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At least 793 records · Page 44Linked to original sources
PubMed1995

Human (MDR1) and mouse (mdr1, mdr3) P-glycoproteins can be distinguished by their respective drug resistance profiles and sensitivity to modulators.

Possible functional differences between P-glycoproteins (P-gps) encoded by the human MDR1 and mouse mdr1 and mdr3 genes with respect to drug resistance profiles and sensitivity to known modulators have been investigated. For this, the three genes were introduced and overexpressed in the same cellular background, that of Chinese hamster LR73 ovary cells, and drug-resistant clones expressing comparable amounts of the corresponding P-gps were selected under the same conditions. Analysis of the specific drug resistance profiles encoded by each P-gp for colchicine, adriamycin, vinblastine, and actinomycin D revealed overlapping but distinct patterns of drug resistance for the three isoforms. While all three P-gps conferred levels of resistance to vinblastine that did not vary by more than 2.5-fold, each isoform could be clearly distinguished by its capacity to confer resistance to colchicine and actinomycin D. Likewise, the study of structurally related and unrelated P-gp modulators indicated strong isoform-specific differences in the capacity of individual modulators to abrogate vinblastine resistance in the corresponding mdr transfectants. The study of several disubstituted piperazine analogs indicated that minor chemical modifications of the linker region of this modulator had strong effects on the sensitivity profile of each isoform to the modulator. Together, these results indicate that the three P-gp isoforms analyzed have specific and distinguishable functional characteristics with respect to interactions with drugs and modulators. These findings also suggest that P-gp positive murine transplantable tumors should be used with caution in the design and in vivo testing of novel P-gp modulators to be used to reverse multidrug resistance to tumor cells expressing human MDR1.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Assembly of fibronectin molecules with mutations or deletions of the carboxyl-terminal type I modules.

Fibronectin is a large modular protein that is assembled into fibrils in a stepwise process that involves the binding of soluble fibronectin to the cell surface and formation of fibronectin multimers that are stabilized by disulfides. Fibronectin contains two types of disulfide-containing repeat modules, types I and II. The type I modules form units that mediate binding to assembly sites (I-1 through I-5), mediate binding to gelatin (I-6 through I-9 plus the type II modules), or have no known function other than fibrin binding (I-10 through I-12). All type I modules contain four cysteines that are disulfide-linked in a 1-3, 2-4 arrangement, except for I-12 that contains six cysteines disulfide-bonded in an unknown arrangement. I-12 contains the consensus sequence Cys-Xaa-Yaa-Cys found in a number of proteins involved in disulfide exchange reactions [Holmgren, A. (1985) Annu. Rev. Biochem. 54, 237; Boniface, J. J., & Reichert, L. E., Jr. (1990) Science 247, 61]. We explored the role of I-12 and adjacent type I modules of fibronectin in matrix assembly. We generated mutant fibronectins in which the second and sixth or fifth and sixth cysteine residues in I-12 were changed to serines (CS mutants) or that contained deletions of the 12th (delta 12) or 10th through 12th (delta 10-12) type I modules. Expression of I-12 as a fusion protein with the gelatin binding part of fibronectin indicated that this module folds independently and that the most likely disulfide pairing is 1-4, 2-6, 3-5.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2000

Role of the beta-strand insert in the central domain of the fibrinogen gamma-module.

The crystal structure of the fibrinogen gamma-module (residues gamma143-411) [Yee, V. C., et al. (1997) Structure 5, 125-138] revealed an unusual feature. Namely, residues gamma381-390 in the functionally important COOH-terminal region form a beta-strand that is inserted into an antiparallel beta-sheet of the central domain (gamma192-286), while the rest (gamma393-411) seems to be flexible. To clarify the structural and functional importance of this beta-strand insert, we analyzed the folding status of the plasmin-derived fibrinogen fragment D(3) and several truncated variants of the gamma-module expressed in Escherichia coli. It was found that D(3), in which most of the COOH-terminal domain of the gamma-module (gamma287-379) is removed proteolytically, retains a gamma374-405 peptide that seems to be associated noncovalently with the bulk of the molecule via its beta-strand insert region. A study of the denaturation-renaturation process of D(3) suggested that without this peptide its truncated gamma-module remains folded but is destabilized. This was confirmed directly with the truncated recombinant variants of the gamma-module, including residues gamma148-392, gamma148-373, and gamma148-286. They all were folded, but those devoid of the beta-strand insert were destabilized. The results indicate that although the beta-strand insert contributes to the stabilization of the gamma-module, it can be removed without destroying the compact structure of the latter. On the basis of this finding and some other observations, we propose a mechanism for the function-related conformational changes in the fibrin(ogen) gamma-modules.

Calorimetry, Differential Scanning↗
PubMed2001

The loading module of rifamycin synthetase is an adenylation-thiolation didomain with substrate tolerance for substituted benzoates.

The rifamycin synthetase is primed with a 3-amino-5-hydroxybenzoate starter unit by a loading module that contains domains homologous to the adenylation and thiolation domains of nonribosomal peptide synthetases. Adenylation and thiolation activities of the loading module were reconstituted in vitro and shown to be independent of coenzyme A, countering literature proposals that the loading module is a coenzyme A ligase. Kinetic parameters for covalent arylation of the loading module were measured directly for the unnatural substrates benzoate and 3-hydroxybenzoate. This analysis was extended through competition experiments to determine the relative rates of incorporation of a series of substituted benzoates. Our results show that the loading module can accept a variety of substituted benzoates, although it exhibits a preference for the 3-, 5-, and 3,5-disubstituted benzoates that most closely resemble its biological substrate. The considerable substrate tolerance of the loading module of rifamycin synthetase suggests that the module has potential as a tool for generating substituted derivatives of natural products.

Actinomycetales↗
PubMed2002

Timing of epimerization and condensation reactions in nonribosomal peptide assembly lines: kinetic analysis of phenylalanine activating elongation modules of tyrocidine synthetase B.

The cyclic decapeptide antibiotic tyrocidine has D-Phe residues at positions 1 and 4, produced during peptide chain growth from L-Phe residues by 50 kDa epimerase (E) domains embedded, respectively, in the initiation module (TycA) and the TycB3 module of the three-subunit (TycABC), 10-module nonribosomal peptide synthetase. While the initiation module clearly epimerizes the aminoacyl thioester Phe1-S-TycA intermediate, the timing of epimerization versus peptide bond condensation at internal E domains has been less well characterized in nonribosomal peptide synthetases. In this study, we use rapid quench techniques to evaluate a three-domain (ATE) and a four-domain version (CATE) of the TycB3 module and a six-domain fragment (ATCATE) of the TycB2(-3) bimodule to measure the ability of the E domain in the TycB3 module to epimerize the aminoacyl thioester Phe-S-TycB3 and the dipeptidyl-S-enzyme (L-Phe-L-Phe-S-TycB3 if L-Phe-D-Phe-S-TycB3). The chiralities of the Phe-S-enzyme and Phe-Phe-S-enzyme species over time were determined by hydrolysis and chiral TLC separations, allowing for the clear conclusion that epimerization in the internal TycB3 module occurs preferentially on the peptidyl-S-enzyme rather than the aminoacyl-S-enzyme, by a factor of about 3000/1. In turn, this imposes constraints on the chiral selectivity of the condensation (C) domains immediately upstream and downstream of E domains. The stereoselectivity of the upstream C domain was shown to be L-selective at both donor and acceptor sites ((L)C(L)) by site-directed mutagenesis studies of an E domain active site residue and using the small-molecule surrogate D-Phe-Pro-L-Phe-N-acetylcysteamine thioester (D-Phe-Pro-L-Phe-SNAC) and D-Phe-Pro-D-Phe-SNAC as donor probes.

Base Sequence↗
PubMed2002

Binding of human complement C8 to C9: role of the N-terminal modules in the C8 alpha subunit.

Human C8 is one of five components of the membrane attack complex of complement (MAC). It is composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. The C8alpha and C8beta subunits contain a pair of N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)] and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. The middle segment of each protein is referred to as the membrane attack complex/perforin domain (MACPF). During MAC formation, C8alpha mediates binding and self-polymerization of C9 to form a pore-like structure on the membrane of target cells. In this study, the portion of C8alpha involved in binding C9 was identified using recombinant C8alpha constructs in which the N- and/or C-terminal modules were either exchanged with those from C8beta or deleted. Those constructs containing the C8alpha N-terminal TSP1 or LDLRA module together with the C8alpha MACPF domain retained the ability to bind C9 and express C8 hemolytic activity. By contrast, those containing the C8alpha MACPF domain alone or the C8alpha MACPF domain and C8alpha C-terminal modules lost this ability. These results indicate that both N-terminal modules in C8alpha have a role in forming the principal binding site for C9 and that binding may be dependent on a cooperative interaction between these modules and the C8alpha MACPF domain.

Animals↗
PubMed2005

Folding and binding integrity of variants of a prototype ligand-binding module from the LDL receptor possessing multiple alanine substitutions.

The LA repeats that comprise the ligand-binding domain of the LDL receptor are among the most common autonomously structured extracellular modules found in the nonredundant protein sequence database. Here, we investigate the information content of the amino acid sequence of a typical LA module by constructing sequences with alanine residues at nonconserved positions in the module. Starting with the sequence of the fifth ligand-binding repeat of the LDL receptor (LA5), we created generic LA modules with alanine substitutions of nonconserved residues in only the N-terminal lobe, only the C-terminal lobe, and throughout both lobes of the module. LA variants with alanine residues at as many as 18 of 37 positions fold to a preferred disulfide isomer in the presence of calcium. Indeed, the six cysteines, the C-terminal calcium coordinating residues, two hydrophobic residues involved in packing, two glycines, and five other residues that form side chain-intramodule hydrogen bonds are alone sufficient to specify the fold of an LA module when alanine residues are present at all other positions. The LA variants with multiple alanines in either the N- or C-terminal lobe were then exploited to identify residues of LA5 that contribute to the binding of apoE-containing ligands in LDL receptor-derived "minireceptors", implicating nonconserved residues of the N-terminal lobe of LA5 in recognition of apoE-DMPC. Our library of LA modules with multiple alanine substitutions should be generally useful for probing the roles of nonconserved side chains in ligand recognition by proteins of the LDL receptor family.

Alanine↗
PubMed2004

Alpha-glucan recognition by a new family of carbohydrate-binding modules found primarily in bacterial pathogens.

TmPul13, a family 13 glycoside hydrolase from Thermotoga maritima, is a four-module protein having pullulanase activity; the three N-terminal modules are of unknown function while the large C-terminal module is likely the catalytic module. Dissection of the functions of the three unknown modules revealed that the 100 amino acid module at the extreme N-terminus of TmPul13 comprises a new family of carbohydrate-binding modules (CBM) that a bioinformatic analysis shows are most frequently found in pullulanase-like sequences from bacterial pathogens. Detailed binding studies of this isolated CBM, here called TmCBM41, reveals a preference for alpha-(1,4)-linked glucans, but occasional alpha-(1,6)-linked glucose residues, such as those found in pullulan, are tolerated. UV difference, isothermal titration calorimetry, and analytical ultracentrifugation binding studies suggest that maltooligosaccharides longer than four glucose residues are able to bind two TmCBM41 molecules per oligosaccharide when sugar concentrations are below the CBM concentration. This is explained in terms of an equilibrium expression involving the formation of both a 1 to 1 sugar to CBM complex and a 1 to 2 sugar to CBM complex (i.e., a CBM dimer ligated by an oligosaccharide). The presence of an alpha-(1-6) linkage in the oligosaccharide appears to prevent this phenomenon.

Amino Acid Motifs↗
PubMed2006

Modulator-induced interference in functional cross talk between the substrate and the ATP sites of human P-glycoprotein.

The human P-glycoprotein (Pgp, ABCB1) is an ATP-dependent efflux pump for structurally unrelated hydrophobic compounds, conferring simultaneous resistance to and restricting bioavailability of several anticancer and antimicrobial agents. Drug transport by Pgp requires a coordinated communication between its substrate binding/translocating pathway (substrate site) and the nucleotide binding domains (NBDs or ATP sites). In this study, we demonstrate that certain thioxanthene-based Pgp modulators, such as cis-(Z)-flupentixol and its closely related analogues, effectively disrupt molecular cross talk between the substrate, and the ATP, sites without affecting the basic functional aspects of the two domains, such as substrate recognition, binding, and hydrolysis of ATP and dissociation of ADP following ATP hydrolysis. The allosteric modulator cis-(Z)-flupentixol has no effect on [alpha-(32)P]-8-azido-ATP binding to Pgp under nonhydrolytic conditions or on the K(m) for ATP during ATP hydrolysis. Both hydrolysis of ATP and vanadate-induced [alpha-(32)P]-8-azido-ADP trapping (following [alpha-(32)P]-8-azido-ATP breakdown) by Pgp are stimulated by the modulator. However, the ability of Pgp substrates (such as prazosin) to stimulate ATP hydrolysis and facilitate vanadate-induced trapping of [alpha-(32)P]-8-azido-ADP is substantially affected in the presence of cis-(Z)-flupentixol. Substrate recognition by Pgp as determined by [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) binding both in the presence and in the absence of ATP is facilitated by the modulator, whereas substrate dissociation in response to vanadate trapping is considerably affected in its presence. In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Finally, cis-(Z)-flupentixol has no effect on dissociation of [alpha-(32)P]-8-azido-ADP (or ADP) from vanadate-trapped Pgp, which is essential for subsequent rounds of ATP hydrolysis. Taken together, our results demonstrate a distinct mechanism of Pgp modulation that involves allosteric disruption of molecular cross talk between the substrate, and the ATP, sites without any direct interference with their individual functions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Folding, calcium binding, and structural characterization of a concatemer of the first and second ligand-binding modules of the low-density lipoprotein receptor.

The ligand-binding domain of the low-density lipoprotein (LDL) receptor is comprised of seven tandemly repeated ligand-binding modules, each being approximately 40 amino acids long and containing six conserved cysteine residues. We have expressed and characterized a concatemer of the first two modules (LB1 and LB2) of the human LDL receptor. Oxidative folding of the recombinant concatemer (rLB1-2), in the presence of calcium ions, gave a single dominant isomer with six disulfide bonds. Peptic cleavage of the short linker region that connects the last cysteine residue of LB1 and the first cysteine residue of LB2 yielded two discrete fragments, thus excluding the presence of intermodule disulfide bonds. The N-terminal module, LB1, reacted with a conformation-specific monoclonal antibody (IgG-C7) made to LB1 in the native LDL receptor. From this, we concluded that the first module was correctly folded, with the same set of disulfide bonds as LB1 of the LDL receptor. The disulfide bond connections of LB2 were identified from mass spectral analysis of fragments formed by digestion of the C-terminal peptic fragment with elastase. These data showed that the disulfide bonds of LB2 connected Cys(I) and Cys(III), Cys(II) and Cys(V), and Cys(IV) and Cys(VI). This pattern is identical to that found for recombinant LB1 and LB2. The concatemer has two high-affinity calcium-binding sites, one per module. An analysis of the secondary chemical shifts of Calpha protons shows that the conformations of LB1 and LB2 in the concatemer are very similar to those of the individual modules, with no evidence for strong interactions between the two modules.

Amino Acid Sequence↗
PubMed2000

Solution structure of the sixth LDL-A module of the LDL receptor.

The low-density lipoprotein receptor (LDLR) is the primary mechanism for uptake of plasma cholesterol into cells and serves as a prototype for an entire class of cell surface receptors. The amino-terminal domain of the receptor consists of seven LDL-A modules; the third through the seventh modules all contribute to the binding of low-density lipoproteins (LDLs). Here, we present the NMR solution structure of the sixth LDL-A module (LR6) from the ligand binding domain of the LDLR. This module, which has little recognizable secondary structure, retains the essential structural features observed in the crystal structure of LDL-A module five (LR5) of the LDLR. Three disulfide bonds, a pair of buried residues forming a hydrophobic "mini-core", and a calcium-binding site that serves to organize the C-terminal lobe of the module all occupy positions in LR6 similar to those observed in LR5. The striking presence of a conserved patch of negative surface electrostatic potential among LDL-A modules of known structure suggests that ligand recognition by these repeats is likely to be mediated in part by electrostatic complementarity of receptor and ligand. Two variants of LR6, identified originally as familial hypercholesterolemia (FH) mutations, have been investigated for their ability to form native disulfide bonds under conditions that permit disulfide exchange. The first, E219K, lies near the amino-terminal end of LR6, whereas the second, D245E, alters one of the aspartate side chains that directly coordinate the bound calcium ion. After equilibration at physiologic calcium concentrations, neither E219K nor D245E folds to a unique disulfide isomer, indicating that FH mutations both within and distant from the calcium-binding site give rise to protein-folding defects.

Amino Acid Sequence↗
PubMed2002

Determination of calpha chemical shift tensor orientation in peptides by dipolar-modulated chemical shift recoupling NMR spectroscopy.

We present a new method for determining the orientation of chemical shift tensors in polycrystalline solids with site resolution and demonstrate its application to the determination of the Calpha chemical shift tensor orientation in a model peptide with beta-sheet torsion angles. The tensor orientation is obtained under magic angle spinning by modulating a recoupled chemical shift anisotropy (CSA) pattern with various dipolar couplings. These dipolar-modulated chemical shift patterns constitute the indirect dimension of a 2D spectrum and are resolved according to the isotropic chemical shifts of different sites in the direct dimension. These dipolar-modulated CSA spectra are equivalent to the projection of a 2D static separated-local-field spectrum onto its chemical shift dimension, except that its dipolar dimension is multiplied with a modulation function. Both (13)C-(1)H and (13)C-(15)N dipolar couplings can modulate the CSA spectra of the Calpha site in an amino acid and yield the relative orientations of the chemical shift principal axes to the C-H and C-N bonds. We demonstrate the C-H and C-N modulated CSA experiments on methylmalonic acid and N-tBoc-glycine, respectively. The MAS results agree well with the results of the 2D separated-local-field spectra, thus confirming the validity of this MAS dipolar-modulation approach. Using this technique, we measured the Val Calpha tensor orientation in N-acetylvaline, which has beta-sheet torsion angles. The sigma(11) axis is oriented at 158 degrees (or 22 degrees) from the C-H bond, while the sigma(22) axis is tilted by 144 degrees (or 36 degrees) from the C-N bond. Both the orientations and the magnitude of this chemical shift tensor are in excellent agreement with quantum chemical calculations.

Anisotropy↗
PubMed2006

Quadruply hydrogen bonded cytosine modules for supramolecular applications.

In view of the range of properties required from supramolecular materials, there is clearly a need for new strong quadruple hydrogen bonded modules, which can be used in polymer or copolymer synthesis via the self- or hetero-association of complimentary units. A cytosine-based module has been prepared for supramolecular applications using a straightforward synthetic approach. The cytosine module was designed such that it does not undergo tautomeric changes observed with ureidopyrimidinones. The cytosine module was capable of forming quadruple hydrogen bonded assemblies both in solution and in the solid state, and the structure of the dimeric self-assembled unit was confirmed by single-crystal X-ray and solution NMR techniques. The dimerization constant was estimated to be greater than 9 x 106 M-1 in deuterated benzene. The capacity of the cytosine-based module to strongly hetero-associate with the ureidopyrimidinone module was demonstrated, and a supramolecular polymer of a bifunctional unit incorporating the cytosine module and PEG-based linker was described.

Cytosine↗
PubMed2006

Structural features of the modulated BiCu2(P(1-x)V(x))O6 solid solution; 4-D treatment of x = 0.87 compound and magnetic spin-gap to gapless transition in new Cu2+ two-leg ladder systems.

The BiCu(2)(P(1-x)V(x))O(6) system shows the appearance of various phenomena that progressively change as a function of the average (P/V)O(4) groups size. Then, from x = 0 to x approximately 0.7, a solid solution exists with respect to the basic orthorhombic unit cell of BiCu(2)PO(6). For greater x values (0.7 < x <0.96), structural modulations with incommensurate q vector that slightly change versus x appear. The 4-D treatment of single-crystal XRD data of the modulated phase corresponding to x = 0.87 at 100 K (orthorhombic, a = 12.379(3)Angstrom, b = 5.2344(9) Angstrom, c = 7.8270(14) Angstrom, q = 0.268(3) b*, super space group: Xbmm(0gamma0) s00, X stands for the nonprimitive centering vector (1/2,0,1/2,1/2), R(obs)(overall) = 5.27%, R(obs)(fundamental) = 4.48%, R(obs)(satellite) = 6.58%) has evidenced strong positional modulated effects within the [BiCu(2)O(2)](3+) ribbons while three XO(4) configurations compete along the x(4) fourth dimension. There is no P/V segregation along x(4) in good agreement with steric-only origins of the modulation. Finally for 0.96 < x <1, two phases coexist, i.e., BiCu(2)VO(6) (X = 1) and a modulated phase of the previous domain.The BiCu(2)VO(6) crystal structure shows a unit cell tripling associated with monoclinic symmetry lowering. The VO(4) orientations between two ribbons proceed with respect to the interribbon distance. Then the full system shows flexible interactions between modulated Bi/M/O-based ribbons and surrounding tetrahedral groups, depending on the average XO(4) size. Furthermore, between two ribbons the Cu(2+) arrangement forms magnetically isolated zigzag copper two-leg ladders. Our preliminary results show a spin-gap behavior likely due to the existence of true S = (1)/(2) Heisenberg two-leg ladders. Modulated compositions are gapless, in good agreement with band-broadening toward a continuum in the magnetic excitation spectrum. The continuous distribution of Cu-Cu distances along the rungs and legs of the ladders should be mainly responsible for this magnetic change.

Journal Article↗
PubMed1998

Third calcium-modulated rod outer segment membrane guanylate cyclase transduction mechanism.

Ca2+-modulated rod outer segment membrane guanylate cyclase (ROS-GC1) has been cloned and reconstituted to show that it is regulated by two processes: one inhibitory, the other stimulatory. The inhibitory process is consistent with its linkage to phototransduction; the physiology of the stimulatory process is probably linked to neuronal transmission. In both regulatory processes, calcium modulation of the cyclase takes place through the calcium binding proteins; guanylate cyclase activating proteins (GCAP1 and GCAP2) in the case of the phototransduction process and calcium-dependent GCAP (CD-GCAP) in the case of the stimulatory process. The cyclase domains involved in the two processes are located at two different sites on the ROS-GC1 intracellular region. The GCAP1-modulated domain resides within the aa 447-730 segment of ROS-GC1 and the CD-GCAP-modulated domain resides within the aa 731-1054 segment. In the present study the GCAP2-dependent Ca2+ modulation of the cyclase activity has been reconstituted using recombinant forms of GCAP2 and ROS-GC1, and its mutants. The results indicate that consistent to phototransduction, GCAP2 at low Ca2+ concentration (10 nM) maximally stimulates the cyclase activity of the wild-type and its mutants: ext (deleted aa 8-408), kin (deleted aa 447-730) and hybrid consisting of the ext, transmembrane and kin domains of ANF-RGC and the C-terminal domain, aa 731-1054, of ROS-GC1. In all cases, it inhibits the cyclase activity with an IC50 of about 140 nM. A previous study has shown that under identical conditions the kin and the hybrid mutant are at best only minimally stimulated. Thus, the GCAP1 and GCAP2 signal transduction mechanisms are different, occurring through different modules of ROS-GC1. These findings also demonstrate that the intracellular region of ROS-GC1 is composed of multiple modules, each designed to mediate a particular calcium-specific signalling pathway.

Animals↗
PubMed2001

Toward a characterization of the connecting module of complex I.

Complex I [NADH-ubiquinone oxidoreductase (complex I, EC 1.6.5.3)] couples electron transfer between NADH and ubiquinone to proton transport across the bacterial cytoplasmic membrane and the mitochondrial inner membrane. This sophisticated enzyme consists of three specialized modules: (1) a hydrophilic NADH-oxidizing module that constitutes the input machinery of the enzyme; (2) a hydrophobic module that anchors the enzyme in the membrane and must take part in proton transport; and (3) a connecting domain that links the two previous modules. Using the complex I of Rhodobacter capsulatus, we developed a genetic study of the structure and function of the connecting module. In the present review, we put together the salient results of these studies, with recent reports of the literature, to try and elucidate the structure of the connecting module and its potential role in the coupling process between electron and proton flux within complex I. From this overview, we conclude that the NUOB-NUOD dimer of the connecting module and a hydrophobic subunit such as NUOH must share a quinone-reduction site. The function of this site in the mechanism of complex I is discussed.

Amino Acid Sequence↗
PubMed1994

Cloned Ca(2+)-dependent K+ channel modulated by a functionally associated protein kinase.

Calcium-dependent potassium (KCa) channels carry ionic currents that regulate important cellular functions. Like some other ion channels, KCa channels are modulated by protein phosphorylation. The recent cloning of complementary DNAs encoding Slo KCa channels has enabled KCa channel modulation to be investigated. We report here that protein phosphorylation modulates the activity of Drosophila Slo KCa channels expressed in Xenopus oocytes. Application of ATP-gamma S to detached membrane patches increases Slo channel activity by shifting channel voltage sensitivity. This modulation is blocked by a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Mutation of a single serine residue in the channel protein also blocks modulation by ATP-gamma S, demonstrating that phosphorylation of the Slo channel protein itself modulates channel activity. The results also indicate that KCa channels in oocyte membrane patches can be modulated by an endogenous PKA-like protein kinase which remains functionally associated with the channels in the detached patch.

Adenosine Triphosphate↗
PubMed1992

Modulation of cis-diamminedichloroplatinum(II) resistance: a review.

In this review an inventory is made of agents used to circumvent cis-diamminedichloroplatinum(II) (CDDP) resistance in vitro and in vivo. Agents that affect CDDP accumulation and membrane related systems, cytoplasmic defense mechanisms, as well as DNA accessibility and repair are reviewed. In resistant cell lines that have decreased accumulation, this can be restored by hyperthermic treatment. With or without effects on accumulation compounds that affect cell signal transduction often increase CDDP cytotoxicity. Calcium channel blockers and calmodulin inhibitors do not seem to be uniformly good modulators of CDDP resistance. For transduction modulators as well as cellular calcium affecting agents mechanisms are mainly unclear or controversial. Glutathione appears, with the now available agents, to be the most promising target for modulation of cytoplasmic defense mechanisms. At the nuclear level the inhibition of DNA repair related enzymes as well as the use of modified nucleosides to interfere with repair is studied in various cell lines. Results with these agents suggest opportunities for clinically feasible cytotoxicity modulation. DNA accessibility could in vitro be affected, but seems to be an unreliable target for modulation. Whenever possible the resistance mechanism affected and the mode of action of the modulator are discussed. As an alternative for modulation another method of overcoming CDDP resistance namely the application of CDDP analogues is considered.

Animals↗