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Biomedical subjects

E Karlsson

Publications and source records attributed to E Karlsson.

At least 55 records · Page 3Linked to original sources
PubMed1996

Effect of fasciculin on hydrolysis of neutral and choline esters by butyrylcholinesterase, cobra venom and chicken acetylcholinesterases.

Acetylcholinesterases (AChEs) very sensitive to fasciculin inhibition (KiS in picomolar range) have a distinctive group of aromatic amino acids in the peripheral region (Y70, Y121, W279 in Torpedo AChE). Enzymes that lack these amino acids like butyrylcholinesterases (BChEs) or one or two of them like cobra venom, insect and chicken AChEs are 1000 to 1,000,000 times less sensitive. Fasciculin is a non-competitive inhibitor of the hydrolysis of choline and neutral esters by very sensitive AChEs. For the other group of enzymes, differences arise according to the type of substrate. Fasciculin still behaves as a non-competitive inhibitor with choline esters. In contrast, hydrolysis of phenylacetate was unaffected or slightly increased with BChEs and a partial competitive inhibition was observed with cobra venom and chicken enzymes.

Acetylcholinesterase↗
PubMed1996

Rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity: potent block by the selective m4 ligand muscarinic toxin 3 (MT3).

1. In rat striatal membranes, muscarinic toxin 3 (MT3), a selective ligand of the cloned m4 receptor subtype, antagonized the acetylcholine (ACh) inhibition of forskolin- and dopamine D1 receptor-stimulated adenylyl cyclase activities with pA2 values of 8.09 and 8.15, respectively. 2. In radioligand binding experiments, MT3 increased the Kd but did not change the Bmax value of [3H]-N-methylscopolamine (3H]-NMS) binding to rat striatal muscarinic receptors. The toxin displaced the major portion of the [3H]-NMS binding sites with a Ki of 8.0 nM. 3. In rat myocardium, MT3 antagonized the ACh inhibition of adenylyl cyclase with a Ki value of 860 nM. 4. In rat cerebral cortical membranes prelabelled with [3H]-myo-inositol, MT3 counteracted the methacholine stimulation of [3H]-inositol phosphates formation with a Ki value of 113 nM. 5. The present study shows that MT3 is a potent antagonist of the striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity. This finding provides strong evidence for the classification of these receptors as pharmacologically equivalent to the m4 gene product (M4). On the other hand, the weaker potencies of MT3 in antagonizing the muscarinic responses in cerebral cortex and in the heart are consistent with the reported lower affinities of the toxin for the cloned m1 and m2 receptor subtypes, respectively.

Acetylcholine↗
PubMed1995

Muscarinic toxins from the black mamba Dendroaspis polylepis.

Three new toxins acting on muscarinic receptors were isolated from the venom of the black mamba Dendroaspis polylepis. They were called muscarinic toxins alpha, beta, and gamma (MT alpha, MT beta, and MT gamma). All of the toxins have four disulphide bonds and 65 or 66 amino acids. The sequences of MT alpha and MT beta were determined. The muscarinic toxins, of which about 12 have been isolated from venoms of green and black mambas, have 60-98% sequence identity with each other, and are similar to many (about 180) other snake venom components, such as alpha-neurotoxins, cardiotoxins, and fasciculins. In contrast to the alpha-neurotoxins, muscarinic toxins do not bind to nicotinic acetylcholine receptors. The binding constants of MT alpha and MT beta were determined for human muscarinic receptors of subtypes m1-m5 stably expressed in Chinese hamster ovary cells. The toxins are less selective than the earlier discovered muscarinic toxins from the green mamba Dendroaspis angusticeps. MT alpha and the muscarinic toxin MT4 from D. angusticeps differ only in a region of three amino acids (residues 31-33), which are Leu-Asn-His in MT alpha and Ile-Val-Pro in MT4. This difference causes a pronounced shift in subtype selectivity. MT alpha has high affinity to all subtypes, with Ki (inhibition constant) values of 23 nM (m1; pKi = 7.64 +/- 0.10), 44 nM (m2; pKi = 7.36 +/- 0.06), 3 nM (m3; pKi = 8.46 +/- 0.14), 5 nM (m4; pKi = 8.32 +/- 0.07), and 8 nM (m5; pKi = 8.09 +/- 0.07). MT4 has high affinity only to m1 (Ki = 62 nM) and m4 (87 nM) receptors, and low (Ki > 1 microM) affinity to m2, m3, and m5. The region at positions 31-33 evidently plays an important role in the toxin-receptor interaction. MT beta has low affinity for m1 and m2 receptors (Ki > 1 microM) and intermediate affinity for m3 (140 nM; pKi = 6.85 +/- 0.03), m4 (120 nM; pKi = 6.90 +/- 0.06), and m5 (350 nM; pKi = 6.46 +/- 0.01). The low affinity of MT beta may reflect a tendency for spontaneous inactivation.

Amino Acid Sequence↗
PubMed1995

Role of arginine residues for the activity of fasciculin.

The West African green mamba, Dendroaspis angusticeps, has two toxins, fasciculins, that are non-competitive inhibitors of acetylcholinesterase. Arginine residues of fasciculin 2 were modified with 1,2-cyclohexanedione. Two of these residues, Arg24 and Arg37, reacted very slowly or not at all. Modification of Arg28 reduced the activity only by 13%. Arg11 and Arg27 are unique for fasciculins; a comparison of the sequences of 175 snake toxins homologous to fasciculins showed that no other toxin has arginine in the corresponding positions. Modification of the two unique arginines had a large effect and decreased the activity by 73% (Arg11) and 85% (Arg27). This was apparently not due to structural perturbations, since the modification did not change the circular dichroic spectra. The two arginine residues probably participate in the binding to acetylcholinesterase. They are located on the same side of the toxin molecule and the distance between their alpha-carbons is 2.7 nm. This may indicate binding to sites that are far apart and suggests that fasciculin covers a large area of the enzyme.

Amino Acid Sequence↗
PubMed1995

A snake toxin against muscarinic acetylcholine receptors: amino acid sequence, subtype specificity and effect on guinea-pig ileum.

The sequence of muscarinic toxin 1 (MT1) from Dendroaspis angusticeps (green mamba) was determined (66 amino acids, M(r) 7509). The central part, peptide 25-40, is rich in hydrophobic amino acids, which is a characteristic of muscarinic toxins. MT1 started to inhibit [3H]-NMS (N-methylscopolamine) binding to synaptosomal membranes of porcine brain (contains all five receptor subtypes) at about 1 nM and to membranes from pig heart muscle (only subtype m2) at about 1 microM. Binding of [3H]-AF-DX 384 to heart was inhibited with an IC50 of 14 microM and to brain in two steps. In the first step (IC50 = 32 nM) binding decreased by 37%, indicating that the toxin acted on m1 or m4 receptors, each accounting for about 40% of total receptor content. The second step was similar to the effect on heart. Pirenzepine inhibited binding of [125I]-MT1 to brain receptors with an IC50 of 6.5 nM, corresponding to a Ki of about 6 nM. Literature values of Ki for pirenzepine are 16-18 nM for m1 and > or = 120 mM for other subtypes. This indicates binding to m1 receptors. mM for other subtypes. This indicates binding to m1 receptors. [125I]-MT1 bound to brain with a Kd of 20 nM and a Hill coefficient of 1.0, i.e. one toxin molecule per receptor. In guinea-pig ileum, MT1 (670 nM) produced a rapid contraction, reversible by atropine. The toxin may be an agonist, but might also cause contraction by inducing acetylcholine release by a different mechanism.

Amino Acid Sequence↗
PubMed1995

Characterization of a potassium channel toxin from the Caribbean Sea anemone Stichodactyla helianthus.

A peptide toxin, ShK, that blocks voltage-dependent potassium channels was isolated from the whole body extract of the Caribbean sea anemone Stichodactyla helianthus. It competes with dendrotoxin I and alpha-dendrotoxin for binding to synaptosomal membranes of rat brain, facilities acetylcholine release at an avian neuromuscular junction and suppresses K+ currents in rat dorsal root ganglion neurones in culture. Its amino acid sequence is R1SCIDTIPKS10RCTAFQCKHS20MKYRLSFCRK30TCGTC35. There is no homology with other K+ channel-blocking peptides, except for BgK from the sea anemone Bunodosoma granulifera. ShK and BgK appear to be in a different structural class from other toxins affecting K+ channels.

Amino Acid Sequence↗
PubMed1995

Purification and sequence determination of a new muscarinic toxin (MT4) from the venom of the green mamba (Dendroaspis angusticeps).

A toxin which partially inhibited [3H]N-methylscopolamine binding to rat brain muscarinic receptors was purified from the venom of green mamba, Dendroaspis angusticeps. The N-terminal sequence (up to 45 amino acids) was determined by automated Edman degradation of the whole molecule. The complete sequence was elucidated after enzymatic cleavage with endoproteinase Arg-C or endoproteinase Lys-C and peptide fragments purification. The identity of the C-terminal amino acid was confirmed by hydrazinolysis. The new toxin (MT4) had eight half-cystines and 66 amino acids. It differed from muscarinic toxin MT1 by a single substitution in position 57 (arginine in MT1, histidine in MT4), proximal to the sixth half-cystine.

Amino Acid Sequence↗
PubMed1995

Effects of insulin-like growth factor-I and growth hormone on the net flux of amino acids across the hind limbs in the surgically traumatized pig.

1. After surgery three groups of six female pigs weighing on average 52.2 kg (SD 3.5) received vehicle, recombinant insulin-like factor-1 (364.4 micrograms day-1 kg-1) or recombinant human growth hormone (467.7 m-i.u. day-1 kg-1) for two post-operative days. Vehicle and peptides were infused intravenously together with total parenteral nutrition providing 129 kJ day-1 kg-1 non-protein calories and 0.35 g N day-1 kg-1. 2. On both post-operative days the mean concentration of insulin-like growth factor-1 in arterial blood samples was clearly below presurgical levels in animals receiving vehicle or recombinant human growth hormone, whereas recombinant human insulin-like growth factor-1 infusions more than restored insulin-like growth factor-1 concentrations. These last samples, however, contained significantly (P < 0.05) less insulin than those from other animals. 3. Infusion of recombinant human growth factor was often associated with higher circulating levels of amino acids compared with recombinant human insulin-like growth factor-1 infusions. Despite this, both hormones significantly (P < 0.05) increased the hind limb net balance of total amino acids on post-operative day 1. Net balances of -44.2, +69.5 and +100.9 mumol/min (pooled SE 35.3) were associated with infusion of vehicle, recombinant human insulin-like growth factor-1 and recombinant human growth hormone respectively. This response was also closely reflected in the group of non-essential amino acids. 4. The net efflux of alanine from the hind limbs was also significantly (P < 0.002) reduced, whereas glutamine was less affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1995

Arm-ankle systolic blood pressure difference at rest and after exercise in the assessment of aortic coarctation.

OBJECTIVE: To evaluate the difference in systolic blood pressure at the arm and ankle at rest and after various exercise tests for the assessment of aortic coarctation. METHODS: 22 patients (mean age 33 years, range 17-66) were investigated on the suspicion of having haemodynamically significant aortic coarctation. Eight had undergone previous coarctation surgery, of whom five had received vascular grafts and three end to end anastomoses. The patients exercised submaximally while supine, seated on a bicycle, and walking on a treadmill, as well as exercising maximally on a treadmill. Arm and ankle blood pressure were measured with a cuff at rest and 1-10 minutes after exercise. Invasive pressures and cardiac output by thermodilution were recorded during catheterisation while patients were at rest and during and after supine bicycle exercise. The degree of constriction was assessed by angiography. Twelve healthy volunteers (mean age 32 years, range 17-56) provided reference values for cuff pressures after exercise. RESULTS: All patients with a difference in cuff pressure at rest of 35 mm Hg or more had a difference in invasive pressure of 35 mm Hg or more. Increasing severity of constriction on angiography correlated with larger pressure gradients at rest and during exercise (P < 0.0001). When cuff measurements after exercise were considered singly or combined to form a predictor they did not improve the prediction of the invasive pressure gradients at rest or after maximal exercise. A pressure gradient between arm and ankle also developed in normal subjects after maximal but not after submaximal exercise. CONCLUSION: In most patients with suspected haemodynamically significant coarctation the difference in cuff pressure between arm and ankle at rest is sufficient to select patients in need of further evaluation. If exercise is performed submaximal exercise is preferable.

Adolescent↗
PubMed1994

Two-step binding of green mamba toxin to muscarinic acetylcholine receptor.

The mechanism of binding of toxin MT2 from venom of green mamba Dendroaspis angusticeps to muscarinic acetylcholine receptors from rat cerebral cortex was investigated by studying the kinetics of the toxin-receptor interaction. The muscarinic antagonist N-methyl-[3H]scopolamine was used as a 'reporter' ligand. Evidence for a mechanism of toxin-receptor interaction comprising at least two steps was obtained. Such a mechanism increases the potency of the toxin. The first step was fast with no competition between the toxin and the antagonist. The second step was slow with formation of a more stable toxin-receptor complex and inhibition of the antagonist binding. It is proposed that the snake toxin is a muscarinic agonist of slow action.

Animals↗
PubMed1994

A toxin from the green mamba Dendroaspis angusticeps: amino acid sequence and selectivity for muscarinic m4 receptors.

Muscarinic toxin 3 (MT3) (65 amino acids, four disulphides, M(r) 7379) was isolated from the venom of the African snake Dendroaspis angusticeps (green mamba) and its amino acid sequence determined. Its ability to inhibit the binding of [3H]N-methylscopolamine ([3H]NMS) to Chinese hamster ovary cells stably expressing subtypes of muscarinic receptors was studied. MT3 displayed high affinity for the m4 receptor (pKi = 8.7 +/- 0.06), 40-fold lower affinity at ml receptors (pKi = 7.11 +/- 0.17) whereas no inhibition of [3H]NMS binding to m2, m3 and m5 receptors was observed at concentrations up to 1 microM. This makes MT3 the most selective m4 receptor ligand known to date.

Alkaloids↗
PubMed1994

Study of structure-activity relationship of fasciculin by acetylation of amino groups.

Dendroaspis angusticeps (green mamba) has two toxins, fasciculins, that are non-competitive inhibitors of acetylcholinesterase. Amino groups of fasciculin 2 were acetylated with acetic anhydride. The monoacetyl derivatives of the epsilon-amino groups (Lys 25, 32, 51 and 58) retained between 28 and 43% of the initial activity and that of the alpha-amino group 72%. Acetylation of Lys 25 that has the most reactive amino group decreased the activity by 65% apparently without producing structural perturbations, since the circular dichroism spectrum was not affected. The three-dimensional structure shows a cationic cluster formed by Lys 32, 51, Arg 24 and 28. A comparison of 175 sequences of homologous toxins shows that Lys 32 is unique for fasciculin. Acetylation of lysine residues in the cluster had a large effect and reduced the activity by 72% (Lys 32) and 57% (Lys 51). This suggests an important role for the cationic cluster. Lys 25 together with Lys 32 and 51 were, therefore, assumed to be in the active site.

Acetylation↗