In this study, we addressed the presence and location of nucleotide-binding sites in the voltage-dependent anion channel (VDAC). VDAC bound to reactive red 120-agarose, from which it was eluted by ATP, less effectively by ADP and AMP, but not by NADH. The photoreactive ATP analog, benzoyl-benzoyl-ATP (BzATP), was used to identify and characterize the ATP-binding sites in VDAC.[␣-32 P]BzATP bound to purified VDAC at two or more binding sites with apparent high and low binding affinities. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of BzATP-labeled VDAC confirmed the binding of at least two BzATP molecules to VDAC. The VDAC BzATP-binding sites showed higher specificity for purine than for pyrimidine nucleotides and higher affinity for negatively charged nucleotide species. VDAC treatment with the lysyl residue modifying reagent, fluorescein 5-isothiocyanate, markedly inhibited VDAC labeling with BzATP. The VDAC nucleotide-binding sites were localized using chemical and enzymatic cleavage. Digestion of [␣-32 P]BzATP-labeled VDAC with CNBr or V8 protease resulted in the appearance of ϳ17-and ϳ14-kDa labeled fragments. Further digestion, high performance liquid chromatography separation, and sequencing of the selected V8 peptides suggested that the labeled fragments originated from two different regions of the VDAC molecule. MALDI-TOF analysis of BzATP-labeled, tryptic VDAC fragments indicated and localized three nucleotide binding sites, two of which were at the N and C termini of VDAC. Thus, the presence of two or more nucleotidebinding sites in VDAC is suggested, and their possible function in the control of VDAC activity, and, thereby, of outer mitochondrial membrane permeability is discussed.It has recently been recognized that there exists a metabolic coupling between the cytosol and the mitochondria, with the outer mitochondrial membrane (OMM), 2 the boundary between these compartments, fulfilling an important function in this coupling (1-4). In the cross-talk between mitochondria and cytosol, mitochondrial ATP and Ca 2ϩ play major roles. As the primary transporter of nucleotides, Ca 2ϩ , and other ions and metabolites across the OMM (reviewed in Refs. 5 and 6), the voltage-dependent anion channel (VDAC) mediates a substantial portion of the OMM molecular traffic. It has been demonstrated that VDAC is permeable to Ca 2ϩ and possesses Ca 2ϩ -binding sites that control its activity (reviewed in Ref. 5). VDAC permeability is also regulated by associated protein(s) (reviewed in Refs. 5-7), and by different ligands that bind to VDAC, such as glutamate, ruthenium red, La 3ϩ , and NADH (reviewed in Refs. 5 and 6). Thus, by providing the pathway for anions, cations, ATP, and other metabolites into and out of the mitochondria, as well as possessing ligand-binding sites, VDAC plays an important role in the regulation of mitochondrial functions. Indeed, VDAC has been suggested to control OMM permeability, coupled respiration, and cell survival (3,4,8).It has been proposed that activati...

Glycosylation is one of the most abundant posttranslation modifications of proteins, and accumulating evidence indicate that the vast majority of proteins in eukaryotes are glycosylated. Glycosylation plays a role in protein folding, interaction, stability, and mobility, as well as in signal transduction. Thus, by regulating protein activity, glycosylation is involved in the normal functioning of the cell and in the development of diseases. Indeed, in the past few decades there has been a growing realization of the importance of protein glycosylation, as aberrant glycosylation has been implicated in metabolic, neurodegenerative, and neoplastic diseases. Thus, the identification and quantification of protein-borne oligosaccharides have become increasingly important both in the basic sciences of biochemistry and glycobiology and in the applicative sciences, particularly biomedicine and biotechnology. Here, we review the state-of-the-art methodologies for the identification and quantification of oligosaccharides, specifically N- and O-glycosylated proteins.

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Ovarian development in the red claw crayfish Cherw quadricarinatus is divided into two major phases, namely primary-and secondary-vitellogenesis. Two anatomically distinctive types of primary-vitellogenic ovary are present: one contains uniform milky white oocytes (in the chromatin, chromatin-nucleolus, early-perinuclear and late-perinuclear stages), while the other contains two diversely colored oocyte populations, the smallest oocytes being uniform milky white and the largest being yellow to orange (i.e., at the lipid stage). Secondary-vitellogenic ovaries are characterized by the presence of a synchronously growing large oocyte group together with oocytes of all the frst four, primary-vitellogenic stages. The synchronous group develops fiom the yolk stage via the prematuration stage into the maturation stage. Polypeptides of relatively low molecular masses (65-95 kDa) are abundant in the primary-vitellogenic ovary containing chromatin to lipid-stage oocytes. Polypeptides of relatively higher molecular masses (>I00 kDa) were detected both in the secondary-vitellogenic ovary (composed mainly of yolkstage oocytes) and in newly laid eggs. During secondary vitellogenesis, the high-density lipoprotein (HDL) fraction of the hemolymph contains four secondary vitellogenic-specific polypeptides (208, 196, 177 and 80 kDa) that are not present in the hemolymph of the primary vitellogenic female. In this study we have elucidated the relationship between the progress of gonad maturation, oocyte development and the sequence of appearance of specific polypeptides in the ovary and the hemolymph of C. quadricarinatus.

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In a previous study, we presented evidence for the existence of a nucleotide-binding site (NBS) in the N-terminal region of the voltage-dependent anion channel (VDAC1). In this study, further localization and possible roles of the proposed VDAC1-NBS were investigated using site-directed mutagenesis. The predicated NBS of murine VDAC1 (mVDAC1) was mutated by replacing two glycine residues with alanines or a conserved lysine residue with a serine. Expression of the G21A,G23A- and K20S-mVDAC1s in human T-REx-293 cells in which endogenous VDAC1 expression had been silenced affected cell growth and cytosolic ATP levels. While G21A,G23A-mVDAC1-expressing cells displayed growth rates similar to native-mVDAC1-expressing cells, the K20S-mVDAC1-expressing cells displayed significantly retarded growth and increased resistance to cell death. Cells expressing either mVDAC1 mutant also displayed significantly reduced cellular ATP levels. When K20S-mutant mVDAC1 was expressed in porin1-less yeast, the transformed cells grew slower on non-fermentable carbon sources, while isolated mitochondria expressing either mVDAC1 mutant showed significant reduction in ATP synthesis. Purified K20S-mVDAC1 displayed a significant decrease in [alpha-(32)P]BzATP-binding and altered channel properties, that is, reduced ion selectivity, while the G21A,G23A-mutant protein displayed only a mild reduction in channel selectivity. These results suggest that mutations in the proposed VDAC1-NBS, particularly the K20S, altered channel activity, thereby interfering with VDAC function as the major pathway for the transport of metabolites and adenine nucleotides across the outer mitochondrial membrane. Finally, involvement of the VDAC1-NBS in the control of mitochondrial ATP synthesis, cell growth and viability is discussed.

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