Animal venoms are important sources of novel pharmacological tools, useful in biochemical characterization of their receptors. Venom quality control, batch-to-batch homogeneity and high reproducibility of venom fractionation and toxin purification are crucial issues for biochemical and pharmacological studies. To address these issues, a study of the variability of tarantula spider venom samples was undertaken. Venom profiles of samples collected from individuals of different age and sex, and from sibling spiders of the same species, were generated by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and analyzed to assess venom variability and method accuracy. Sex-linked venom variation was studied on eight species. Clear qualitative differences were observed for six out of eight species, as well as quantitative differences. Age-related variation studied in Poecilotheria rufilata showed essentially age-related quantitative differences between adults of both sexes and immature juveniles. The venoms of nine siblings and three wild-collected Pterinochilus murinus were studied for individual variation, showing only very minor quantitative differences. On the same samples, the quality of MALDI-TOFMS venom fingerprinting was demonstrated to be highly reproducible. Our results show that tarantula venom peptide fingerprinting is a highly reliable identification method, that pooled batches of venom from several animals can be used for venom purification, that venom composition does not appear to be qualitatively related to ontogenesis in the spiders studied, and that qualitative sex-linked variation occurs across most species and may be important in activity studies.

Abstract. Treatment of cells with tumor-promoting phorbol diesters, which causes activation of protein kinase C, leads to phosphorylation of the epidermal growth factor (EGF) receptor at threonine-654. Addition of phorbol diesters to intact cells causes inhibition of the EGF-induced tyrosine-protein kinase activity of the EGF receptor and it has been suggested that this effect of phorbol diesters is mediated by the phosphorylation of the receptor by protein kinase C. We measured the activity of protein kinase C in A431 cells by determining the incorporation of [32p]phosphate into peptides containing threonine-654 obtained by trypsin digestion of EGF receptors. After 3 h of exposure to serum-free medium, A431 cells had no detectable protein kinase C activity. Addition of EGF to these cells resulted in [32p] incorporation into threonine-654 as well as into tyrosine residues. This indicates that EGF promotes the activation of protein kinase C in A431 cells. The phosphorylation of threonine-654 induced by EGF was maximal after only 5 min of EGF addition and the [32p] incorporation into threonine-654 reached 50% of the [32p] in a tyrosine-containing peptide. This indicates that a significant percentage of the total EGF receptors are phosphorylated by protein kinase C. A variety of external stimuli activate Na÷/H ÷ exchange, including EGF, phorbol diesters, and hypertonicity. To ascertain whether activation of protein kinase C is an intracellular common effector of all of these systems, we measured the activity of protein kinase C after exposure of A431 cells to hyperosmotic conditions and observed no effect on phosphorylation of threonine-654, therefore, activation of Na+/H ÷ exchange by hypertonic medium is independent of protein kinase C activity. Since stimulation of protein kinase C by phorbol diesters results in a decrease in EGF receptor activity, the stimulation of protein kinase C a¢tivity by addition of EGF to A431 cells contributes to a feedback mechanism which results in the attenuation of EGF receptor function.T UMOR-PROMOTING phorbol diesters cause rapid and significant alterations of epidermal growth factor (EGF) 1 receptor biochemistry in intact cells. Within minutes of phorbol 12-myristate 13-acetate (PMA) addition to human epidermoid carcinoma A431 cells (39) or mouse fibroblast Swiss 3T3 cells (4), the apparent affinity of the EGF receptor decreases and the EGF-induced tyrosinespecific protein kinase activity of the cytoplasmic domain of the receptor is attenuated (9,12). This has been associated with rapid phosphorylation of EGF receptors at serine and threonine residues (8,9,24).Biochemical investigations suggest that the Ca 2+-and phospholipid-dependent protein kinase (protein kinase C) is the major target for PMA action (for review see reference 33)1. Abbreviations used in this paper: DAG, diacylglycerol; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; PI, phosphatidylinositol; PMA, phorbol 12-myristate 13-acetate. and constitutes the cellular phorbol diester "receptor" (1,30...

Previous studies have documented the activation of Na+/H+ exchange in A431 cells by the addition of epidermal growth factor or serum (Rothenberg et al., 1983b). Here we show that exposure of A4 31 cells to medium of increased osmolarity also leads to activation of Na+/H+ exchange and to an increase in intracellular pH (pHi), which under a variety of conditions displays similar kinetics to that observed upon addition of mitogens to the cells. Measurements of cell volume using the 3-0-methylglucose equilibration technique clearly show that mitogens do not activate Na+/H+ exchange by an osmotic mechanism (i.e., a decrease in cell volume). In fact, mitogens can induce further intracellular alkalinization if added to cells which have been shrunken in hypertonic medium. Activation of the Na+/H+ antiport does not lead to an obligatory change in pHi. Addition of epidermal growth factor of hypertonic solution to A431 cells in bicarbonate buffer activates Na+/H+ exchange without a concomitant increase in pHi. Under these conditions the increased proton efflux via Na+/H+ exchange must therefore be compensated by other mechanisms that control cytoplasmic pH.

The search for novel pharmacological tools in spider venoms involves the need for precise and reproducible species identification methods. As an addition to morphological analysis, we have developed venom fingerprinting by reversed-phase chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) as an efficient and precise venom identification tool. In order to compare the possible use of liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) as an additional venom characterization tool, we have applied both methodologies to the study of several tarantula venom samples in the Pterinochilus murinus group. These species possess highly active venoms yet their taxonomy remains difficult. We demonstrate that both methodologies can be successfully applied to tarantula venom characterization. MALDI-TOFMS and ESI-MS gave similar overall profiles and allowed fine discrimination of samples. At least one venom sample was proven to belong to a completely different venom group. Coupling of ESI-MS with HPLC separation afforded a new dimension in venom analysis, with clear discrimination between components of similar Mr and gave a finer picture of venom composition, number of molecular species and molecular weight distribution.

Addition of polypeptide growth factors to cultured cells results in a rapid stimulation of Na+/H ÷ exchange, which leads to cytoplasmic alkalinization. We studied the effects of the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) on the Na+/H ÷ exchange system of A431 cells. Stimulation of Na+/H + exchange by epidermal growth factor (EGF) and serum as well as by vanadate ions is strongly inhibited after treatment of cells with nanomolar concentrations of PMA. Phorbol esters that have no activity as tumor promotors also do not modulate the activation of Na+/ H ÷ exchange. By contrast, the stimulation of Na+/H ÷ exchange that is produced upon exposure of cells to hypertonic solution is only slightly inhibited by PMA treatment, indicating that PMA treatment does not directly block the activity of the Na+/H + antiporter. Furthermore, incubation of cells with PMA causes a weak stimulation of Na+/H + exchange, although this effect is mostly observed at relatively high PMA concentrations and appears to require external Ca 2+. The inhibition BY PMA of EGF-promoted Na+/H + exchange is not due to inhibition of EGF-binding to the EGF receptor. Since PMA activates protein kinase C, our observations are consistent with the hypothesis that protein kinase C functions to attenuate the stimulation of Na+/H + exchange by polypeptide growth factors.Stimulation of an amiloride-sensitive Na ÷ uptake is a ubiquitous consequence of the addition of polypeptide growth factors (mitogens) to cells. This increased Na ÷ uptake is primarily due to activation ofNa+/H + exchange (1-7). Recent studies have demonstrated that stimulation of Na+/H + exchange by mitogens leads to an increase in cytoplasmic pH (8)(9)(10)(11). This cytoplasmic alkalinization is likely to have a permissive role in the mitogenic response of cells to growth factors (12).Phorbol diester derivatives that are potent tumor promoters in the mouse skin model system have recently been found to exert a wide variety of biological responses in cells. Phorbol 12-myristate 13-acetate (PMA),~ the most potent compound from this series, is mitogenic for a number of fibroblasts, acting synergistically with growth factors (see reference 13 and references therein), while in other cells PMA induces differentiation (for a recent review, see reference 14). Biochemical investigations suggest that the Ca 2+ and phospholipid-dependent protein kinase (protein kinase C) is the major i Abbreviations used in this paper. EGF, epidermal growth factor; PMA, phorbol 12-myristate 13-acetate. target for PMA action. PMA is a potent protein kinase C activator in vitro, apparently substituting for the natural activator diacylglycerol, which is produced upon hormonal stimulation of phosphatidylinositol turnover (reviewed in reference 15). Binding studies ( 16-18) suggest that protein kinase C is also the cellular phorbol diester "receptor." One of the substrates of activated protein kinase C is the epidermal growth factor (EGF) receptor. Activated protein kinase C catalyzes the phosphor...