Isotherms for the binding of dodecyltrimethylammonium (DTA+) ions by sodium dextran sulfate (NaDxS) and sodium poly(styrenesulfonate) (NaPS) in the presence of added NaCl are reported. The binding isotherms were determined by using a potentiometric technique based on surfactant ion selective solid-state electrodes. The solid membranes used in the electrodes consist of poly(vinyl chloride) (PVC) plasticized by bis(2-ethylhexyl) phosphate with a DTA-dodecyl sulfate carrier complex. The electrodes exhibit Nernstian response for DTA+ down to concentrations as low as 1 X "5 mol kg"1 even in the presence of a large excess of NaCl, allowing for sensitive and accurate free surfactant ion determinations. The binding of DTA+ to both polyanions is shown to be highly cooperative. The cooperativity parameter from the Zimm-Bragg theory may be estimated at 650 ± 100 and 200 ± 100 for the NaDxS and NaPS cases, respectively, and is independent of the NaCl concentration in both cases. The binding constant K of DTA+ to an isolated site on the polyanion is considerably larger in the PS-DTA system than in the DxS-DTA system, presumably because of differences in the hydrophobic/ hydrophilic properties of the two polymers. K is found to decrease strongly with increasing NaCl concentration; this decrease is similar in magnitude to the decrease in the critical micelle concentration (cmc) of dodecyltrimethylammonium bromide (DTABr) with increasing total counterion concentration in the presence of added NaCl.

Binding isotherms are reported for dodecyl-and tetradecyltrimethylammonium (DTA+ and TTA+) ion binding to polyacrylate (PA), alginate, pectate, and (carboxymethyl)cellulose (CMC) in the presence and absence of added NaCl at 30 °C, determined by using a potentiometric technique based on surfactant cation selective solid-state membrane electrodes. The results indicate a highly cooperative nature of the binding process of DTA+ and TTA+ by PA, alginate, and pectate but a lower cooperativity in the CMC system. TTA+ binding results in a larger cooperative binding constant (Ku) in all systems. The differences between DTA+ and TTA+ in the free energy of surfactant binding are 2.19kT for PA, 2.50kT for alginate, 2.56kT for pectate, and 2.64kT for CMC without NaCl and 2A2kT for PA with 0.01 m NaCl. These differences are comparable to the free energy of transfer of two methylene groups from water to a hydrocarbon medium or to a micelle. The presence of 0.01 m NaCl results in smaller Ku values and higher cooperativity.

1 The possible existence of a b 3 -adrenergic receptor (b 3 -AR) in the human detrusor muscle was investigated by in vitro functional studies and analysis of mRNA expression. 2 Isoprenaline, noradrenaline and adrenaline each produced a concentration-dependent relaxation of the human detrusor. The rank order for their relaxing potencies was isoprenaline (pD 2 6.37+0.07) 5 noradrenaline (pD 2 6.07+0.12) 5 adrenaline (pD 2 5.88+0.11). 3 Neither dobutamine (b 1 -and b 2 -AR agonist) nor procaterol (b 2 -AR agonist) produced any signi®cant relaxation at concentrations up to 10 75 M. BRL37344A, CL316243 and CGP-12177A (b 3 -AR agonists), relaxed the preparations signi®cantly at concentrations higher than 10 76 M. The pD 2 values for BRL37344A, CL316243 and CGP-12177A were 6.42+0.25, 5.53+0.09 and 5.74+0.14, respectively. 4 CGP-20712A (10 77 ± 10 75 M), a b 1 -AR antagonist, did not a ect the isoprenaline-induced relaxation. On the other hand, ICI-118,551, a b 2 -AR antagonist, produced a rightward parallel shift of the concentration-relaxation curve for isoprenaline only at the highest concentration used (10 75 M) and its pK B value was 5.71+0.19. Moreover, SR58894A (10 77 ± 10 75 M), a b 3 -AR antagonist, caused a rightward shift of the concentration-relaxation curve for isoprenaline in a concentration-dependent manner. The pA 2 value and slope obtained from Schild plots were 6.24+0.20 and 0.68+0.31. 5 The b 1 -, b 2 -and b 3 -AR mRNAs were all positively expressed in detrusor smooth muscle preparations in a reverse transcription polymerase chain reaction assay. 6 In conclusion, the present results provide the ®rst evidence for the existence of the b 3 -AR subtype in the human detrusor. They also suggest that the relaxation induced by adrenergic stimulation of the human detrusor is mediated mainly through b 3 -AR activation.

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In addition to its kinase activity, the myosin light chain kinase (MLCK) of smooth muscle has an actin binding activity through which it can regulate the actinmyosin interaction of smooth muscle (Kohama, K., Okagaki, T., Hayakawa, K., Lin, Y., Ishikawa, R., Shimmen In addition to this kinase activity, MLCK can act as an actin-binding protein; MLCK is present in association with the sarcomeric I-band (2), and it binds to actin filaments with a high affinity (3-5). We have shown that MLCK can inhibit the ATP-dependent interaction between actin and myosin by binding to actin filaments. This inhibition can be relieved by Ca 2ϩ /CaM, as was demonstrated by avoiding the complication derived from its kinase activity (6 -8), although the demonstration was limited to in vitro only.Such an inhibition, however, is not peculiar to MLCK. Caldesmon (see Ref. 9 for a review) and calponin (10) The relationship between the structure of MLCK and the function of the kinase activity in phosphorylating the myosin light chain and how Ca 2ϩ /CaM modulates the activity has been well established (see Ref. 13 for a review). However, there has been little study of the structure-function relationship of the actin binding activity except for the purification of an actinbinding fragment from MLCK (14).In this study, we have investigated which sequence of MLCK is responsible for the actin binding activity, which sequence inhibits the actin-myosin interaction, and which sequence binds CaM to relieve the inhibition. Our approach has been to cleave MLCK to prepare native fragments containing the actin binding activity (14), to design MLCK cDNA to express the recombinant fragments of actin binding activity in Escherichia coli, and then to analyze them biochemically. A few peptides have been synthesized to confirm these analyses. We have shown that: (i) MLCK has two actin-binding sites on the Nterminal side away from the central kinase domain, (ii) the binding site responsible for the inhibitory effect is at the Met MATERIALS AND METHODSPreparation of Proteins-All procedures were carried out at 0 -4°C. The purity of proteins was routinely monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (see below) so * This work was supported in part by grants from the Fujisawa Foundation and the Mitsubishi Foundation and by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.ʈ To whom correspondence should be addressed. Tel.: 81-27-220-7960; Fax: 81-27-235-1401 or 81-27-220-7962. 1 The abbreviations used are: MLCK, myosin light chain kinase; CaM, calmodulin; PAGE, polyacrylamide gel electrophoresis; NTCB fragment, chicken gizzard MLCK fragment produced by the cleavage with 2-nitro-5-thiocyanatobenzoic acid; NN fragment, expressed fragment (amino acids 1-337 ...