure of the reductase is not yet established. The second, crude protein fraction is believed to contain several ancillary enzymes required for the reaction. One of the low molecular weight components is S-adenosylmethionine; a second component is a loosely bound metal. We propose that S-adenosylmethionine together with a metal participates in the generation of the radical required for the reduction of carbon 2' of the ribosyl moiety of CTP.

Anaerobiosis↗
PubMed1989

Enzymatic regulation of the radical content of the small subunit of Escherichia coli ribonucleotide reductase involving reduction of its redox centers.

The active form of protein B2, a homodimeric subunit of Escherichia coli ribonucleotide reductase, contains a diferric iron center and a cationic free radical localized to tyrosine 122 of one of the two polypeptide chains. Hydroxyurea scavenges this radical but leaves the iron center intact. The resulting metB2 (earlier named B2/HU) is enzymatically inactive. Crude extracts of E. coli catalyze the interconversion of metB2 and B2. Radical introduction into metB2 requires a flavin reductase together with a second poorly defined protein fraction ("Fraction b") as well as dioxygen, NAD(P)H, and a flavin (Fontecave, M., Eliasson, R., and Reichard, P. (1987) J. Biol. Chem. 262, 12325-12331). We now find that ferrous ions can substitute for Fraction b and that the diferric center of metB2 is reduced during anaerobic incubation of the system with reduced flavin and ferrous ions. Spectroscopic evidence and isotope experiments suggest an in situ reduction of the diferric to a diferrous center. Admission of oxygen then results in the instantaneous oxidation of tyrosine 122 to the cationic radical coupled to the reformation of the diferric center, giving enzymatically active B2. These data suggest that reduced diferrous B2 is an intermediate between metB2 and B2 during radical introduction. In addition, we find that anaerobic incubation of B2 with reduced flavin results in the loss of the tyrosyl radical and the formation of metB2. This reaction occurs in the absence of Fraction b or ferrous ions. Our experiments reconstitute with defined reagents the interconversion between metB2 and B2 observed earlier in the E. coli extract. The flavin reductase system catalyzes the interconversion in both directions with dioxygen as the critical factor deciding whether activation or inactivation of ribonucleotide reductase occurs.

Animals↗
PubMed1989

Oxygen-sensitive ribonucleoside triphosphate reductase is present in anaerobic Escherichia coli.

Ribonucleoside diphosphate reductase from Escherichia coli and mammalian cells provides the deoxyribonucleoside triphosphates for DNA synthesis. The active enzyme contains a tyrosyl free radical whose formation requires oxygen. Earlier genetic evidence suggested that the enzyme is not required for anaerobic growth of E. coli, implicating the activity of a different enzyme or enzyme system for deoxyribonucleotide synthesis in the absence of oxygen. We now conclude from isotope incorporation experiments that E. coli during anaerobiosis obtains its deoxyribonucleotides by reduction of ribonucleotides. Extracts from anaerobically grown bacteria contain a different enzyme activity capable of reducing CTP to dCTP. To obtain an active enzyme, strict anaerobiosis must be maintained during extract preparation and during assay of the enzyme. The reaction is stimulated by NADPH, Mg2+, and ATP. Inhibition by deoxyribonucleoside triphosphates suggests that the anaerobic enzyme has allosteric properties. Antibodies raised against the aerobic enzyme do not inhibit the new activity, and hydroxyurea, a potent scavenger of the tyrosyl radical of the aerobic enzyme, only weakly inhibits the anaerobic enzyme. The anaerobic enzyme has interesting evolutionary aspects since it might reflect on an activity that in the absence of oxygen made possible the transition from an "RNA world" into a "DNA world."

Aerobiosis↗
PubMed1987

NAD(P)H:flavin oxidoreductase of Escherichia coli. A ferric iron reductase participating in the generation of the free radical of ribonucleotide reductase.

The active form of one subunit of Escherichia coli ribonucleotide reductase (protein B2) contains an organic free radical localized to tyrosine 122 of its polypeptide chain. When this radical is scavenged, e.g. by treatment with hydroxyurea, the enzyme is inactivated (protein B2/HU). E. coli contains an enzyme system consisting of at least three proteins that in the presence of NADPH, FMN, dithiothreitol, and oxygen introduce the tyrosyl radical into B2/HU (Eliasson, R., Jörnvall, H., and Reichard, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2373-2377). One of the three proteins was identified as superoxide dismutase. We now identify a second protein, previously provisionally named Fraction c, as an NAD(P)H:flavin oxidoreductase (flavin reductase). After 4,000-fold purification the protein moved as a single band on sodium dodecyl sulfate gel electrophoresis with a molecular weight of 28,000-29,000. The enzyme contained no flavin but reduced riboflavin, FMN, and FAD by NADH, or riboflavin and FMN by NADPH. It is a powerful ferric iron reductase. We propose that its complementing activity during radical generation involves participation in the reduction of the ferric iron center of protein B2/HU. Radical formation is then linked to the reoxidation of iron by oxygen. The flavin reductase may also participate in other aspects of iron metabolism of E. coli.

Chromatography, High Pressure Liquid↗
PubMed1987

Evaluation of a commercially available kit for the colorimetric determination of zinc in human seminal plasma.

A kit from Wako Pure Chemical Industries for colorimetric determination of zinc has been evaluated for its possible use in the determination of zinc in human seminal plasma. The within-assay variation for 15 replicates of each of two seminal plasma samples having zinc concentrations (mM) of 0.43 +/- 0.025 and 6.06 +/- 0.125 (mean +/- SD) was 5.7% and 2.1%, respectively. The between-assay variation after analysis of 15 replicates of a seminal plasma sample (zinc conc. 5.6 mM) on different days was 2.3%. No interference from other metal ions present in seminal plasma was observed. The average % recovery of zinc added to seminal plasma was 102.7 +/- 1.77 (mean +/- SD). A close correlation (r = 0.996, n = 105) was found between the levels of zinc determined by the colorimetric method and that determined by atomic absorption spectrophotometry as reference method. It is concluded that the present colorimetric method, which is fast, sensitive and linear over the entire concentration range of zinc present in human seminal plasma, can be recommended for use in semen analysis laboratories.

Colorimetry↗
PubMed1986

Superoxide dismutase participates in the enzymatic formation of the tyrosine radical of ribonucleotide reductase from Escherichia coli.

One of the two nonidentical subunits of Escherichia coli ribonucleotide reductase, protein B2, contains in its active form two antiferromagnetically coupled Fe(III) ions and an organic free radical that arises by the one-electron oxidation of tyrosine-122 of the polypeptide chain. Protein B2 lacking the tyrosine radical but with the iron center intact (called protein B2/HU because it is produced by treatment with hydroxyurea) is enzymatically inactive. Previously, it was found that a crude extract from E. coli transforms B2/HU into B2 in the presence of dithiothreitol, Mg2+, and oxygen. On purification of the enzyme system, we now find that radical introduction requires three separate proteins as well as NADPH and FMN. One of the proteins is superoxide dismutase. We hypothesize that the overall reaction involves a reduction of the iron center followed by the oxidation of iron and tyrosine-122. Superoxide dismutase may then be involved in the second step to protect an oxidation-sensitive intermediate. Alternatively, the enzyme might be directly involved in the oxidation step.

Enzyme Activation↗
PubMed1985

LDH-C4 in human seminal plasma and its relationship to testicular function. II. Clinical aspects.

Lactate dehydrogenase (EC 1.1.1.27) isoenzyme LDH-C4 (LDH-X) has been analyzed quantitatively in semen from men with different fertility status, such as men with pregnant wives, men with primary or secondary infertility, volunteers, men taking salazopyrine and men exposed to very hot baths. LDH-C4 activity per ml or per ejaculate was not related to the fertility status of the men. The situation was different when the LDH-C4/sperm ratio was used as a variable. The median (p50) LDH-C4 activity in semen was (in nanokat/10(8) spermatozoa) 9.4 for samples from men (N = 34) whose wives were in early pregnancy, 24.7 for men (N = 102) with primary infertility and 28.8 for men (N = 18) taking sulphasalazine. The differences in median values between the former group and the latter two groups were highly significant (P less than 0.001). In addition, semen samples from 3 infertile men who took daily hot baths as a habit, had a significantly higher LDH-C4/sperm ratio than samples collected during periods when they did not take hot baths. Semen samples were subdivided according to the number of spermatozoa in the ejaculate after correction for days of abstinence and the size of the testes. There was an inverse correlation between the LDH-C4/sperm ratio and the adjusted sperm count (million per day and per ml testes). Men with an adjusted sperm count of 0.5 or less had a median (p50) LDH-C4 activity of 38 nanokat/10(3) spermatozoa compared to 14.5 in samples from men with an adjusted sperm count of more than 1.0 (P less than 0.001). The LDH-C4/sperm ratio in seminal plasma may therefore serve as an indicator of the function of the seminiferous epithelium, and its assessment may provide a new means for the study of spermatogenesis and male reproduction.

Adult↗
PubMed1985

LDH-C4 in human seminal plasma and testicular function. III. Relationship to other semen variables.

Lactic dehydrogenase C4 was analysed in human seminal plasma, expressed in nanokatals and related to the sperm count. The LDH-C4/sperm ratio has previously been shown to reflect the functional integrity of the seminiferous epithelium. In the present study this ratio was related to several other semen variables that reflect various functional or biochemical properties of the spermatozoa or other cell systems. For none of these variables was there a significant linear correlation with the LDH-C4/sperm ratio, but interesting differences became apparent when the semen samples were subdivided into two groups, those with normal (less than 20) and those with a high (greater than 20) ratio. For nuclear chromatin stability, oxygen consumption, succinate-induced increase in oxygen consumption and lipid peroxidation, there were significant differences between the two groups. There was no difference between the groups for the staining properties of the DNA-DNP complex, zinc, fructose and free 1-carnitine content in the ejaculate, and the zinc/fructose ratio. The results indicate that functional properties of the spermatozoa can be related to the status of the seminiferous epithelium.

Cell Nucleus↗
PubMed1984

Evidence for the prostatic origin of immunoreactive inhibin-like material in human seminal plasma.

Inhibin is defined as a gonadal peptide exerting an inhibitory effect on the secretion of follicle-stimulating hormone (FSH) by the pituitary. Using a radioimmunoassay (RIA) procedure developed for a homogeneous inhibin-like peptide with a molecular weight of 14 000 daltons isolated from human seminal plasma, immunoreactive inhibin-like matrial (ILM) was quantitated in serum, urine and semen of men in order to investigate its origin. Vasectomy did not result in a significant reduction in seminal plasma ILM. Determination of ILM immunoreactivity in ejaculates form normal men and semen samples characterized by prostate-rich and prostate-deficient secretions, indicated high levels of ILM in the prostatic secretions. Immunoreactive ILM levels estimated in different fractions of split ejaculates from normal men paralleled those of zinc and acid phosphatase activity and were significantly higher in fractions representing prostatic secretions compared to those representing the secretions of seminal vesicles. Estimation of ILM in semen, serum and urine from bilaterally gonadectomized men showed that immunoreactive ILM levels remained high after gonadectomy. It is concluded that the bulk of the immunoreactive ILM present in the semen, blood and urine of men is not secreted by the testes. The principal site of origin of this material, at least in semen, appears to be the prostate.

Acid Phosphatase↗
PubMed1983

Enzymic modification of a tyrosine residue to a stable free radical in ribonucleotide reductase.

Protein B2, a subunit of ribonucleotide reductase from Escherichia coli, contains in its active form a tyrosyl free radical as part of the polypeptide chain and a dimeric iron center that stabilizes the radical. The enzyme depends on this radical for its catalytic activity. Treatment with hydroxyurea scavenges the radical without disturbing the iron center and, thereby, results in an inactive form of the subunit, B2/HU. A second inactive form, apoB2, lacking both the radical and the iron center, is obtained by treatment of B2 with 8-hydroxyquinoline. Here we describe an enzyme activity in extracts from E. coli that transforms the catalytically inactive B2/HU form into the active B2 subunit by regeneration of the tyrosyl radical. This reaction requires the presence of oxygen, dithiothreitol, and Mg2+ and does not proceed through apoB2. Under anaerobic conditions, we obtained evidence for a second activity in the bacterial extract that destroys the free radical and transforms B2 into B2/HU. We suggest that this novel type of protein modification is functionally related to the synthesis of deoxyribonucleotides and DNA.

Apoproteins↗
PubMed1983

Male and female infertility problems in the immotile-cilia syndrome.

The immotile-cilia syndrome is a heterogeneous disease. The dynein arms were missing totally or almost totally, and both spermatozoa and cilia were immotile in most cases examined by us. In other cases, the ciliary axoneme displayed other defects and the spermatozoa had motility although restricted; there was no progressive motility. Fourteen men with this syndrome were all sterile. Fifteen women had the syndrome and twelve tried to become pregnant. This had been successful (one, one, and two children) in three cases only. Thus, it appears that female fertility is also impaired in this syndrome, although not completely. No case of ectopic pregnancy has been reported.

Cilia↗