Peter Presek

Alpha adrenoceptors in rat brain direct identification with prazosin

SummaryTritiated prazosin was used to characterize high affinity binding sites with characteristics similar to α1 adrenoceptors in rat brain membranes. These sites were compared with α2 adrenoceptors labeled with tritiated clonidine. The prazosin sites had an association constant of 2 nM−1 and bound the ligand optimal around pH 7.0. The density of the sites was 300 fmoles per mg of protein; the half time of dissociation of prazosin was 7 min at 30° C. The order or potencies of agonists, determined from binding-inhibition experiments with labeled prazosin, was: naphazoline > clonidine > adrenaline > noradrenaline > phenylephrine > α-methylnoradrenaline > dophamine. The order of potencies of antagonists was: prazosin > phenoxybenzamine > phentolamine > clozapine > yohimbine. Sodium ions and divalent cations as well as guanyl nucleotides have little or no effect on the binding of the labeled antagonist. This is in contrast to the binding of the labeled agonist clonidine (Glossmann and Presek, 1979a, 1979b). Labeled prazosin may be a useful tool to characterize α1 adrenoceptors.

Alpha noradrenergic receptors in brain membranes sodium magnesium and guanyl nucleotides modulate agonist binding

SummaryBinding of [3H]clonidine to alpha noradrenergic receptors in rat brain is inhibited by monovalent cations (Na+>Li+>K+), stimulated by magnesium ion and inhibited by guanyl nucleotides. In the presence of 1 mM EDTA the receptors bind tritiated clonidine in a noncooperative fashion at a single site with a Ka(association constant) of 0.12 nM−1. In the presence of magnesium the affinity of the receptors increases by a factor of two (Ka=0.23 nM−1). The increase of affinity is attributed to a two-fold decrease in the dissociation rate constant. In the presence of sodium ions the concentration of binding sites is not changed but Scatchard plots are now curvilinear indicating either heterogeneity of the receptors or negative cooperativity in ligand binding. This effect of sodium ions is not influenced by the presence of magnesium. The conversion into the sodium-liganded state is rapid; it is complete within 60 s at 30° C.The effects of the guanyl nucleotides on clonidine binding are complex: In the presence saturating concentrations of sodium ions they cannot inhibit clonidine binding except when free magnesium (>1 mM) is present. Without added sodium and in the presence of 1 mM EDTA the rank order of potencies is: GDP≧GTP>Gpp(NH)p. In the presence of 10 mM magnesium the rank order is reversed: Gpp(NH)p ≫ GTP≧GDP. The apparent affinity of the nucleotides for inhibition of clonidine binding is also changed by magnesium. The affinity of Gpp(NH)p increases about 100-fold by addition of magnesium ion.

Pyruvate kinase type M2 is phosphorylated at tyrosine residues in cells transformed by Rous sarcoma virus

Chicken embryo cells (CECs) contain pyruvate kinase (PK) type M2 (M2‐PK). Transformation of CECs by Rous sarcoma virus (RSV) leads to a reduction in the affinity of PK for the substrate phosphoenolpyruvate. In vitro, M2‐PK can be phosphorylated at tyrosine residues by pp60v‐src, the transforming protein of RSV. To study tyrosine phosphorylation of M2‐PK in intact RSV‐transformed cells, the protein was immunoprecipitated from 32P‐labeled normal and RSV‐SR‐A‐transformed CECs. Phosphoamino acid analysis of immunoprecipitated M2‐PK revealed that M2‐PK of both normal and transformed CECs contained phosphoserine and small amounts of phosphothreonine. Only M2‐PK of transformed CECs contained phosphotyrosine in addition. For enzyme kinetic studies M2‐PK was partially purified by chromatography upon DEAE‐Sephacel and hydroxyapatite. A decreased affinity for phosphoenolpyruvate was observed 3 h after the onset of transformation using the temperature‐sensitive mutant of RSV, ts‐NY 68. The kinetic changes were correlated with tyrosine phosphorylation of M2‐PK, but there is no direct evidence that they are caused by post‐translational modification of the enzyme.

Quercetin inhibits tyrosine phosphorylation by the cyclic nucleotide-independent, transforming protein kinase, pp60src

SummaryThe bioflavonoid quercetin is a potent inhibitor of a cyclic nucleotide-independent, tumor virus-coded protein kinase which phosphorylates tyrosine residues and acts as a cellular transforming protein. Half-maximal inhibition of the protein kinase occurred at 3–4 μM quercetin whereas rutin was much less effective. The finding, that quercetin inhibits a cyclic nucleotide-independent protein kinase activity, may provide clues to the diverse pharmacological effects of the bioflavonoids.

The Use of Ligand Binding for the Characterisation of α-Adrenoceptors

&NA; Binding sites with characteristics of &agr;2‐ and &agr;1‐adrenoceptors were found on rat brain membranes with the help of labelled clonidine and prazosin, respectively. Prazosin also identified &agr;1‐adrenoceptors in rat myocardium. Two classes of modulators, mono‐ and divalent cations, are shared by the &agr;2‐and &agr;1‐adrenoceptors. Divalent cations (Ni2+, Mn2+, Mg2+) potentiate clonidine (&agr;2‐receptor) binding in a temperature‐dependent manner. The potentiation by Mg2+ is due to increased availability of sites and an increase in the association constant. The effects of Mg2+ can be reversed by chelation or by lowering the temperature from 30 to 2°C; guanosine triphosphate (half‐maximal effect at 2.5 &mgr;M) prevents the potentiation. Mg2+ also decreases the affinity of rat brain &agr;2‐adrenoceptors for the &agr;1‐antagonist prazosin. &agr;2‐Adrenoceptors decrease their affinity for certain agonists by addition of sodium ions. The “sodium shift” is pronounced for adrenaline, &agr;‐methylnoradrenaline, and noradrenaline relative to clonidine. There is little or no sodium shift for phenylephrine, yohimbine, and prazosin. In the presence of Na+, the Kd values of certain agonists may be similar as found for the &agr;1‐adrenoceptors in the presence of Mg2+. It is suggested that &agr;2‐adrenoceptors in vivo are under constraint, most likely by sodium ions. Characterisation of these receptors in the absence of sodium ions in vitro reveals a conformation through which the receptor may cycle during excitation. It is suggested that a thermodynamic approach should be taken to explain the driving forces for coupling initiated by agonists. &agr;1‐Adrenoceptors in rat brain and rat heart membranes are influenced with respect to agonist binding by magnesium and sodium ions. Mg2+ increases the affinity of adrenaline, noradrenaline, and phenylephrine for the rat heart &agr;1‐adrenoceptors. If sodium is added together with magnesium, noradrenaline and &agr;‐methylnoradrenaline lose, whereas naphazoline and clonidine gain, affinity. The rank order of potencies (measured by the respective KD values) was reversed for &agr;‐methylnoradrenaline and phenylephrine when sodium was added. In addition to monovalent and divalent cations, &agr;2‐adrenoceptors are modulated in vitro by guanyl nucleotides. A heat‐labile factor confers relatively high affinity for the agonist clonidine to &agr;2‐adrenoceptors in rat brain membranes.

Tetanus Toxin Inhibits Depolarization-Stimulated Protein Phosphorylation in Rat Cortical Synaptosomes: Effect on Synapsin I Phosphorylation and Translocation

Abstract: Synapsin I, a prominent phosphoprotein in nerve terminals, is proposed to modulate exocytosis by interaction with the cytoplasmic surface of small synaptic vesicles and cytoskeletal elements in a phosphorylation‐dependent manner. Tetanus toxin (TeTx), a potent inhibitor of neurotransmitter release, attenuated the depolarization‐stimulated increase in synapsin I phosphorylation in rat cortical particles and in synaptosomes. TeTx also markedly decreased the translocation of synapsin I from the small synaptic vesicles and the cytoskeleton into the cytosol, on depolarization of synaptosomes. The effect of TeTx on synapsin I phosphorylation was both time and TeTx concentration dependent and required active toxin. One‐ and two‐dimensional peptide maps of synapsin I with V8 proteinase and trypsin, respectively, showed no differences in the relative phosphorylation of peptides for the control and TeTx‐treated synaptosomes, suggesting that both the calmodulin‐and the cyclic AMP‐dependent kinases that label this protein are equally affected. Phosphorylation of synapsin IIb and the B‐50 protein (GAP43), a known substrate of protein kinase C, was also inhibited by TeTx. TeTx affected only a limited number of phosphoproteins and the calcium‐dependent decrease in dephosphin phosphorylation remained unaffected. In vitro phosphorylation of proteins in lysed synaptosomes was not influenced by prior TeTx treatment of the intact synaptosomes or by the addition of TeTx to lysates, suggesting that the effect of TeTx on protein phosphorylation was indirect. Our data demonstrate that TeTx inhibits neurotransmitter release, the phosphorylation of a select group of phosphoproteins in nerve terminals, and the translocation of synapsin I. These findings contribute to our understanding of the basic mechanism of TeTx action.

Zn2+ enhances protein tyrosine kinase activity of human platelet membranes

In human platelet membranes enhanced tyrosine phosphorylation of certain proteins was observed when Zn2+ instead of Mg2+ or Mn2+ was used as a divalent cation for the kinase reaction. An enhanced level of phosphate incorporation into tyrosine residues occurred into a 68 kDa polypeptide besides the 45 kDa and 105 kDa proteins. Preincubation of platelet membranes with TBR‐IgG showed a concentration‐dependent inhibition of the phosphorylation of the 45, 68 and 105 kDa proteins. Moreover, pp60c‐src, representing the major protein tyrosine kinase activity in platelets, was found to be stimulated by Zn2+. The data, thus, support the assumption that pp60c‐src kinase is responsible for Zn2+ stimulated tyrosine phosphorylation.

High-yield purification of a pp60c-src related protein-tyrosine kinase from human platelets

A protein-tyrosine kinase (PTK, EC 2.7.1.112) from human platelets was purified with high yield. Purification of the enzyme involved sequential chromatography on casein-agarose, tyrosine-agarose, heparin-Sepharose and hydroxylapatite. The procedure resulted in substantially enriched 54/52 kDa polypeptides on SDS-polyacrylamide gel electrophoresis and a yield of about 25% in PTK activity. About 250 micrograms of purified protein could be obtained from 1 g of cell protein. The purification factor varied between 1000 and 1500. Determination of the molecular mass of the purified PTK under nondenaturating conditions by molecular sieve chromatography revealed that the enzyme is a monomer of about 50 kDa. Among various protein substrates tested, casein was most prominently phosphorylated. All substrates were exclusively phosphorylated at tyrosine residues. Autophosphorylation at tyrosine residues of the 54/52 kDa proteins was observed in the presence of Mg2+ or Mn2+. At each purification step, the 54/52 kDa proteins were precipitated by sera from tumor-bearing rabbits immunoprecipitating pp60src, but not by control sera. The amount of the immunoprecipitated purified 54/52 kDa phosphoproteins was directly proportional to the amount of antiserum used. Partial peptide mapping by V8 proteinase showed a 26 kDa tyrosine-phosphorylated fragment for the 54 and the 52 kDa proteins as well as for the pp60c-src molecules of intact platelets. All these data indicated that purified PTK is closely related to pp60c-src of human platelets. Using casein as a substrate for the purified enzyme, the Km for ATP was 4 microM and the Vmax for the reaction was 2.0 nmol/min per mg.

Cation sensitivity of [125I]heat binding to α1-adrenoceptors in rat cerebral cortex membranes☆

The α1-adrenoceptor antagonist 2-[β-(4-hydroxyphenyl)-ethylaminomethyl]tetralone (HEAT) was radiolabeled with 125I and purified to the maximal specific radioactivity of 2200 Ci/mmol. [125I]HEAT selectively labels α1-adrenoceptors (KD ∼ 0.1 nM at 30°C) in rat cerebral cortex membranes. Divalent cations stimulated [125I]HEAT binding to the α1-adrenoceptors. The rank order of potencies was Ni2+ > Mn2+ > Mg2+ > Ca2+ ≃ Sr2+. The effect of Mg2+ was mainly on the Bmax of the α1-adrenoceptors. Monovalent cations also stimulated [125I]HEAT binding. Maximal stimulation (up to 3-fold) was seen with Na+ (optimal concentration at − 150 mM). Li+ and NH4+ were less effective whereas K+ was ineffective. The stimulation of [125I]HEAT binding to α1-adrenoceptors by Na+ was temperature-dependent.

Serum levels of pantropic p53 protein and EGF-receptor, and detection of anti-p53 antibodies in former uranium miners (SDAG Wismut).

BACKGROUND The oncogene product EGF-receptor (EGF-R), the tumor suppressor gene product p53, and anti-p53 antibodies are detectable in serum of certain cancer patients. Increased levels of some of these products were reported in lung cancer patients after occupational asbestos exposure, after exposure to polycyclic aromatic hydrocarbons or vinyl chloride. This molecular epidemiological study investigated the use of serum EGF-R, p53-protein, and anti-p53 antibodies as biomarkers for detection of effects of radon and its decay products. METHODS Serum EGF-R, p53-protein, and anti-53 antibodies were measured using ELISA in former uranium miners of SDAG Wismut without lung disease (n=106) and miners with Schneeberg lung cancer (n=22). They were compared with healthy subjects (n=23), patients with lung cancer not due to ionizing radiation (n=88), and patients with non-malignant lung or pleural diseases (n=50). RESULTS No significantly elevated or decreased serum values for p53 protein, EGF-R, or anti-p53 antibodies could be found. There was no correlation of these with Working Level Months (WLM). CONCLUSIONS p53 protein, EGF-R, or anti-p53 antibodies in serum are not useful as biomarkers for detection of lung cancer related to ionizing radiation (i.e., Schneeberg lung cancer).