A Thermodynamic Approach to Hormone-Receptor Interaction; Application to Insulin Binding to Adipocytes,Adipocyte Plasma Membranes and Liposomes Incorporating Adipocyte Insulin Receptors

Abstract

The binding of insulin to its receptor in rat adipocyte and isolated plasma membranes has been measured. The adipocyte insulin receptor has been reconstituted in lecithin liposomes and the binding of insulin investigated. A method of interpreting binding data presented as binding vs. the logarithm of free insulin concentration (binding isotherms) in terms of the binding potential concept of Wyman (1965) is described, and the results are compared with the commonly used Scatchard analysis of binding. The binding potential approach enables binding constants and Gibbs energies of formation of the insulin-receptor complex to be determined as a function of insulin bound. The limiting Gibbs energies of binding at 15°C to intact cells, membranes and liposomes were found to be -55, -52 and -49 kJ mol^?1 respectively. The affinity of the receptor for insulin decreases smoothly with increase in binding in all three systems. For intact adipocytes the number of insulin receptors per cell is found to be approximately 43,000.     

alpha-Galactoside Binding Proteins from Plant Membranes: Isolation, Characterization, and Relation to Helminthosporoside Binding Proteins of Sugarcane

α-Galactoside binding proteins were isolated from cellular membranes of mint and tobacco as well as two clones of sugarcane which differ in their sensitivity to helminthosporoside, a toxic galactoside. Sodium trichloroacetate was used to disrupt membranes after which the proteins were purified using a melibiose-Sepharose-6B affinity column. Proteins from mint, tobacco, and susceptible sugarcane had equal electrophoretic mobilities, whereas resistant sugarcane protein migrated more slowly. Pretreatment of the proteins with fluorescamine caused them to migrate with the tracking dye. Each of the proteins had molecular weights of about 100,000 and each was shown to be oligomeric. Gel filtration revealed that aqueous solutions of these membrane proteins contained a mixture of size species which included a high molecular weight multimer and lower molecular weight oligomers. The relative abundance of the oligomers was dependent upon protein concentration: the lower concentrations yielded higher relative amounts of oligomers (Kenfield and Strobel 1980 Biochim Biophys Acta 600: 705-712). Also, the binding activity of these receptors was inversely proportional to protein concentration. At low protein concentration (4 micrograms per milliliter), the K_d's of each of the proteins for galactinol, raffinose, and helminthosporoside was about 10 micromolar. At high protein concentrations (100 micrograms per milliliter), mint and resistant sugarcane proteins failed to bind α-galactosyl ligands, whereas proteins from tobacco and susceptible sugarcane exhibited a markedly decreased binding activity compared to that at lower protein concentrations. Binding proteins from susceptible sugarcane were mixed with receptors from either resistant sugarcane or mint at low protein concentrations, then assayed for binding activity. Such mixtures showed a concentration-dependent decrease in binding activity analogous to the activity of homogeneous protein solutions. Bovine serum albumin, a nonsubunit protein, had no effect on the binding activity of the protein from susceptible sugarcane. Thus, receptors from diverse plants can associate in vitro and form functional oligomers. The amino acid composition of each of the binding proteins was similar but not identical. The significance of these results is discussed in regard to regulation of carbohydrate transport and sensitivity to phytotoxins.     

小鼠肝质膜蛋白质的生物信息学研究

随着高通量蛋白质组研究技术的发展,使用生物信息学方法对鉴定出的蛋白质进行批量的物理化学性质和功能属性的研究显得越来越重要．对2-DE分离的小鼠肝质膜中鉴定的209个蛋白质运用生物信息学方法进行了一系列的功能属性分析,包括统计分析ProtParam软件计算出的209个蛋白质的理论相对分子质量、等电点以及疏水值的分布情况,使用TMHMM预测蛋白质的跨膜区数目,运用系统发生谱方法预测蛋白质相互作用网络,根据其相互作用网络预测部分未知蛋白质的功能．     

Isolation and Characterization of Two Fatty Acid Binding Proteins from Mouse Brain

Abstract: Two fatty acid binding proteins (FABPs) were isolated from Swiss Webster mouse brains. Neither protein cross-reacted with antisera to recombinant liver L-FABP. One protein, designated brain H-FABP, migrated on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a single band at 14.5 kDa with pl 4.9. Brain H-FABP bound NBD-stearic acid and cis -parinaric acid with K _D values near 0.02 and 0.5 µ M , respectively. Brain H-FABP cross-reacted with affinity-purified antisera to recombinant heart H-FABP. The second protein, mouse brain B-FABP, migrated on tricine SDS-PAGE gels as a doublet at 16.0 and 15.5 kDa with pl values of 4.5 and 4.7, respectively. Brain B-FABP bound NBD-stearic acid and cis -parinaric acid with K _D values near 0.01 and 0.7 µ M , respectively. The brain B-FABP doublet was immunoreactive with affinity-purified antibodies against recombinant mouse brain B-FABP, but not with affinity-purified antibodies against heart H-FABP. [³H]Oleate competition binding indicated that the two brain FABPs had distinct ligand binding specificities. Both bound fatty acids, fatty acyl CoA, and lysophosphatidic acid. Although both preferentially bound unsaturated fatty acids, twofold differences in specific saturated fatty acid binding were observed. Brain B-FABP and brain H-FABP represented 0.1 and 0.01% of brain total cytosolic protein, respectively. In summary, mouse brain contains two native fatty acid binding proteins, brain H-FABP and brain B-FABP.     

Specific Binding of Insulin to Membranes from Dendrodendritic Synaptosomes of Rat Olfactory Bulb

The external plexiform layer of the olfactory bulb is among the brain regions where insulin receptors are most abundant. In vitro binding of porcine 125I-insulin to membranes of dendrodendritic synaptosomes isolated from adult rat olfactory bulbs was studied to test the hypothesis that dendrodendritic synapses are major insulin-receptive sites in the external plexiform layer of olfactory bulbs. Of the specific insulin binding sites present in a total particulate fraction from the olfactory bulbs, approximately half were recovered in the dendrodendritic synaptosome fraction. The only other subcellular fraction to which substantial insulin binding was observed was the conventional (axodendritic/axosomatic) synaptosome fraction. Analysis of equilibrium binding of insulin to dendrodendritic synaptosomal membranes, at total insulin concentrations of 0.5-1,000 nM, revealed binding site heterogeneity consistent with a two-site model for insulin binding to a high-affinity (KD = 6 nM), low-capacity (Bmax = 110 fmol/mg of protein) site and a low-affinity (KD = 190 nM), high-capacity (Bmax = 570 fmol/mg of protein) site. The results indicate that the intense labeling of the external plexiform layer of the olfactory bulb in autoradiographic studies of insulin binding can be attributed to insulin receptors on dendrodendritic synaptic membranes in this region.     

Expression of Calcium Binding Proteins in Mouse Type II Taste Cells

It is well established that calcium is a critical signaling molecule in the transduction of taste stimuli within the peripheral taste system. However, little is known about the regulation and termination of these calcium signals in the taste system. The authors used Western blot, immunocytochemical, and RT-PCR analyses to evaluate the expression of multiple calcium binding proteins in mouse circumvallate taste papillae, including parvalbumin, calbindin D28k, calretinin, neurocalcin, NCS-1 (or frequenin), and CaBP. They found that all of the calcium binding proteins they tested were expressed in mouse circumvallate taste cells with the exception of NCS-1. The authors correlated the expression patterns of these calcium binding proteins with a marker for type II cells and found that neurocalcin was expressed in 80% of type II cells, whereas parvalbumin was found in less than 10% of the type II cells. Calretinin, calbindin, and CaBP were expressed in about half of the type II cells. These data reveal that multiple calcium binding proteins are highly expressed in taste cells and have distinct expression patterns that likely reflect their different roles within taste receptor cells.     

Glucagon Affinity Absorbents: Selective Binding of Receptors of Liver Cell Membranes

AFFINITY chromatography has been used in the rapid isolation of enzymes, antibodies, antigens and other ligand-binding proteins^1–6. Selective adsorbents with biological specificity perhaps may best be used in the resolution and isolation of complex biological structures and important regulatory macromolecules present in cells in very low amounts. For example, polypeptide and steroid hormone receptors, drug receptors, transport proteins and repressor molecules may be well suited for study by this technique because they display specific binding functions with a high degree of affinity.     

In Vitro and In Vivo Binding of S-Adenosyl-L-Homocysteine to Membranes from Rat Cerebral Cortex

Membranes from rat cerebral cortex are able to bind S-adenosyl-L-homocysteine (SAH) with a KD of 5 . 10(-7) M and n of 170 pmol/g fresh tissue (i.e. 20 mg protein). The binding is enhanced by Mg2+ and Ca2+ but not K+ and Na+. gamma-Aminobutyric acid, diazepine, noradrenaline and alpha antagonists are without any effect; S-adenosyl-L-methionine, adenosine and adenosine triphosphate inhibit SAH binding. Linkage with an adenosine receptor has not been expressly demonstrated by our method. SAH binding proteins are more abundant in the crude synaptosomal pellet (P2). A similar fixation seems to occur on brain membranes after [3H]SAH administration to rat. The binding might be linked to a methylase activity or an adenosine receptor.     

Interactions of Malondialdehyde and 4-Hydroxynonenal with Rat Liver Plasma Membranes and their Effect on Binding of Prostaglandin E2 by Specific Receptors

We investigated effect of aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydrox-ynonenal (HNE) on prostaglandin (PG) E₂ receptors of liver plasma membranes. The modification of the membranes by MDA diminished PGE₂ binding, decreasing receptor affinity for PGE₂ and receptor density whereas HNE increased PGE₂ binding, enhanced receptor density but did not changed receptor affinity. ESR study showed the decrease of the whole membrane fluidity after modification by MDA whereas HNE lowered membrane fluidity only in the internal zone of lipid bilayer and increased it in the surface area. The possible effects of membrane changes caused by MDA and HNE on PGE₂ receptor parameters are discussed.     

人血浆HDL对动脉粥样硬化家兔肝细胞膜 LDL受体活性影响的研究

高胆固醇饲料喂养造成的动脉粥样硬化（Ａｓ） 模型家兔通过静脉注射人血浆ＨＤＬ 制剂, 观察ＨＤＬ 对Ａｓ家兔肝细胞膜ＬＤＬ受体活性的影响． 结果发现, 摄取高胆固醇饲料的Ａｓ 家兔, 其肝细胞膜ＬＤＬ 受体 Ｋｄ 值虽无明显变化但Ｂｍａｘ 值显著减小（ Ｐ＜ ０－０１ , 与正常对照组比较） ; 注射ＨＤＬ 制剂后, Ａｓ 家兔肝细胞膜ＬＤＬ受体Ｋｄ 值仍无明显改变, 但Ｂｍａｘ 值却显著回升（ Ｐ＜ ０－０１ , 与高脂组比较） ． 表明人血浆ＨＤＬ 具有增加Ａｓ 家兔肝细胞膜ＬＤＬ 受体活性的作用．     

Calcium Binding to Plasma Membranes from Normal and SV40 Transformed Hamster Lymphocytes

Abstract

The calcium binding properties of isolated plasma membranes from normal and SV40 transformed hamster lymphocytes were compared over the Ca²⁺ concentration range of 10^?5M to 5 × 10^?3M and at physiological ionic strength. At all Ca²⁺ concentrations, normal membranes bound more Ca²⁺ than tumor membranes; at blood Ca²⁺ levels (1–2 mM) plasma membranes of normal cells bind twice as much as membranes from tumor cells. Normal plasma membranes demonstrated positive cooperative Ca²⁺ binding whereas tumor membranes displayed non-interacting Ca²⁺-binding sites. Ca²⁺ binding to both membranes was insensitive to Mg²⁺ (0.1 to 2.5 mM). A pH shift from 7 to 6 resulted in a 70% decrease of normal membrane-bound Ca²⁺ compared to a 40% decrease observed with tumor membranes. Extracellular surface Ca²⁺ binding to intact cells was also studied after a 72-hour equilibration of cells with ⁴⁵Ca²⁺ and with ethylene-glycol-bis-(β-amino-ethyl ether) N, N′-tetraacetate chelation as marker for surface Ca²⁺. Tumor cell surface Ca²⁺ binding was only 10% of that observed with quiescent lymphocytes. Normal lymphocytes stimulated to divide with phytohemagglutinin also showed a decreased level of surface Ca²⁺ (50%). However, plasma membranes isolated from non-dividing and phytohemagglutinin-stimulated lymphocytes exhibited equivalent Ca²⁺ binding.     

Evidence for Differential Binding of Growth Hormones to Membrane and Cytosolic GH Binding Proteins of Rabbit Liver

Abstract

The existence of three GH binding proteins in rabbit liver membranes has been adduced from binding studies with a panel of monoclonal antibodies (1)˙ Immunologically cross-reactive analogues of ‘type 2’ binding proteins were shown to exist in rabbit liver cytosol and in affinity purified receptor from liver microsomes. We now report differences in the binding of human and ovine GH with respect to two antigenic determinants on the ‘type 1″ GH binding protein. The discovery of these differences has enabled the detection of cross-reactive analogues of both binding protein types ‘1″ and ‘2’ in liver cytosol and in affinity purified preparations from liver membranes. These findings show a) a close structural relationship between the pool of cytosolic GH binding proteins and those present in the membranes; and b) differential ligand binding to, as well as absolute ligand selection by GH binding proteins, which could reflect the ability of GH to trigger a range of biological responses either through different receptors or differential interaction with particular receptor subtypes.     

Insulin Binding and Effects on Pyrimidine Nucleoside Uptake and Incorporation in Cultured Mouse Astrocytes

Binding of 125I-insulin to primary cultures of differentiated mouse astrocytes was time-dependent, reaching equilibrium after 2 h at 22 degrees C, with equilibrium binding corresponding to 20.79 fmol/mg of protein, representing approximately 5,000 occupied binding sites/cell. The half-life of 125I-insulin dissociation at 22 degrees C was 2 min, with an initial dissociation rate constant of 4.12 X 10(-2) s-1. Dissociation of bound 125I-insulin was not accelerated significantly in the presence of unlabeled insulin (16.7 microM). Porcine and desoctapeptide insulins competed for specific 125I-insulin binding in a dose-dependent manner, whereas growth hormone, glucagon, and somatostatin did not. For porcine insulin, Scatchard analysis suggested multiple-affinity binding sites (high-affinity Ka = 4.92 X 10(8) M-1; low-affinity Ka = 0.95 X 10(7) M-1). After incubation with insulin (0.5 microM) for 2 h at 37 degrees C, increases above basal values of 254 +/- 23 and 189 +/- 34% for [3H]uridine uptake and incorporation, respectively, were observed. After incubation with insulin (0.5 microM) for 24 h at 37 degrees C, there were increases of 145 +/- 6% for [3H]thymidine uptake and 166 +/- 11% for thymidine incorporation. Basal and stimulated uridine and thymidine uptake and incorporation were inhibited by 50 microM dipyridamole. These studies confirm that mouse astrocytes in vitro possess specific insulin receptors and demonstrate an effect of insulin on pyrimidine nucleoside uptake and incorporation.     

Relationships of Plasma Leptin Levels to Changes in Plasma Free Fatty Acids in Women Who Are Lean and Women Who Are Abdominally Obese

HENNES, MAGDA MI, ARNAVAZ DUA, DIANA L MAAS, GABRIELE E SONNENBERG, GLENN R KRAKOWER, AHMED H KISSEBAH. Relationships of plasma leptin levels to changes in plasma free fatty acids in women who are lean and women who are abdominally obese. Regulation of leptin production by the hormonal and metabolic milieu is poorly understood. Because abdominal obesity is commonly associated with elevated plasma free fatty acid (FFA) flux, we examined the effects of augmenting FFA on plasma leptin levels in women who were lean and of suppressing FFA in women with abdominal obesity. In study 1, nine subjects who were lean, after a 12-hour overnight fast, received either intravenous saline or Intralipid plus heparin to increase the plasma FFA concentration to approximately 1000 μmol/ L. After 3 hours of additional fasting, subjects underwent 3-hour hyperglycemic clamps. In study 2, seven subjects with abdominal obesity were evaluated by a similar protocol, but lipolysis and plasma FFA flux were instead maximally suppressed by acipimox. In the individuals who were lean, leptin levels were unchanged during clamping. Increasing plasma FFA reduced plasma leptin from 7.66 ± 0.66 to 7.05 ±0 0.66 (p=0.03), but 3 hours of hyperglycemia plus hyperinsulinemia had no additional effect on leptin levels (7.15 ± 0.71). Basal leptin levels, 4-fold higher in the subjects with obesity, were reduced from 34.6 ± 2.4 μg/L to 32.3 ± 1.1 μg/L (p=0.004) during the clamp period. When plasma FFA flux was suppressed, however, plasma leptin levels after clamped hyperglycemia/hyperinsulinemia were increased to 38.9 ± 1.2 μg/L (p=0.014 vs. time 0 and 0.001 vs. saline protocol). Changes in leptin concentrations are not correlated with changes in FFA. These results suggest that plasma FFA concentration does not regulate plasma leptin levels in basal, extended fasting, or hyperglycemic/hyperinsulinemic states.