Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

Small conductance Ca²⁺-sensitive potassium (SK2) channels are voltage-independent, Ca²⁺-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca²⁺ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca²⁺ and Ca²⁺-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca²⁺ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.     

Dependence on blood acetate concentration of the metabolic effects of ethanol in perfused rat liver

Unprotected oligonucleotides and oligodeoxynucleotides terminated with an unhindered 5'-phosphate group react with nucleoside 5'- phosphorimidazolides in aqueous solution to give 'capped' pyrophosphates in at least 70% yield. If adenosine 5'- phosphorimidazolide is used as a substrate in the reaction, ligase intermediates are obtained as products.     

Effects of haematocrit value and glucagon on the metabolism of perfused rat liver

Liver from adult male rats were perfused in situ for 30 min with either undiluted, defibrinated rat blood (haematocrit value 38%) or the same blood diluted with buffer to give a haematocrit of 20%. Perfusion with diluted blood lowered the PO2 of the effluent perfusate but this was insufficient to prevent the fall in O2 consumption due to the reduction in haematocrit. Glucagon (5 X 10(-9) M) increased hepatic O2 consumption with whole blood but not with diluted blood. perfusate K+ was increased by perfusion with diluted blood and glucagon. Bile flow was depressed and biliary K+ increased by glucagon but only in experiments with whole blood. Perfusate glucose was raised by lowering of hepatic O2 consumption but the hormonal stimulation of glucose output was the same at both haematocrits. Net ketogenesis was increased with perfusion with diluted blood and by glucagon. In the absence of glucagon there was a net secretion of triacylglycerols which was depressed by lowering of the haematocrit. Glucagon inhibited triacylglycerol secretion and the effect was greater with whole blood so that there was net uptake. While effects of glucagon were obtained during perfusion at a lower haematocrit, it would appear that whole blood was the medium that allowed their fullest expression.     

Effect of oxygen pressure on synthesis and export of nitrogenous solutes by nodules of cowpea

Nodules of cowpea plants (Vigna unguiculata (L.) Walp. cv. Vita 3 :Bradyrhizobium CB756) cultured for periods of 23 d with their root systems maintained in atmospheres containing a range of partial pressures of O₂ (pO₂; 1–80%, v/v, in N₂) formed and exported ureides (allantoin and allantoic acid) as the major products of fixation at all pO₂ tested. In sub-ambient pO₂ (1 and 2.5%) nodules contained specific activities of uricase (urate: O₂ oxidoreductase; EC 1.7.3.3) and allantoinase (allantoin hydrolyase; EC 3.5.2.5) as much as sevenfold higher than in those from air. On a cell basis, uninfected cells in nodules from 1% O₂ contained around five times the level of uricase. Except for NAD: glutamate synthase (EC 1.4.1.14), which was reduced in sub-ambient O₂, the activities of other enzymes of ureide synthesis were relatively unaffected by pO₂. Short-term effects of pO₂ on assimilation of fixed nitrogen were measured in nodules of air-grown plants exposed to subambient pO₂ (1, 2.5 or 5%, v/v in N₂) and¹⁵N₂. Despite a fall in total¹⁵N₂ fixation, ureide synthesis and export was maintained at a high level except in 1% O₂ where formation was halved. The data indicate that in addition to the structural and diffusional adaptations of cowpea nodules which allow the balance between O₂ supply and demand to be maintained over a wide range of pO₂, nodules also show evidence of biochemical adaptations which maintain and enhance normal pathways for the assimilation of fixed nitrogen. This work was supported by a grant from the Australian Research Council (to C.A.A.) and an Australian Development Assistance Bureau postgraduate fellowship (to F.D.D.).     

Potent slow-binding inhibition of cathepsin B by its propeptide.

A peptide (PCB1) corresponding to the proregion of the rat cysteine protease cathepsin B was synthesized and its ability to inhibit cathepsin B activity investigated. PCB1 was found to be a potent inhibitor of mature cathepsin B at pH 6.0, yielding a Ki = 0.4 nM. This inhibition obeyed slow-binding kinetics and occurred as a one-step process with a k1 = 5.2 x 10(5) M-1 s-1 and a k2 = 2.2 x 10(-4) s-1. On dropping from pH 6.0 to 4.7, Ki increased markedly, and whereas k1 remained essentially unchanged, k2 increased to 4.5 x 10(-3) s-1. Thus, the increase in Ki at lower pH is due primarily to an increased dissociation rate for the cathepsin B/PCB1 complex. At pH 4.0, the inhibition was 160-fold weaker (Ki = 64 nM) than at pH 6.0, and the propeptide appeared to behave as a classical competitive inhibitor rather than a slow-binding inhibitor. Incubation of cathepsin B with a 10-fold excess of PCB1 overnight at pH 4.0 resulted in extensive cleavage of the propetide whereas no cleavage occurred at pH 6.0, consistent with the formation of a tight complex between cathepsin B and PCB1 at the higher pH. The synthetic propeptide of cathepsin B was found to be a much weaker inhibitor of papain, a structurally similar cysteine protease, and no pH dependence was observed. Inhibition constants of 2.8 and 5.6 microM were obtained for papain inhibition by PCB1 at pH 4.0 and 6.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibiosis and antixenosis of chrysanthemum cultivars to the aphidAphis gossypii

An assessment was made of the antibiotic and antixenotic resistance of three cultivars of autumn flowering chrysanthemum (derived fromDendranthema morifolium (Ramat) Tzvelev andD. indicum (L.) Desmoulins) to the aphidAphis gossypii Glover. Antibiosis was also assessed at the top, middle and lower leaves of the plant. The three cultivars showed a range of resistance with Hero being the most susceptible, Surfine the most resistant and Purple Anne intermediate. There was a trend for antibiosis to improve with position down the plant, however there were cultivar by position interactions. Antixenosis was assessed at two levels, the first being under conditions where the aphid had complete access to the plant in caged choice assays and the second being under conditions where the aphid only had host odour to assess under olfactometer choice conditions. Preferences under the caged choice conditions found Hero to be the most preferred cultivar while there was no preference shown between Purple Anne and Surfine. However, olfactometer assessments showed Hero to be the least preferred of the three cultivars and, again, no preferences could be found between Purple Anne and Surfine.     

An in vivo-in vitro alkaline DNA unwinding assay for hepatic DNA damage: comparison with the alkaline sucrose gradient centrifugation technique

The addition of glycerol, sucrose, or other diol-containing reagents to solutions of aminoacyl-tRNA (aa-tRNA) substantially increased the rate of hydrolysis of the aminoacyl ester bond. Glycerol at 4.9% (v/v) doubled the rate of deacylation for several aa-tRNAs and peptidyl-tRNAs, including fMet-tRNAMetf, while 1% (v/v) glycerol increased the deacylation rate by 20%. This effect was not caused by a nuclease contamination, and tRNA deacylated in the presence of glycerol could be fully recharged. The deacylation of aa-tRNA was accelerated by glycerol and sucrose even in the presence of EF-Tu X GTP. In addition, the extent of tRNA aminoacylation was reduced when glycerol was present at concentrations above 2% (v/v). Thus, glycerol and sucrose are not necessarily inert or neutral additions to an in vitro incubation.     

Cooperativity of papain-substrate interaction energies in the S2 to S2' subsites

Enzyme-substrate contacts in the hydrolysis of ester substrates by the cysteine protease papain were investigated by systematically altering backbone hydrogen-bonding and side-chain hydrophobic contacts in the substrate and determining each substrate's kinetic constants. The observed specificity energies [defined as delta delta G obs = -RT ln [(kcat/KM)first/(kcat/KM)second)]] of the substrate backbone hydrogen bonds were -2.7 kcal/mol for the P2 NH and -2.6 kcal/mol for the P1 NH when compared against substrates containing esters at those sites. The observed binding energies were -4.0 kcal/mol for the P2 Phe side chain, -1.0 kcal/mol for the P1' C=O, and -2.3 kcal/mol for the P2' NH. The latter three values probably all significantly underestimate the incremental binding energies. The P2 NH, P2 Phe side-chain, and P1 NH contacts display a strong interdependence, or cooperativity, of interaction energies that is characteristic of enzyme-substrate interactions. This interdependence arises largely from the entropic cost of forming the enzyme-substrate transition state. As favorable contacts are added successively to a substrate, the entropic penalty associated with each decreases and the free energy expressed approaches the incremental interaction energy. This is the first report of a graded cooperative effect. Elucidation of favorable enzyme-substrate contacts remote from the catalytic site will assist in the design of highly specific cysteine protease inhibitors.     

Importance of hydrogen-bonding interactions involving the side chain of Asp158 in the catalytic mechanism of papain

In a previous study, it was shown that replacing Asp158 in papain by Asn had little effect on activity and that the negatively charged carboxylate of Asp158 does not significantly stabilize the active site thiolate-imidazolium ion pair of papain (Ménard et al., 1990). In this paper, we report the kinetic characterization of three more mutants at this position: Asp158Gly, Asp158Ala, and Asp158Glu. From the pH-activity profiles of these and other mutants of papain, it has been possible to develop a model that enables us to dissect out the contribution of the various mutations toward (i) intrinsic activity, (ii) ion pair stability, and (iii) the electrostatic potential at the active site. Results obtained with mutants that place either Gly or Ala at position 158 indicate that the hydrogen bonds involving the side chain of Asp158 in wild-type papain are indirectly important for enzyme activity. When CBZ-Phe-Arg-MCA is used as a substrate, the (kcat/KM)obs values at pH 6.5 are 3650 and 494 M-1 s-1 for Asp158Gly and Asp158Ala, respectively, as compared to 119,000 M-1 s-1 for papain. Results with the Asp158Glu mutant suggest that the side chain of Glu moves closer to the active site and cannot form hydrogen bonds similar to those involving Asp158 in papain. From the four mutations introduced at position 158 in papain, we can conclude that it is not the charge but the hydrogen-bonding interactions involving the side chain of Asp158 that contribute the most to the stabilization of the thiolate-imidazolium ion pair in papain. However, the charge and the hydrogen bonds of Asp158 both contribute to the intrinsic activity of the enzyme.     

Detection of covalent enzyme-substrate complexes of nitrilase by ion-spray mass spectroscopy

Nitrilase from Rhodococcus ATCC 39484 was found to consist of two species of Mr 40,258 +/- 2 and 40,388 +/- 2 Da. When the enzyme was incubated with nitrile substrates and the reaction quenched with acid, higher Mr species were observed. The mass differences were consistent with addition of a substrate molecule to each species. These results represent the first reported demonstration that this, or any other nitrilase forms a covalent intermediate with its substrates. The observation that the intermediate, suggested to be either a thioimidate or an acylenzyme, can be trapped by acidification indicates that the rate of breakdown of the intermediate is rate-limiting.