scholarly journals Light-dependent de-activation/re-activation of Anabaena variabilis ferredoxin: NADP+ reductase

1991 ◽  
Vol 274 (3) ◽  
pp. 781-786 ◽  
Author(s):  
M F Fillat ◽  
D E Edmondson ◽  
C Gomez-Moreno
Keyword(s):  
Anabaena Variabilis ◽  
Disulphide Bond ◽  
Activation Process ◽  
Thiol Groups ◽  
Native Form ◽  
Steady State Kinetic ◽  
Significant Difference ◽  
Cyanobacterium Anabaena ◽  
State Kinetic

The activity of ferredoxin: NADP+ reductase (FNR) was found to decline to approximately 20% maximal levels with little or no loss in enzyme levels when cultures of the cyanobacterium Anabaena variabilis were maintained in the stationary phase of growth. Re-activation of enzyme activity occurred when cells were diluted into either fresh or re-utilized media and illuminated. This reversible de-activation/re-activation process was found, in vivo, to be dependent on the intensity of light illuminating the cells. The de-activated form of FNR was purified to homogeneity and exhibited the same molecular mass, isoelectric-focusing pattern and N-terminal amino acid sequence as the native form. Both de-activated and native FNR preparations each exhibited three reactive thiol groups on denaturation in urea; however, the rate of reaction with Ellman's reagent was much faster with the de-activated form than with the native form. Both preparations contain a single disulphide bond. Upon reduction of the disulphide bond in either form of the enzyme, the five reactive thiol groups exhibited identical reactivities in the presence of urea. Steady-state kinetic analysis of the de-activated form showed a marked increase in Km values for NADPH in diaphorase assays and an increase in Km for ferredoxin in the ferredoxin-mediated reduction of cytochrome c. No significant difference in kcat. was observed in comparison of the de-activated with the native form in any of the above assays; however, the de-activated form did exhibit a lower kcat. value in the transhydrogenase assay. The de-activated form of FNR bound ferredoxin with a 16-fold lower affinity than the native enzyme. These data suggest that the de-activation of FNR in vivo in response to low light intensity involves an alteration in protein structure, possibly via an intramolecular thiol disulphide interchange, which influences the interaction of the enzyme with its substrates.

scholarly journals Pre-Steady-State Kinetic Analysis of the Incorporation of Anti-HIV Nucleotide Analogs Catalyzed by Human X- and Y-Family DNA Polymerases

2010 ◽  
Vol 55 (1) ◽  
pp. 276-283 ◽  
Author(s):  
Jessica A. Brown ◽  
Lindsey R. Pack ◽  
Jason D. Fowler ◽  
Zucai Suo
Keyword(s):  
Mitochondrial Dna ◽  
Steady State ◽  
Substrate Specificity ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Steady State Kinetic ◽  
Y Family ◽  
State Kinetic

ABSTRACTNucleoside reverse transcriptase inhibitors (NRTIs) are an important class of antiviral drugs used to manage infections by human immunodeficiency virus, which causes AIDS. Unfortunately, these drugs cause unwanted side effects, and the molecular basis of NRTI toxicity is not fully understood. Putative routes of NRTI toxicity include the inhibition of human nuclear and mitochondrial DNA polymerases. A strong correlation between mitochondrial toxicity and NRTI incorporation catalyzed by human mitochondrial DNA polymerase has been established bothin vitroandin vivo. However, it remains to be determined whether NRTIs are substrates for the recently discovered human X- and Y-family DNA polymerases, which participate in DNA repair and DNA lesion bypassin vivo. Using pre-steady-state kinetic techniques, we measured the substrate specificity constants for human DNA polymerases β, λ, η, ι, κ, and Rev1 incorporating the active, 5′-phosphorylated forms of tenofovir, lamivudine, emtricitabine, and zidovudine. For the six enzymes, all of the drug analogs were incorporated less efficiently (40- to >110,000-fold) than the corresponding natural nucleotides, usually due to a weaker binding affinity and a slower rate of incorporation for the incoming nucleotide analog. In general, the 5′-triphosphate forms of lamivudine and zidovudine were better substrates than emtricitabine and tenofovir for the six human enzymes, although the substrate specificity profile depended on the DNA polymerase. Our kinetic results suggest NRTI insertion catalyzed by human X- and Y-family DNA polymerases is a potential mechanism of NRTI drug toxicity, and we have established a structure-function relationship for designing improved NRTIs.

scholarly journals Phosphorylation of Ribavirin and Viramidine by Adenosine Kinase and Cytosolic 5′-Nucleotidase II: Implications for Ribavirin Metabolism in Erythrocytes

2005 ◽  
Vol 49 (6) ◽  
pp. 2164-2171 ◽  
Author(s):  
Jim Zhen Wu ◽  
Gary Larson ◽  
Heli Walker ◽  
Jae Hoon Shim ◽  
Zhi Hong
Keyword(s):  
Catalytic Efficiency ◽  
Adenosine Kinase ◽  
Common Belief ◽  
Physiological Concentration ◽  
Steady State Kinetic ◽  
Phosphorylation Mechanism ◽  
Phosphate Buffered Saline ◽  
State Kinetic

ABSTRACT Many nucleoside analog drugs, such as ribavirin and viramidine, are activated or metabolized in vivo through 5′-phosphorylation. In this report, we determined the steady-state kinetic parameters for 5′-monophosphorylation of ribavirin and viramidine by adenosine kinase. The apparent Km for ribavirin is 540 μM, and k cat is 1.8 min−1. Its catalytic efficiency of 3.3 × 10−3 min−1 · μM−1 is 1,200-fold lower than that of adenosine. In contrast to the common belief that ribavirin is exclusively phosphorylated by adenosine kinase, cytosolic 5′-nucleotidase II was found to catalyze ribavirin phosphorylation in vitro. The reaction is optimally stimulated by the physiological concentration of ATP or 2,3-bisphosphoglycerate. In phosphate-buffered saline plus ATP and 2,3-bisphosphoglycerate, the apparent Km for ribavirin is 88 μM, and k cat is 4.0 min−1. These findings suggest that cytosolic 5′-nucleotidase II may be involved in ribavirin phosphorylation in vivo. Like ribavirin, viramidine was found to be phosphorylated by either adenosine kinase or cytosolic 5′-nucleotidase II, albeit with a much lower activity. The catalytic efficiency for viramidine phosphorylation is 10- to 330-fold lower than that of ribavirin, suggesting that other nucleoside kinase(s) may be involved in viramidine phosphorylation in vivo. Both ribavirin and viramidine are not phosphorylated by deoxycytidine kinase and uridine-cytidine kinase. The coincidence of presence of high concentrated 2,3-bisphosphoglycerate in erythrocytes suggests that cytosolic 5′-nucleotidase II could play an important role in phosphorylating ribavirin and contribute to anabolism of ribavirin triphosphate in erythrocytes. Elucidation of ribavirin and viramidine phosphorylation mechanism should shed light on their in vivo metabolism, especially the ribavirin-induced hemolytic anemia in erythrocytes.

scholarly journals Bovine lens aldehyde dehydrogenase. Kinetics and mechanism

1983 ◽  
Vol 215 (2) ◽  
pp. 361-368 ◽  
Author(s):  
H H Ting ◽  
M J C Crabbe
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Esterase Activity ◽  
Ternary Complex ◽  
Succinic Semialdehyde ◽  
Complex Mechanism ◽  
Thiol Groups ◽  
Bovine Lens ◽  
Steady State Kinetic ◽  
State Kinetic

Bovine lens cytoplasmic aldehyde dehydrogenase exhibits Michaelis-Menten kinetics with acetaldehyde, glyceraldehyde 3-phosphate, p-nitrobenzaldehyde, propionaldehyde, glycolaldehyde, glyceraldehyde, phenylacetylaldehyde and succinic semialdehyde as substrates. The enzyme was also active with malondialdehyde, and exhibited an esterase activity. Steady-state kinetic analyses show that the enzyme exhibits a compulsory-ordered ternary-complex mechanism with NAD+ binding before acetaldehyde. The enzyme was inhibited by disulfiram and by p-chloromercuribenzoate, and studies with with mercaptans indicated the involvement of thiol groups in catalysis.

scholarly journals Pre-Steady-State Kinetic Analysis of Truncated and Full-LengthSaccharomyces cerevisiaeDNA Polymerase Eta

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Jessica A. Brown ◽  
Likui Zhang ◽  
Shanen M. Sherrer ◽  
John-Stephen Taylor ◽  
Peter M. J. Burgers ◽  
...  
Keyword(s):  
Steady State ◽  
Dna Polymerase ◽  
Dna Lesions ◽  
Base Substitution ◽  
Polymerase Domain ◽  
Pyrimidine Dimers ◽  
C Terminus ◽  
Steady State Kinetic ◽  
State Kinetic

Understanding polymerase fidelity is an important objective towards ascertaining the overall stability of an organism's genome.Saccharomyces cerevisiaeDNA polymeraseη(yPolη), a Y-family DNA polymerase, is known to efficiently bypass DNA lesions (e.g., pyrimidine dimers) in vivo. Using pre-steady-state kinetic methods, we examined both full-length and a truncated version of yPolηwhich contains only the polymerase domain. In the absence of yPolη's C-terminal residues 514–632, the DNA binding affinity was weakened by 2-fold and the base substitution fidelity dropped by 3-fold. Thus, the C-terminus of yPolηmay interact with DNA and slightly alter the conformation of the polymerase domain during catalysis. In general, yPolηdiscriminated between a correct and incorrect nucleotide more during the incorporation step (50-fold on average) than the ground-state binding step (18-fold on average). Blunt-end additions of dATP or pyrene nucleotide5′-triphosphate revealed the importance of base stacking during the binding of incorrect incoming nucleotides.

In-vivo evaluation of the effect of cyanoacrylate on prosthetic vascular graft infection – does cyanoacrylate increase the severity of infection?

VASA ◽  
2020 ◽  
Vol 49 (4) ◽  
pp. 281-284
Author(s):  
Atıf Yolgosteren ◽  
Gencehan Kumtepe ◽  
Melda Payaslioglu ◽  
Cuneyt Ozakin
Keyword(s):  
Vascular Graft ◽  
Graft Infection ◽  
Vascular Graft Infection ◽  
Group 4 ◽  
Significant Difference ◽  
Group 3 ◽  
Group 2 ◽  
Prosthetic Vascular Graft Infection ◽  
Group 1

Summary. Background: Prosthetic vascular graft infection (PVGI) is a complication with high mortality. Cyanoacrylate (CA) is an adhesive which has been used in a number of surgical procedures. In this in-vivo study, we aimed to evaluate the relationship between PVGI and CA. Materials and methods: Thirty-two rats were equally divided into four groups. Pouch was formed on back of rats until deep fascia. In group 1, vascular graft with polyethyleneterephthalate (PET) was placed into pouch. In group 2, MRSA strain with a density of 1 ml 0.5 MacFarland was injected into pouch. In group 3, 1 cm 2 vascular graft with PET piece was placed into pouch and MRSA strain with a density of 1 ml 0.5 MacFarland was injected. In group 4, 1 cm 2 vascular graft with PET piece impregnated with N-butyl cyanoacrylate-based adhesive was placed and MRSA strain with a density of 1 ml 0.5 MacFarland was injected. All rats were scarified in 96th hour, culture samples were taken where intervention was performed and were evaluated microbiologically. Bacteria reproducing in each group were numerically evaluated based on colony-forming unit (CFU/ml) and compared by taking their average. Results: MRSA reproduction of 0 CFU/ml in group 1, of 1410 CFU/ml in group 2, of 180 200 CFU/ml in group 3 and of 625 300 CFU/ml in group 4 was present. A statistically significant difference was present between group 1 and group 4 (p < 0.01), between group 2 and group 4 (p < 0.01), between group 3 and group 4 (p < 0.05). In terms of reproduction, no statistically significant difference was found in group 1, group 2, group 3 in themselves. Conclusions: We observed that the rate of infection increased in the cyanoacyrylate group where cyanoacrylate was used. We think that surgeon should be more careful in using CA in vascular surgery.

In-vivo anti-oxidant screening of Tectona grandis extract

2020 ◽  
Vol 9 (1) ◽  
pp. 1-4
Author(s):  
Tamilarasi G P ◽  
Sabarees G
Keyword(s):  
Biochemical Parameters ◽  
Tectona Grandis ◽  
The Body ◽  
Altered Expression ◽  
Physiological Conditions ◽  
Synthetic Drugs ◽  
Significant Difference ◽  
Anti Oxidant ◽  
Radical Generation

Oxidation is an essential reaction in the human body, which determines the expression of proteins in the body. This results in the altered expression like rapid growth resulting in cancers and other disorders. Many synthetic drugs are available in the market that is effective in limiting the free radical generation and the reaction of radicals with cells. Unfortunately, all those synthetic drugs were found to cause side effects and adverse effects in the body. But given the accuracy of the predictability of the results and administration, this research focuses on testing the anti-oxidant efficiency in rat models testing the biochemical parameters. Investigations have also been done on the anti-oxidant activity of Tectona, but every research was concentrated to prove the anti-oxidant activity only. extract had been tested for anti-oxidant activity by estimating various tissue parameters and it showed better activity. As predicted, there is a significant difference in the and results which can be explained are due to the physiological conditions that exist inside the body.

Alternativas farmacéuticas: ¿pueden ser intercambiables?

2020 ◽  
Vol 2 (2) ◽  
pp. 38-42
Author(s):  
Miriam del Carmen Carrasco-Portugal ◽  
Francisco Javier Flores-Murrieta
Keyword(s):  
Generic Substitution ◽  
In Vivo Studies ◽  
Regulatory Agencies ◽  
European Medicines Agency ◽  
Reference Formulation ◽  
Active Moiety ◽  
Significant Difference ◽  
Pharmaceutical Form ◽  
Extent Of Absorption

Pharmaceutical alternatives are products with the same active moiety, but different salt, ester or pharmaceutical form. Regulatory agencies have different criteria for this kind of drug. The European Medicines Agency (EMA) accepts the generic substitution using these alternatives, whereas the Food and Drug Administration (FDA) only authorizes generic substitution of pharmaceutical equivalents. The objective of this paper is to describe some relevant aspects that should be considered before deciding on making a generic substitution with pharmaceutical alternatives. It is important to note that a pharmaceutical alternative must show no significant difference in the rate and extent of absorption (bioequivalence) in a well-conducted in vivo study when compared with the reference formulation. Current Mexican regulations state that generic substitution is possible using pharmaceutical alternatives when bioequivalence is demonstrated in in vivo studies conducted under the NOM-177-SSA1-2013 criteria. In conclusion, generic substitution with pharmaceutical alternatives is possible if these products demonstrate in vivo bioequivalence when compared with the reference product.

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