Mechanisms of ATPases--a multi-disciplinary approach

ATPases are important molecular machines that convert the chemical energies stored in ATP to mechanical actions within the cell. ATPases are among the most abundant proteins with diverse functions involved in almost every cellular pathway. The well characterised ATPases include the various motor proteins responsible for cargo transfers, cell motilities, and muscle contractions; the protein degradation machinery - the proteasome; the ATP synthase, F-ATPase; and the chaperone systems. Other ATPases include DNA helicases and DNA replication complex; proteins responsible for protein/complex disassembly; and certain gene regulators. It is beyond the scope of this review to cover the complete range of ATPases. Instead, we will focus on a few representative ATPases, chosen based on their diverse mechanisms and properties. Furthermore, this review is by no means trying to cover comprehensively the literature for each ATPase nor the historical aspects in each field. We will focus on describing the various techniques being employed to derive the mechanisms and properties of the chosen ATPases. Among them, high and low resolution structural studies combined with biochemical assays seem to be the dominant technical advances adapted to reveal mechanisms for most of the ATPases except the bacterial sigma54 activators, whose mechanism of action is mostly derived from large amount of biochemical studies. A number of them, especially the F-ATPase and motor proteins, have been studied successfully by various single molecule and imaging techniques. We will therefore discuss them in greater details in order to describe the wide range techniques being utilised.     

Diastereoselective inter- and intramolecular pinacol coupling of aldehydes promoted by monomeric titanocene(II) complex Cp(2)TiPh

A monomeric titanocene(III) derivative, Cp(2)TiPh, effectively promoted the pinacol coupling of both an aromatic aldehyde, benzaldehyde, and an aliphatic aldehyde, 3-phenylpropionaldehyde. The same reactive complex was successfully generated by a catalytic amount of a precursor, Cp(2)Ti(Ph)Cl, and its stoichiometric amount of Zn. The Cp(2)TiPh-catalyzed pinacol coupling of benzaldehyde derivatives and aliphatic aldehydes afforded the corresponding 1,2-diols in high yields with moderate to good threo-selectivity. On the other hand, Cp(2)TiPh-catalyzed pinacol cyclization of dials gave cyclic 1,2-diols with excellent diastereoselectivity. The extension of this protocol to chiral dials demonstrated that the phenyltitanium complex catalytically transmitted an axial chirality or a central chirality of the starting dials to the central chirality of the resultant 1,2-diols.     

Stereoselectivity in the collision-activated reactions of gas phase salt complexes

The collision-activated dissociations (CAD) of gas phase salt complexes composed of chiral ions were studied in a quadrupole ion trap mass spectrometer. Because both partners in the salt are chiral, diastereomeric complexes can be formed (e.g., RR, RS). Two general types of complexes were investigated. In the first, the complex was composed of deprotonated binaphthol and a chiral bis-tetraalkylammonium dication. CAD of these complexes leads to the transfer of a proton or an alkyl cation to the binaphtholate leading to a singly-charged tetraalkylammonium cation. During CAD, diastereomeric complexes give significantly different product distributions indicating reasonable stereoselectivity in the process. In the second system, the complexes involved a peptide dianion and a chiral tetraalkylammonium cation. These systems may be viewed as very simple models for the interactions of peptides/proteins with small chiral molecules. Again, stereoselectivity was evident during CAD, but the extent was dependent on the nature of the peptide and not observable in some cases. To better understand the structural features needed to achieve stereoselectivity in gas phase salt complexes, representative transition states were modeled computationally. The results suggest that it is critical for the asymmetry of the nucleophile (i.e., anion) to be well represented in the vicinity of its reactive center.     

Solid sampling technique for direct detection of condensed tannins in bark by matrix-assisted laser desorption/ionization mass spectrometry

A novel method for the direct analysis of condensed tannin components in bark was developed on the basis of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with a simple solid sampling technique. The MALDI mass spectra obtained from the wood (bark) powder sample clearly showed a series of peaks corresponding to the sodium ion adducts of condensed tannin oligomers up to around m/z 3000. The results indicate that the condensed tannins in the bark sample used in this work mostly consist of combinations of flavan-3-ol units such as profisetinidin (PF), prorobinetinidin (PR) and prodelphinidin (PD), at least up to 10-mers (m/z approximately 3000).     

CpRuIIPF6/quinaldic acid-catalyzed chemoselective allyl ether cleavage. A simple and practical method for hydroxyl deprotection

A cationic CpRu(II) complex in combination with quinaldic acid shows high reactivity and chemoselectivity for the catalytic deprotection of hydroxyl groups protected as allyl ethers. The catalyst operates in alcoholic solvents without the need for any additional nucleophiles, satisfying the practical requirements of operational simplicity, safety, and environmental friendliness. The wide applicability of this deprotection strategy to a variety of multifunctional molecules, including peptides and nucleosides, may provide new opportunities in protective group chemistry. [structure: see text]     

High-performance liquid chromatography of coenzyme Q-related compounds and its application to biological materials

A convenient and precise method for the separation and determination of coenzyme Q (CoQ)-related compounds (CoQ homologues, plastoquinone-9, ubichromenol-9, etc.) was developed using high-performance liquid chromatography (HPLC). All compounds tested were separated using a reverse-phase column with a suitable mobile phase and detected at a wavelength of 275 nm. CoQ extracts in plasma and erythrocytes were purified by thin-layer chromatography prior to HPLC analysis, but such purification was not necessary when determining CoQ in urine and tissues. Hydroquinone forms of CoQ existing in animal tissues were oxidized to the corresponding quinone forms with potassium hexacyanoferrate(III). This HPLC method was applied satisfactorily to the determination of the contents of CoQ homologues in human and animal samples. CoQ10 was the only homologue detected in human samples, and CoQ8, CoQ9 and CoQ10 were native homologues of CoQ in rat tissues. Ubichromenol-9 and plastoquinone-9 were not detected in these samples.     

Ligand Substitution Reaction of Bis(ethylenediamine)glycinatocobalt(III) Complex with Ethylenediamine Catalyzed by the Photo-excited Tris(2,2′-bipyridine)ruthenium(II) Complex

Substitution reaction with ethylenediamine of coordinated glycinate ligand in bis(ethylenediamine)-glycinatocobalt(III) complex has been studied in the presence of photo-excited tris(2,2′-bipyridine)ruthenium(II) complex in alkaline aqueous solution (buffered around pH 12) containing 1.0M chloride ion at 25°C. VIS absorption and CD spectra were used for the racemate and the optically active isomers of the Co(III) complexes, respectively. The reaction was catalyzed by the excited Ru(II) complex to give tris(ethylenediamine)cobalt(III) complex. Mechanism of the ligand-substitution reaction and role of the excited Ru(II) complex were discussed.     

Scale-up of high shear granulation based on the internal stress measurement

Scale-up of wet granulation in a vertical high shear mixer was conducted. Pharmaceutical excipient powders composed of lactose, cornstarch and micro-crystallinecellulose, and hydroxypropylcellulose as a binder were mixed together and then granulated with purified water under various operating conditions and vessel scales. A novel internal stress measurement system was developed and stress of normal and tangential directions that granules received from the agitator blade during the granulation was continuously measured. The results indicated that granules received stress mainly from the tangential direction, which also showed the largest value near at the vessel wall. The effects of the agitator tip speed and the centrifugal acceleration on the measured stress was investigated. It was found that the tip speed of the agitator blade could be the main factor for the granule growth. The physical properties such as strength, size distribution and compressibility of granules prepared by changing the operating conditions and the vessel scales were evaluated and the scale-up characteristics of high shear granulation were investigated experimentally. The results showed that these physical properties had linear correlations with the tip speed. It was finally concluded that the scale-up of high shear granulation could be well conducted by means of the tip speed of the agitator blade.     

Rate of spontaneous deactivation of styryl and α-methylstyryl carbanions

The deactivation of α-methylstyryl and styryl carbanions followed by spectral change and titration was studied kinetically. First-order rate constants were obtained for the deactivation of oligo-α-methylstyryl lithium, disodium, and dipotassium in tetrahydrofuran and of polystyryl potassium in varying degrees of polymerization in benzene that contained a small amount of tetrahydrofuran and n-hexane. It was observed that the addition of cryptate [222] exerts a significant effect on the system of oligo-α-methylstyryl disodium in tetrahydrofuran. The effect of dilution, degree of polymerization of the polymer, and counterions on the rate of deactivation of carbanions was discussed.     

A glucose electrode based on carbon paste chemically modified with a ferrocene-containing siloxane polymer and glucose oxidase,coated with a poly(ester-sulfonic acid) cation-exchanger

A carbon-paste chemically modified with glucose oxidase and a ferrocene-containing siloxane polymer was further modified by coating the electrode surface with a poly(ester-sulfonic acid) cation-exchanger, Eastman AQ-29D. The polymer is obtained as a homogeneous aqueous dispersion at pH 5–6; when dried, the polymer coating is not water-soluble. The coating was shown not to be detrimental to the enzyme activity but to prevent electrochemically active anionic interferents such as ascorbate and urate from reaching the electrode surface. The polymer coating also prevented glucose oxidase from leaking out of the carbon paste into the contacting solution and protected the electrode surface from fouling agents present in urine and bovine serum albumin. Uncoated electrodes lost some 10-2-15% of their original response to glucose after storage in buffer for three weeks whereas the response of the coated electrodes remained constant. Calibration curves for glucose were strictly linear up to about 5 mM for uncoated and up to 20 mM for coated electrodes. The response current to glucose was not decreased after coating.