Biomedical subjects

M Wessling-Resnick

Publications and source records attributed to M Wessling-Resnick.

45 records · Page 3Linked to original sources

PubMed1993

Characterization of transferrin-independent iron transport in K562 cells. Unique properties provide evidence for multiple pathways of iron uptake.

The present study characterizes the transport of nontransferrin (non-Tf) iron by K562 cells. Accumulation of radiolabel by cells incubated with 55Fe-nitrilotriacetate (NTA) is a saturable process that is time and temperature dependent (Ea approximately 20 kcal/mol). Initial rate analysis of iron influx yields values of Vmax = 855 fmol/min/10(6) cells and apparent Km = 0.54 microM. NHCL4 and chloroquine, agents that block cellular acquisition of iron from Tf, do not interfere with assimilation from FeNTA, demonstrating that uptake is truly independent of the Tf-mediated pathway. Furthermore, the inactivation of this transport mechanism by limited proteolytic digestion on ice indicates that specific cell surface proteins are involved. The extent of radiolabel incorporation into heme and ferritin is the same regardless of whether K562 cells acquire iron from 55FeNTA via the cell surface mechanism or from 55Fe-Tf via receptor-mediated endocytosis. Unlike other Tf-independent iron transport pathways that have been described, the K562 cell transport mechanism is not inhibited by divalent cations such as Ni2+, Co2+, or Mn2+. Uptake from 55FeNTA can be blocked by Cu2+ but at concentrations > 1500-fold molar excess. However, Cd2+ is a fairly specific inhibitor of 55Fe uptake by K562 cells (IC50 approximately 50 microM). Additionally, the K562 cell transport mechanism is not Ca2+ dependent and does not appear to be regulated by extracellular iron salts, in contrast to features noted for non-Tf iron uptake by fibroblasts (Sturrock, A., Alexander, J., Lamb, J., Craven, C. M., and Kaplan, J. (1991) J. Biol. Chem. 265, 3139-3145; Kaplan, J., Jordan, I., and Sturrock, A. (1991) J. Biol. Chem. 266, 2997-3004). These unique characteristics of the K562 cell uptake mechanism suggest that multiple transport systems function in Tf-independent iron assimilation.

Ascorbic Acid↗

PubMed1990

Characterization of the mechanism of endocytic vesicle fusion in vitro.

A cell-free assay to monitor receptor-mediated endocytic processes has been developed that uses biotinylated transferrin and avidin-linked beta-galactosidase as receptor-associated and fluid-phase probes, respectively (Wessling-Resnick, M., and Braell, W. A. (1990) J. Biol. Chem. 265, 690-699). The fusion of vesicles from heterologous sources can be detected in this assay: endocytic vesicles from K562 cells (a human cell line) will fuse with vesicles from Chinese hamster ovary cells. Fusion between endocytic vesicles is inhibited upon treatment with N-ethylmaleimide but can be restored by the addition of untreated cytosol from either cell type. The in vitro fusion reaction is also inhibited by the nonhydrolyzable nucleotide analogs guanosine 5'-(3-thiotriphosphate) (GTP gamma S) and adenosine 5'-(3-thiotriphosphate) (ATP gamma S). Other nonhydrolyzable guanine nucleotides are found to inhibit the in vitro reaction in the following order of potency: GTP gamma S greater than 5'-guanylyl imidodiphosphate (GTP-PNP) greater than alpha,beta-methylene GTP (GTP-PCP). The inhibitory effects of the nonhydrolyzable analogs of GTP and ATP are not additive. Moreover, excess GTP relieves the inhibition by GTP gamma S more than it relieves the inhibition by ATP gamma S, while excess ATP preferentially alleviates ATP gamma S (not GTP gamma S) inhibition. These properties suggest that the two nucleotides exert their effects at distinct points in the fusion process. Although micromolar levels of excess Ca2+ also inhibit vesicle fusion, the inhibition exerted by GTP gamma S appears to proceed via a pathway independent of the divalent cation. The GTP gamma S-sensitive step in endocytic vesicle fusion is found to occur at a mechanistic stage prior to and distinct from the N-ethylmaleimide-sensitive step of the reaction. This situation permits the accumulation of a membrane vesicle intermediate in the presence of GTP gamma S; subsequent incubation of these vesicles with cytosol and GTP restores their fusion competence. Characteristics of in vitro endocytic vesicle fusion suggest that similarities exist with steps of the fusion mechanism involved with membrane traffic events of the secretory pathway.

Animals↗

PubMed1990

The sorting and segregation mechanism of the endocytic pathway is functional in a cell-free system.

A cell-free system which reconstitutes early stages of receptor-mediated endocytosis has been developed, based on detection of the association between avidin-beta-galactosidase (Av beta Gal) and biotin-transferrin (B-Tf). Initially, Av beta Gal (a fluid-phase marker) and B-Tf (receptor-bound) are internalized and delivered to a common endosomal compartment in vivo and in vitro. Subsequently, these two probes enter divergent intracellular pathways: Av beta Gal is sorted from the endosome and directed for delivery to lysosomes, whereas B-Tf is segregated away from the fluid-phase marker, remaining bound to the transferrin receptor for return to the cell surface. Using the avidin-biotin association reaction to monitor the co-localization of these two probes, we have been able to reconstruct this sorting and segregation process in a cell-free system. The in vitro reaction is time-, temperature-, and ATP-dependent, and is not affected by NH4Cl; cell-free segregation of the two probes is also sensitive to N-ethylmaleimide. As these characteristics are also properties of in vitro endocytic vesicle fusion, it is likely that the latter event is a prerequisite for the sorting and segregation process. Both the in vivo and in vitro sorting of Av beta Gal and B-Tf to their respective and distinct destinations can be followed by subcellular fractionation on Percoll gradients. Our observations provide the first evidence that the cellular mechanism to identify, sort, and sequester endocytosed material can be reconstituted in a cell-free system.

Avidin↗

PubMed1989

Evidence for oligomeric forms of transducins alpha subunit: formation of intermolecular alpha-alpha disulfide linkages.

Transducin, the retinal G-protein, is a heterotrimeric protein composed of alpha, beta and gamma subunits. Intermolecular disulfide linkages between the alpha-subunits of transducin molecules are spontaneously formed when the purified G-protein is placed in a non-reducing buffer system. The beta and gamma subunits do not participate in the intermolecular disulfide bridge formation. The alpha-alpha subunit disulfide bonds result in the inhibition of transducin activation by bleached rhodopsin which is restored by reducing the disulfides with dithiothreitol. The trapping of oligomers by disulfide bond formation provides physical evidence for specific intermolecular interactions between alpha-subunits of transducin.

Animals↗

PubMed1988

Mapping of the carboxyl terminus within the tertiary structure of transducin's alpha subunit using the heterobifunctional cross-linking reagent, 125I-N-(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) cysteine.

A heterobifunctional cross-linking reagent, 125I-N-(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) cysteine (125-ACTP), has been synthesized. 125I-ACTP has been used to derivative reduced sulfhydryls of the retinal G protein, transducin (Gt), to form a mixed disulfide bond under mild, nondenaturing conditions (pH 7.4, 4 degrees C). The resulting disulfide was easily cleaved using reducing reagents. A 200-fold molar excess of 125I-ACTP relative to Gt resulted in the incorporation of 1-1.3 mol of the 125I-N-(3-iodo-4-azidophenylpropionamido)cysteine moiety of ACTP into Gt alpha. In contrast to 125I-ACTP, dithionitrobenzoate and dithiopyridone derivatized six sulfhydryls in native Gt. Incubation of a 10-fold molar excess of 125I-ACTP relative to Gt resulted in the derivatization of 0.75-0.9 and 0.1 mol of reduced sulfhydryls/mol Gt alpha and beta, respectively. Gt gamma was not derivatized by 125I-ACTP. Thus, Gt alpha was preferentially derivatized by 125I-ACTP. Tryptic digestion and amino acid sequencing of Gt alpha indicated that both Cys-347 near the carboxyl terminus and Cys-210 between the second and third consensus sequences forming the GTP-binding site were derivatized by 125I-ACTP in a ratio of approximately 70 and 30%, respectively. Thus, both Cys-210 and Cys-347 are labeled, even though derivatization by 125I-ACTP does not exceed 1 mol of SH/mol Gt alpha. It appears that derivatization of one sulfhydryl, either Cys-210 or Cys-347, excludes labeling of the second cysteine either by steric hindrance or induced conformational change making the second cysteine inaccessible to 125I-ACTP. Consistent with this finding was the observation that pertussis toxin-catalyzed ADP-ribosylation of Cys-347 inhibited 125I-ACTP derivatization of Cys-210. Derivatization of Gt alpha at either Cys-210 or Cys-347 by 125I-ACTP inhibited rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate binding to Gt, mimicking the effect of ADP-ribosylation of Cys-347 by pertussis toxin. ACTP contains a radioiodinated phenylazide moiety which, upon activation, can cross-link the derivatized cysteine to an adjacent polypeptide domain. Following reduction of the disulfide, the [125I] iodophenyl moiety will be transferred to the azide-inserted polypeptide. When photoactivation of the phenylazide moiety of 125I-ACTP after sulfhydryl derivatization was performed, insertion of the Cys-347 which contains Cys-210, was found.(ABSTRACT TRUNCATED AT 400 WORDS)

Azides↗

PubMed1987

Transducin interactions with rhodopsin. Evidence for positive cooperative behavior.

Transducin and rhodopsin belong to a family of guanine nucleotide-binding (G) protein-coupled receptor systems that provide signal transduction mechanisms resulting in numerous metabolic responses in a variety of cell types. A simple, direct binding assay has been developed to investigate the molecular interactions between transducin and rhodopsin. The binding curves generated by these studies are sigmoidal, indicating an allosteric response. The Scatchard plots of this data display an asymptotic, bell-shaped character representative of the positive cooperative behavior. A Hill coefficient, nH = 1.92, was determined and found to be in close agreement with previous kinetic studies of allosterism described for rhodopsin's catalytic mechanism (Wessling-Resnick, M., and Johnson G.L. (1987) J. Biol. Chem. 262, 3697-3705). The value for Kd app was determined to be 0.05 microM. Bmax values obtained from the binding studies suggest that oligomeric complexes of rhodopsin may be involved in interactions with transducin to form multiple high affinity binding sites for the G protein. The positive cooperative behavior demonstrated in this investigation can provide insight into the molecular basis for regulation of other G protein-coupled receptor systems.

Allosteric Regulation↗

PubMed1987

Kinetic and hydrodynamic properties of transducin: comparison of physical and structural parameters for GTP-binding regulatory proteins.

Transducin is a member of the family of GTP-binding regulatory proteins that interact with cell surface receptors and that include Gs, Gi, and Go. Kinetic and physical properties of purified bovine transducin were characterized by the following results: (1) Initial rate analysis demonstrates a dissociative-type mechanism for the guanine nucleotide exchange process of transducin in the absence of rhodopsin. A second-order rate constant of kf = (1.7-2.7) X 10(-7) M-1 s-1 was determined for this reaction. (2) Equilibrium binding measurements indicated a Kd of 0.05-0.10 microM for guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to transducin. (3) Neither the rate nor the extent of GTP gamma S binding was affected in the presence of up to 50 mM Mg2+, as compared to values obtained in the presence of excess ethylenediaminetetraacetic acid. (4) Sucrose density gradient ultracentrifugation gave S20,w values for transducin, its alpha subunit, and its beta gamma subunit complex of 4.23 +/- 0.25, 3.42 +/- 0.37, and 4.04 +/- 0.2, respectively. (5) Incubation of transducin in the presence of up to 20 mM Mg2+ did not alter its sedimentation behavior; however, the presence of guanine nucleotides did produce a shift in transducin's migration in the sucrose gradient. (6) Gel filtration over Sephacryl S-300 indicated that transducin elutes at a Stokes radius of 37.5 A and that transducin's alpha subunit displays a Stokes radius of 24 A. (7) A molecular mass of 68 kDa for transducin is derived from the determined hydrodynamic parameters. These results are compared with properties known for other G proteins, and functional differences between transducin and Gs, Gi, and Go are proposed in relation to the proteins' primary sequences.

Animals↗

PubMed1987

Allosteric behavior in transducin activation mediated by rhodopsin. Initial rate analysis of guanine nucleotide exchange.

Photolyzed rhodopsin acts in a catalytic manner to mediate the exchange of GTP for GDP bound to transducin. We have analyzed the steady-state kinetics of this activation process in order to determine the molecular mechanism of interactions between rhodopsin, transducin, and guanine nucleotides. Initial velocities (Vo) of the exchange reaction catalyzed by rhodopsin were measured for various transducin concentrations at several fixed levels of the GTP analog, [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S). The initial rate data analysis rigorously demonstrates that rhodopsin mediates the activation of transducin by a double-displacement catalytic mechanism. The Michaelis-Menten curves determined as a function of [transducin] reveal remarkable allosteric behavior; analysis of this data yields a Hill coefficient of 2. Lineweaver-Burk plots of Vo-1 versus [transducin]-1 display curvilinearity indicative of positive cooperativity and a series of parallel lines are generated by plotting Vo-1 as a function of [transducin]-2. The plots of Vo-1 versus [GTP gamma S]-1 show no evidence of allosterism and are a parallel series. Furthermore, the allosteric behavior observed in the activation of transducin is also witnessed in the rhodopsin-catalyzed guanine nucleotide exchange of the G protein's purified alpha subunit in the absence of the beta X gamma subunit complex. The latter observation implies that the molecular basis for allosterism in the activation process resides in the interactions between the photoreceptor and transducin's alpha subunit.

Allosteric Regulation↗

PubMed1986

Dipeptide metalloendoprotease substrates are glucose transport inhibitors and membrane structure perturbants.

Dipeptide substrates for metalloendoproteases have previously been shown to block biological processes requiring membrane fusion. Thus, we employed such compounds as potential inhibitors of the insulin-dependent activation of glucose transport in fat cells. This event is thought to involve vesicle movement from an intracellular site to the cell surface and would therefore require membrane fusion during the activation step. We find that synthetic dipeptides which are metalloendoprotease substrates rapidly and reversibly inhibit insulin-activated glucose oxidation in a dose-dependent manner but exhibit essentially no effect on basal levels. A similar result is obtained when glucose transport is measured directly in intact fat cells, in metabolically poisoned cells, and in isolated membrane vesicles derived from insulin-activated or untreated fat cells. That is, the dipeptide substrates inhibit insulin-activated glucose uptake to a greater extent than basal transport, and they do so even when vesicle translocation and fusion have already taken place as in ATP-depleted cells and isolated vesicles. Onset of transport inhibition after dipeptide addition is rapid, but not instantaneous, with a t 1/2 of 15-30 s. The metalloendoprotease substrates also inhibit glucose uptake and cytochalasin B binding in human erythrocytes but not in human placental microsomes. Finally, light microscopic examination of substrate-treated red cells reveals marked cupping and/or echinolation of the cell membrane. We conclude the following from these observations: Metalloendoprotease substrates are inhibitors of adipocyte glucose transport.(ABSTRACT TRUNCATED AT 250 WORDS)

3-O-Methylglucose↗