Enrichment of ligands with molecular dockings and subsequent characterization for human alcohol dehydrogenase 3

Alcohol dehydrogenase 3 (ADH3) has been assigned a role in nitric oxide homeostasis due to its function as an S-nitrosoglutathione reductase. As altered S-nitrosoglutathione levels are often associated with disease, compounds that modulate ADH3 activity might be of therapeutic interest. We performed a virtual screening with molecular dockings of more than 40,000 compounds into the active site of human ADH3. A novel knowledge-based scoring method was used to rank compounds, and several compounds that were not known to interact with ADH3 were tested in vitro. Two of these showed substrate activity (9-decen-1-ol and dodecyltetraglycol), where calculated binding scoring energies correlated well with the logarithm of the k _cat/K _m values for the substrates. Two compounds showed inhibition capacity (deoxycholic acid and doxorubicin), and with these data three different lines for specific inhibitors for ADH3 are suggested: fatty acids, glutathione analogs, and cholic acids.     

Kinetik der hepatischen Farbstoffaufnahme von Indocyaningrün. Einfluss von Bilirubin und Natriumglykocholat

Summary Kinetics of hepatic uptake of indocyanine green, a dye which is used for evaluation of liver function, were studied in the rat. The results indicate that the relationship between ICG-dose and initial hepatic dye uptake obeys Michaelis-Menten kinetics suggesting an interaction of the dye with a carrier or fixed site in the liver cell. Thus it was possible to calculate maximum ICG-uptake (v _max ) and the Michaelis constant (K _m ) of this transport system from several submaximal values.v _max was 7.65 (6-06-9.65)²² mg per 100 g liver/min and K _m 0.56 (0.31–0.81)²². Under the influence of substances which inhibit the elimination of dyes by the liver the parametersv _max and K _m showed changes which allowed characterization of the type of inhibition. While sodium glycocholate had no influence on maximum hepatic ICG-uptake and the Michaelis constant bilirubin caused a significant increase of K _m to 1.29 (0.68–1.90)²² without significantly changingv _max . These data suggest that bilirubin interferes with hepatic uptake of indocyanine green by competitive inhibition and that uptake of bile acids is dependent on a different mechanism.     

Isoform specific phosphorylation of p53 by protein kinase CK1

The ability of three isoforms of protein kinase CK1 (α, γ₁, and δ) to phosphorylate the N-terminal region of p53 has been assessed using either recombinant p53 or a synthetic peptide reproducing its 1–28 sequence. Both substrates are readily phosphoylated by CK1δ and CK1α, but not by the γ isoform. Affinity of full size p53 for CK1 is 3 orders of magnitude higher than that of its N-terminal peptide (K _m 0.82 μM vs 1.51 mM). The preferred target is S20, whose phosphorylation critically relies on E17, while S6 is unaffected despite displaying the same consensus (E-x-x-S). Our data support the concept that non-primed phosphorylation of p53 by CK1 is an isoform-specific reaction preferentially affecting S20 by a mechanism which is grounded both on a local consensus and on a remote docking site mapped to the K²²¹RQK²²⁴ loop according to modeling and mutational analysis.     

Normetanephrine and metanephrine oxidized by both types of monoamine oxidase

Summary Both normetanephrine and metanephrine were found to be oxidized by both types of monoamine oxidase in mouse liver mitochondria. Both K_m and V_max values of type B MAO for both substrates were higher than those of type A MAO, which caused the shift of inhibition curves with clorgyline and deprenyl according to the increase in substrate concentration.     

Genetically engineered avidins and streptavidins

Chicken avidin and bacterial streptavidin, (strept)avidin, are proteins widely utilized in a number of applications in life science, ranging from purification and labeling techniques to diagnostics, and from targeted drug delivery to nanotechnology. (Strept)avidin-biotin technology relies on the extremely tight and specific affinity between (strept)avidin and biotin (dissociation constant, K_d≈10⁻¹⁴–10⁻¹⁶ M). (Strept)avidins are also exceptionally stable proteins. To study their ligand binding and stability characteristics, the two proteins have been extensively modified both chemically and genetically. There are excellent accounts of this technology and chemically modified (strept)avidins, but no comprehensive reviews exist concerning genetically engineered (strept)avidins. To fill this gap, we here go through the genetically engineered (strept)avidins, summarizing how these constructs were designed and how they have improved our understanding of the structural and functional characteristics of these proteins, and the benefits they have provided for (strept)avidin-biotin technology. Received 22 June 2006; received after revision 1 August 2006; accepted 21 September 2006     

Trypsinogen-kinase fromAspergillus fumigatus

Summary The activation of bovine trypsinogen by an extracellular acid proteinase fromA. fumigatus is described. The enzyme activates trypsinogen optimally at pH 3.5 and 32°C. The effect of substrate and enzyme concentrations on the activation has been studied and the K_m-value has been determined.Acknowledgments. We are grateful to Dr. N. Ramanathan for the permission to publish this paper and to CSIR, New Delhi, for the financial assistance to M.P.     

Kinetics and subcellular distribution of S35-taurine uptake in rat cerebral cortex slices

Zusammenfassung Die Kinetik der Aufnahme von S³⁵-Taurin in Rattencortex-Schnittchen wird im Konzentrationsbereich von 9×10^–8 M bis 5×10^–3 M untersucht. Nach Abzug des Transportes durch Diffusion (K _D ×S) findet man einen Mechanismus, der Michaelis-Menten Kinetik folgt (v _sat ), mitK _m =1,73×10^–4 M. Ein solcher Transport liegt nicht im Bereich des spezifischen «uptake» der Neurotransmitter. Auch die sehr niedrige Aufnahme-Rate und die subzelluläre Verteilung nach «uptake» sprechen gegen eine Neurotransmitter-Funktion von Taurin.     

Acyl-CoA thioesterase 9 (ACOT9) in mouse may provide a novel link between fatty acid and amino acid metabolism in mitochondria

Acyl-CoA thioesterase (ACOT) activities are found in prokaryotes and in several compartments of eukaryotes where they hydrolyze a wide range of acyl-CoA substrates and thereby regulate intracellular acyl-CoA/CoA/fatty acid levels. ACOT9 is a mitochondrial ACOT with homologous genes found from bacteria to humans and in this study we have carried out an in-depth kinetic characterization of ACOT9 to determine its possible physiological function. ACOT9 showed unusual kinetic properties with activity peaks for short-, medium-, and saturated long-chain acyl-CoAs with highest V _max with propionyl-CoA and (iso) butyryl-CoA while K _cat/K _m was highest with saturated long-chain acyl-CoAs. Further characterization of the short-chain acyl-CoA activity revealed that ACOT9 also hydrolyzes a number of short-chain acyl-CoAs and short-chain methyl-branched CoA esters that suggest a role for ACOT9 in regulation also of amino acid metabolism. In spite of markedly different K _ms, ACOT9 can hydrolyze both short- and long-chain acyl-CoAs simultaneously, indicating that ACOT9 may provide a novel regulatory link between fatty acid and amino acid metabolism in mitochondria. Based on similar acyl-CoA chain-length specificities of recombinant ACOT9 and ACOT activity in mouse brown adipose tissue and kidney mitochondria, we conclude that ACOT9 is the major mitochondrial ACOT hydrolyzing saturated C₂-C₂₀-CoA in these tissues. Finally, ACOT9 activity is strongly regulated by NADH and CoA, suggesting that mitochondrial metabolic state regulates the function of ACOT9.     

Purification and properties of glutamine synthetase I fromRhizobium sp. UMKL 20

Summary Glutamine synthetase I was purified fromRhizobium sp. UMKL 20 following polyethylene glycol precipitation. The enzyme had a subunit molecular weight of 58 kd. Apparent K_m values for ammonia and glutamate were 5.6 and 15.2 mM, respectively. Glutamine synthetase I activity was inhibited by several end products of glutamine metabolism. The purified enzyme was highly adenylylated (E _n ^– =8.5).Acknowledgment. I would like to thank Mr J. C. Lai for technical assistance. This work was carried out with the support of Vote F 153/79 from the University of Malaya.     

Conotoxins of the O-superfamily affecting voltage-gated sodium channels

The venoms of predatory cone snails harbor a rich repertoire of peptide toxins that are valuable research tools, but recently have also proven to be useful drugs. Among the conotoxins with several disulfide bridges, the O-superfamily toxins are characterized by a conserved cysteine knot pattern: C-C-CC-C-C. While ω-conotoxins and κ-conotoxins block Ca²⁺ and K⁺ channels, respectively, the closely related δ- and μO-conotoxins affect voltage-gated Na⁺ channels (Na_v channels). δ-conotoxins mainly remove the fast inactivation of Na_v channels and, thus, functionally resemble long-chain scorpion α-toxins. μO-conotoxins are functionally similar to μ-conotoxins, since they inhibit the ion flow through Na_v channels. Recent results from functional and structural assays have gained insight into the underlying molecular mechanisms. Both types of toxins are voltage-sensor toxins interfering with the voltage-sensor elements of Na_v channels. Received 27 December 2006; received after revision 30 January 2007; accepted 19 February 2007     

Stability analysis of the bacteriophage ϕKMV lysin gp36C and its putative role during infection

The kinetic, thermodynamic and structural stability of gp36C, the virion-associated peptidoglycan hydrolase domain of bacteriophage ϕKMV, is analyzed. Recombinant gp36C is highly thermoresistant (k = 0.595 h⁻¹ at 95°C), but not thermostable (T_m = 50.2°C, ΔH_cal = 6.86 × 10⁴ cal mol⁻¹). However, aggregation influences kinetic stability in an unusual manner since aggregation is more pronounced at 55°C than at higher temperatures. Furthermore, gp36C reversibly unfolds in a two-state endothermic transition, and circular dichroism analysis shows that gp36C almost completely refolds after a 3-h heat treatment at 85°C. These properties are in agreement with gp36C being part of the extensible tail which is ejected in an unfolded state during phage infection. Received 24 April 2006; received after revision 26 May 2006; accepted 10 June 2006     

A new method for determining the Michaelis-Menten constant viscometrically for enzyme reactions of high-molecular substrates

Zusammenfassung In Übereinstimmung mit der neu entwickelten viskosimetrischen Methode wurde die Michaelis-Menten-KonstanteK _m =3,6×10^–5 mmol/ml der Hydrolysenreaktion der Natriumcarboxymethylcellulose unter der katalytischen Mitwirkung von Cx-Cellulasen-Enzymen bestimmt.     

Baker's yeast cytidine deaminase: Substrate and inhibitor specificity,and a hypothesis on metabolic and regulatory significance

Riassunio La citidina deaminasi, parzialmente purificata da lievito di pane, è capace di deaminare sia la citidina che la deossicitidina. I valori delleK _m per ambedue i 2 substrati sono 25×10^–5 M e 9.1×10^–5 M rispettivamente. Inoltre l'enzima è inibito da numerosi nucleosidi monofosfati, difosfati e trifosfati. È molto significativa il tipo di inibizione allosterica esercitata dal dTTP. Si riporta una ipotesi sul ruolo metabolico della citidina deaminasi.     

Influence of castration and testosterone replacement on hypothalamic tyrosine hydroxylase activity in the rat

Summary Hypothalamic tyrosine hydroxylase (TH) activity of castrate rats is modulated by testosterone propionate (TP) in vivo. Kinetic studies revealed that bothV _max andK _m were virtually unaltered for substrate tyrosine in the presence of an excess of DMPH₄ cofactor. TP replacement to castrate rats increased theK _m for added DMPH₄ cofactor, whileV _max decreased. These results suggest that TP decreases TH activity of castrate rats by inhibiting the enzymereduced pteridine cofactor complex.     

Ergot alkaloids and phosphodiesterase; ‘in vitro’ activities in several rat brain areas

Summary The ‘in vitro’ activity of ergotamine, dihydroergocristine, dihydroergocornine and dihydroergocryptine on the phosphodiesterase system at low and high K_m in several rat brain areas was examined. These drugs were found to exert an inhibitory effect in all the areas examined with regard to both systems, and particularly on low substrate concentration phosphodiesterases. Acknowledgments. Ergot alkaloids were a generous gift of Poli Industria Chimica, Milan.     

Induction of the in vitro p-hydroxylation of14C-amphetamine stereoisomers in phenobarbital-treated rats

Summary The rate of p-hydroxylation of¹⁴C-(-)-amphetamine by liver microsomes was higher than that of (+)-isomer in phenobarbital-treated male rats. The apparent K_m values for (-)- and (+)-amphetamine hydroxylation were 4.54×10^–5 M and 2.27×10^–5 M respectively, in both treated and control animals.     

Biochemical effects and growth inhibition in MCF-7 cells caused by novel sulphonamido oxa-polyamine derivatives

The novel polyamine derivatives sulphonamido oxa-spermine (oxa-Spm) and sulphonamido oxa-spermidine (oxa-Spd) exhibited rapid cytotoxic action towards MCF-7 human breast cancer cells with IC₅₀ values of 4.35 and 6.47 μM, respectively, after 24-h drug exposure. Neither compound is a substrate of serum amine oxidase. Both oxa-Spm and oxa-Spd caused cell shrinkage, as determined by phase-contrast microscopy. After incubation with 10 μM of either compound for 8 h, the cells underwent chromatin condensation and nuclear fragmentation. However, no clear DNA ladder was obtained by electrophoresis. The sulphonamido oxa-polyamine derivatives and especially oxa-Spd enhanced the activity of polyamine oxidase (PAO), an enzyme capable of oxidising N¹-acetylated spermine and spermidine to spermidine and putrescine, respectively, generating cytotoxic H₂O₂ and 3-acetamidopropanal as by-products. The intracellular polyamine content was only marginally reduced in response to drug treatment. In conclusion, our data show that these novel sulphonamido oxa-polyamine derivatives possess high cytotoxic activity against MCF-7 cells and indicate that induction of PAO may mediate their cytotoxicity via apoptosis. Received 17 January 2002; received after revision 22 February 2002; accepted 22 February 2002     

The diterpenoid alkaloid noroxoaconitine is a Mapkap kinase 5 (MK5/PRAK) inhibitor

The mitogen-activated protein kinase-activated protein kinase MK5 is ubiquitously expressed in vertebrates and is implicated in cell proliferation, cytoskeletal remodeling, and anxiety behavior. This makes MK5 an attractive drug target. We tested several diterpenoid alkaloids for their ability to suppress MK5 kinase activity. We identified noroxoaconitine as an ATP competitor that inhibited the catalytic activity of MK5 in vitro (IC₅₀ = 37.5 μM; K _i = 0.675 μM) and prevented PKA-induced nuclear export of MK5, a process that depends on kinase active MK5. MK5 is closely related to MK2 and MK3, and noroxoaconitine inhibited MK3- and MK5- but not MK2-mediated phosphorylation of the common substrate Hsp27. Molecular docking of noroxoaconitine into the ATP binding sites indicated that noroxoaconitine binds more strongly to MK5 than to MK3. Noroxoaconitine and derivatives may help in elucidating the precise biological functions of MK5 and may prove to have therapeutic values.     

Monoclonal antibodies to L-asparaginase

Summary Five hybridomas secreting monoclonal antibody toE. coli L-asparaginase were isolated. These monoclonal antibodies were classified into 3 different subclasses; Ig G₁ (1 clone), Ig G₂ (2 clones) and Ig G₃ (2 clones). One of them possessed anti-L-asparaginase neutralizing activity. Four antibodies examined demonstrated a linear Langmuir binding plot and binding affinities, with equilibrium dissociation constant (K_d) ranging between 2.5×10^–9M and 6.3×10^–10 M. The monoclonal antibodies should be useful probes for investigation of the enzyme activity.     

<Emphasis Type="Italic">Anopheles gambiae</Emphasis> odorant binding protein crystal complex with the synthetic repellent DEET: implications for structure-based design of novel mosquito repellents

Insect odorant binding proteins (OBPs) are the first components of the olfactory system to encounter and bind attractant and repellent odors emanating from various sources for presentation to olfactory receptors, which trigger relevant signal transduction cascades culminating in specific physiological and behavioral responses. For disease vectors, particularly hematophagous mosquitoes, repellents represent important defenses against parasitic diseases because they effect a reduction in the rate of contact between the vectors and humans. OBPs are targets for structure-based rational approaches for the discovery of new repellent or other olfaction inhibitory compounds with desirable features. Thus, a study was conducted to characterize the high resolution crystal structure of an OBP of Anopheles gambiae, the African malaria mosquito vector, in complex with N,N-diethyl-m-toluamide (DEET), one of the most effective repellents that has been in worldwide use for six decades. We found that DEET binds at the edge of a long hydrophobic tunnel by exploiting numerous non-polar interactions and one hydrogen bond, which is perceived to be critical for DEET’s recognition. Based on the experimentally determined affinity of AgamOBP1 for DEET (K _d of 31.3 μΜ) and our structural data, we modeled the interactions for this protein with 29 promising leads reported in the literature to have significant repellent activities, and carried out fluorescence binding studies with four highly ranked ligands. Our experimental results confirmed the modeling predictions indicating that structure-based modeling could facilitate the design of novel repellents with enhanced binding affinity and selectivity.