Pulsatile secretion of bioactive luteinizing hormone in adult male rhesus macaques: acute and chronic effects of orchidectomy

Glycosaminoglycans (GAGs) were purified from bovine follicular fluid, and their effectiveness to compete for heparin-binding sites in granulosa cells was evaluated. The GAGs dermatan sulfate (DS) and heparan sulfate (HS) were purified by anion-exchange high-performance liquid chromatography. Approximately 5 micrograms of protein from suspensions of bovine granulosa cells were incubated with 101 pmoles of [3H]heparin and 0.01-5.0 mg/ml of HS or DS for 2 h at 37 degrees C in 40 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.35. Heparan sulfate obtained from small and medium follicles displaced [3H]heparin in a dose-dependent manner from 0.1 to 5 mg/ml, but HS from large follicles did not displace [3H]heparin. The DS obtained from small, medium, and large follicles displaced [3H]heparin in a dose-dependent manner, and the potency of the DS to displace [3H]heparin increased as the size of the follicles from which the DS was purified increased. Those results were independent of the maturational state of the granulosa cells. In a separate experiment, heparin (17.1% sulfate) was N-desulfated (11.8%), and the desulfated heparin did not displace [3H]heparin. It was concluded that the effectiveness of follicular HS and DS to compete for heparin-binding sites on granulosa cells was dependent on the maturation of the follicle from which the fluid was obtained rather than on the source of granulosa cells. The binding interaction of the GAGs relies, to some extent, on the presence and positions of sulfate moieties.     

Identification of common and specific growth factor binding sites in heparan sulfate proteoglycans

The structural complexity within heparan sulfate has suggested that it contains multiple protein-specific binding sites. To evaluate the selectivity of growth factor binding to heparan sulfate, we conducted a detailed study of the intercompetition of fibroblast growth factor-2 (FGF-2) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) binding to heparan sulfate (HS) on bovine aortic smooth muscle cells. Radioligand binding assays were conducted, and an analytical method was developed for determining the apparent binding constants and numbers of specific and shared binding sites within HS. These studies revealed the presence of two general classes of HS-binding sites for FGF-2 and HB-EGF. The major class (approximately 10(6) sites per cell) was able to bind to either growth factor with relatively low affinity (K(d) = 12 and 44 nM for FGF-2 and HB-EGF, respectively) and was termed "common" binding sites. However, both FGF-2 and HB-EGF also showed specific high affinity (0.6 and 6.1 nM for FGF-2 and HB-EGF, respectively) binding to a minor subset (118,000 and 28,000 sites per cell for FGF-2 and HB-EGF, respectively) of "unique" binding sites, which were unable to bind the other growth factor. These studies indicate that growth factor binding to HS involves multiple binding sites of variable affinity, density, and selectivity. The approach outlined in this study could be applied to aid in the evaluation of the relative biological roles of these selective and nonselective growth factor binding domains within HS.     

Phosphate transport and proteins with SH groups in rat liver mitochondria

Phosphate transport in rat liver mitochondria was studied by following [32P] phosphate uptake within physiological concentrations. Transport inhibition due to mersalyl and protection by mersalyl against N-ethylmaleimide measured in those conditions corresponded to earlier results obtained by the swelling technique. When mitochondria were incubated with [3H] N-ethylmaleimide in the presence of mersalyl, the radioactive labeling in proteins of particles obtained after sonication was decreased in all fractions, but three proteins were both highly alkylated and also highly protected by mersalyl (M.W. 48,000 - 36,000 - 31,000). Two of these (M.W. 36,000 and 31,000) were partially purified by ultrogel chromatography in the presence of sodium dodecyl sulfate. Furthermore, it was shown that both phosphate and nigericin diminished labeling by N-ethylmaleimide in the final supernatant fraction. Two proteins (M.W. 98,000 and 31,000) were significantly alkylated by [3H] N-ethylmaleimide and protected by phosphate and nigericin.     

Photoaffinity Labeling of Benzodiazepine Receptor Proteins with the Partial Inverse Agonist [3H]Ro 15–4513: A Biochemical and Autoradiographic Study

Photolabeling of the benzodiazepine receptor, which to date has been done with benzodiazepine agonists such as flunitrazepam, can also be achieved with Ro 15-4513, a partial inverse agonist of the benzodiazepine receptor. [3H]Ro 15-4513 specifically and irreversibly labeled a protein with an apparent molecular weight of 51,000 (P51) in cerebellum and at least two proteins with apparent molecular weights of 51,000 (P51) and 55,000 (P55) in hippocampus. Photolabeling was inhibited by 10 microM diazepam but not by 10 microM Ro 5-4864. The BZ1 receptor-selective ligands CL 218872 and beta-carboline-3-carboxylate ethyl ester preferentially inhibited irreversible binding of [3H]Ro 15-4513 to protein P51. Not only these biochemical results but also the distribution and density of [3H]Ro 15-4513 binding sites in rat brain sections were similar to the findings with [3H]flunitrazepam. Thus, the binding sites for agonists and inverse agonists appear to be located on the same proteins. In contrast, whereas [3H]flunitrazepam is known to label only 25% of the benzodiazepine binding sites in brain membranes, all binding sites are photolabeled by [3H]Ro 15-4513. Thus, all benzodiazepine receptor sites are associated with photolabeled proteins with apparent molecular weights of 51,000 and/or 55,000. In cerebellum, an additional protein (MW 57,000) unrelated to the benzodiazepine receptor was labeled by [3H]Ro 15-4513 but not by [3H]flunitrazepam. In brain sections, this component contributed to higher labeling by [3H]Ro 15-4513 in the granular than the molecular layer.     

Characterization of photoaffinity labeling of benzodiazepine binding sites

The specific photoaffinity labeling of membrane-bound and detergent-solubilized benzodiazepine binding sites has been investigated using UV irradiated [3H] flunitrazepam as a photochemical probe. The time course and the regional and pharmacological specificity of the photolabeling reaction has been determined for "brain-specific" benzodiazepine binding sites; "peripheral-type" binding sites treated in an identical manner were not specifically labeled. Comparison of the number of sites labeled and blocked by [3H]flunitrazepam photolabeling of detergent-solubilized preparations indicated that about one site was blocked and unavailable for reversible binding for each site photolabeled. In contrast, when membrane-bound sites were photolabeled, about four sites were inactivated for each site photolabeled. Examination of photolabeled binding sites from various brain regions including cortex, striatum, and hippocampus using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave only a single labeled band of apparent Mr = 48,000.     

CD44 isoforms containing exon V3 are responsible for the presentation of heparin-binding growth factor

Glycosaminoglycan-modified isoforms of CD44 have been implicated in growth factor presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding growth factors, basic-fibroblast growth factor (b-FGF), heparin binding-epidermal growth factor (HB-EGF), and amphiregulin, showed that the HS-modified CD44 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS- modified CD44 immunoprecipitated from keratinocytes, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS- modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding growth factors to leukocytes during an inflammatory response. PCR and antibody-binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. Keratinocytes, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 35SO4(-2)-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS-binding proteins. Furthermore, it is probable that HS- modified CD44 is involved in the presentation of HS-binding proteins by keratinocytes in inflamed skin. However, our data suggests that CD44 is not likely to be the proteoglycan principally involved in presenting HS- binding growth factors to leukocytes on the vascular cell wall.     

Transport of fibroblast growth factor 2 in the pericellular matrix is controlled by the spatial distribution of its binding sites in heparan sulfate

The heparan sulfate (HS) chains of proteoglycans are a key regulatory component of the extracellular matrices of animal cells, including the pericellular matrix around the plasma membrane. In these matrices they regulate transport, gradient formation, and effector functions of over 400 proteins central to cell communication. HS from different matrices differs in its selectivity for its protein partners. However, there has been no direct test of how HS in the matrix regulates the transport of its partner proteins. We address this issue by single molecule imaging and tracking in fibroblast pericellular matrix of fibroblast growth factor 2 (FGF2), stoichiometrically labelled with small gold nanoparticles. Transmission electron microscopy and photothermal heterodyne imaging (PHI) show that the spatial distribution of the HS-binding sites for FGF2 in the pericellular matrix is heterogeneous over length scales ranging from 22 nm to several μm. Tracking of individual FGF2 by PHI in the pericellular matrix of living cells demonstrates that they undergo five distinct types of motion. They spend much of their time in confined motion (～110 nm diameter), but they are not trapped and can escape by simple diffusion, which may be slow, fast, or directed. These substantial translocations (μm) cover distances far greater than the length of a single HS chain. Similar molecular motion persists in fixed cells, where the movement of membrane PGs is impeded. We conclude that FGF2 moves within the pericellular matrix by translocating from one HS-binding site to another. The binding sites on HS chains form non-random, heterogeneous networks. These promote FGF2 confinement or substantial translocation depending on their spatial organisation. We propose that this spatial organisation, coupled to the relative selectivity and the availability of HS-binding sites, determines the transport of FGF2 in matrices. Similar mechanisms are likely to underpin the movement of many other HS-binding effectors.     

Mapping general anesthetic binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [³H]TDBzl-etomidate, a photoreactive etomidate analogue

The γ-aminobutyric acid type A receptor (GABA(A)R) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABA(A)Rs purified from bovine brain, [3H]azietomidate photolabeling of αMet-236 and βMet-286 in the αM1 and βM3 transmembrane helices identified an etomidate binding site in the GABA(A)R transmembrane domain at the interface between the β and α subunits [Li, G. D., et.al. (2006) J. Neurosci. 26, 11599-11605]. To further define GABA(A)R etomidate binding sites, we now use [3H]TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-α1β3 GABA(A)Rs and more extensively identify photolabeled GABA(A)R amino acids. [3H]TDBzl-etomidate photolabeled in an etomidate-inhibitable manner β3Val-290, in the β3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [3H]azietomidate, while no photolabeling of amino acids in the αM2 and βM2 helices that also border the etomidate binding site was detected. The location of these photolabeled amino acids in GABA(A)R homology models derived from the recently determined structures of prokaryote (GLIC) or invertebrate (GluCl) homologues and the results of computational docking studies predict the orientation of [3H]TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA(A)R intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of β3Met-227 in βM1 by [3H]TDBzl-etomidate and [3H]azietomidate also provides evidence of a homologous etomidate binding site at the β3-β3 subunit interface in the α1β3 GABA(A)R.     

Cooperation of binding sites at the hydrophilic domain of cell-surface sulfatase Sulf1 allows for dynamic interaction of the enzyme with its substrate heparan sulfate

Background

Sulf1 is a cell-surface sulfatase removing internal 6-O-sulfate groups from heparan sulfate (HS) chains. Thereby it modulates the activity of HS-dependent growth factors. For HS interaction Sulf1 employs a unique hydrophilic domain (HD).

Methods

Affinity-chromatography, AFM-single-molecule force spectroscopy (SMFS) and immunofluorescence on living cells were used to analyze specificity, kinetics and structural basis of this interaction.

Results

Full-length Sulf1 interacts broadly with sulfated glycosaminoglycans (GAGs) showing, however, higher affinity toward HS and heparin than toward chondroitin sulfate or dermatan sulfate. Strong interaction depends on the presence of Sulf1-substrate groups, as Sulf1 bound significantly weaker to HS after enzymatic 6-O-desulfation by Sulf1 pretreatment, hence suggesting autoregulation of Sulf1/substrate association. In contrast, HD alone exhibited outstanding specificity toward HS and did not interact with chondroitin sulfate, dermatan sulfate or 6-O-desulfated HS. Dynamic SMFS revealed an off-rate of 0.04/s, i.e., ~ 500-fold higher than determined by surface plasmon resonance. SMFS allowed resolving the dynamics of single dissociation events in each force–distance curve. HD subdomain constructs revealed heparin interaction sites in the inner and C-terminal regions of HD.

Conclusions

Specific substrate binding of Sulf1 is mediated by HD and involves at least two separate HS-binding sites. Surface plasmon resonance K_D-values reflect a high avidity resulting from multivalent HD/heparin interaction. While this ensures stable cell–surface HS association, the dynamic cooperation of binding sites at HD and also the catalytic domain enables processive action of Sulf1 along or across HS chains.

General significance

HD confers a novel and highly dynamic mode of protein interaction with HS.     

A focused sulfated glycoconjugate Ugi library for probing heparan sulfate-binding angiogenic growth factors

A library of small molecule heparan sulfate (HS) mimetics was synthesized by employing the Ugi four-component condensation of d-mannopyranoside-derived isocyanides with formaldehyde as the carbonyl component and a selection of carboxylic acids and amines, followed by sulfonation. The library was used to probe the subtle differences surrounding the ionic binding sites of three HS-binding angiogenic growth factors (FGF-1, FGF-2 and VEGF). Each compound features 3 or 4 sulfo groups which serve to anchor the ligand to the HS-binding site of the protein, with a diverse array of functionality in place extending from C-1 or C-6 to probe for adjacent favorable binding interactions. Selectivity of binding to these proteins was clearly observed and supported by molecular docking calculations.     

Herpes simplex virus types 1 and 2 differ in their interaction with heparan sulfate

Cell surface heparan sulfate (HS) serves as an initial receptor for many different viruses, including herpes simplex virus types 1 and 2 (HSV-1 and 2, respectively). Glycoproteins C and B (gC and gB) are the major components of the viral envelope that mediate binding to HS. In this study, purified gB and gC homologous proteins as well as purified HSV-1 and HSV-2 virions were compared for the ability to bind isolated HS receptor molecules. HSV-1 gC and HSV-2 gC bound comparable amounts of HS. Similarly, HSV-1 gB and its HSV-2 counterpart showed no difference in the HS-binding capabilities. Despite the similar HS-binding potentials of gB and gC homologs, HSV-1 virions bound more HS than HSV-2 particles. Purified gC and gB proteins differed with respect to sensitivity of their interaction with HS to increased concentrations of sodium chloride in the order gB-2 > gB-1 > gC-1 > gC-2. The corresponding pattern for binding of whole HSV virions to cells in the presence of increased ionic strength of the medium was HSV-2 gC-neg1 > HSV-1 gC(-)39 > HSV-1 KOS 321 > HSV-2 333. These results relate the HS-binding activities of individual glycoproteins with the cell-binding abilities of whole virus particles. In addition, these data suggest a greater contribution of electrostatic forces for binding of gB proteins and gC-negative mutants compared with binding of gC homologs and wild-type HSV strains. Binding of wild-type HSV-2 virions was the least sensitive to increased ionic strength of the medium, suggesting that the less extensive binding of HS molecules by HSV-2 than by HSV-1 can be compensated for by a relatively weak contribution of electrostatic forces to the binding. Furthermore, gB and gC homologs exhibited different patterns of sensitivity of binding to cells to inhibition with selectively N-, 2-O-, and 6-O-desulfated heparin compounds. The O-sulfate groups of heparin were found to be more important for interaction with gB-1 than gB-2. These results indicate that HSV-1 and HSV-2 differ in their interaction with HS.     

Expression of externally-disposed heparin/heparan sulfate binding sites by uterine epithelial cells

A class of high-affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time-dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]-Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 x 10(6) sites per cell. Approximately 9-14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo-uterine and epithelial-basal lamina interactions.     

Dendritic cell internalization of foot-and-mouth disease virus: influence of heparan sulfate binding on virus uptake and induction of the immune response

Dendritic cells (DC), which are essential for inducing and regulating immune defenses and responses, represent the critical target for vaccines against pathogens such as foot-and-mouth disease virus (FMDV). Although it is clear that FMDV enters epithelial cells via integrins, little is known about FMDV interaction with DC. Accordingly, DC internalization of FMDV antigen was analyzed by comparing vaccine virus dominated by heparan sulfate (HS)-binding variants with FMDV lacking HS-binding capacity. The internalization was most efficient with the HS-binding virus, employing diverse endocytic pathways. Moreover, internalization relied primarily on HS binding. Uptake of non-HS-binding virus by DC was considerably less efficient, so much so that it was often difficult to detect virus interacting with the DC. The HS-binding FMDV replicated in DC, albeit transiently, which was demonstrable by its sensitivity to cycloheximide treatment and the short duration of infectious virus production. There was no evidence that the non-HS-binding virus replicated in the DC. These observations on virus replication may be explained by the activities of viral RNA in the DC. When DC were transfected with infectious RNA, only 1% of the translated viral proteins were detected. Nevertheless, the transfected cells, and DC which had internalized live virus, did present antigen to lymphocytes, inducing an FMDV-specific immunoglobulin G response. These results demonstrate that DC internalization of FMDV is most efficient for vaccine virus with HS-binding capacity, but HS binding is not an exclusive requirement. Both non-HS-binding virus and infectious RNA interacting with DC induce specific immune responses, albeit less efficiently than HS-binding virus.     

Granule-associated serine neutral proteases of the mouse bone marrow-derived mast cell that degrade fibronectin: their increase after sodium butyrate treatment of the cells

Mouse bone marrow-derived mast cells, differentiated in vitro with concanavalin A splenocyte-conditioned medium and sensitized with monoclonal IgE, release neutral serine proteases after activation with specific antigen. Sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis of the supernatants from immunologically activated mast cells revealed the presence of four prominent proteins of 27,000, 29,000, 30,000 and 31,000 m.w. When the supernatants and sonicated residual cells from antigen-challenged or nonactivated IgE-sensitized mast cells were incubated with [3H]diisopropylfluorophosphate ([3H]DFP) and the proteins were subjected to SDS-PAGE followed by autoradiography, proteins of 27,000 to 31,000 m.w. were labeled with [3H]DFP. The antigen-dependent release of labeled proteins was accompanied by a corresponding depletion of similarly sized [3H]DFP-labeled proteins from these cell pellets relative to unactivated cells. The SDS gels were also stained with Coomassie Blue and were sectioned to separate the individual proteins for measurement of their incorporated radioactivity; the net percent antigen-dependent release of all four [3H]DFP-labeled proteins ranged from 64 to 68% and was comparable to that of the secretory granule markers, beta-hexosaminidase and histamine. That the [3H]DFP-labeled proteins were derived from the secretory granules of the cells was supported by studies in which mast cells were cultured for 4 days in the presence of 1 mM sodium butyrate. This treatment produced a differential increase in their cellular content of histamine (10-fold), [3H]DFP binding proteins (two- to fourfold), and beta-hexosaminidase (minimally), while the net percent antigen-dependent release of each of these constituents was unchanged. After sensitization and antigen activation, the net percent release of histamine, beta-hexosaminidase, and the four [3H]DFP-labeled proteins was 51, 59, and 53 to 61%, respectively, for sodium butyrate-treated cells, and 53, 60, and 64 to 68%, respectively, for cells not exposed to sodium butyrate. Human plasma fibronectin was used as a substrate to demonstrate that the exocytosed proteins possessed proteolytic activity. As assessed by optical density scanning of stained SDS-PAGE gels of the substrate, the proteases present in the supernatants of antigen-activated cells, but not of sensitized unchallenged cells, rapidly degraded native fibronectin at pH 7.0. This degradation was prevented by pretreatment of the exocytosed proteins from immunologically activated cells for 90 min at 37 degrees C with 2 mM DFP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Clearance, metabolic fate and tissue distribution of an injected bolus of photoaffinity-labeled rat androgen binding protein

An injected bolus of tritiated photolabeled rat androgen binding protein (ABP) is cleared from the circulation in a biphasic manner. The rapid component of the clearance curve indicates a clearance half time (t1/2) of 4.24 +/- 0.20 h and the slow component indicates a t1/2 of 13.27 +/- 2.92 h. After injection, photolabeled ABP is rapidly degraded as determined by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. This method dissociates ABP into subunits of approximately 48,000 daltons. One-half hour after injection of photolabeled ABP, 90% of the radioactivity migrates as a 20,000- rather than as a 48,000-dalton moiety; at the 24-h point only 20,000- and 16,000-dalton species were detectable. Chromatography of plasma on Sephadex G-150 indicated that, under nondenaturing conditions, radioactivity was still associated with proteins having a molecular weight similar to the native ABP dimer. These results suggest that the dimer is substrate for the proteolytic processes affecting ABP. Small (congruent to 7000 dalton) fragments of ABP could be detected in urine by G-150 chromatography 2 h after injection. The control protein [3H] bovine serum albumin (BSA) was cleared from the blood with a time course identical to that of photolabeled ABP (t1/2's 4.69 +/- 0.09 h and 13.56 +/- 1.03 h). [3H] BSA was degraded to peptides that were similar in size to those formed from photolabeled ABP. The uptake of photolabeled ABP and [3H] BSA by potential target tissues (testis, epididymis and ventral prostate) was equivalent, suggesting that the uptake of ABP was a nonspecific phenomenon rather than a receptor-mediated event. The liver and kidneys contained more radioactivity that did the other tissues and there was a differential uptake and clearance of photolabeled ABP and [3H] BSA by these organs. Whether this difference is related to specific uptake processes or to differences in the size and/or structure of the proteins or other factors remains to be determined.     

Light microscopic autoradiographic localization of [3H]pirenzepine and [3H](-)quinuclidinyl benzilate binding in human stellate ganglia

We have utilized the LKB Ultrofilm method of autoradiography to anatomically localize putative M1 and M2 muscarinic receptor subtypes in human stellate ganglia. Ten micron sections were labeled in vitro with either 1 nM of the classical antagonist [3H](-)quinuclidinyl benzilate ([3H](-)QNB) or 20 nM of the non-classical antagonist [3H]pirenzepine ([3H]PZ), using 1 microM atropine sulfate to define non-specific binding for both ligands. Our results indicate that [3H](-)QNB and [3H]PZ binding sites are distributed within the principal ganglion cells and nerve bundles.     

Photoaffinity labeling of serotonin-binding proteins

A photosensitive arylazide derivative of serotonin (nitroaryl-azidophenyl serotonin, NAP-serotonin) has been synthesized for use in studying the biochemical nature of serotonin binding sites. [³H]-NAP-serotonin possesses a similar ability to bind to the crude membranes of rat brains does [³H]-serotonin and therefore seems suitable for use as a photoaffinity labeling probe for serotonin binding sites. Upon irradiation with ultraviolet light, [³H]-NAP-serotonin covalently attaches to protein components of the brain homogenate. Several distinct radioactively labeled proteins have been separated by sodium dodecyl sulfate polyacrylamide gel electro-phoresis. Their apparent molecular weights were 80,000, 49,000, and 38,000 (±5%). When 1 μM of unlabeled serotonin or d-lysergic acid diethylamide (d-LSD) was added prior to photolysis, the incorporation of [³H]-NAP-serotonin into these proteins was inhibited significantly. No inhibitory effect was observed when dopamine was used. These observations suggest that the photoaffinity labeled proteins are specific for serotonin binding.     

Characterization of the proteins from Melanoplus bivittatus that bind juvenile hormone, and a refined EFDA photolabeling technique.

1. Juvenile hormone (JH) is specifically bound by a protein from hemolymph and fat body cytosol of the grasshopper, Melanoplus bivittatus. 2. This protein has a native molecular weight of 331,000 and subunits of 77,000. 3. Proteins that bind JH were covalently photolabeled with a JH analog, epoxyfarnesyl diazoacetate (EFDA). Samples were irradiated in spot plates and hydroxyapatite was used to separate bound from free [3H]EFDA. Differential solubilization was used to extract unlinked [3H]EFDA and solubilize [3H]EFDA linked to protein. 4. Hemolymph proteins of M(r) 479,000, 240,000 and 77,000 also bound [3H]EFDA. 5. Proteins that bound [3H]EFDA were not vitellogenins.     

Effects of inhibition of N-linked glycosylation by tunicamycin on nucleoside transport polypeptides of L1210 leukemia cells

Membrane polypeptides (relative mass (Mr) 48,000--55,000) associated with the equilibrative transport of nucleosides were identified in cultured murine leukemia (L1210/C2) cells by site-specific photolabeling with [3H]nitrobenzylthioinosine ([3H]NBMPR). Growth of cells in the presence of tunicamycin resulted in the gradual conversion of 3H-labeled polypeptides to a form that migrated more rapidly (Mr 42,000--47,000) during sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis. When plasma membrane fractions were photolabeled and incubated with O-glycanase or endoglycosidase F, the [3H]NBMPR-labeled polypeptides migrated in SDS-polyacrylamide gels with the same mobility as native NBMPR-binding polypeptides, whereas incubation with either N-glycanase or trifluoromethane sulfonic acid converted [3H]NBMPR-labeled polypeptides to the more rapidly migrating form (Mr 41,000--48,000). These observations are consistent with the presence of N-linked oligosaccharides of the complex type on the NBMPR-binding polypeptides of L1210/C2 cells. Tunicamycin exposures that reduced incorporation of [3H]mannose into plasma membrane fractions by greater than 95% had little, if any, effect on either the affinity (Kd values, 0.1-0.2 nM) or abundance (Bmax values, 200,000--220,000 sites/cell) of NBMPR-binding sites, whereas uridine transport kinetics at 37 degrees C were altered in a complex way. Thus, although N-linked glycosylation is not required for insertion of the NBMPR-binding protein into the plasma membrane or for interaction of NBMPR with the high-affinity binding sites, it is important for function of at least one of the three nucleoside transporters expressed by L1210/C2 cells.     

Metabolic labeling of glycosylphosphatidylinositol-anchor of heparan sulfate proteoglycans in rat ovarian granulosa cells.

The glycosylphosphatidylinositol (GPI)-anchor of the plasma membrane-associated heparan sulfate (HS) proteoglycan was metabolically radiolabeled with [3H]myristic acid, [3H]palmitic acid, [3H]inositol, [3H]ethanolamine, or [32P]phosphate in rat ovarian granulosa cell culture. Cell cultures labeled with [3H]myristic acid or [3H]palmitic acid were extracted with 4 M guanidine HCl buffer containing 2% Triton X-100 and the proteoglycans were purified by ion exchange chromatography after extensive delipidation. Specific incorporation of 3H into GPI-anchor was demonstrated by removing the label with a phosphatidylinositol-specific phospholipase C (PI-PLC). Incorporation of 3H activity into glycosaminoglycans and core glycoproteins was also demonstrated. However, the specific activity of 3H in these structures was approximately 2 orders of magnitude lower than that in the GPI-anchor, suggesting that 3H label was the result of the metabolic utilization of catabolic products of the 3H-labeled fatty acids. PI-PLC treatment of cell cultures metabolically labeled with [3H]inositol, [3H]ethanolamine, or [32P]phosphate specifically released radiolabeled cell surface-associated HS proteoglycans indicating the presence of GPI-anchor in these proteoglycans. GPI-anchored HS proteoglycans accounted for 20-30% of the total cell surface-associated HS proteoglycans and virtually all of them were removed by PI-PLC. These results further substantiate the presence of GPI-anchored heparan sulfate proteoglycan in ovarian granulosa cells and its cell surface localization.