Assessment of uncoupling by amiloride analogs.

The amiloride analogs N5-methyl-N5-isobutylamiloride, N5-ethyl-N5-isopropylamiloride, and N5,N5-hexamethyleneamiloride are frequently used to investigate NaH exchange on the premise that they are highly specific inhibitors of the NaH-antiporters. We assessed the relative protonophoric activity of these compounds in reconstituted and native membrane vesicles, using acridine orange fluorescence to measure intravesicular pH. All the compounds tested were found to be potent protonophores at concentrations which are normally used to demonstrate inhibition of NaH exchange. Uncoupling was dependent on both the pH of the assay system and the total amount of lipid present. At the pH optima, which lay in a range from 7.5 to 8.5, these amiloride analogs were more potent uncouplers than the classical protonophore carbonyl cyanide m-chlorophenylhydrazone. Therefore, extreme care must be taken in the interpretation of results obtained using these or similar derivatives of amiloride.     

A comparative spectroscopic study of tryptophan probes engineered into high- and low-affinity domains of recombinant chicken troponin C.

The spectral properties of three tryptophan-substituted mutants of recombinant chicken troponin C are compared. Site-specific mutagenesis was used to introduce a tryptophan residue into the high-affinity (Ca2+/Mg2+) domain of troponin C at residue position 105, thereby creating the mutant phenylalanine-105 to tryptophan (F105W). The spectral properties of F105W and a double mutant, F29W/F105W, were compared with the mutant phenylalanine-29 to tryptophan (F29W). Since wild-type chicken troponin C does not naturally contain either tyrosine or tryptophan residues, the tryptophan substitutions behaved as site-specific reporters of metal ion binding and conformational change. The residues that occupy positions 29 and 105 are at homologous locations in low-affinity and high-affinity domains, preceding the first liganding residues of binding loops I and III, respectively. Mutant proteins were examined by a combination of absorbance and fluorescence methods. Calcium induced significant changes in the near-UV absorbance spectra, fluorescence emission spectra, and far-UV circular dichroism of all three mutant proteins. Magnesium induced significant changes in the spectral properties of only F105W and F29W/F105W proteins. Tryptophan substitutions allowed Ca(2+)-specific and Ca(2+)/Mg(2+) sites to be titrated independently of one another. Results indicate that there is no interaction between the two binding domains under conditions where troponin C is isolated from the troponin complex. Magnesium-induced changes in the environment of the tryptophan reporter at position 105 were significantly different from those induced by calcium. This suggests that calcium and magnesium differ in their influence on the conformation of the high-affinity, Ca(2+)/Mg(2+) sites.     

Folding topology of the disulfide-bonded dimeric DNA-binding domain of the myogenic determination factor MyoD.

The myogenic determination factor MyoD is a member of the basic-helix-loop-helix (bHLH) protein family. A 68-residue fragment of MyoD encompassing the entire bHLH region (MyoD-bHLH) is sufficient for protein dimerization, sequence-specific DNA binding in vitro, and conversion of fibroblasts into muscle cells. The circular dichroism spectrum of MyoD-bHLH indicates the presence of significant alpha-helical secondary structure; however, the NMR spectrum lacks features of a well-defined tertiary structure. There is a naturally occurring cysteine at residue 135 in mouse MyoD that when oxidized to a disulfide induces MyoD-bHLH to form a symmetric homodimer with a defined tertiary structure as judged by sedimentation equilibrium ultracentrifugation and NMR spectroscopy. Oxidized MyoD-bHLH retains sequence-specific DNA-binding activity, albeit with an apparent 100-1000-fold decrease in affinity. Here, we report the structural characterization of the oxidized MyoD-bHLH homodimer by NMR spectroscopy. Our findings indicate that the basic region is unstructured and flexible, while the HLH region consists of two alpha-helices of unequal length connected by an as yet undetermined loop structure. Qualitative examination of interhelical NOEs suggests several potential arrangements for the two helix 1/helix 2 pairs in the symmetric oxidized dimer. These arrangements were evaluated for whether they could incorporate the disulfide bond, satisfy loop length constraints, and juxtapose the two basic regions. Only a model that aligns helix 1 parallel to helix 1' and antiparallel to helix 2 was consistent with all constraints. Thus, an antiparallel four-helix bundle topology is proposed for the symmetric dimer. This topology is hypothesized to serve as a general model for other bHLH protein domains.     

Regulation mechanisms of intracellular pH of Xenopus laevis oocyte.

Intracellular pH values (pHi) of Xenopus oocytes were optically measured using a fluorescent dye, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The oocytes were loaded with dye by incubation with a membrane-permeable form (BCECF-AM). Mean pHi of the oocytes in pH 7.6 solution was 7.69. Increasing ambient pCO2 rapidly decreased pHi and estimated buffering power was 23.8 mM/pH unit. Changing ambient HCO3- from 5 to 30 mM did not alter pHi. After incubation in a Na(+)-free solution, Na+ addition to the bath rapidly increased pHi and this response was blocked by amiloride (ED50 2 microM). The addition of NH4Cl to the bath caused an initial transient increase in PHi followed by a secondary decrease. The secondary decrease was greatly inhibited by a histidine specific reagent, diethylpyrocarbonate. It was also slightly inhibited by ouabain, Ba2+ and furosemide, but not by amiloride. These data suggest that (1), fluorescence technique is applicable to PHi measurements of Xenopus oocytes; (2), Xenopus oocytes have an amiloride sensitive Na+/H(+)-exchange, and permeabilities to CO2, NH3, and NH+4. These observation may be useful in studying the relationship between pHi and oocytes development, and the expression of acid/base transporters in Xenopus oocytes.     

Extracellular lipase from Pseudomonas aeruginosa is an amphiphilic protein.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) secreted by Pseudomonas aeruginosa PAC1R was purified from cell-free growth medium by preparative isoelectric focusing. After blotting the N-terminal amino acid sequence and the amino acid composition were determined and compared to P. fragi and P. cepacia lipases yielding significant homology between all three species. Additionally, a consensus sequence K-Y-P-i-v-l-V-H-G was identified residing at the N-terminus of Pseudomonas lipases and in the central part of Staphylococcus lipases. Treatment of lipase with the serine-specific inhibitor diethyl p-nitrophenyl phosphate caused a rapid and complete inhibition of enzyme activity indicating the presence of a serine at the catalytic site as expected from lipase consensus sequences. Upon charge-shift electrophoresis the electrophoretic mobility of purified lipase was shifted either anodally or cathodally in the presence of sodium deoxycholate and cetyltrimethylammoniumbromide, respectively. This result demonstrates that extracellular lipase of P. aeruginosa exhibits an amphiphilic character like intrinsic membrane proteins.     

The compact domain conformation of human Glu-plasminogen in solution.

A complete understanding of the accelerating mechanisms of plasminogen activation and fibrinolysis necessarily requires structural information on the conformational forms of plasminogen. Given the absence of high-resolution structural data on plasminogen the use of lower resolution approaches has been adopted. Two such approaches have previously indicated a compact conformation of Glu-plasminogen (Tranqui, L., Prandini, M., and Chapel, A. (1979) Biol. Cellulaire, 34, 39-42; Bányai, L. and Patthy, L. (1985) Biochim. Biophys. Acta, 832, 224-227) whereas a third has suggested a fairly extended conformation (Mangel, W., Lin, B. and Ramakrishnan, V. (1990) Science, 248, 69-73). Native Glu-plasminogen has been investigated using small-angle X-ray scattering (SAXS) experiments. It is concluded that this molecule in solution is compact (radius of gyration, RG 3.05 +/- 0.02 nm and maximum intramolecular distance, Im 9.1 +/- 0.3 nm) and that the data are consistent with the right-handed spiral structure observed using electron microscopy by Tranqui et al. (1979). A spiral structure of native plasminogen would have important implications for the conformational response of plasminogen to fibrin and concomitant stimulation of plasminogen activation.     

Cloning of two adult hamster globin cDNAs (alpha and beta major).

A cDNA library was prepared from poly(A) mRNA extracted from adult anemic hamster spleen erythroid cells. cDNA clones containing inserts coding for adult alpha and beta major globin chains were isolated. Their identity was confirmed by (a) translation of hybrid selected mRNA and (b) nucleotide sequence analysis of the inserts and comparison to the adult globin cDNAs of mouse, rabbit and human. Availability of sequences for embryonic (Li et al. (1992) Biochim. Biophys. Acta 1130, 218-220) and adult globin cDNAs (this report) will aid in investigations of the molecular mechanisms involved in the globin ontogeny of hamsters.     

Chemical modification of alpha-thrombin and in vitro characterization of its anticoagulant activity

Acetylthrombin, succinylthrombin, maleylthrombin, polyethylene glycol (PEG)-thrombin, and acetylated PEG-thrombin were prepared and characterized. Although clotting activities could be reduced remarkably by the modification, the potencies of all the modified thrombins for protein C activation also decreased. However, in the case of acetylthrombin, succinylthrombin, and acetylated PEG-thrombin, the remaining potencies for protein C activation exceeded largely the remaining clotting activities. Moreover, by the addition of thrombodulin, the potencies for protein C activation were enhanced 16 - 60 fold for succinylthrombin, maleylthrombin, and PEG-thrombin.