Purification of a Src family tyrosine protein kinase from bovine retinas

Since tyrosine phosphorylation appears to play important functions in photoreceptor cells, we searched here for retinal nonreceptor tyrosine kinases of the Src family. We demonstrated that Src family tyrosine kinases were present in the cytosolic fraction of extracted bovine retinas. A Src family tyrosine kinase with an apparent molecular mass of about 62 kDa was purified to homogeneity from the soluble fraction of dark-adapted bovine retinas after three consecutive purification steps: ω-aminooctyl-agarose hydrophobic chromatography, Cibacron blue 3GA-agarose pseudo-affinity chromatography, and α-casein-agarose affinity chromatography. The purified protein was subjected to N-terminal amino acid sequencing and the sequence Gly-Ile-Ile-Lys-Ser-Glu-Glu was obtained, which displayed homology with the first seven residues of the Src family tyrosine kinase c-Yes from Bos taurus (Gly-Cys-Ile-Lys-Ser-Lys-Glu). Although the cytosolic fraction from dark-adapted retinas contained tyrosine kinases of the Src family capable of phosphorylating the α-subunit of transducin, which is the heterotrimeric G protein involved in phototransduction, the purified tyrosine kinase was not capable of using transducin as a substrate. The cellular role of this retinal Src family member remains to be found.

The interaction of Schistosoma japonicum glutathione transferase with Cibacron blue 3GA and its fragments

Background: The 26kDa glutathione transferase (GST, EC 2.5.1.18) from Schistosoma japonicum (SjGST) is recognized as the major detoxification enzyme of S. japonicum, a pathogenic helminth causing schistosomiasis. Objective: In the present study, the interaction of the chlorotriazine dye Cibacron blue 3GA (CB3GA) and its structural analogues with SjGST was investigated. The work aimed to shine light on the non-substrate ligand-binding properties of the enzyme. Methods: Kinetic inhibition analysis, affinity labelling experiments and molecular modelling studies were employed. Results: The results showed that CB3GA is a potent inhibitor (IC50 0.057 ± 0.003μM) towards SjGST. The enzyme was specifically and irreversibly inactivated by the dichlorotriazine-analogue of CB3GA (IC50 0.190 ± 0.024 μM), following a biphasic pseudo-first-order saturation kinetics with approximately 1 mol of inhibitor per mol of dimeric enzyme being incorporated. All other monochlorotriazine analogues behave as reversible inhibitors with lower inhibition potency (IC50 5.2-82.3 μM). Kinetic inhibition studies together with molecular modelling and molecular dynamics simulations established that the CB3GA binding site overlaps both the G- and H-sites. Both hydrophobic/polar interactions as well as steric effects have decisive roles in determining the inhibitory strength of CB3GA and its analogues. Conclusion: The results of the present study might be useful in future drug design and development efforts towards SjGST.

Structure-guided design of a novel class of benzyl-sulfonate inhibitors for influenza virus neuraminidase

Influenza NA (neuraminidase) is an antiviral target of high pharmaceutical interest because of its essential role in cleaving sialic acid residues from cell surface glycoproteins and facilitating release of virions from infected cells. The present paper describes the use of structural information in the progressive design from a lead binding ion (a sulfate) to a potent submicromolor inhibitor (Ki 0.13 μM). Structural information derived from the X-ray structure of an NA complexed with several sulfate ions, in combination with results derived from affinity labelling and molecular modelling studies, was used to guide design of potent sulfonic acid-based inhibitors. These inhibitors are structural fragments of the polysulfonate triazine dye Cibacron Blue 3GA and represent novel lead scaffolds for designing non-carbohydrate inhibitors for influenza neuraminidases.

Role of ADP-ribose in 11,12-EET-induced activation of KCa channels in coronary arterial smooth muscle cells

We recently reported that cADP-ribose (cADPR) and ADP-ribose (ADPR) play an important role in the regulation of the Ca2+-activated K+ (KCa) channel activity in coronary arterial smooth muscle cells (CASMCs). The present study determined whether these novel signaling nucleotides participate in 11,12-epoxyeicosatrienoic acid (11,12-EET)-induced activation of the KCa channels in CASMCs. HPLC analysis has shown that 11,12-EET increased the production of ADPR but not the formation of cADPR. The increase in ADPR production was due to activation of NAD glycohydrolase as measured by a conversion rate of NAD into ADPR. The maximal conversion rate of NAD into ADPR in coronary homogenate was increased from 2.5 ± 0.2 to 3.4 ± 0.3 nmol · min−1 · mg protein−1 by 11,12-EET. The regioisomers of 8,9-EET, 11,12-EET, and 14,15-EET also significantly increased ADPR production from NAD. Western blot analysis and immunoprecipitation demonstrated the presence of NAD glycohydrolase, which mediated 11,12-EET-activated production of ADPR. In cell-attached patches, 11,12-EET (100 nM) increases KCa channel activity by 5.6-fold. The NAD glycohydrolase inhibitor cibacron blue 3GA (3GA, 100 μM) significantly attenuated 11,12-EET-induced increase in the KCa channel activity in CASMCs. However, 3GA had no effect on the KCa channels activity in inside-out patches. 11,12-EET produced a concentration-dependent relaxation of precontracted coronary arteries. This 11,12-EET-induced vasodilation was substantially attenuated by 3GA (30 μM) with maximal inhibition of 57%. These results indicate that 11,12-EET stimulates the production of ADPR and that intracellular ADPR is an important signaling molecule mediating 11,12-EET-induced activation of the KCa channels in CASMCs and consequently results in vasodilation of coronary artery.