Characterization of Luteinizing Hormone and Luteinizing Hormone Receptor and Their Indispensable Role in the Ovulatory Process of the Medaka

The molecular properties and roles of luteinizing hormone (Lh) and its receptor (Lhcgrbb) have not been studied for the medaka (Oryzias latipes), which is an excellent animal model for ovulation studies. Here, we characterized the medaka Lh/Lhcgrbb system, with attention to its involvement in the ovulatory process of this teleost fish. In the medaka ovary, follicle-stimulating hormone receptor mRNA was expressed in small and medium-sized follicles, while lhcgrbb mRNA was expressed in the follicle layers of all growing follicles. Experiments using HEK 293T cells expressing medaka Lhcgrbb in vitro revealed that gonadotropin from pregnant mare’s serum and medaka recombinant Lh (rLh) bound to the fish Lhcgrbb. The fish gonadotropin subunits Gtha, Fshb, and Lhb were essentially expressed at fairly constant levels in the pituitary of the fish during a 24-h spawning cycle. Using medaka rLh, we developed a follicle culture system that allowed us to follow the whole process of oocyte maturation and ovulation in vitro. This follicle culture method enabled us to determine that the Lh surge for the preovulatory follicle occurred in vivo between 19 and 15 h before ovulation. The present study also showed that oocyte maturation and ovulation were delayed several hours in vitro compared with in vivo. Treatment of large follicles with medaka rLh in vitro significantly increased the expression of Mmp15, which was previously demonstrated to be crucial for ovulation in the fish. These findings demonstrate that Lh/Lhcgrbb is critically involved in the induction of oocyte maturation and ovulation.     

Polyphosphoinositides Are Enriched in Plant Membrane Rafts and Form Microdomains in the Plasma Membrane

In this article, we analyzed the lipid composition of detergent-insoluble membranes (DIMs) purified from tobacco (Nicotiana tabacum) plasma membrane (PM), focusing on polyphosphoinositides, lipids known to be involved in various signal transduction events. Polyphosphoinositides were enriched in DIMs compared with whole PM, whereas all structural phospholipids were largely depleted from this fraction. Fatty acid composition analyses suggest that enrichment of polyphosphoinositides in DIMs is accompanied by their association with more saturated fatty acids. Using an immunogold-electron microscopy strategy, we were able to visualize domains of phosphatidylinositol 4,5-bisphosphate in the plane of the PM, with 60% of the epitope found in clusters of approximately 25 nm in diameter and 40% randomly distributed at the surface of the PM. Interestingly, the phosphatidylinositol 4,5-bisphosphate cluster formation was not significantly sensitive to sterol depletion induced by methyl-β-cyclodextrin. Finally, we measured the activities of various enzymes of polyphosphoinositide metabolism in DIMs and PM and showed that these activities are present in the DIM fraction but not enriched. The putative role of plant membrane rafts as signaling membrane domains or membrane-docking platforms is discussed.Polyphosphoinositides are phosphorylated derivatives of phosphatidylinositol (PtdIns) implicated in many aspects of cell function. They control a surprisingly large number of processes in animal, yeast, and plant cells, including exocytosis, endocytosis, cytoskeletal adhesion, and signal transduction not only as second-messenger precursors but also as signaling molecules on their own by interacting with protein partners, allowing spatially selective regulation at the cytoplasm-membrane interface (for review, see Di Paolo and De Camilli, 2006). Polyphosphoinositides also control the activity of ion transporters and channels during biosynthesis or vesicle trafficking (Liu et al., 2005; Monteiro et al., 2005b). In plants, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] is present in very small quantities (for review, see Stevenson et al., 2000; Meijer and Munnik, 2003) and was visualized in vivo by expressing a fluorescent protein (GFP or yellow fluorescent protein) fused to the pleckstrin homology (PH) domain of the human phospholipase C δ1 (PLCδ1) that specifically binds PtdIns(4,5)P₂. The fused protein yellow fluorescent protein-PHPLCδ1 was present in the cytoplasm but concentrated at the plant plasma membrane (PM) in response to salt stress or upon treatment with the PLC inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (van Leeuwen et al., 2007). In pollen tubes and root hairs, where spatially focused cell expansion occurs, highly localized PtdIns(4,5)P₂ has been evidenced at the membrane tip (Braun et al., 1999; Kost et al., 1999). PtdIns(4,5)P₂ likely functions as an effector of small G proteins at the apex of cells influencing membrane fusion events (Monteiro et al., 2005a). In guard cells, the level of PtdIns(4,5)P₂ increases at the PM upon illumination (Lee et al., 2007). Combining imaging, patch clamp, and genetic evidence, Lee et al. (2007) further proposed that PtdIns(4,5)P₂ is important for stomatal opening. Stomatal guard cells have also been reported to contain phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidylinositol 4-phosphate (PtdIns4P), the products of PtdIns 3-kinase and PtdIns 4-kinase activities, respectively. Jung et al. (2002) demonstrated that PtdIns3P and PtdIns4P play an important role in the modulation of stomatal closing and that reductions in the levels of functional PtdIns3P and PtdIns4P enhance stomatal opening. Recently, the hyperosmotic stress response was studied in Arabidopsis (Arabidopsis thaliana). Several groups (Pical et al., 1999; DeWald et al., 2001; Konig et al., 2007, 2008b) have shown that plants exhibit a transient increase in polyphosphoinositides after hyperosmotic stress, providing a model for comparing constitutive and stress-inducible polyphosphoinositide pools. Under nonstress conditions, structural phospholipids and PtdIns contained 50 to 70 mol % polyunsaturated fatty acids (PUFA), whereas polyphosphoinositides were more saturated (10–20 mol % PUFA; Konig et al., 2007). Upon hyperosmotic stress, polyphosphoinositides with up to 70 mol % PUFA were formed that differed from constitutive species and coincided with a transient loss in unsaturated PtdIns. These patterns indicate the inducible turnover of an unsaturated PtdIns pool and the presence of distinct polyphosphoinositide pools in plant membranes (Konig et al., 2007).Since these biological phenomena are likely to occur in distinct regions of the PM, it has been our working hypothesis that in plant cells polyphosphoinositides are localized in various microdomains to participate in different cellular functions. Two decades ago, Metcalf et al. (1986) already suggested that the plant PM contains stable immiscible domains of fluid and gel-like lipids using fluorescent lipid and phospholipid probes incorporated into soybean (Glycine max) protoplasts prepared from cultured soybean cells. To this day, it has been generally accepted that lipids and proteins of the PM are not homogeneously distributed within membranes but rather form various domains of localized enrichment. The best-characterized membrane domains are membrane rafts (MRs; Pike, 2006). MRs are liquid-ordered subdomains within eukaryotic membranes that are hypothesized to play important roles in a variety of biological functions by coordinating and compartmentalizing diverse sets of proteins to facilitate signal transduction mechanisms, focal regulation of cytoskeleton, and membrane trafficking (for review, see Rajendran and Simons, 2005; Brown, 2006). Both evidenced in plants and animals, MRs are enriched in sphingolipids and sterols and largely deprived in phospholipids (for review, see Brown and London, 2000; Bhat and Panstruga, 2005). Sterols interact preferentially, although not exclusively, with sphingolipids due to their structure and the saturation of their hydrocarbon chains. Because of the rigid nature of the sterol group, sterols have the ability to pack in between the lipids in rafts, serving as molecular spacers and filling voids between associated sphingolipids (Binder et al., 2003). Acyl chains of MR lipids tend to be more rigid and in a less fluid state (Roche et al., 2008). In agreement, the hydrophobic chains of the phospholipids within the raft are more saturated and tightly packed than those of lipids in the surrounding bilayer (Mongrand et al., 2004). MRs can be isolated from PM by extraction with nonionic detergents such as Triton X-100 (TX100) or Brij-98 at low temperatures. Fluid nonraft domains will solubilize while the MRs remain intact and can be enriched after centrifugation, floating in a Suc density gradient. Floating purified fractions, therefore, are called detergent-insoluble membranes (DIMs) or detergent-resistant membranes and are thought to be the biochemical counterpart of in vivo MRs.In plants, a few results suggest the role in vivo of dynamic clustering of PM proteins, and they refer to plant-pathogen interaction. A cell biology study reported the pathogen-triggered focal accumulation of components of the plant defense pathway in the PM, a process reminiscent of MRs (Bhat et al., 2005). The proteomic analysis of tobacco (Nicotiana tabacum) DIMs led to the identification of 145 proteins, among which a high proportion were linked to signaling in response to biotic stress, cellular trafficking, and cell wall metabolism (Morel et al., 2006). Therefore, these domains are likely to constitute, as in animal cells, signaling membrane platforms concentrating lipids and proteins necessary for the generation of signaling molecules of physiological relevance. This hypothesis was confirmed by a quantitative proteomic study describing the dynamic association of proteins with DIMs upon challenge of tobacco cells with an elicitor of defense reaction (Stanislas et al., 2009). Recently, Raffaele et al. (2009) showed that a group of proteins specific to vascular plants, called remorins (REMs), share the biochemical properties of other MR proteins and are clustered into microdomains of approximately 70 nm in diameter in the PM and plasmodesmata in tobacco, providing a link between biochemistry (DIM purification) and imaging (membrane microdomain observation).Several investigators have previously suggested that PtdIns(4,5)P₂-rich raft assemblies exist in animal cell membranes to provide powerful organizational principles for tight spatial and temporal control of signaling in motility. Laux et al. (2000) demonstrated that PtdIns(4,5)P₂ formed microdomains in the PM of animal cells, and at least part of these microdomains was colocalized with the myristoylated Ala-rich type C kinase substrate, a protein enriched in MRs, and involved in the regulation of the actin cytoskeleton. The relationship between the spatial organization of PtdIns(4,5)P₂ microdomains and exocytotic machineries has been evidenced in rat. Both PtdIns(4,5)P₂ and syntaxin, a protein essential for exocytosis, exhibited punctate clusters in isolated PM. PtdIns(4,5)P₂ also accumulated at sites of cell surface motility together with a Rho-type GTPase. Therefore, PtdIns(4,5)P₂ may coordinate membrane dynamics and actin organization as well as integrate signaling (Aoyagi et al., 2005). These results provide evidence of compartmentalization of PtdIns(4,5)P₂-dependent signaling in cell membranes.Little is known in plants about whether and how separate pools of polyphosphoinositides come about and how they are regulated. In this article, we have analyzed the lipid composition of DIMs enriched from tobacco PM, with a particular focus on phospholipids involved in signaling events, such as polyphosphoinositides. We showed that polyphosphoinositides were enriched in DIMs, whereas structural phospholipids were largely excluded. We were able to calculate that almost half of the PtdInsP and PtdIns(4,5)P₂ were present in MR domains. Fatty acid composition analyses demonstrate that this enrichment is accompanied by the presence of more saturated fatty acids in polyphosphoinositides. Consistently, using an electron microscopy approach with immunogold labeling and a pattern-identifying statistical analysis, we showed that more than half of the PtdIns(4,5)P₂ labeling is clustered into microdomains of approximately 25 nm in diameter in the PM. Finally, we measured the activities of lipid-using enzymes present in DIMs/PM and showed that activities responsible for polyphosphoinositide metabolism are present in the DIM fraction.     

Plasma Luteinizing Hormone Response to Increasing doses of Synthetic Gonadotrophin-Releasing Hormone in Heifers

The dosenresponse relationship for a synthetic gonadotrophin-releasing hormone (GnRH) was studied in normally cycling heifers using the area under the luteinizing hormone (LH) curve as a response parameter. Oestrus was synchronized by an injection of 0.5 mg cloprostenol before the experiment started and after the 3rd treatment with GnRH. Treatment with GnRH as assigned in a Latin square included 5 dose levels (0, 10, 50, 100, 250 μg) and 5 treatment days over a period of 22 days. GnRH was capable of inducing an increase of plasma LH within 30 min after injection. Plasma LH response increased with increasing doses of GnRH, the largest increase being observed when the dose was raised from 50 μg to 100 μg. One heifer did not respond to any of the doses applied. The existence of an individual treshold dose of GnRH is suggested.     

Alterations in Detergent-Resistant Plasma Membrane Microdomains in Arabidopsis thaliana During Cold Acclimation

Microdomains in the plasma membrane (PM) have been proposedto be involved in many important cellular events in plant cells.To understand the role of PM microdomains in plant cold acclimation,we isolated the microdomains as detergent-resistant plasma membranefractions (DRMs) from Arabidopsis seedlings and compared lipidand protein compositions before and after cold acclimation.The DRM was enriched in sterols and glucocerebrosides, and theproportion of free sterols in the DRM increased after cold acclimation.The protein-to-lipid ratio in the DRM was greater than thatin the total PM fraction. The protein amount recovered in DRMsdecreased gradually during cold acclimation. Cold acclimationfurther resulted in quantitative changes in DRM protein profiles.Subsequent mass spectrometry and Western blot analyses revealedthat P-type H⁺-ATPases, aquaporins and endocytosis-related proteinsincreased and, conversely, tubulins, actins and V-type H⁺-ATPasesubunits decreased in DRMs during cold acclimation. Functionalcategorization of cold-responsive proteins in DRMs suggeststhat plant PM microdomains function as platforms of membranetransport, membrane trafficking and cytoskeleton interaction.These comprehensive changes in microdomains may be associatedwith cold acclimation of Arabidopsis.     

NMDA Receptors in the Medial Zona Incerta Stimulate Luteinizing Hormone and Prolactin Release

1. The aim of the present work is to demonstrate the interaction between the glutamatergic/NMDA and dopaminergic systems in the medial zona incerta on the control of luteinizing hormone and prolactin secretion and the influence of reproductive hormones. 2. Proestrus and ovariectomized rats were primed with estrogen and progesterone to induce high or low levels of luteinizing hormone and prolactin. 2-Amino-7-phosphonoheptanoic acid, an NMDA receptor antagonist, and dopamine were injected in the medial zona incerta. Blood samples were withdrawn every hour between 1,600 and 2,000 hours or 2,200 hours via intracardiac catheter from conscious rats. Additional groups of animals injected with the NMDA receptor antagonist were killed 1 or 4 h after injection. Dopamine and its metabolite 3,4-dihydroxyphenylacetic acid were measured in different hypothalamic regions. 3. 2-Amino-7-phosphonoheptanoic acid blocked the ovulatory luteinizing hormone surge in proestrus rats. 2-Amino-7-phosphonoheptanoic acid also blocked the increase in luteinizing hormone induced by ovarian hormones in ovariectomized rats, an effect that was partially reversed by dopamine injection. Conversely, the increased release of luteinizing hormone and prolactin induced by dopamine was prevented by 2-amino-7-phosphonoheptanoic acid. We found that the NMDA antagonist injection decreased the dopaminergic activity--as evaluated by the 3,4-dihydroxyphenylacetic acid/dopamine ratio--in the medio basal hypothalamus and increased in the preoptic area. 4. Our results show an stimulatory role of NMDA receptors on the ovulatory luteinizing hormone release and on luteinizing hormone release induced by sexual hormones and demonstrate that the stimulatory effect of dopamine on luteinizing hormone and prolactin is mediated by the NMDA receptors. These results suggest a close interaction between the glutamatergic and dopaminergic incertohypothalamic systems on the control of luteinizing hormone and prolactin release.     

大鼠脑突触质膜糖皮质激素受体的纯化

本文利用抗大鼠肝细胞内糖皮质激素受体的单克隆抗体制备的免疫亲和层析柱,将大鼠脑突触质膜糖皮质激素受体纯化了约1150倍,SDS聚丙烯酰胺簿层梯度凝胶电泳显示,在约67kD处有一较明显的染色条带。     

Plasma and Urinary Luteinizing Hormone Levels in the Diagnosis of Endocrine Disease

The diagnostic value of measurements of plasma and urinary luteinizing hormone (LH) has been studied in 209 patients with endocrine disease. In 44 patients puberty was either delayed or had failed to occur. In those with chromosomal abnormalities the LH levels were often within the normal range, whereas those with a pituitary cause usually had low levels. In boys with delayed puberty plasma LH levels rose before physical changes occurred and had prognostic value. In patients with later gonadal failure, men with impotence or infertility, and women with secondary amenorrhoea LH assays proved of little value, although in one case a premature menopause was suspected and six patients with anorexia nervosa had low LH levels.Sixty patients with disorders of the hypothalamicpituitary area were studied. Levels of LH were measured and considered in relation to the other anterior pituitary hormones. Impairment of LH secretion was one of the first effects on hormone production of disease affecting this area, and this was, of course, most readily detected in postmenopausal women.The normal ranges of both plasma and urine LH are wide and there seems to be considerable day-to-day variation, especially of urinary output. Several samples should, therefore, be measured if therapeutic decisions are involved.     

Effects of Alleles at the W Locus on Testicular Luteinizing Hormone Receptors in Adult Mice

Abstract

The autoregulation of testicular LH receptors was studied in W^x/W^v mice with germ cell aplasia and in normal (±/±) mice. To assess the effects of each individual allele, W^x/± and W^v/± mice were also examined. Basal testicular LH receptor concentration was higher in W^x/W^v mice than in all other genotypes, and higher in W^x/± than in ±/± mice. Twenty-four h after injection of 0.3 IU hCG/g bw, LH receptor concentration was decreased in ±/± and W^v/± mice, but not in W^x/W^v or W^x/± animals. Administration of hCG caused a significant increase in plasma testosterone levels in all genotypes. However, injection of the highest dose of hCG used (0.9 IU/g bw) caused a significantly greater elevation in plasma testosterone in W^x/W^v than in ±/± mice. Plasma gonadotropin levels were significantly higher in W^x/W^v mice than in all other genotypes. The present results indicate that the W^x allele is responsible for the changes in testicular function observed in W^x/W^v mice, and suggest that this allele may be involved in the genetic regulation of testicular LH receptors in the mouse.     

雌性哺乳动物促卵泡素受体与促黄体素受体的结构与表达

本文从雌性哺乳动物促卵泡素受体(FSHR)和促黄体素受体(LHR)的结构及在卵泡发育过程中的表达和功能加以综述,指出FSHR及LHR在卵泡的生长发育、优势化及排卵等方面具有重要的功能。     

H-Ras Distribution and Signaling in Plasma Membrane Microdomains Are Regulated by Acylation and Deacylation Events

H-Ras must adhere to the plasma membrane to be functional. This is accomplished by posttranslational modifications, including palmitoylation, a reversible process whereby H-Ras traffics between the plasma membrane and the Golgi complex. At the plasma membrane, H-Ras has been proposed to occupy distinct sublocations, depending on its activation status: lipid rafts/detergent-resistant membrane fractions when bound to GDP, diffusing to disordered membrane/soluble fractions in response to GTP loading. Herein, we demonstrate that H-Ras sublocalization is dictated by its degree of palmitoylation in a cell type-specific manner. Whereas H-Ras localizes to detergent-resistant membrane fractions in cells with low palmitoylation activity, it locates to soluble membrane fractions in lineages where it is highly palmitoylated. Interestingly, in both cases GTP loading results in H-Ras diffusing away from its original sublocalization. Moreover, tilting the equilibrium between palmitoylation and depalmitoylation processes can substantially alter H-Ras segregation and, subsequently, its biochemical and biological functions. Thus, the palmitoylation/depalmitoylation balance not only regulates H-Ras cycling between endomembranes and the plasma membrane but also serves as a key orchestrator of H-Ras lateral diffusion between different types of plasma membrane and thereby of H-Ras signaling.     

Association of Cellular Prion Protein with Gangliosides in Plasma Membrane Microdomains of Neural and Lymphocytic Cells

In this report we demonstrated that cellular prion protein is strictly associated with gangliosides in microdomains of neural and lymphocytic cells. We preliminarily investigated the protein distribution on the plasma membrane of human neuroblastoma cells, revealing the presence of large clusters. In order to evaluate its possible role in tyrosine signaling pathway triggered by GEM, we analyzed PrP^c presence in microdomains and its association with gangliosides, using cholera toxin as a marker of GEM in neuroblastoma cells and anti-GM3 MoAb for identification of GEM in lymphoblastoid cells. In neuroblastoma cells scanning confocal microscopical analysis revealed a consistent colocalization between PrP^c and GM1 despite an uneven distribution of both on the cell surface, indicating the existence of PrP^c-enriched microdomains. In lymphoblastoid T cells PrP^c molecules were mainly, but not exclusively, colocalized with GM3. In addition, PrP^c was present in the Triton-insoluble fractions, corresponding to GEM of cell plasma membrane. Additional evidence for a specific PrP^c-GM3 interaction in these cells was derived from the results of TLC analysis, showing that prion protein was associated with GM3 in PrP^c immunoprecipitates. The physical association of PrP^c with ganglioside GM3 within microdomains of lymphocytic cells strongly suggests a role for PrP^c-GM3 complex as a structural component of the multimolecular signaling complex involved in T cell activation and other dynamic lymphocytic plasma membrane functions.     

Caveolae Are Highly Immobile Plasma Membrane Microdomains, Which Are not Involved in Constitutive Endocytic Trafficking

To investigate whether caveolae are involved in constitutive endocytic trafficking, we expressed N- and C- terminally green fluorescent protein (GFP)-tagged caveolin- 1 fusion proteins in HeLa, A431, and Madin-Darby canine kidney cells. The fusion proteins were shown by immunogold labeling to be sorted correctly to caveolae. By using confocal microscopy and photobleaching techniques, it was found that although intracellular structures labeled with GFP-tagged caveolin were dynamic, GFP-labeled caveolae were very immobile. However, after incubation with methyl- beta-cyclodextrin, distinct caveolae disappeared and the mobility of GFP-tagged caveolin in the plasma membrane increased. Treatment of cells with cytochalasin D caused lateral movement and aggregation of GFP-labeled caveolae. Therefore, both cholesterol and an intact actin cytoskeleton are required for the integrity of GFP-labeled caveolae. Moreover, stimulation with okadaic acid caused increased mobility and internalization of the labeled caveolae. Although the calculated mobile fraction (for t = infinity) of intracellular, GFP-tagged caveolin- associated structures was 70-90%, GFP-labeled caveolae in unstimulated cells had a mobile fraction of <20%, a value comparable to that previously reported for E-cadherin in junctional complexes. We therefore conclude that caveolae are not involved in constitutive endocytosis but represent a highly stable plasma membrane compartment anchored by the actin cytoskeleton.     

Bicarbonate Induces Membrane Reorganization and CBR1 and TRPV1 Endocannabinoid Receptor Migration in Lipid Microdomains in Capacitating Boar Spermatozoa

Mammalian spermatozoa acquire full fertilizing ability only after a morphofunctional maturation called "capacitation." During this process the high level of bicarbonate present within the upper female genital tract or in culture medium induces a marked reorganization of sperm membranes characterized by a biphasic behavior: In a few minutes, it promotes membrane phospholipid scrambling preliminary to the apical translocation of sterol that, 2-4?h later, enables spermatozoa to recognize zona pellucida after albumin-mediated cholesterol extraction. In the present research it was demonstrated that spermatozoa incubated with bicarbonate in protein-free media underwent a marked reorganization of lipid microdomains present in a detergent-resistant membrane fraction (DRM) isolated by ultracentrifugation on sucrose density gradient. In fact, bicarbonate exposed sperm (ES) cells, compared with ejaculated spermatozoa (nonexposed sperm [nES] cells), displayed an increase in protein DRM content and, in particular, in Cav-1 and CD55, markers of caveolae and lipid rafts, as well in acrosin-2, a marker of the outer acrosomal membrane (OAM). Moreover, the amount of certain proteins involved in capacitation, such as the endocannabinoid system receptors cannabinoid receptor type 1 (CBR1) and transient receptor potential cation channel 1 (TRPV1), increased in DRM obtained from ES. These data allow us to hypothesize that sperm membrane reorganization takes place even in the absence of extracellular proteins; that not only the plasma membrane but also the OAM participate in this process; and that important molecules playing a key role in inside-out signaling, such as the endocannbinoid receptors TRPV1 and CBR1, are involved in this event, with potentially important consequences on sperm function.     

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H-ATPase: I. Characteristics and Intracellular Localization of the Fusicoccin Receptor in Microsomes from Radish Seedlings

The characteristics of fusicoccin binding were investigated in microsomes from 24-h-old radish (Raphanus sativus L.) seedlings. The time course of fusicoccin binding depended on fusicoccin concentration: equilibrium was reached much faster at 10 nanomolar fusicoccin than at 0.3 nanomolar fusicoccin. Scatchard analysis of equilibrium binding as a function of fusicoccin concentration indicated a single class of receptor sites with a K_d of 1.8 nanomolar and a site density of 6.3 picomoles per milligram protein. Similar values (K_d 1.7 nanomolar and site density 7 picomoles per milligram protein) were obtained from the analysis of the dependence of equilibrium binding on membrane concentration at fixed fusicoccin concentrations. Fusicoccin binding comigrated with the plasma membrane H⁺-ATPase in an equilibrium sucrose density gradient: both activities formed a sharp peak (1.18 grams per milliliter) clearly distinct from that of markers of other membranes which all peaked at lower densities. The saturation profiles of fusicoccin binding and of fusicoccin-induced activation of the plasma membrane H⁺-ATPase, measured under identical conditions, were similar, supporting the view that fusicoccin-induced activation of the plasma membrane H⁺-ATPase is mediated by fusicoccin binding to its plasma membrane receptor.     

Glycoprotein Receptors for Concanavalin A isolated from Pig Lymphocyte Plasma Membrane by Affinity Chromatography in Sodium Deoxycholate

CURRENT interest in the structure of cell plasma membranes arises partly from demonstrations that specific receptors for hormones (for example, insulin and glucagon¹), drugs and other biologically active molecules (for example, Phaseolus vulgaris phytohaemagglutinin², concanavalin A³ and acetylcholine^4,5) are frequently located at the cell surface. Isolation of these receptors has, however, been impeded by the insolubility of cell membranes in the more orthodox biochemical solvents, whereas more drastic solubilization procedures have usually destroyed biological activity⁶.     

The Effect of Metformin Therapy on Luteinizing Hormone Receptor Agonist-Mediated Stimulation of Testosterone Production and Spermatogenesis in Diabetic Rats

Journal of Evolutionary Biochemistry and Physiology - The functional restoration of the male reproductive system in type 2 diabetes mellitus (DM2) is one of the urgent global challenges in modern...     

Plasma Steroid and Luteinizing Hormone Levels during Prostaglandin F2α Administration in Luteal Phase of Menstrual Cycle

An intravenous infusion of prostaglandin F₂α (12.5-250μg/min) was administered in four volunteers in the mid-late luteal phase and three in the early luteal phase of the menstrual cycle.Frequent measurement of plasma progesterone, oestrogens, and luteinizing hormone (LH) showed that administration of high doses depressed plasma progesterone levels in the late luteal phase and caused concomitant side effects. Levels of progesterone in the early luteal phase were unaffected. In both phases oestrogen and LH levels were little altered. In two subjects, hourly progesterone levels measured throughout the day at a similar time in a subsequent control menstrual cycle showed an appreciable variation in one but steady levels in the second. This variation may contribute to the magnitude of the fall in progesterone noted during the infusion of prostaglandins.     

Plasma Membrane Receptors for Exopolysaccharides of the Ring Rot Causal Agent in Potato Cells

By means of differential centrifugation, microsomal fractions enriched in the plasma membrane were isolated from suspension cell cultures of two cultivars of potato (Solanum tuberosum L.) contrasting in their resistance to the causal agent of ring rot (Clavibacter michiganensis subsp. sepedonicus) (Cms). Electrophoresis of the fractions showed that they comprised a wide range of proteins from 15 to 75 kD. The protein bands were more brightly expressed in the microsomal membranes of the cells of susceptible cultivar. The proteins of 70 and 42 kD were present only in the cellular membranes of the resistant cultivar. In order to visualize the binding of exopolysaccharides (EPS) produced by Cms to the receptors of membrane fractions, a conjugate of EPS with a fluorescent marker was used. The membrane fraction isolated from the cells of the susceptible cultivar was found to be richer in receptors for EPS Cms than the membrane fraction from the resistant cultivar. It is supposed that numerous receptors for EPS present on the plasma membrane may partially account for potato susceptibility to Cms. These receptors may facilitate the binding of bacteria to the plant cells, the formation of colonies, and the development of the disease.