MALDI-TOF MS suggested that the high molecular weight proanthocyanidin (condensed tannin) from persimmon (Diospyros kaki L.) pulp comprised a heteropolyflavanol series with flavan-3-O-galloylated extenders, flavan-3-ol and flavonol terminal units, and A-type interflavan linkages. Thiolysis-HPLC-ESI-MS with DAD, electrochemical, and ESI-MS detection confirmed a previously unreported terminal unit, the flavonol myricetin, in addition to the typical flavan-3-ols catechin and epigallocatechin gallate. The extender units were epicatechin, epigallocatechin, (epi)gallocatechin-3-O-gallate, and (epi)catechin-3-O-gallate. The crude tannin had a high prodelphinidin content (65%) and a high degree of 3-O-galloylation (72%). The material was fractionated on Toyopearl TSK-HW-50-F to yield fractions distinguished by degree of polymerization (DP). Thiolysis suggested that the persimmon tannin was composed of polymers ranging from 7 to 20 kDa (DP 19-47), but sizes estimated by GPC were 50-70% smaller. The crude material was chemically degraded with acid to yield products that were amenable to NMR and ESI-MS analysis, which were used to establish for the first time that persimmon tannin has a mixture of B-type and A-type linkages.

Vacuolar proton-translocating ATPase pumps consist of two domains, V 1 and V o . Subunit d is a component of V o located in a central stalk that rotates during catalysis. By generating mutations, we showed that subunit d couples ATP hydrolysis and proton transport. The mutation F94A strongly uncoupled the enzyme, preventing proton transport but not ATPase activity. C-terminal mutations changed coupling as well; ATPase activity was decreased by 59 -72%, whereas proton transport was not measurable (E328A) or was moderately reduced (E317A and C329A). Except for W325A, which had low levels of V 1 V o , mutations allowed wild-type assembly regardless of the fact that subunits E and d were reduced at the membrane. N-and C-terminal deletions of various lengths were inhibitory and gradually desta- Viral infections, cancer, osteoporosis, and renal tubular acidosis are some of the human disease states associated with the V-ATPase 2 function. V-ATPases are ATP-driven proton pumps present in Golgi, endosomes, lysosomes, and vacuoles, where they are responsible for maintaining the acidic luminal pH essential for receptor-mediated endocytosis, zymogen activation, and protein sorting (1-4). In addition to the endogenously distributed V-ATPases, some cells contain V-ATPases at the plasma membrane, where they pump protons from the cytosol to the extracellular milieu. V-ATPase proton transport across the plasma membrane is essential for bone resorption, urinary acidification, sperm maturation, and neurotransmitter sequestration (3).V-ATPases are related to F-ATP synthases (5), and both protein complexes work as molecular motors (6 -9). V-ATPases, however, work exclusively in the direction of ATP hydrolysis in vivo. V-ATPases consist of two domains, V 1 and V o , similar to the F 1 and F o domains found in ATP synthases (1-4). Eight different subunits (A-H) compose V 1 , which is peripherally attached to the membrane and hydrolyzes ATP (1-4). Six different subunits (a, c, cЈ, cЉ, d, and e) associate to form V o , which holds V 1 at the membrane and forms the path to transport protons (1-4). V 1 and V o subunits contribute to the formation of one central stalk and two or three peripheral stalk structures that connect a proteolipid ring (made of subunits c, cЈ, and cЉ) in V o and the catalytic core of V 1 (a hexamer of three subunits A alternating with three subunits B) (10, 11).The organization of the central and peripheral stalks is essential for structural and functional coupling of ATP hydrolysis and proton transport. It is proposed that during catalysis, ATP hydrolysis at the V 1 hexamer A 3 B 3 drives rotation of a rotor (central stalk made of subunits D, F, and d connected to the proteolipid ring) (6, 12). Six essential glutamates are protonated in the ring when protons are transferred from the cytosol via two half-tunnel structures formed in the stationary subunit a at the membrane (13-15). By connecting the stationary subunits (A 3 B 3 and a), the peripheral stalk(s) (subunits C, E, G, H, and the N terminus of subunit a) wo...

We have developed a simple method for preparing and verifying suitable standards for the acid butanol assay from a readily available source. Phenolics were extracted from fresh apples with methanol, and sugars were removed from the crude extract by treatment with Amberlite resin before fractionating the proanthocyanidins into ethyl acetate. The ethyl acetate fraction was chromatographed on Toyopearl TSK HW-50F to yield about 50 mg of procyanidin dimer and 35 mg of trimer from 1 kg fresh apple fruit. The purity and identity of the standards was easily confirmed by using ESI-MS. In the acid butanol assay, the pure dimer, trimer and purified Sorghum procyanidin had similar color yields on a mass basis, and produced about three times more color than purified quebracho tannin. This new standard overcomes problems associated with overestimation of tannin due to use of the unreactive quebracho tannin standard. Use of the new standard will enable accurate comparisons of tannin levels between laboratories and will standardize comparisons between species, thus promoting our understanding of the role of condensed tannins in plants.

The bioactivities and bioavailability of plant polyphenols including proanthocyanidins and other catechin derivatives may be affected by covalent reaction between polyphenol and proteins. Both processing conditions and gastrointestinal conditions may promote formation of covalent complexes for polyphenol-rich foods and beverages such as wine. Little is known about covalent reactions between proteins and tannin, because suitable methods for quantitating covalent complexes have not been developed. We established capillary electrophoresis methods that can be used to distinguish free protein from covalently bound protein-polyphenol complexes and to monitor polyphenol oxidation products. The methods are developed using the model protein bovine serum albumin and the representative polyphenol (-)epigallocatechin gallate. By pairing capillaries with different diameters with appropriate alkaline borate buffers, we are able to optimize resolution of either the protein-polyphenol complexes or the polyphenol oxidation products. This analytical method, coupled with purification of the covalent complexes by diethylaminoethyl cellulose chromatography, should facilitate characterization of covalent complexes in polyphenol-rich foods and beverages such as wine.

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