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PCR analysis and immunoblotting with isoform specific antibodies was used to identify the presence of type I, II and III inositol 1,4,5-trisphosphate receptors (InsP3Rs) in rat testis. PCR analysis also revealed that rat testis express both forms of the S1 splice variant (S1+ and S1-), but only the S2- from of the S2 splice variant of the type I InsP3 receptor. PCR analysis was also used to identify InsP3R isoform expression at a cellular level using myoid, Sertoli and germ cells derived from the testis of Wistar rats. The extent of [3H]-InsP3 binding was found to be 9 times lower for testicular microsomes than for cerebellar microsomes, with a Bmax of 1.4 pmoles/mg protein compared to 12.5 pmoles/mg protein for cerebellar microsomes. The Kd for InsP3 binding to its receptor in testicular microsomes was 60 +/- 10 nM which was similar to that found for cerebellar microsomes (80 +/- 20 nM). InsP3-induced Ca2+ release (IICR) in testicular microsomes was found to have an EC50 (concentration which causes a half-maximal response) of 0.5 +/- 0.03 microM, also similar to that seen for cerebellar microsomes (0.3 microM). Maximal IICR occurred at about 20 microM InsP3, with up to 4% of total intracellular Ca2+ stores being mobilized as compared to between 10-30% for cerebellar microsomes. Time resolved IICR using stopped-flow spectrofluorimetry, showed the kinetics of IICR for this testis preparation to be monophasic with a maximum rate constant of 0.15 s-1 at 30 microM InsP3. The rate constants are 7 times slower than values for cerebellar microsomes under similar conditions (approximately 1 s-1) and taken together with the binding data support the proposal that the receptor density/Ca2+ store is approximately 8 times lower than seen in cerebellar microsomal vesicles. The pharmacological properties as assessed using heparin and InsP3 analogues also confirmed similar behaviour for testicular InsP3Rs and cerebellar InsP3Rs.

Three endogenous molecules have now been shown to release Ca2+ in the sea urchin egg: inositol trisphosphate (InsP3), cyclic adenosine 5'-diphosphate ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP), a derivative of NADP. While the mechanism through which the first two molecules are able to release Ca2+ is established and well characterized with InsP3 and cADPR-activating InsP3 and ryanodine receptors, respectively, the newly described NAADP has been shown to release Ca2+ via an entirely different mechanism. The most striking feature of this novel Ca2+ release mechanism is its inactivation, since subthreshold concentrations of NAADP are able to fully and irreversibly desensitize the channel. In the present study we have investigated the fast kinetics of activation and inactivation of NAADP-induced Ca2+ release. NAADP was found to release Ca2+ in a biphasic manner, and such release was preceded by a pronounced latent period, which was inversely dependent on concentration. Moreover, the kinetic features of NAADP-induced Ca2+ release were not altered by pretreatment with low concentrations of NAADP, although the extent of Ca2+ release was greatly affected. Our data suggest that the inactivation of NAADP-induced Ca2+ release is an all-or-none phenomenon, and while some receptors have been fully inactivated, those that remain sensitive to NAADP do so without any change in kinetic features.

Bee venom PLA2 possesses a binding site for long-chain fatty acids that can be acylated by long-chain fatty acid imidazolides [Drainas, D. and Lawrence, A.J. (1978) Eur. J. Biochem. 91, 131-138]. Occupation of the site either by oleic acid or the oleoyl residue enhances the catalytic activity by 45.7-fold in the presence of 20% 1-propanol and occupation of the site by the oleoyl residue increases the lytic activity against rabbit erythrocytes by 60-fold. Treatment of the enzyme with oleic acid and glutaraldehyde is known to produce irreversible activation [Lawrence, A.J. and Moores, G.R. (1975) FEBS Lett. 49, 287-291]. Here we show that reduction of the glutaraldehyde-treated enzyme with borohydride stabilizes the activated state and enables the fatty acid to be removed, revealing that a large proportion of the induced activation does not require the presence of oleic acid and indicating that activation is due to a change in the conformation rather than the hydrophobicity of the protein. A kinetic study of enzyme activated by oleoyl imidazolide showed that this modification stabilizes the protein against reversible inactivation by 1-propanol. Comparison of the CD spectra of native and oleoyl imidazolide-activated enzyme shows a change in secondary structure with apparent increase in both alpha-helix and beta-sheet content. During reaction of the enzyme with oleoyl imidazolide, the protein fluorescence shows a biphasic response with an initial fall attributed to occupation of the binding site followed by a progressive decrease with a shift of the emission maximum from 341 to 348 nm. The rate of the second phase closely matched the rate of increase in catalytic activity of the enzyme. Free oleic acid caused a rapid fall in fluorescence emission without the subsequent slow change. These results support the proposal that oleic acid or the oleoyl residue occupy a very similar site on the protein and that occupation of this site increases the exposure of one or both of the Trp residues to the aqueous environment. Binding studies show that activation by oleoyl imidazolide does not increase the affinity of the enzyme for the erythrocyte membrane. It is proposed that occupation of a long-chain fatty acid binding site on the enzyme enhances catalytic activity by changing the conformation of the protein rather than acting as a hydrophobic anchor to the substrate surface.

The effects of ethanol and other aliphatic alcohols on the endoplasmic reticulum Ca2+ pump and the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ channel were studied in pig cerebellar microsomes. Methanol, ethanol and propanol all stimulated ATP-dependent Ca2+ uptake, whereas butanol inhibited this process. Ethanol inhibited InsP3-induced Ca2+ release [half-maximal inhibition at 3.5%, v/v (600 mM)]. However, ethanol affected only the amount of InsP3-releasable Ca2+, without affecting the concentration of InsP3 required to induce half-maximal release. Other alcohols of longer chain length were more potent than ethanol at inhibiting InsP3-induced Ca2+ release, but none of the alcohols tested affected [3H]InsP3 binding to its receptor. Using stopped-flow techniques, measurements of the rate of InsP3-induced Ca2+ release in the preparation of pig cerebellar microsomes used in this study showed the kinetics to be monophasic, with a rate constant of 0.93 s-1 at 20 μM InsP3. This rate constant was dependent upon InsP3 concentration, decreasing to 0.38 s-1 at 0.25 μM InsP3. Ethanol was shown to reduce the fractional amount of InsP3-induced Ca2+ release without significantly affecting the rate constant for this process.

The effects of the alkali metal ions Na+, K+, Rb+, and Cs+ on ATP-dependent Ca2+ uptake, [3H]Inositol 1,4,5-trisphosphate (InsP3) binding, and quantal InsP3-induced Ca2+ release were investigated using rat cerebellar microsomes. Both the ion species and concentration affected the ability of the microsomes to support Ca2+ uptake with K+ being mot effective (3.8 nmol of Ca2+/min/mg at 100 mM K+). The order of efficacy of the other ions was as follows: K+ > Na+ > Rb+ = Cs+ >> Li+. The binding of [3H]InsP3 to cerebellar microsomes was, however, affected little by the presence of these ions. All these alkali metal ions (except Li+) supported InsP3-induced Ca2+ release at concentrations above 25 mM; however, the extent of Ca2+ release (expressed as a percent Ca2+ release compared with that released by the ionophore A23187) was dependent upon the ion species present. Again K+ was more potent than the other ions at facilitating InsP3-induced Ca2+ release (order of efficacy: K+ > Rb+ > Na+ > Cs+), although the concentration of InsP3 required to induce half-maximal Ca2+ release (IC50) was not significantly altered. Over the ion concentration range tested (25-100 mM), the extent of InsP3-induced Ca2+ release with both K+ and Rb+ increased in a linear fashion, while Na+ showed only a slight increase and Cs+ showed no increase over this range. The effect of K+ concentration on quantal Ca2+ release was to alter the extent of release rather than the IC50 InsP3 concentration. Using stopped-flow techniques, the effects of InsP3 and K+ concentrations on the kinetics of InsP3-induced Ca2+ release were shown to exhibit a monoexponential process in this microsomal preparation. The rate constants for Ca2+ release increased with InsP3 concentration (0.11 s-1 at 0.02 microM InsP3 to 0.5 s-1 at 40 microM InsP3); however, the relationship between the fractional extent of release and rate constants for release did not change in a similar way with InsP3 concentration. Although the fractional extent of Ca2+ release increased with K+ concentration, the rate constants for release over this K+ concentration range were unaffected. This observation leads us to question the role of K+ as a counter ion required for Ca2+ release, and we therefore postulate a role for K+ (and the other alkali metal ions) as a "co-factor" required for channel opening.

Elevation of cytosolic calcium concentrations, induced by many neurotransmitters, plays a crucial role in neuronal function. Some neurotransmitters produce the second messenger InsP3 which activates an intracellular calcium channel (InsP3 receptor) usually located in the endoplasmic reticulum. This article undertakes a comprehensive survey of most pharmacological modulators of the InsP3 receptor so far reported. This review discusses in detail competitive antagonists, non-competitive antagonists and thiol reactive reagents, highlighting their modes of action and in some cases indicating drawbacks in their use.

The mode of inhibition of the phospholipase A2 (PLA2) enzyme from the Chinese cobra (Naja naja atra) by Zn2+ is qualitatively different from inhibition by Ba2+. Inhibition by Ba2+ shows the kinetic characteristics of a conventional competitive inhibitor acting to displace Ca2+ from a single essential site, but Zn2+ has the paradoxical property of being more inhibitory at high than at low Ca2+ concentration. Kinetic analysis of the Ca(2+)-dependence of enzymic activity shows a bimodal response, indicating the presence of two Ca(2+)-binding sites with affinities of 2.7 microM and 125 microM respectively, and we propose that these can be identified with the two Ca(2+)-binding sites revealed by crystallographic analysis [White, Scott, Otwinowski, Gleb and Sigler (1990) Science 250, 1560-1563]. The results are consistent with the model that the enzyme is activated by two Ca2+ ions, one that is essential and can be displaced by Ba2+, and one that modulates the activity by a further 5-10-fold and which can be displaced by Zn2+. An alternative model is also presented in which the modulating Zn(2+)-binding site is a phenomenon of the lipid/water interface.

Measurement of solution electrical conductance (conductimetry) is a simple direct assay method for the protogenic, hydrolytic reactions catalyzed by all phospholipase enzymes. The technique is especially suitable for assay of phospholipase D (PLD) enzymes where cleavage of zwitterionic substrates reinforces the pH dependent conductance change and allows the method to be used over a much wider pH range than the equivalent titrimetric assay. The ability to detect zwitterion cleavage enables the method to assay reactions in which phospholipase D transfers neutral, or anionic, alcohol species to the zwitterionic substrates phosphatidyl choline and phosphatidyl ethanolamine. The method can follow the sequential attack by different phospholipases and provides a simple technique for investigating the effect of substrate structure on susceptibility to various phospholipase enzymes. The results confirm that PLD from Streptomyces chromofuscus can attack lysophospholipids, but cannot transfer primary alcohols to the phosphatidyl residue, while the PLD from savoy cabbage is an efficient transferase, but cannot attack lysophospholipids. The data suggest that the bacterial PLD fails to act as a transferase because it hydrolyzes the transphosphatidylation products. Some phosphatidyl alcohols are more highly susceptible to PLA2 attack than the parent phosphatidyl choline derivatives.

Graphical abstract, Based on previous reports of certain 5-deazaflavin derivatives being capable of activating the tumour suppressor p53 in cancer cells through inhibition of the p53-specific ubiquitin E3 ligase HDM2, we have conducted an structure–activity relationship (SAR) analysis through systematic modification of the 5-deazaflavin template. This analysis shows that HDM2-inhibitory activity depends on a combination of factors. The most active compounds (e.g., 15) contain a trifluoromethyl or chloro substituent at the deazaflavin C9 position and this activity depends to a large extent on the presence of at least one additional halogen or methyl substituent of the phenyl group at N10. Our SAR results, in combination with the HDM2 RING domain receptor recognition model we present, form the basis for the design of drug-like and potent activators of p53 for potential cancer therapy.

The main difficulty in the development of ATP antagonist kinase inhibitors is target specificity, since the ATP-binding motif is present in many proteins. We introduce a strategy that has allowed us to identify compounds from a kinase inhibitor library that block the cyclin-dependent kinases responsible for regulating transcription, i.e., CDK7 and especially CDK9. The screening cascade employs cellular phenotypic assays based on mitotic index and nuclear p53 protein accumulation. This permitted us to classify compounds into transcriptional, cell cycle, and mitotic inhibitor groups. We describe the characterization of the transcriptional inhibitor class in terms of kinase inhibition profile, cellular mode of action, and selectivity for transformed cells. A structural selectivity rationale was used to optimize potency and biopharmaceutical properties and led to the development of a transcriptional inhibitor, 3,4-dimethyl-5-[2-(4-piperazin-1-yl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one, with anticancer activity in animal models. Refereed/Peer-reviewed

Polo-like kinases (PLKs) are key enzymes that control mitotic entry of proliferating cells and regulate many aspects of mitosis necessary for successful cytokinesis. Of the four known human PLKs, PLK1 is the best characterized and is overexpressed in many tumour types with aberrant elevation frequently constituting a prognostic indicator of poor disease outcome. Despite the fact that PLK1 has been regarded as a validated mitotic cancer target for a number of years, very few reports of small-molecule PLK inhibitors have appeared to date. In order to provide a starting point for the discovery and development of selective PLK inhibitors, we have characterized a number of known generic kinase inhibitors with hitherto unknown activity against PLK1, as well as discovering novel inhibitors through structure-guided design. Previously, the only characterized biochemical PLK1 inhibitor was scytonemin, a symmetric indolic marine natural product that is a micromolar non-specific ATP competitor. In addition to the progress in the development of ATP-competitive small-molecule PLK inhibitors, recent reports on the use of antisense oligonucleotides (ASONs) and small interfering RNAs (siRNAs) directed against PLK1 have shown selective antiproliferative effects on cancer cells both in vitro and in vivo, producing phenotypes consistent with known PLK functions, and confirming that targeting PLKs with conventional small-molecule agents may be a valid and effective anticancer strategy. Here we present a progress update on the approaches taken so far in developing PLK inhibitors.