Isolation of Stable Human Ceruloplasmin and Its Interaction with Salmon Protamine

An interaction was discovered between ceruloplasmin (CP, a ferro-O₂-oxidoreductase, EC 1.16.3.1), a copper-containing protein of human blood plasma, and salmon protamine (PR), a cationic polypeptide of vertebrates that provides a compact structure of spermatozoid DNA. Addition of PR to CP at a molar ratio of 2 : 1 decreases the CP electrophoretic mobility. Two types of CP binding centers for PR were determined: two centers with a high (K _d1 of 5.31 × 10⁻⁷ M) and four centers with a low affinity (K _d2 of 1.56 × 10⁻⁵ M). PR was shown to form complexes with CPs of various animal species. The CP-PR complex dissociates at an increased ionic strength (0.3 M NaCl), at pH decreased below 4.7, and/or in the presence of added polyanions (DNA, lipopolysaccharides, or heparin) or polylysine, which indicates the electrostatic nature of the interaction. The CP-PR interaction increased 1.5-fold the rate of CP-catalyzed oxidation of Fe²⁺. The preliminary treatment of blood plasma with arginine-Sepharose and heparin-Sepharose (to remove the blood coagulation factors) and affinity chromatography on PR-Sepharose allowed us to isolate the practically unproteolyzed monomeric CP in 90% yield; it remained stable for more than two months at 37°C.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 269–279.Original Russian Text Copyright © 2005 by Sokolov, Zakharova, Shavlovskii, Vasil’ev.     

The Na+-independentd-glucose transporter in the enterocyte basolateral membrane: Orientation and cytochalasin B binding characteristics

Summary Phloridzin-insensitive, Na⁺-independentd-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll^® gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed aK _t of 48mm and aV _max of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lowerK _t of 23mm and aV _max of 0.20 nmol glucose/mg protein/sec.d-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. TheK _t for cytochalasin B as an inhibitor of glucose transport was 0.11 m and theK _D for binding to the carrier was 0.08 m.d-glucose-sensitive binding of cytochalasin B to the membrane preparation was maximized withl- andd-glucose concentrations of 1.25m. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na⁺-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and theK _t for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.     

Characterization and solubilization of the cytochalasin B binding component from human placental microsomes

Human placental microsomes exhibit uptake of d-[³H]glucose which is sensitive to inhibition by cytochalasin B (apparent K_i = 0.78 /gm M). Characterization of [³H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by d-glucose with an ED₅₀ = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a K_d = 0.15μM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60–70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [³H]cytochalasin B to the soluble protein displays a pattern of inhibition by d-glucose similar to that observed for intact membranes, and the measurement of an ED₅₀ = 37.5 mM d-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [³H]cytochalasin B incorporation into soluble protein (M_r range 42 000-68 000) is prevented by the presence of d-glucose. An identical photolabelling pattern is observed for incorporation of [³H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions.     

Interaction of cisplatin and analogues with a Met-rich protein site

The chaperone protein CopC from Pseudomonas syringae features high-affinity binding sites (K _D ~ 10⁻¹³ M) for both Cu^I (Met-rich) and Cu^II (His-rich). When presented with these sites in the apoprotein, electrospray ionisation mass spectrometry confirmed that cis-Pt(NH₃)₂Cl₂ (cisplatin) and the fragments [Pt^IIL]²⁺ (L is 1,2-diaminoethane, 2,2′-bipyridine) occupied the Cu^I site specifically in the 1:1 Pt–CopC adducts (purified by cation-exchange chromatography). The cis-Pt(NH₃)₂ fragment was not present in these adducts (the dominant product for cisplatin was Pt–CopC in which all original ligands were displaced), while bidentate ligands L were retained in LPt–CopC adducts. In the context of the Met-rich Cu^I pump Ctr1 as a significant entry point for cisplatin into mammalian cells, the present work confirms the ability of Met-rich sites in proteins to remove all ligands from cisplatin. It focuses attention on the potential of proteins that are part of the natural copper transport pathways to sequester the drug. These pathways are worthy of further study at the molecular level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mediated uptake of folate by a high-affinity binding protein in sublines of L1210 cells adapted to nanomolar concentrations of folate

Summary An L1210 cell line (JT-1), which can grow in medium supplemented with 1nm folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37°C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23±0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [³H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37°C remained as unmetabolized folic acid. Binding was also rapid at 0°C but uptake at the plateau was only one-half the value obtained at 37°C. Half-maximal saturation of the binding component (K _D) occurred at a folate concentration of 0.065nm at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 (K _D=2.0nm). 5-Methyltetrahydrofolate was also bound by this component (K _i=13nm at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate (K _i=45nm) and methotrexate (K _i=325nm). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500nm caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein. An additional low-affinity, high-capacity transport system for folate that had been proposed previously was not observed under a variety of experimental conditions in either the adapted or parental cells.     

Pressure perturbation calorimetry of apolipoproteins in solution and in model lipoproteins

High‐density lipoproteins (HDLs) are complexes of lipids and proteins (termed apolipoproteins) that remove cell cholesterol and protect from atherosclerosis. Apolipoproteins contain amphipathic α‐helices that have high content (≥1/3) and distinct distribution of charged and apolar residues, adopt molten globule‐like conformations in solution, and bind to lipid surfaces. We report the first pressure perturbation calorimetry (PPC) study of apolipoproteins. In solution, the main HDL protein, apoA‐I, shows relatively large volume contraction, ΔV_unf = ?0.33%, and an apparent reduction in thermal expansivity upon unfolding, Δα_unf ≤ 0, which has not been observed in other proteins. We propose that these values are dominated by increased charged residue hydration upon α‐helical unfolding, which may result from disruption of multiple salt bridges. At 5°C, apoA‐I shows large thermal expansion coefficient, α(5°) = 15·10^?4 K^?1, that rapidly declines upon heating from 5 to 40°C, α(40°) ? α(5°) = ?4·10^?4 K^?1; apolipoprotein C‐I shows similar values of α(5°) and α(40°). These values are larger than in globular proteins. They indicate dominant effect of charged residue hydration, which may modulate functional apolipoprotein interactions with a broad range of their protein and lipid ligands. The first PPC analysis of a protein–lipid complex is reported, which focuses on the chain melting transition in model HDL containing apoA‐I or apoC‐I, dimyristoyl phosphatidylcholine, and 0–20% cholesterol. The results may provide new insights into volumetric properties of HDL that modulate metabolic lipoprotein remodeling during cholesterol transport. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Reconstitution of (Na+ + K+)-ATPase into phospholipid vesicles with full recovery of its specific activity

(1) ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from rectal glands of the spiny dogfish has been reconstituted into phospholipid vesicles. The nonionic detergent octaethyleneglycoldodecyl monoether (C₁₂E₈) is used to dissolve both the enzyme and the lipids and reconstitution is accomplished by subsequent removal of the detergent by adsorption to polystyrene beads. (2) About 60% of the enzyme incorporates in the right-side-out orientation (r/o). The fraction of molecules in the inside-out orientation (i/o) increases from about 10% to about 30% with a parallel decrease in the fraction of ‘non-oriented’ (n-o) molecules (both sides exposed) when the protein/lipid ratio decreases from 1:10 to 1:75. (3) The orientation of enzyme molecules detected from vanadate binding is the same as measured from activity, i.e., the turnover of the enzyme molecule in the diffrent orientations is the same. (4) The recovery of the specific activity of the incorporated enzyme increases with an increase in the protein/lipid ratio and is 100% with a protein/lipid ration of about 1:20 or higher. Full recovery is only obtained provided a proper lipid composition is chosen which includes both negatively charged phospholipids, preferably phosphatidylinositol, and cholesterol. (5) The ATP-dependent, K⁺-stimulated Na⁺-influx is found to be about 35 μmol Na⁺ per mg (i/o)-protein per min at 22°C in 1:10 protein/lipid liposomes. The specific activity corresponds to 3 Na⁺ transported per ATP molecule hydrolyzed.     

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.     

Generation of receptor-active, globotriaosyl ceramide/cholesterol lipid 'rafts' in vitro: A new assay to define factors affecting glycosphingolipid receptor activity

Purified renal globotriaosyl ceramide (Gb₃)/cholesterol mixtures, sonicated and heated in a Triton-containing buffer and placed below a discontinuous sucrose gradient, form glycosphingolipid (GSL)-containing dense lipid structures at the 30/5% sucrose interface after centrifugation. Inclusion of fluorescein-labeled verotoxin 1 B subunit (FITC-VT1 B) within the most dense sucrose layer results in the fluorescent labeling of this Gb₃-containing raft structure. Alternatively, inclusion of ¹²⁵I-labeled VT1 and fractionation allows quantitation of binding. FITC-VT1 B effectively competes for ¹²⁵I-VT1/Gb₃ raft binding. This assay will allow the definition of the optimal raft composition for VT1 (or any other ligand) binding. The effect of several potential cellular raft components are reported. Increased cholesterol content increased VT1 binding. Addition of phosphatidylethanolamine had minimal effect while phosphatidylserine was inhibitory. Although inclusion of sphingomyelin increased the Gb₃ content of the 'raft', reduced VT1 binding was seen. Inclusion of other glycolipids can also be inhibitory. The addition of globotetraosyl ceramide had no effect; however, addition of sulfogalactosyl ceramide, but not sulfogalactoglycerolipid, inhibited VT1/Gb₃ raft binding. These results suggest that certain GSLs can disfavour the formation of the appropriate 'raft' structure for ligand binding and that this is dependent on both their carbohydrate and lipid structure. Such “deceptor” GSLs may provide an as yet, unappreciated mechanism for the regulation of cellular GSL receptor activity. This model is an effective tool to approach the dynamics and ligand-binding specificity of GSL/cholesterol-containing lipid microdomains. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Sulphydryl groups in the maintenance of the activity of binding protein for N-1-naphthylphthalamic acid fromAcer pseudoplatanus cells

Binding protein for N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, was studied by analysis of the effects of reactions which modify particular amino acid side chains upon their binding activity. Na₂SO₃, N-ethylmaleimide (NEM) and dithiobisnitrobenzoic acid all inhibited the specific binding of NPA to its binding protein fromAcer pseudoplatanus L. cells. The presence of 10^-6 M Na₂SO₃ in the binding assay reduced the affinity of the binding protein to NPA from K_d of 1.5 ￡ 10^-8 M to K_d of 2.1 ￡ 10_-8 M, while concentration of the binding protein was not significantly changed. When the same analysis was applied to NPA binding to the NEM-treated membrane particles, it was found that NEM inactivated binding without changing the affinity for NPA. This study revealed the importance of sulphydryl group(s) in the maintenance of NPA binding protein activity.     

The interaction of proteins with hydroxyapatite: II. Role of acidic and basic groups

The elution behavior from hydroxyapatite columns of the modification products of seven basic and three acidic proteins has been investigated. Three classes of NH₂ derivatives were prepared. These consisted of (1) replacement by a guanidyl group with no change in charge; (2) blocking with loss of charge; and (3) replacement of positive charges by negative ones. Two types of COOH derivatives were prepared: (1) blocking with loss of charge; and (2) replacement of COOH by SO₃H with no change in charge. The elution behavior of the derivatives in PO₄, F^?, Cl^?, ClO₄^?, and Ca²⁺ ion eluants showed that (1) the elution patterns are determined by the isoelectric points of the proteins, there being no symmetry between the binding or elution behavior of acidic and basic proteins; (2) the binding of basic proteins requires the presence of a high density of positively charged groups; (3) the binding of all proteins to hydroxyapatite equilibrated with phosphate buffer is enhanced by a decrease in the number of their negative charges; and (4) calcium ions affect the binding of proteins to hydroxyapatite at the level of carboxyls, since clusters of carboxyls strengthen both the interaction with Ca²⁺ and the binding to hydroxyapatite.     

Regulation of the high‐affinity choline transporter activity and trafficking by its association with cholesterol‐rich lipid rafts

The sodium‐coupled, hemicholinium‐3‐sensitive, high‐affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol‐rich lipid rafts in both SH‐SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH‐SY5Y cells expressing rat CHT with filipin, methyl‐β‐cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol‐saturated MβC. Kinetic analysis of binding of [³H]hemicholinium‐3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (K_D) and maximum number of binding sites (B_max); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol‐rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis.

     

Characterization of glutamate transport system in hydrophobic protein (H protein) of Bacillus subtilis

Hydrophobic protein (H protein) was isolated from membrane fractions of Bacillus subtilis and constituted into artificial membrane vesicles with lipid of B. substilis. Glutamate was accumulated into the vesicle when a Na⁺ gradient across the membrane was imposed. The maximum effect of Na⁺ on the transport was achieved at a concentration of about 40 mM, while the apparent K_m for Na⁺ was approximately 8 mM. On the other hand, K_m for glutamate in the presence of 50 mM Na⁺ was about 8 μM. Increasing the concentration of Na⁺ resulted in a decrease in K_m for glutamate, maximum velocity was not affected. The transport was sensitive to monensin (Na⁺ ionophore).Glutamate was also accumulated when pH gradient (interior alkaline) across the membrane was imposed or a membrane potential was induced with K⁺-diffusion potential. The pH gradient-driven glutamate transport was sensitive to carbonylcyanide m-chlorophenylhydrazone and the apparent K_m for glutamate was approximately 25 μM.These results indicate that two kinds of glutamate transport system were present in H protein: one is Na⁺ dependent and the other is H⁺ dependent.     

Characterization and properties of steroid binding protein in Bufo arenarum serum

Serum steroid binding properties of mature Bufo arenarum females were studied. Binding data obtained using charcoal adsorption assay and equilibrium dialysis methods indicates a single protein, named Bufo arenarum sex binding protein (Ba SBP), which binds 5 α-dihydrotestosterone (DHT), testosterone (T), and estradiol-17β (E₂) with high affinity (10⁷ M^?1 – 10⁸ M^?1) and fair capacity (10^?6 M). Scatchard plot analysis demonstrated the coexistence of two binding sites. Ba SBP has a sedimentation coefficient of 5.2 S in sucrose gradient centrifugation in low salt and under steady-state conditions. The specificity of this protein, determined by competitive binding experiments, is comparable to human SBP. DHT and T bind with higher affinity than E₂. Estriol and estrone competed poorly, while diethylstilbestrol and C₂₁ steroids did not compete. The binding capacity of this protein is under estrogenic control. © 1994 Wiley-Liss, Inc.     

The effect of histidine modification on copper-dependent lipid peroxidation in human low-density lipoprotein

Summary

Lipid peroxidation and subsequent oxidative modification of low-density lipoprotein (LDL) have been implicated as causal events in atherosclerosis. Cu²⁺ may play an important role in LDL oxidation by binding to histidine residues of apolipoprotein B-100 (apo B) and initiating and propagating lipid peroxidation. To investigate the role of histidine residues, we used diethylpyrocarbonate (DEPC), a lipid-soluble histidine-specific modifying reagent. When LDL (0.1 mg protein/ml, or 0.2 µM) was incubated with DEPC (1 mM), at least 76 ± 7% of the histidine residues in apo B were modified. Treatment of LDL with DEPC led to an increase in the rate of Cu²⁺-induced initiation of lipid peroxidation (R_i), but a significant decrease in the rate of propagation. These changes resulted in an overall increased resistance of LDL to oxidation, with a significantly increased lag phase preceding the propagation phase of lipid peroxidation. In contrast to DEPC, ascorbate completely prevented the initiation of LDL oxidation (R_i = 0). Our data indicate that there are two types of copper/histidine binding sites on apo B: those facing the lipid core of the LDL particle, which mediate the propagation of lipid peroxidation and are modified by DEPC; and those found on the surface of the LDL particle exposed to the aqueous environment, which are responsible for mediating the initiation of lipid peroxidation and are modifiable by ascorbate in the presence of Cu²⁺.     

Two substrate sites in the renal Na+-d-glucose cotransporter studied by model analysis of phlorizin binding and-d-glucose transport measurements

Summary Time courses of phlorizin binding to the outside of membrane vesicles from porcine renal outer cortex and outer medulla were measured and the obtained families of binding curves were fitted to different binding models. To fit the experimental data a model with two binding sites was required. Optimal fits were obtained if a ratio of low and high affinity phlorizin binding sites of 1:1 was assumed. Na⁺ increased the affinity of both binding sites. By an inside-negative membrane potential the affinity of the high affinity binding site (measured in the presence of 3 mM Na⁺) and of the low affinity binding site (measured in the presence of 3 or 90 mM Na⁺) was increased. Optimal fits were obtained when the rate constants of dissociation were not changed by the membrane potential. In the presence of 90 mM Na⁺ on both membrane sides and with a clamped membrane potential,K _D values of 0.4 and 7.9 M were calculated for the low and high affinity phlorizin binding sites which were observed in outer cortex and in outer medulla. Apparent low and high affinity transport sites were detected by measuring the substrate dependence ofd-glucose uptake in membrane vesicles from outer cortex and outer medulla which is stimulated by an initial gradient of 90 mM Na⁺(out>in). Low and high affinity transport could be fitted with identicalK _m values in outer cortex and outer medulla. An inside-negative membrane potential decreased the apparentK _m ofhigh affinity transport whereas the apparentK _m of low affinity transport was not changed. The data show that in outer cortex and outer medulla of pighigh and low affinity Na⁺-d-glucose cotransporters are present which containlow and high affinity phlorizin binding sites, respectively. It has to be elucidated from future experiments whether equal amounts of low and high affinity transporters are expressed in both kidney regions or whether the low and high affinity transporter are parts of the same glucose transport moleculc.     

Storage and mobilisation of nutriment for uterine milk synthesis by Glossina morsitans

Nitrogen content of eggs and larvae of Glossina morsitans was a constant proportion of dry weight and equivalent to ca. 55% protein assuming tsetse proteins contain 16% nitrogen. The larval gut content (uterine milk) contained 40% protein. Fatty acid composition of lipids in the milk and in the larval body was similar, with Palmitic (35–38%), Palmitoleic (31–35%) and Oleic acid (23–25%) predominating. Results support the hypothesis that uterine milk contains both protein and lipid and that its composition is relatively constant throughout the period of its synthesis and secretion.Patterns of incorporation of radioactivity by fertilized adult females from injected [¹⁴C]-leucine changed throughout a pregnancy cycle. High levels of incorporation into lipid (22–30%) during early pregnancy fell to around 10% during late pregnancy. Over the same period low levels of incorporation into protein (5%) increased to 15%. Results support the hypothesis that uterine milk is synthesized from a lipid store laid down in early pregnancy coupled with protein derived largely from blood meals ingested later. Such a system would not require the insect to store proteins for larval growth and is economical in terms of energy expenditure.     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affiity (K_d=10⁻⁹ M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [³H]tamoxifen binding capacity is thermolabile, being most stable at 4°C and rapidly lost at 37°C. More [³H]tamoxifen, however, is specifically bound at incubation temperatures of 25°C and 37°C than at 4°C although prewarming nuclei has no effect, suggesting exchange of [³H]tamoxifen for an unidentified endogenous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (K_d=10⁻⁹ M) [³H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78±0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000×g, 30 min) contains high-capacity (955±405 (S.D.) fmol/mg protein), low-affinity (K_d=10.9±4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000×g, 60 min) contains low-capacity (46±15 (S.D.) fmol/mg protein), high-affinity (K_d=0.61± 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%–3.2%, and nuclear sites less than 0.5% of total sites.     

Regulation of Na+-coupled glucose carrier SGLT1 by AMP-activated protein kinase

Abstract

AMP-activated protein kinase (AMPK), a serine/threonine kinase activated upon energy depletion, stimulates energy production and limits energy utilization. It has previously been shown to enhance cellular glucose uptake through the GLUT family of facilitative glucose transporters. The present study explored the possibility that AMPK may regulate Na⁺-coupled glucose transport through SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with and without AMPK and electrogenic glucose transport determined by dual electrode voltage clamping experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or expressing constitutively active ^γR70QAMPK (α1β1γ1(R70Q)) alone, the addition of glucose to the extracellular bath generated a current (I_g), which was half maximal (K_M) at ≈ 650 μM glucose concentration. Coexpression of ^γR70QAMPK did not affect K_M but significantly enhanced the maximal current (≈ 1.7 fold). Coexpression of wild type AMPK or the kinase dead ^αK45RAMPK mutant (α1(K45R)β1γ1) did not appreciably affect I_g. According to confocal microscopy and Western Blotting, AICAR (1 mM), phenformin (1 mM) and A-769662 (10 μM) enhanced the SGLT1 protein abundance in the cell membrane of Caco2 cells suggesting that AMPK activity may increase membrane translocation of SGLT1. These observations support a role for AMPK in the regulation of Na⁺-coupled glucose transport.     

Purification and characterization of folate binding proteins from rat placenta

Rat placenta contains virtually no unsaturated (i.e., apo-form) folate binding protein. However, by lowering the pH of a solubilized membrane preparation of this tissue to 3.5, the endogenous bound folate was dissociated from the protein and adsorbed to charcoal. The apo-form of the folate binding protein thus obtained was purified by affinity chromatography using pteroylglutamic acid covalently coupled to Sepharose 4B. A single protein band with an apparent M_r of 36 000 was observed by SDS-polyacrylamide gel electrophoresis of the eluate from the affinity matrix. Western blot of this preparation using a rabbit antiserum raised with the affinity eluate also identified a single 36 kDa protein band. However, peptide sequencing of the N-terminal region of the proteins in the affinity eluate established that it contained two homologous proteins. Computer alignment of the first 22 N-terminal amino acids of each rat placental protein with human, bovine milk and mouse folate binding proteins showed 50–64% identical homology and 27% homology when the eight proteins were aligned together. The affinity of both rat proteins is highest for pteroylglutamic acid (K_a = 1.6 − 109 l/mol) lower for N⁵-methyltetrahydrofolate and substantially lower for N⁵-formyltetrahydrofolate. In the dose-response range studied there was no apparent affinity for methotrexate. The folate binding proteins could be released from a preparation of placental membranes using phospholipase C indicating that these proteins belong to the class of proteins anchored to the plasma membrane by a glycosyl phosphatidylinositol adduct.