Mechanism of action of α-galactosidase

The effect ofpH on K_m and V_max values of coconut α-galactosidase indicates the involvement of two ionizing groups with pK_a values of 3.5 and 6.5 in catalysis. Chemical modification has indicated the presence of two carboxyl groups, a tryptophan and a tyrosine, at or near the active site of α-galactosidase. Based on these facts a new mechanism of action for α-galactosidase is proposed in which the ionizing group with a pK_a of 3.5 is a carboxyl group involved in stabilizing a carbonium ion intermediate and the ionizing group with a pK_a of 6.5 is a carboxyl group perturbed due to the presence of a hydrophobic residues in its vicinity which donates a H⁺ ion in catalysis.     

Looking back on the discovery of α-bungarotoxin

This review is a personal narration by a retiring pharmacologist from Taiwan who looks back at his discovery of -bungarotoxin from the historical perspective of Taiwan during the last 50 years, with accounts of his experiences and his efforts to overcome hardship. How the -toxin was isolated and characterized as an irreversible specific nicotinic acetylcholine (ACh) receptor antagonist,and how it subsequently became a useful experimental probe are presented here. The dilemma of differentiating the actions of tubocurarine and -bungarotoxin is analyzed. The author also outlines findings based on work done in his laboratory using -bungarotoxin as a tool on particular aspects of synaptic transmission. These include presynaptic receptor for positive feedback of transmitter release, explosive release of ACh, up- and down-regulation of ACh receptors after chronic drug treatment, autodesensitization of junctional ACh receptors, differences in action between natural transmitter and exogenous agonists and that between junctional and extra-junctional ACh receptors. Some experimental pitfalls, in which biomedical scientists are frequently trapped, are raised. Finally, some anecdotes are appended from which the reader may further understand scientific life in the 20th century, including its joys and regrets.     

Mechanisms of α-latrotoxin action

The major component of black widow spider venom, alpha-latrotoxin, triggers massive exocytosis in a variety of neurosecretory cells. An important trigger for exocytosis is the calcium influx via alpha-latrotoxin-induced channels in biological membranes. However, this mechanism fails to explain exocytosis which occurred in the complete absence of extracellular calcium. Recently, sophisticated biochemical and molecular techniques have led to the discovery of novel alpha latrotoxin-binding membrane receptors: neurexins and latrophilin/CIRL (calcium-independent receptor for alpha-latrotoxin). Neurexins are single transmembrane proteins which bind to alpha-latrotoxin in a calcium-dependent manner and also interact with the synaptic vesicle protein, synaptotagmin. On the other hand, latrophilin is a seven-transmembrane protein and belongs to the family of G-protein-coupled receptors. The multitude of effects of alpha-latrotoxin on exocytosis in different cell systems and the nature of its membrane targets are discussed in this article. The molecular details of how alpha-latrotoxin binding is transduced eventually to exocytosis remain to be elucidated.     

The action pattern of sorghum α-amylase

The action pattern of sorghum α-amylase was examined with four substrates: amylose, soluble starch, Naegeli dextrin, and waxy maize. The products were examined by paper chromatography and by spectrophotometric measurements of their iodine-staining properties. High concentrations of maltohexaose, maltoheptaose, and maltooctaose were produced, in addition to lesser amounts of other oligosaccharides. The oligosaccharides of eight or less glucose units persisted in the digest for some time after the achroic point, but maltooctaose was hydrolyzed more rapidly than maltohexaose or maltoheptaose. The significance of the iodine-staining characteristics are discussed in terms of possible mechanisms for amylases.     

Heterogeneity of neuromuscular junctions in striated muscle of human esophagus demonstrated by triple staining for the vesicular acetylcholine transporter, α-bungarotoxin, and acetylcholinesterase

During studies on enteric co-innervation in the human esophagus, we found that not all acetylcholinesterase (AChE)-positive motor endplates stained for α-bungarotoxin (α-BT) and the vesicular acetylcholine transporter (VAChT), respectively. Therefore, we probed for differences in neuromuscular junctions in human esophagus by using triple staining for VAChT, α-BT, and AChE followed by qualitative and quantitative analysis. To exclude that the results were caused by processing artifacts, we additionally examined the influence of a number of factors including post-mortem changes and the type and duration of fixation on the staining results. Four types of neuromuscular junction could be distinguished in human esophagus: type I with VAChT-positive and type II with VAChT-negative nerve terminals on a α-BT-positive and AChE-positive endplate area, type III with VAChT-positive nerve terminals on a α-BT-negative but AChE-positive endplate area, and type IV with VAChT-negative nerve terminals on a α-BT-negative but AChE-positive endplate area. On average, 32% of evaluated AChE-positive motor endplates were type I, 6% type II, 24% type III, and 38% type IV. Based on these results, we suggest that, in human esophagus, (1) the most reliable method for staining motor endplates is presently AChE histochemistry, (2) α-BT-sensitive and α-BT-resistant nicotinic acetylcholine receptors exist in neuromuscular junctions, and (3) different types of VAChT or transport mechanisms for acetylcholine probably exist in neuromuscular junctions.This study was supported by the “Johannes und Frieda Marohn-Stiftung”, Erlangen.     

Preparation and characterization of 3H-labeled α-bungarotoxin

α-Bungarotoxin has been labeled with [³H]pyridoxamine phosphate, by reaction with pyridoxal phosphate followed by reduction with sodium boro[³H]hydride. Specific activities of up to 27 Ci/mmol have been obtained. Mono- and dilabeled toxins bind irreversibly to the acetylcholine receptor from Torpedo electroplax, despite a change in isoelectric point from 9.2 for native toxin to 6.2 for dilabeled toxin. The ³H-labeled α-bungarotoxin is usable for over a year.     

Mechanisms of action of the α-amylase of Micromonospora melanosporea

The -amylase of Micromonospora melanosporea was produced extracellularly during batch fermentation in a 5.0-1 fermentor. The absence of an organic nitrogen source in its growth medium facilitated subsequent purification of the enzyme by ammonium sulphate fractionation and two consecutive Superose-12 gel-filtration steps. The enzyme exhibited maxima for activity at pH 7.0 and 55° C and was 72% stable at pH 6.0–12.0 for 30 min at 40° C. It had a relative molecular mass of 45 000 and an isoelectric point at pH 7.6. The enzyme catalyses the conversion of starch to maltose (53%, w/w) as the predominant final end-product. Initial hydrolysis of this substrate, however, gave rise to the formation of maltooligosaccharides in the range maltotriose to maltohexaose. Maximum yields of these intermediate sugars accumulated to between 31 and 42% (w/w) as the reaction proceeded. The action of the M. melanosporea amylase on high concentrations of saccharides larger than maltotriose resulted in the formation of mainly maltose and maltotriose without concomitant glucose production. A combination of hydrolytic and transfer events is postulated to be responsible for this phenomenon and for the high maltose levels achieved. Correspondence to: C. T. Kelly     

Mechanism of action of βadrenergic receptor blocking agents in angina pectoris: Comparison of action of propranolol with dexpropranolol and practolol

The effect on exercise tolerance of racemic propranolol has been assessed in eight angina pectoris patients and compared with that of dexpropranolol (the dextro isomer of propranolol), practolol (I.C.I. 50172), and saline. Dexpropranolol has the same local anaesthetic action as propranolol with negligible β-adrenergic receptor blocking activity, while practolol is a cardio-selective β-adrenergic blocking agent which does not have local anaesthetic activity.Saline and dexpropranolol had no significant effect on exercise time; racemic propranolol and practolol improved exercise tolerance in six subjects, the response to the two drugs being very similar in individual patients. It was concluded that the beneficial effect of propranolol in angina pectoris results from its action as a β-adrenergic receptor blocking agent and is not due to its local anaesthetic, or quinidine-like, activity.     

Binding of α-bungarotoxin to the cholinergic receptor proteolipid from Electrophorus electroplax

• 1.1. This investigation was initiated to study the similarities or differences between the cholinergic proteolipid first isolated in our laboratory from Electrophorus electroplax and the protein-detergent-α-bungarotoxin complex isolated by others.
• 2.2. The binding of α-[¹³¹I]bungarotoxin to the proteolipid extracted from electric tissue or electroplax membranes was studied using column chromatography and a partition method.
• 3.3. The saturation curve obtained revealed a single site of binding per molecule of proteolipid of molecular weight 37000.
• 4.4. The binding of α-bungarotoxin could not be displaced with acetylcholine or decamethonium.
• 5.5. It is concluded that the cholinergic proteolipid extracted with organic solvents is similar to the protein-α-bungarotoxin complex separated by the use of strong detergents.

     

Effects of α-bungarotoxin on acetylcholine-induced response at the Helix neuronal membrane

The effects of -bungarotoxin (-BT) on two patterns of acetylcholine (ACh)-induced response differing in desensitization rate were investigated in isolated mollusk neurons using intracellular dialysis and concentration clamping techniques. It was found that -BT depressed both types of ACh-induced response — a reversible action in the majority of experiments performed. It also exerted a blocking effect on ACh-induced currents dependent on the presence of albumin, although albumin itself produced no noticeable change in ACh-induced response. Concentration dependence of -BT-induced blockade on both types of currents evoked by 1 and 10 µM ACh was investigated. The -BT concentrations producing a 50% suppression of the current evoked by 1 µM ACh were calculated by approximating concentration plots as (13.85±1.25)×10^–9 and (5.56±1.0)×10^–8 g/ml for type A and B cells respectively.Institute of Experimental Biology. Academy of Sciences of the Armenian SSR, Erevan. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 729–735, November–December, 1989.     

Comparison of acetylcholine and α-bungarotoxin binding sites in insects and vertebrates

1. The nervous tissue of locusts contains high affinity as well as low affinity binding sites for acetylcholine which display a similar nicotinic pharmacology.2. Hill plot analysis indicated a non-cooperative binding of acetylcholine.3. In membrane preparations from locust ganglia and mouse brain the number of binding sites for ACh was about ten fold lower than for BGTX, whereas in membranes from electric tissue both sites occurred in similar concentrations.4. Drug binding studies suggest that the high affinity binding sites for ACh and BGTX in preparations from insect and mouse are different; whereas in electric tissue both sites are very similar.5. Precipitation experiments using immobilized BGTX and specific antibodies indicated that in insect nervous tissue as in electric tissue the ACh and BGTX binding sites are located on the same receptor molecule and occupy distinct partially overlapping binding sites, whereas in the vertebrate brain both sites are located on distinct binding proteins.     

A fluorescent coumarin derivative of α-bungarotoxin. Synthesis and spectroscopic characterization

The preparation and spectroscopic characteristics of a novel coumarin derivative of α-bungarotoxin are described. The suitability of this fluorescent conjugate as a probe of cell surface topography and dynamics of acetylcholine receptors is discussed. Data are presented that show this coumarin-α-bungarotoxin conjugate to be an ideal donor fluorophore for time-resolved resonance energy transfer studies employing fluorescein as an acceptor.     

Extrasynaptic localization of α-bungarotoxin receptors in the rat superior cervical ganglion

Binding of [¹²⁵I]α-bungarotoxin to nicotinic cholinergic receptors (α-bungarotoxin receptors) was investigated in the rat superior cervical ganglion by light and electron microscope autoradiography. Both techniques indicated that labelling, which was inhibited by d-tubocurarine, occurred around and/or over neuronal perikarya. In particular, ultrastructural autoradiography showed that the synapses were devoid of radioactivity, suggesting that α-bungarotoxin receptors in the rat superior cervical ganglion are molecules distinct from the nicotinic (postsynaptic) receptors normally involved in ganglionic transmission. By contrast, specific labelling was found in extrasynaptic areas of the neuronal membrane in contact with satellite cells (neuron-satellite cell boundary). Quantitative analysis indicated that at that level silver grains were present on both the neuronal membrane and satellite cells. Furthermore, beside neuronal perikarya, radioactivity was also found around nerve fibres, probably in relation to both the axonal and interstitial sides of the ensheathing Schwann cells. Only a few grains were clearly accumulated inside nerve fibres. Finally, significant amounts of specific radioactivity were detected in the neuronal cytoplasm, especially at the level of rough endoplasmic reticulum and Golgi apparatus. However, parallel diffusion experiments with [¹²⁵I]α-bungarotoxin and [³H]inulin (a marker for the extracellular space) provided no evidence that the toxin enters the neuronal cytoplasm. Thus, the intraneuronal (specific) labeling was probably a reflection of α-bungarotoxin binding to membrane receptors and the subsequent internalization of the toxin-receptor complex in the neurons. We conclude that in the rat superior cervical ganglion extrasynaptic nicotinic acetylcholine receptors (α-bungarotoxin receptors) may be widely located on the neuronal membrane as well as on the plasma membrane of satellite and Schwann cells. The physiological significance of this molecular architecture is discussed.     

Mode of action of Chrysosporium lucknowense C1 α-l-arabinohydrolases

The mode of action of four Chrysosporium lucknowense C1 α-L-arabinohydrolases was determined to enable controlled and effective degradation of arabinan. The active site of endoarabinanase Abn1 has at least six subsites, of which the subsites -1 to +2 have to be occupied for hydrolysis. Abn1 was able to hydrolyze a branched arabinohexaose with a double substituted arabinose at subsite -2. The exo acting enzymes Abn2, Abn4 and Abf3 release arabinobiose (Abn2) and arabinose (Abn4 and Abf3) from the non-reducing end of reduced arabinose oligomers. Abn2 binds the two arabinose units only at the subsites -1 and -2. Abf3 prefers small oligomers over large oligomers. It is able to hydrolyze all linkages present in beet arabinan, including the linkages of double substituted residues. Abn4 is more active towards polymeric substrate and releases arabinose monomers from single substituted arabinose residues. Depending on the combination of the enzymes, the C1 arabinohydrolases can be used to effectively release branched arabinose oligomers and/or arabinose monomers.     

The action mode of the ribosome-inactivating protein α-sarcin

Based on the tertiary structure of the ribosome-inactivating protein alpha-sarcin, domains that are responsible for hydrolyzing ribosomes and naked RNA have been dissected. In this study, we found that the head-to-tail interaction between the first amino beta-strand and the last carboxyl beta-strand is not involved in catalyzing the hydrolysis of ribosomes or ribonucleic acids. Instead, a four-strand pleated beta-sheet is indispensable for catalyzing both substrates, suggesting that alpha-sarcin and ribonuclease T1 (RNase T1) share a similar catalytic center. The integrity of an amino beta-hairpin and that of the loop L3 in alpha-sarcin are crucial for recognizing and hydrolyzing ribosomes in vitro and in vivo. However, a mutant protein without the beta-hairpin structure, or with a disrupted loop L3, is still capable of digesting ribonucleic acids. The functional involvement of the beta-hairpin and the loop L3 in the sarcin stem/loop RNA of ribosomes is demonstrated by a docking model, suggesting that the two structures are in essence naturally designed to distinguish ribosome-inactivating proteins from RNase T1 to inactivate ribosomes.