In vitro gibberellin a(4) binding to extracts of cucumber hypocotyls

Cucumber hypocotyls were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [(3)H]-gibberellin(4) (GA(4)) to soluble macromolecular components present in the cytosol was demonstrated at 0 C by Sephadex chromatography. Binding assays performed with cytosol that had been preheated or incubated with protease, DNase, RNase, or phospholipase A or C indicated that heat and protease treatments disrupted the binding, which suggests that binding occurred to a protein. Equilibrium dialysis of a protein-enriched fraction prepared by ammonium sulfate precipitation also indicated binding of [(3)H]GA(4) to macromolecular components. [(3)H]GA(4) binding was pH-sensitive, saturable, reversible, and significantly affected by biologically active gibberellins, but not by inactive gibberellins or other plant hormones such as indoleacetic acid, abscisic acid, or kinetin. Thin layer chromatography indicated that [(3)H]GA(4), and not a metabolite, was the species bound. A kinetic analysis indicated that specific binding of [(3)H]GA(4) was due to a single class of binding sites having an estimated K(d) of 10(-7) molar and a concentration of 0.8 x 10(-12) moles gram(-1) fresh weight or 0.4 x 10(-12) moles milligram(-1) soluble protein.     

In Vitro Gibberellin A(1) Binding in Zea mays L

The first and second leaf sheaths of Zea mays L. cv Golden Jubilee were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [³H]gibberellin A₁ (GA₁) to a soluble macromolecular component present in the cytosol was demonstrated at 4°C by Sephadex G-200 chromatography. The binding component was of high molecular weight (HMW) and greater than 500 kilodaltons. The HMW component was shown to be a protein and the ³H-activity bound to this protein was largely [³H]GA₁ and not a metabolite. Binding was pH sensitive but only a small percentage (20%) appeared to be exchangeable on addition of unlabeled GA₁. Both biologically active and inactive GAs and non-GAs were able to inhibit GA₁ binding. [³H]GA₁ binding to an intermediate molecular weight (IMW) fraction (40-100 kilodaltons) was also detected, provided cytosol was first desalted using Sephadex G-200 chromatography. Gel filtration studies suggest that the HMW binding component is an aggregate derived from the IMW fraction. The HMW binding fraction can be separated into two components using anion exchange chromatography.     

Competition for in vitro [h]gibberellin a(4) binding in cucumber by substituted phthalimides: comparison with in vivo gibberellin-like activity

Certain N-substituted phthalimides (NSPs) have gibberellin (GA)-like activity in a number of GA bioassays. The interaction between representative NSPs and a protein fraction from cucumber (Cucumis sativus L.) hypocotyls that has GA-binding characteristics consistent with those expected of GA receptors was studied. Analysis of in vitro equilibrium saturation data indicated the presence of only one class of high affinity [³H]GA₄ binding sites (K_d ~ 30 nanomolar, n = 0.25 picomole per milligram of protein). In the presence of 6 or 60 micromolar 1-[3-chlorophthalimido]-cyclohexanecarboximide (AC-94,377), the K_d for [³H]GA₄ increased, whereas the maximum number of saturable [³H]GA₄ binding sites did not change significantly. The dissociation of [³H]GA₄ from its binding sites was complex and was best described by a bi-exponential equation. AC-94,377 did not affect the rates of [³H]GA₄ dissociation from its binding sites. These results implied that AC-94,377 and [³H]GA₄ compete for binding to the same sites. A correlation was observed between the activity of over 20 NSPs in the cucumber hypocotyl bioassay and their in vitro affinity for the GA binding sites. Our observations lend further support to the notion that certain GA binding proteins in cucumber cytosol are GA receptors and also provide a molecular explanation for the GA-like in vivo activity of some NSPs.     

Vernalization and Gibberellin Physiology of Winter Canola (Endogenous Gibberellin (GA) Content and Metabolism of [3H]GA1 and [3H]GA20

Winter canola (Brassica napus cv Crystal) is an oilseed crop that requires vernalization (chilling treatment) for the induction of stem elongation and flowering. To investigate the role of gibberellins (GAs) in vernalization-induced events, endogenous GA content and the metabolism of [3H]GAs were examined in 10-week vernalized and nonvernalized plants. Shoot tips were harvested 0, 8, and 18 d postvernalization (DPV), and GAs were purified and quantified using 2H2-internal standards and gas chromatography-selected ion monitoring. Concentrations of GA1, GA3, GA8, GA19, and GA20 were 3.1-, 2.3-, 7.8-, 12.0-, and 24.5-fold higher, respectively, in the vernalized plants at the end of the vernalization treatment (0 DPV) relative to the nonvernalized plants. Thermoregulation apparently occurs prior to GA19 biosynthesis, since vernalization elevated the concentration of all of the monitored GAs. [3H]GA20 or [3H]GA1 was applied to the shoot tips of vernalized and nonvernalized plants, and after 24 h, plants were harvested at 6, 12, and 15 DPV. Following high-performance liquid chromatography analyses, vernalized plants showed increased conversion of [3H]GA20 to a [3H]GA1-like metabolite and reduced conversion of [3H]GA1 or [3H]GA20 to polar 3H-metabolites, putative glucosyl conjugates. These results demonstrate that vernalization influences GA content and GA metabolism, with GAs serving as probable regulatory intermediaries between chilling treatment and subsequent stem growth.     

Binding of [3H]oxytocin to cells isolated from the mammary gland of the lactating rat.

More than 90 percent of the cells isolated from the mammary gland of lactating rats with 0.1 percent collagenase were viable by dye exclusion. Myoepithelial cells comprised about one-third of the mammary cells and appeared to be morphologically intact in electron micrographs. [(3)H]Oxytocin-binding activity was localized in an enriched myoepitheial cell fraction obtained by density gradient centrifugation of the isolated cells. The amount of [(3)H] oxytocin bound at 20 degree C and pH 7.6 was proportional to the concentration of oxytocin and the number of cells, reaching a steady state by 40 min. About 0.45 fmol of oxytocin were bound per 10(6) cells. There was a single class of independent binding sites with an apparent K(d), estimated from equilibrium conditions, of 5 nM. This value agrees within experimental error with the value calculated from the ratio of reverse to forward rate constants (5.8 x 10(-4)s(-1) and 2.2 x 10(5) M(-1)s(-1), respectively), consistent with a single-step model for the interaction of oxytocin with binding sites on the cells. Erythrocytes bound only 3.5 percent of the amount of oxytocin bound by an equal number of mammary cells. Oxytocin analogues competed with [(3)H]oxytocin for binding sites in the following order: [deamino]oxytocin > [4-threonine]oxytocin > oxytocin > [O- methyltyrosine]oxytocin > [8-lysine]vasopressin; [lysine]-bradykinin and [4-proline]oxytocin were not inhibitory in the dose ranges tested. These results demonstrate that isolated mammary cells possess oxytocin receptors with properties comparable to those found in broken mammary cell preparations.     

Taxol binds to polymerized tubulin in vitro

Taxol, a natural plant product that enhances the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells, was labeled with tritium by catalytic exchange with (3)H(2)O. The binding of [(3)H]taxol to microtubule protein was studied by a sedimentation assay. Microtubules assembled in the presence of [(3)H]taxol bind drug specifically with an apparent binding constant, K(app), of 8.7 x 19(-7) M and binding saturates with a calculated maximal binding ration, B(max), of 0.6 mol taxol bound/mol tubulin dimer. [(3)H]Taxol also binds and assembles phosphocellulose-purified tubulin, and we suggest that taxol stabilizes interactions between dimers that lead to microtubule polymer formation. With both microtubule protein and phosphocellulose- purified tubulin, binding saturation occurs at approximate stoichiometry with the tubulin dimmer concentration. Under assembly conditions, podophyllotoxin and vinblastine inhibit the binding of [(3)H]taxol to microtubule protein in a complex manner which we believe reflects a competition between these drugs, not for a single binding site, but for different forms (dimer and polymer) of tubulin. Steady-state microtubules assembled with GTP or with 5’-guanylyl-α,β-methylene diphosphonate (GPCPP), a GTP analog reported to inhibit microtubule treadmilling (I.V. Sandoval and K. Weber. 1980. J. Biol. Chem. 255:6966-6974), bind [(3)H]taxol with approximately the same stoichiometry as microtubules assembled in the presence of [(3)H]taxol. Such data indicate that a taxol binding site exists on the intact microtubule. Unlabeled taxol competitively displaces [(3)H]taxol from microtubules, while podophyllotoxin, vinblastine, and CaCl(2) do not. Podophyllotoxin and vinblastine, however, reduce the mass of sedimented taxol-stabilized microtubules, but the specific activity of bound [(3)H]taxol in the pellet remains constant. We conclude that taxol binds specifically and reversibly to a polymerized form of tubulin with a stoichiometry approaching unity.     

Purified NPC1 protein: II. Localization of sterol binding to a 240-amino acid soluble luminal loop

Defects in Niemann-Pick, Type C-1 protein (NPC1) cause cholesterol, sphingolipids, phospholipids, and glycolipids to accumulate in lysosomes of liver, spleen, and brain. In cultured fibroblasts, NPC1 deficiency causes lysosomal retention of lipoprotein-derived cholesterol after uptake by receptor-mediated endocytosis. NPC1 contains 1278 amino acids that form 13 membrane-spanning helices and three large loops that project into the lumen of lysosomes. We showed earlier that NPC1 binds cholesterol and oxysterols. Here we localize the binding site to luminal loop-1, a 240-amino acid domain with 18 cysteines. When produced in cultured cells, luminal loop-1 was secreted as a soluble dimer. This loop bound [(3)H]cholesterol (K(d), 130 nM) and [(3)H]25-hydroxycholesterol (25-HC, K(d), 10 nM) with one sterol binding site per dimer. Binding of both sterols was competed by oxysterols (24-, 25-, and 27-HC). Unlabeled cholesterol competed strongly for binding of [(3)H]cholesterol, but weakly for [(3)H]25-HC binding. Binding of [(3)H]cholesterol but not [(3)H]25-HC was inhibited by detergents. We also studied NPC2, a soluble protein whose deficiency causes a similar disease phenotype. NPC2 bound cholesterol, but not oxysterols. Epicholesterol and cholesteryl sulfate competed for [(3)H]cholesterol binding to NPC2, but not NPC1. Glutamine 79 in luminal loop-1 of NPC-1 is important for sterol binding; a Q79A mutation abolished binding of [(3)H]cholesterol and [(3)H]25-HC to full-length NPC1. Nevertheless, the Q79A mutant restored cholesterol transport to NPC1-deficient Chinese hamster ovary cells. Thus, the sterol binding site on luminal loop-1 is not essential for NPC1 function in fibroblasts, but it may function in other cells where NPC1 deficiency produces more complicated lipid abnormalities.     

Auxin from the developing inflorescence is required for the biosynthesis of active gibberellins in barley stems

Multiple gibberellins (GAs) were quantified in the stems of intact, decapitated, and decapitated auxin-treated barley (Hordeum vulgare) plants. Removal of the developing inflorescence reduced the endogenous levels of indole-3-acetic acid (IAA), GA(1), and GA(3) and increased the level of GA(29) in internodal and nodal tissues below the site of excision. Application of IAA to the excised stump restored GA levels to normal in almost all cases. The conversion of [(14)C]GA(20) to bioactive [(14)C]GA(1) and of [(14)C]GA(5) to bioactive [(14)C]GA(3) was reduced by decapitation, and IAA application was able to restore conversion rates back to the levels found in intact plants. The amount of mRNA for the principal vegetative 3-oxidase (converting GA(20) to GA(1), and GA(5) to GA(3)) was decreased in decapitated plants and restored by IAA application. The results indicate that the inflorescence of barley is a source of IAA that is transported basipetally into the internodes and nodes where bioactive GA(1) and GA(3) are biosynthesized. Thus, IAA is required for normal GA biosynthesis in stems, acting at multiple steps in the latter part of the pathway.     

Preparation and characterization of adrenocortical plasma membrane angiotensin II receptors.

A bovine adrenocortical particulate fraction prepared by zonal ultracentrifugation and banding between rho20 1.08 and 1.101 in a linear sucrose gradient bound 7.3 times more [3H]angiotensin II (ATII) per milligram protein than the original homogenate. Enzyme marker and electron microscope studies indicated that this fraction was largely devoid of mitochondria while being enriched in smooth membranes of predominantly plasmalemmal origin. The binding of labeled ATII was maximal after 10 min incubation (22degreesC) and remained at equilibrium for at least 20 min thereafter. [3H]ATII binding was completely inhibited by saturating concentrations of nonradioactive ATII. The high-affinity binding site in the preparation had a specific binding capacity of 2.38 pmol-mg-1, with an equilibrium constant of 2.36 x 10(8) M(-1). Inhibition-displacement studies with unlabeled ATI,ATII,ATII fragments, analogs, and antagonists show that the receptor fraction has the highest affinity for the intact native octapeptide. ACTH and bradykinin had no specific effects on [3H]ATII binding. The current study suggests that the receptor fraction may be of use in a highly sensitive ATII radioligand assay.     

Photoperiodic control of gibberellin metabolism in spinach

[(3)H]GA(20) applied to spinach plants (Spinacia oleracea L.) was metabolized to several products. Two of these were identified by combined gasliquid chromatography-radio counting as [(3)H]GA(29) and [(3)H]3-epi-GA(1). Inasmuch as both GA(20) and GA(29) are endogenous gibberellins in spinach (Metzger, Zeevaart 1980 Plant Physiol 65: 623-626), it was concluded that the conversion of GA(20) to GA(29) is a natural process. However, 3-epi-GA(1) was not detected in extracts of spinach shoots analyzed by combined gas chromatography-mass spectrometry. This indicates that the conversion of exogenous [(3)H]GA(20) to [(3)H]3-epi-GA(1) may be an artifact.Long-day pretreatment of spinach shoots caused a 2-fold increase in the rate of [(3)H]GA(20) metabolism over the rate of metabolism in plants maintained under short-day conditions. Furthermore, [(3)H]GA(29) accumulated more rapidly under long than under short days, whereas photoperiodic treatment had no effect on the accumulation of [(3)H]3-epi-GA(1). Thus, the long-day-induced increase in the level of endogenous GA(29) in spinach shoots (Metzger, Zeevaart 1980 Plant Physiol 66: 844-846) appears to be the result of an increased capability to convert GA(20) to GA(29).     

The gamma-aminobutyric-acid/benzodiazepine-receptor protein from rat brain. Large-scale purification and preparation of antibodies

The gamma-aminobutyric-acid-receptor protein complex from rat brain was solubilized in high yield, purified in milligram amounts by benzodiazepine affinity chromatography and used to generate a high-titer rabbit antiserum. High concentrations of Triton X-100 detergent plus KCl solubilized about 90% of the membrane-bound gamma-aminobutyric acid receptor (assayed by [3H]muscimol binding) and benzodiazepine receptor (assayed by [3H]flunitrazepam binding) activities. Both activities were retained on an affinity column using an immobilized benzodiazepine ligand, and most of the column-absorbed receptor could be eluted by a solution of free benzodiazepine plus 4 M urea. The purified protein bound [3H]muscimol and [3H]flunitrazepam with receptor-like pharmacological specificity and specific activities of about 1700 pmol and 700 pmol bound/mg protein, respectively, for the two ligands. This corresponds to a purification of over 600-fold and a near theoretical purity, with a yield of milligram quantities from 100 g brain. Four peptide bands were observed on gel electrophoresis in sodium dodecyl sulfate, with molecular mass values of 31, 47, 52 and 57 kDa. The latter two were most significantly stained, and identified as receptor subunits by photolabeling with [3H]flunitrazepam (52 kDa) and [3H]muscimol (57 kDa), and by reaction on Western blots with monoclonal antibodies to this protein produced by Schoch et al. [(1985) Nature (Lond.) 314, 168-171]. Rabbit antiserum was raised to the purified protein and could, at high dilutions, both coprecipitate soluble gamma-aminobutyric-acid/benzodiazepine-receptor-binding activities and stain the receptor subunits (principally 52-kDa band) on Western blots.     

Pre-formed xyloglucans and xylans increase in molecular weight in three distinct compartments of a maize cell-suspension culture

Cultured cells of maize ( Zea mays L.) were pulse-labelled with l-[1-(3)H]arabinose (Ara) and then monitored for 7 days. The (3)H-hemicelluloses present in three compartments (protoplasm, cell wall and culture medium) were size-fractionated and the fractions assayed for [(3)H]xyloglucans and [(3)H]xylans. Protoplasmic [(3)H]xylans and [(3)H]xyloglucans initially (15 min after [(3)H]Ara-feeding) had weight-average relative molecular masses ( M(w)) approximately 0.5x10(6) and 0.3x10(6), respectively, both rising to 2x10(6) by 30 min. Thus, newly formed hemicellulose molecules were joined to other polymers, or to each other, presumably within Golgi vesicles. New (3)H-hemicelluloses very rapidly bound to the cell wall; however, after 1 day, some [(3)H]xyloglucan and [(3)H]xylan was sloughed from the wall into the medium. The wall-bound [(3)H]xyloglucans were present in the form of extremely large complexes, of M(w)>17x10(6), even as early as 15 min after [(3)H]Ara-feeding. This M(w) is >70-fold greater than that observed by similar methods in cultures of a dicotyledon ( Rosa sp.). Thus, during wall-binding, newly secreted xyloglucans greatly increased in size, possibly by transglucosylation. Some modest degradation (trimming) of wall-bound [(3)H]xyloglucan occurred later. The earliest wall-bound [(3)H]xylan had M(w) approximately 2x10(6), similar to the protoplasmic [(3)H]xylan; this increased to approximately 4x10(6) by 6 h. For the first 2 days after [(3)H]Ara-feeding, the soluble extracellular (3)H-hemicelluloses present in the culture medium had M(w) approximately 1x10(6)-2x10(6), comparable to the protoplasmic hemicelluloses. However, between 2 and 3 days after [(3)H]Ara-feeding, the M(w) of the soluble extracellular [(3)H]xylans increased abruptly to approximately 10x10(6); the soluble extracellular [(3)H]xyloglucans underwent a similar but more gradual increase in M(w). Maize (3)H-hemicelluloses thus underwent increases in M(w) in three episodes: (i) intra-protoplasmically, (ii) during wall-binding (especially xyloglucans), and (iii) after sloughing into the medium. Possible mechanisms and roles of these increases are discussed.     

Evidence for a tandem two-site model of ligand binding to muscarinic acetylcholine receptors

After short preincubations with N-[(3)H]methylscopolamine ([(3)H]NMS) or R(-)-[(3)H]quinuclidinyl benzilate ([(3)H]QNB), radioligand dissociation from muscarinic M(1) receptors in Chinese hamster ovary cell membranes was fast, monoexponential, and independent of the concentration of unlabeled NMS or QNB added to reveal dissociation. After long preincubations, the dissociation was slow, not monoexponential, and inversely related to the concentration of the unlabeled ligand. Apparently, the unlabeled ligand becomes able to associate with the receptor simultaneously with the already bound radioligand if the preincubation lasts for a long period, and to hinder radioligand dissociation. When the membranes were preincubated with [(3)H]NMS and then exposed to benzilylcholine mustard (covalently binding specific ligand), [(3)H]NMS dissociation was blocked in wild-type receptors, but not in mutated (D99N) M(1) receptors. Covalently binding [(3)H]propylbenzilylcholine mustard detected substantially more binding sites than [(3)H]NMS. The observations support a model in which the receptor binding domain has two tandemly arranged subsites for classical ligands, a peripheral one and a central one. Ligands bind to the peripheral subsite first (binding with lower affinity) and translocate to the central subsite (binding with higher affinity). The peripheral subsite of M(1) receptors may include Asp-99. Experimental data on [(3)H]NMS and [(3)H]QNB association and dissociation perfectly agree with the predictions of the tandem two-site model.     

Molecular cloning of a cDNA for a small GTP binding protein, BRho, from the embryo of Bombyx mori and its characterization after expression and purification

A cDNA clone encoding a small GTP binding protein (Brho) was isolated from an embryonic cDNA library of Bombyx mori that encoded a polypeptide with 202 amino acids sharing 60-80% similarity with the Rho1 family of GTP binding proteins. The effector site and one of the guanine nucleotide binding sites differed from other members of the Rho family. To characterize the biochemical properties of Brho, the clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The fusion protein bound [(35)S] GTPgammaS and [(3)H] GDP with association constants of 11x10(6) M(-1) and 6.2x10(6) M(-1), respectively. The binding of [(35)S] GTPgammaS was inhibited by GTP and GDP, but by no other nucleotides. The calculated GTP-hydrolysis activity was 89.6 m mol/min/mol of Brho. Bound [(35)S] GTPgammaS and [(3)H] GDP were exchanged with GTPgammaS most efficiently in the presence of 6 mM MgCl(2). These results suggest that Brho has a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolytic activity, and returns to the GTP-bound state with the exchange of GDP with GTP. Arch.     

Effect of Ethylene Treatment on Polar IAA Transport, Net IAA Uptake and Specific Binding of N-1-Naphthylphthalamic Acid in Tissues and Microsomes Isolated from Etiolated Pea Epicotyls

The effect of ethylene treatment on polar indole-3-acetic acid (IAA) transport, net IAA uptake in the presence and absence of N-1-naphthylphthalamic acid (NPA) and [³H]NPA binding characteristics was investigated in tissue segments or microsomes isolated from etiolated pea (Pisum sativum L. cv Alaska) epicotyls. Basipetal IAA transport in 5 millimeter segments isolated from ethylene-treated seedlings was inhibited by ethylene in a dose-dependent manner. Threshold, half-maximal and saturating concentrations of ethylene were 0.01, 0.55, 10.0 microliters per liter, respectively. This inhibition became apparent after 6 to 8 hours of ethylene treatment. Transport velocity in both control and ethylene-treated tissues was estimated to be 5 millimeters per hour. Net IAA uptake was stimulated in ethylene-treated tissues and the relative ability of the phytotropin NPA to enhance net IAA uptake was reduced in treated tissues. Specific binding of [³H]NPA to microsomes prepared from both control and ethylene-treated tissues was saturable and consistent with the existence of a single class of binding sites with an apparent affinity (K_d) toward NPA of 8 to 9 nanomolar. The density of these binding sites (per milligram protein) was lower (36% of control) in ethylene-treated tissues. Direct application of ethylene to microsomal preparations isolated from untreated seedlings had no effect on the level of specific [³H]NPA binding.     

Binding of platelet activating factor by isolated membranes from U937 cells

Platelet activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acetyl-GPC) has been known to have biological effect on cells. The mechanisms of the effect of the potent phospholipid on cells has not been established. We have used 1-O-[3H]alkyl-2-acetyl-GPC [( 3H]PAF) to study the interaction on the isolated membranes of U937 cells. The binding process was time, protein concentration, temperature dependent and reversible. The binding of [3H]PAF to the U937 cell membranes was slightly inhibited by the addition of PAF analogue, 3-O-Hexadecyl-2-acetyl-sn-glycerol-1-phosphorylcholine. U937 cell membranes showed high affinity binding sites for PAF with equilibrium dissociation constant (Kd) of 5 x 10(-9) M. The displacement of bound [3H]PAF with 500-fold excess of nonlabeled PAF was not altered suggesting that the bound [3H]PAF was not degraded during the binding. Binding of [3H]PAF on U937 cell membranes was inhibited by PAF antagonist, 59227RP. The kinetic of the inhibition by PAF antagonist is competitive suggesting that PAF and PAF antagonist bind at the same site.     

Interaction of gibberellins and phytochrome in the control of cowpea epicotyl elongation

The physiological response of cowpea ( Vigna sinensis L.) epicotyl explants to far‐red light (FR) and its interaction with gibberellins (GAs) have been investigated. The effect of FR and GA₁ varied with the age of the seedlings from which the explants were made: for FR, it decreased progressively with age (though the sensitivity of the epicotyls to FR did not change significantly until at least day 11), whereas it remained essentially constant for applied GA₁ between days 5 and 9 after sowing. This indicates that the loss of response to FR may be due to a decrease in endogenous GA levels in the epicotyl. For a range of GA₁ and GA₂₀ (0.01–1 µg explant⁻¹), both hormones were more active in FR than in R irradiated epicotyls, suggesting that phytochrome may affect GA sensitivity besides GA metabolism. The location of the epicotyl region most sensitive to FR (between 5 and 20 mm below the apex) was different from that to GAs (the upper 10 mm). Nevertheless, FR extended the region responsive to applied GAs, even in paclobutrazol‐treated epicotyls where elongation was due entirely to exogenous GAs. This means that modulation of epicotyl elongation by phytochrome, that occurs in a zone different from though overlapping with the GA‐sensitive subapical zone, is also mediated by GAs. Growth in the most FR‐sensitive region of the epicotyl stimulated by FR or GA₁ was due to cell elongation, and in the most GA‐sensitive region to both cell division and elongation. The effect of FR and GA₁ was negated by colchicine, indicating that microtubules may be involved in the response to both factors.     

Characterization of the asialoglycoprotein receptor on isolated rat hepatocytes

Rat hepatocytes, freshly isolated by a collagenase perfusion technique, bound [3H]asialo-orosomucoid in a sugar-specific and calcium-dependent manner as expected for the hepatic asialoglycoprotein receptor. At least 90% of the total cell surface-bound [3H]asialo-orosomucoid represented specific binding and could be removed by washing with EDTA. Freshly isolated cells had about 7 x 10(4) surface receptors per cell. However, when cells were incubated at 37 degrees C, the number of surface receptors per cell rapidly increased 2- to 3-fold to about 2.2 x 10(5). This increase in receptor number occurred in the absence of serum and began within minutes, depending on the particular conditions used to keep the cells in suspension. (The maximal rate of appearance of new receptors at 37 degrees C was about 70 receptors per cell per s.) When cells were first exposed to a brief EDTA treatment at 4 degrees C, before measuring the binding of [3H]asialo-orosomucoid, the number of surface receptors per cell was found to increase by about 45%. Therefore, about 30% of the surface receptors on freshly isolated cells have already bound endogenous asialoglycoproteins or are present in the membrane in a cryptic form. At 4 degrees C the binding of [3H]asialo-orosomucoid was rapid (kon greater than or equal to 1.8 x 10(4) M-1s-1), whereas the dissociation of bound [3H]asialo-orosomucoid, measured in the presence of excess nonradioactive glycoprotein, was extremely slow (koff less than or equal to 0.9 x 10(-5) s-1). The association constant calculated from these data (Ka = 2.0 x 10(9) M-1) agreed well with that obtained from equilibrium binding experiments (Ka = 2.4 x 10(9) M-1) using untreated cells or cells which had first been treated with EDTA or incubated at 37 degrees C. In all cases, when the concentration of [3H]asialo-orosomucoid was higher than about 600 ng/ml, the Scatchard plots were curvilinear. The data are, however, consistent with the conclusion that there is a single high affinity receptor on the hepatocyte surface. The additional receptors that appear on the surface when cells are incubated at 37 degrees C or exposed to EDTA are identical with those on untreated cells,     

Glutaric acid stimulates glutamate binding and astrocytic uptake and inhibits vesicular glutamate uptake in forebrain from young rats

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by glutaryl-CoA dehydrogenase deficiency, which leads to accumulation in body fluids and in brain of predominantly glutaric acid (GA), and to a lesser extent of 3-hydroxyglutaric and glutaconic acids. Neurological presentation is common in patients with GA I. Although the mechanisms underlying brain damage in this disorder are not yet well established, there is growing evidence that excitotoxicity may play a central role in the neuropathogenesis of this disease. In the present study, preparations of synaptosomes, synaptic plasma membranes and synaptic vesicles, as well as cultured astrocytes from rat forebrain were exposed to various concentrations of GA for the determination of the basal and potassium-induced release of [(3)H]glutamate by synaptosomes, Na(+)-independent glutamate binding to synaptic membranes and vesicular glutamate uptake and Na(+)-dependent glutamate uptake into astrocytes, respectively. GA (1-100 nM) significantly stimulated [(3)H]glutamate binding to brain plasma membranes (40-70%) in the absence of extracellular Na(+) concentrations, reflecting glutamate binding to receptors. Furthermore, this stimulatory effect was totally abolished by the metabotropic glutamate ligands DHPG, DCG-IV and l-AP4, attenuated by the ionotropic non-NMDA glutamate receptor agonist AMPA and had no interference of the NMDA receptor antagonist MK-801. Moreover, [(3)H]glutamate uptake into synaptic vesicles was inhibited by approximately 50% by 10 and 100 nM GA and Na(+)-dependent [(3)H]glutamate uptake by astrocytes was significantly increased (up to 50%) in a dose-dependent manner (maximal stimulation at 100 microM GA). In contrast, synaptosomal glutamate release was not affected by the acid at concentrations as high as 1 mM. These results indicate that the inhibition of glutamate uptake into synaptic vesicles by low concentrations GA may result in elevated concentrations of the excitatory neurotransmitter in the cytosol and the stimulatory effect of this organic acid on glutamate binding may potentially cause excitotoxicity to neural cells. Finally, taken together these results and previous findings showing that GA markedly decreases synaptosomal glutamate uptake, it is possible that the stimulatory effect of GA on astrocyte glutamate uptake might indicate that astrocytes may protect neurons from excitotoxic damage caused by GA by increasing glutamate uptake and therefore reducing the concentration of this excitatory neurotransmitter in the synaptic cleft.     

Thermodynamic characterization of OsGID1-gibberellin binding using calorimetry and docking simulations

Gibberellins (GAs) are phytohormones regulating various developmental processes in plants. In rice, the initial GA-signaling events involve the binding of a GA to the soluble GA receptor protein, GID1. Although X-ray structures for certain GID1/GA complexes have recently been determined, an examination of the complexes does not fully clarify how GID1s discriminate among different GAs. Herein, we present a study aimed at defining the types of forces important to binding via a combination of isothermal titration calorimetry (ITC) and computational docking studies that employed rice GID1 (OsGID1), OsGID1 mutants, which were designed to have a decreased possible number of hydrogen bonds with bound GA, and GA variants. We find that, in general, GA binding is enthalpically driven and that a hydrogen bond between the phenolic hydroxyl of OsGID1 Tyr134 and the C-3 hydroxyl of a GA is a defining structural element. A hydrogen-bond network that involves the C-6 carboxyl of a GA that directly hydrogen bonds the hydroxyl of Ser198 and indirectly, via a two-water-molecule network, the phenolic hydroxyl of Tyr329 and the NH of the amide side-chain of Asn255 is also important for GA binding. The binding of OsGID1 by GA(1) is the most enthalpically driven association found for the biologically active GAs evaluated in this study. This observation might be a consequence of a hydrogen bond formed between the hydroxyl at the C-13 position of GA(1) and the main chain carbonyl of OsGID1 Phe245. Our results demonstrate that by combining ITC experiments and computational methods much can be learned about the thermodynamics of ligand/protein binding.