Solubilized Membrane Preparations Research Articles

Ethanol inhibits γ-secretase proteolytic activity in vascular smooth muscle cells.

Ethanol (EtOH) inhibits Notch-mediated vascular smooth muscle cell (SMC) proliferation, an event that is key in vessel remodeling and atherogenesis. The object of this study was to determine whether EtOH inhibits Notch signaling in SMC at the level of γ-secretase, a protease that in concert with α-secretase catalyzes the release of the intracellular domain of the Notch receptor necessary for signaling. Human coronary artery SMCs (HCASMCs) were treated with a recombinant soluble Notch ligand, Delta-like ligand 4 (DLL4) (2 μg/ml), or transfected with a constitutively active Notch 1 intracellular domain (N1ICD), in the absence or presence of EtOH. EtOH (25 mM) treatment inhibited DLL4-stimulated CBF-1/RBP-Jk-dependent promoter activity (determined by luciferase assay) and downstream target gene HRT-3 mRNA levels. In contrast, EtOH had no effect on N1ICD-driven CBF-1/RBP-Jk-dependent promoter activity or HRT-3 expression. These data suggest that EtOH inhibits Notch signaling at, or prior to, Notch intracellular domain (NICD) generation. γ-Secretase activity was determined in solubilized membrane preparations from HCASMC treated with/without EtOH (25 mM) or the γ-secretase inhibitor DAPT (20 μM) using (i) a fluorometric assay and (ii) Western blot detection of cleavage products using a Flag-tagged Notch-based substrate, N100Flag. EtOH inhibited basal and DLL4-stimulated γ-secretase activity, and SMC growth to a similar extent as DAPT, whereas it had no effect on α-secretase (TACE/ADAM17) activity also determined by fluorometric assay. Moreover, EtOH treatment inhibited the expression of caveolin-1, a lipid raft protein implicated in regulating γ-secretase activity, and altered its cellular distribution in HCASMC. EtOH inhibits Notch signaling in vascular SMCs at the level of γ-secretase activity, possibly by affecting lipid raft function. Such a response might be expected to result in attenuation of pathologic vessel remodeling and thus may contribute to moderate alcohols' cardioprotective effects.

Functional interaction of nicotinic acetylcholine receptors and Na+/K+ ATPase from Locusta migratoria manilensis (Meyen).

Associated proteins are important for the correct functioning of nicotinic acetylcholine receptors (nAChRs). In the present study, a neonicotinoid-agarose affinity column was used to isolate related proteins from a solubilized membrane preparation from the nervous system of Locusta migratoria manilensis (Meyen). 1530 peptides were identified and most of them were involved in the membranous structure, molecular interaction and cellular communication. Among these peptides, Na+/K+ ATPase had the highest MASCOT score and were involved in the molecular interaction, which suggested that Na+/K+ ATPase and nAChRs might have strong and stable interactions in insect central nervous system. In the present study, functional interactions between nAChRs and Na+/K+ ATPase were examined by heterologous expression in Xenopus oocytes. The results showed that the activated nAChRs increased pump currents of Na+/K+ ATPase, which did not require current flow through open nAChRs. In turn, Na+/K+ ATPase significantly increased agonist sensitivities of nAChRs in a pump activity-independent manner and reduced the maximum current (Imax) of nAChRs. These findings provide novel insights concerning the functional interactions between insect nAChRs and Na+/K+ ATPase.

Improved Mass Spectrometric Analysis of Membrane Proteins Based on Rapid and Versatile Sample Preparation on Nanodiamond Particles

We have developed a novel streamlined sample preparation procedure for mass spectrometric (MS) analysis of membrane proteins using surface-oxidized nanodiamond particles. The platform consists of solid-phase extraction and elution of the membrane proteins on nanodiamonds, concentrating the membrane proteins on the nanodiamonds and separating out detergents, chaotropic agents, and salts, and other impurities that are often present at high concentrations in solubilized membrane preparations. In this manner, membrane-protein extracts are transformed into MS-ready samples in minutes. The protocol is not only fast, but also widely adaptable and highly effective for preparing generic membrane protein samples for both MALDI-MS studies of membrane-protein complexes and shotgun membrane proteomics studies. As proof of concept, we have demonstrated substantial improvements in the MALDI-MS analysis of the particulate methane monooxygenase (pMMO) complex, a three-subunit transmembrane protein solubilized in various detergent buffers. Enzymatic digestions of membrane proteins are also greatly facilitated since the proteins extracted on to the nanodiamonds are exposed on the surface of the nanoparticles rather than in SDS gels or in detergent solutions. We illustrate the effectiveness of nanodiamonds for SDS removal in the preparation of membrane proteins for MS analysis on the proteome level by examining the quality of the tryptic peptides prepared by on-surface nanodiamond digestion of an E. coli membrane fraction for shotgun proteomics.

Influence of MT7 toxin on the oligomerization state of the M1 muscarinic receptor1

The idea that GPCRs (G-protein-coupled receptors) may exist as homo- or hetero-oligomers, although still controversial, is now widely accepted. Nevertheless, the functional roles of oligomerization are still unclear and gaining greater insight into the mechanisms underlying the dynamics of GPCR assembly and, in particular, assessing the effect of ligands on this process seems important. We chose to focus our present study on the effect of MT7 (muscarinic toxin 7), a highly selective allosteric peptide ligand, on the oligomerization state of the hM1 (human M1 muscarinic acetylcholine receptor subtype). We analysed the hM1 oligomerization state in membrane preparations or in live cells and observed the effect of MT7 via four complementary techniques: native-PAGE electrophoresis analysed by both Western blotting and autoradiography on solubilized membrane preparations of CHO-M1 cells (Chinese-hamster ovary cells expressing muscarinic M1 receptors); FRET (fluorescence resonance energy transfer) experiments on cells expressing differently tagged M1 receptors using either an acceptor photobleaching approach or a novel fluorescence emission anisotropy technique; and, finally, by BRET (bioluminescence resonance energy transfer) assays. Our results reveal that MT7 seems to protect the M1 receptor from the dissociating effect of the detergent and induces an increase in the FRET and BRET signals, highlighting its ability to affect the dimeric form of the receptor. Our results suggest that MT7 binds to a dimeric form of hM1 receptor, favouring the stability of this receptor state at the cellular level, probably by inducing some conformational rearrangements of the pre-existing muscarinic receptor homodimers.

A Miniaturized 1536-Well Format γ-Secretase Assay

gamma-Secretase is an aspartyl protease that cleaves multiple substrates including the amyloid precursor protein (APP) and the Notch proteins. Abnormal proteolysis of APP is involved in the pathogenesis of Alzheimer's disease (AD) and overactive Notch signaling plays an oncogenic role in a variety of cancers. gamma-Secretase has emerged as a promising target for drug development in the treatment of AD and cancer. Assays with increased capacity for high-throughput screening would allow for quicker screening of chemical libraries and facilitate inhibitor development. We have developed a homogeneous time-resolved fluorescence (HTRF)-based assay that makes use of a novel biotinylated recombinant APP substrate and solubilized membrane preparation as the source of the gamma-secretase enzyme. The assay was miniaturized to a 1536-well format and validated in a pilot screen against a library of approximately 3,000 compounds. The overall assay performance was robust due to a calculated Z' factor of 0.74 and its demonstrated ability to identify known gamma-secretase inhibitors such as pepstatin A. This validated assay can readily be used for primary screening against large chemical libraries searching for novel inhibitors of gamma-secretase activity that may represent potential therapeutics for AD and a variety of neoplasms.

Elucidating the Mechanisms of Assembly and Subunit Interaction of the Cellulose Synthase Complex of Arabidopsis Secondary Cell Walls

Cellulose is the most abundant biopolymer in nature; however, questions relating to the biochemistry of its synthesis including the structure of the cellulose synthase complex (CSC) can only be answered by the purification of a fully functional complex. Despite its importance, this goal remains elusive. The work described here utilizes epitope tagging of cellulose synthase A (CESA) proteins that are known components of the CSC. To avoid problems associated with preferential purification of CESA monomers, we developed a strategy based on dual epitope tagging of the CESA7 protein to select for CESA multimers. With this approach, we used a two-step purification that preferentially selected for larger CESA oligomers. These preparations consisted solely of the three known secondary cell wall CESA proteins CESA4, CESA7, and CESA8. No additional CESA isoforms or other proteins were identified. The data are consistent with a model in which CESA protein homodimerization occurs prior to formation of larger CESA oligomers. This suggests that the three different CESA proteins undergo dimerization independently, but the presence of all three subunits is required for higher order oligomerization. Analysis of purified CESA complex and crude extracts suggests that disulfide bonds and noncovalent interactions contribute to the stability of the CESA subunit interactions. These results demonstrate that this approach will provide an excellent framework for future detailed analysis of the CSC.

γ-Secretase Complex Assembly within the Early Secretory Pathway

gamma-Secretase is an aspartyl protease complex composed of the four core components APH-1, nicastrin (NCT), presenilin (PS), and PEN-2. It catalyzes the final intramembranous cleavage of the beta-secretase-processed beta-amyloid precursor protein to liberate the neurotoxic amyloid beta-peptide. Whereas unassembled complex components appear to be unstable and/or to be retained within the endoplasmic reticulum (ER), the fully assembled complex is known to exert its biological function in late secretory compartments, including the plasma membrane. We thus hypothesized that the gamma-secretase complex undergoes a stepwise assembly within the ER. We demonstrate that gamma-secretase-associated NCT can be actively retained within the ER by the addition of a retention signal. Under these conditions, complex assembly occurred in the absence of maturation of NCT, and ER-retained immature NCT associated with APH-1, PEN-2, and PS fragments. Moreover, a biotinylated transition state gamma-secretase inhibitor allowed the preferential isolation of the fully assembled complex containing immature NCT. Furthermore, we observed a conformational change in immature NCT, which is known to be selectively associated with complete gamma-secretase complex assembly. This was also observed for a small amount of immature endogenous NCT. ER-retained NCT also rescued the biochemical phenotype observed upon RNA interference-mediated NCT knockdown, viz. reduced amyloid beta-peptide production; instability of PS, PEN-2, and APH-1; and accumulation of beta-amyloid precursor protein C-terminal fragments. Finally, we demonstrate that dimeric (NCT/APH-1) and trimeric (NCT/APH-1/PS) intermediates of gamma-secretase complex assembly containing endogenous NCT are retained within the ER and that the incorporation of the fourth and last binding partner (PEN-2) also occurs on immature NCT, suggesting a complete assembly of the gamma-secretase complex within the ER.

The SH-3 and SH-4 Antibodies Recognize Distinct Epitopes on CD73 from Human Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are pluripotent cells in the bone marrow that have the capacity to differentiate along a number of connective tissue lineages, including cartilage, bone, adipose tissue, and stroma. The SH-3 and SH-4 monoclonal antibodies recognize epitopes present on the surface of human MSCs. This study describes the isolation and characterization of the antigen that is recognized by these antibodies. A protein of molecular weight approximately 67 kDa was immunoprecipitated from a solubilized membrane preparation of human MSCs using the SH-3 antibody. Analysis of peptides derived from this protein by mass spectrometry and sequencing identified it as CD73 (ecto-5′-nucleotidase). The SH-4 antibody was also shown to react with purified bovine CD73 by immunoblotting, but the SH-3 antibody failed to react with the bovine protein. These results indicate that both SH-3 and SH-4 epitopes are present on CD73, but they are distinct. CD73, present in lymphoid tissue, plays a role in the activation of B-lymphocytes and in signal transduction in the hematopoietic compartment of bone marrow. The role that CD73 may play in bone marrow stromal interactions and in the differentiation of MSCs is discussed.

Activation of Helicobacter pylori VacA Toxin by Alkaline or Acid Conditions Increases Its Binding to a 250-kDa Receptor Protein-tyrosine Phosphatase β

Helicobacter pylori, a Gram-negative gastric bacterium, secretes VacA, a cytotoxin that causes vacuolar degeneration of susceptible cells. Velocity sedimentation analysis showed that treatment of VacA at alkaline pH led to disassembly of VacA oligomers, an observation reported previously for acid-treated VacA. Exposure of VacA to acid or alkali increased its binding to AZ-521 cells, as shown by indirect immunofluorescence and flow cytometry. Moreover, immunoprecipitates with polyclonal antibodies against VacA from AZ-521 cells previously exposed to acid- or alkali-treated VacA had a 250-kDa glycoprotein containing galactose-beta(1-3)-N-acetylgalactosamine and galactose-beta(1-4)-N-acetylglucosamine. p250, purified by chromatography on peanut agglutinin affinity and Superose 6 columns, contained N-terminal and internal amino acid sequences of YRQQRKLVEEIGWSYT and LIIQDHILEATQDDY, respectively. These sequences are identical to those of a receptor protein-tyrosine phosphatase (RPTPbeta/PTPzeta); in agreement, p250 reacted with anti-human RPTPbeta monoclonal antibody. Immunoprecipitation with anti-human RPTPbeta antibody of solubilized membrane preparations previously incubated with VacA or heat-inactivated VacA demonstrated that RPTPbeta bound native, but not denatured, VacA. Acidic and alkaline treatments were associated with activation of VacA and increased binding to the cell surface RPTPbeta.

Squalene–hopene cyclase from Methylococcus capsulatus (Bath): a bacterium producing hopanoids and steroids

We report the cloning and characterisation of the Methylococcus capsulatus shc gene, which encodes the squalene–hopene cyclase (SHC). This enzyme catalyses the complex cyclization of squalene to the pentacyclic triterpene skeleton of hopanoids and represents the key reaction in this biosynthesis. Using a combination of PCR amplification and DNA hybridization, two overlapping 2.6 kb PstI and 3.3 kb SalI DNA fragments were cloned bearing a 1962 bp open reading frame encoding a 74 kDa protein with 654 amino acids and a predicted isoelectric point at about pH 6.3. The deduced amino acid sequence of the M. capsulatus shc gene showed significant similarity to known prokaryotic SHCs and to a lesser degree to the related eukaryotic oxidosqualene cyclases (OSCs). Like other triterpene cyclases, the M. capsulatus SHC contains seven so-called QW-motifs as well as an aspartate-rich domain. The recombinant M. capsulatus SHC was expressed in Escherichia coli and in vitro activity of the recombinant cyclase was demonstrated using crude cell-free lysate or solubilized membrane preparation. The cyclization products hop-22-ene and hopan-22-ol (diplopterol) were identified by GC and GC-MS.

Insulin-like growth factor binding protein-2 binds to cell surface proteoglycans in the rat brain olfactory bulb.

A family of six insulin-like growth factor binding proteins (IGFBPs) bind IGF-I and modulate its biological activity. IGFBPs may bind to macromolecules on the cell surface or pericellular extracellular matrix, and this interaction may modulate their effect on IGF activity. To date, little is known about the specificity of IGFBPs in the regulation of IGF action in the brain. We therefore explored whether IGFBPs were associated with cell membrane or extracellular matrix components in the rat brain. IGF-I binding sites with the characteristics of an IGFBP were found in the olfactory bulb mitral cell layer. This IGFBP was identified as IGFBP-2 by immunoprecipitation of both solubilized membrane preparations and cross-linked 125I-IGF: IGFBP complexes. While binding of IGFBP-2 to cell membranes was unaffected by RGD-containing peptide, it was inhibited by high salt concentration, suggesting interaction with proteoglycans. IGFBP-2 bound in vitro to the glycosaminoglycans chondroitin-4 and -6-sulfate, keratan sulfate, and heparin. IGFBP-2 also bound the proteoglycan aggrecan, an effect reduced by digestion of its glycosaminoglycans. Binding of IGFBP-2 to chondroitin-6-sulfate decreased the binding affinity of IGFBP-2 for IGF-I approximately 3-fold. Finally, an IGFBP-2 antibody coimmunoprecipitated IGFBP-2 and an approximately 200 kDa proteoglycan containing chondroitin-sulfate side chains from the rat olfactory bulb, providing definitive evidence for IGFBP-2 binding to olfactory bulb proteoglycans. These findings indicate that IGFBP-2 binds to proteoglycans in cell membranes of the rat olfactory bulb. Because we have previously shown that IGFs are highly expressed in the rat olfactory bulb, cell associated IGFBP-2 may have an important role in directing IGFs to specific sites in this brain region.

Conversion of hydrophilic tubulin into a hydrophobic compound. Evidence for the involvement of membrane proteins.

Brain membranes contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. We have previously postulated: (a) that this kind of tubulin is a peripheral membrane protein that arises from microtubules that in vivo interact with membranes and (b) that the hydrophobic behaviour is due to the interaction of tubulin with a membrane component. Here we report the in vitro conversion of hydrophilic into hydrophobic tubulin by incubating microtubule associated proteins (MAPs) free taxol-stabilized microtubules with Triton X-100 solubilized membranes. After incubation, the microtubules were sedimented, depolymerized and subjected to partition into Triton X-114. Part of the tubulin was isolated in the detergent phase and contained, as observed in native membranes, a high proportion of the acetylated isotype. Because of the high proportion of acetylated tubulin the 'in vitro' conversion resembles the 'in vivo' interaction. Electrophoretic analysis of the detergent phase shows, besides tubulin, two major protein bands of 29 and 100 kDa molecular mass. The ability of the solubilized membranes to convert hydrophilic into hydrophobic tubulin is greatly diminished if the solubilized membrane preparation is preincubated in the presence of trypsin or heated at 90 degrees C for 5 min, indicating that the membrane component that confers the hydrophobic behaviour to tubulin is of proteinaceous nature.

Anti-Erythropoietin Receptor Monoclonal Antibody: Epitope Mapping, Quantification of the Soluble Receptor, and Detection of the Solubilized Transmembrane Receptor and the Receptor-Expressing Cells

A hybridoma cell line producing the monoclonal antibody against erythropoietin receptor (EpoR) was established using the soluble ectodomain of mouse erythropoietin receptor (sEpoR) as an antigen. The monoclonal antibody termed 1G3 bound to the denatured sEpoR. Epitope mapping with peptide library revealed that 1G3 recognized the amino terminal region including the hexapeptide (positions 6 to 11; LeuProAspProLysPhe). The amino acid sequence in this hexapeptide was identical in mice, rats, and humans, and therefore 1G3 bound to EpoR from all of these sources. Using 1G3, we evaluated sEpoR by a sandwich enzyme-linked immunoassay, and EpoR in the solubilized membrane preparation was detected by Western blotting. The cells expressing EpoR were identified with immunochemical staining. We confirmed the presence of EpoR in a neuronal cell line and PC12 cells, and EpoR was expressed in primary cultured hippocampal neurons.

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 N 5-methyltetrahydrofolate and substantially lower for N 5-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.

Cloning of a Coconut Endosperm cDNA Encoding a 1-Acyl-sn-Glycerol-3-Phosphate Acyltransferase That Accepts Medium-Chain-Length Substrates

Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (H.M. Davies, D.J. Hawkins, J.S. Nelsen [1995] Phytochemistry 39:989-996). Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions. Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library. Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 34.8 kD. Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences. Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme.

Escherichia coli Expression of Site-Directed Mutants of Cytochrome P450 2B1 from Six Substrate Recognition Sites: Substrate Specificity and Inhibitor Selectivity Studies

Cytochrome P450 2B1 wild-type and eight site-directed mutations at positions 114, 206, 236, 302, 363, 367, and 478 have been expressed in an Escherichia coli system. Solubilized membrane preparations yielded 100-180 nmol of P450/L of culture. The metabolism of a number of substrates including androstenedione, progesterone, (benzyloxy)resorufin, pentoxyresorufin, and benzphetamine was analyzed. The E. coli-expressed enzymes displayed the same androstenedione metabolite profiles previously observed with a COS cell expression system. Several of the mutants exhibited an increased rate of progesterone hydroxylation, possibly as the result of an enlarged substrate binding pocket and increased D-ring alpha-face binding. (Benzyloxy)resorufin and pentoxyresorufin O-dealkylation by the P450 2B1 mutants exhibited activities ranging from 10% to 99% and 3% to 71% of wild-type, respectively. Interestingly, the Val-363-->Leu mutant showed markedly suppressed pentoxyresorufin but unaltered (benzyloxy)resorufin dealkylase activity. Benzphetamine N-demethylase activities ranged from 28% to 110% of wild-type. Mechanism-based inactivation of the P450 2B1 mutants showed that susceptibility to inactivation by chloramphenicol and D-erythro- and L-threo-chloramphenicol was abolished in the Val-367-->Ala mutant. The Val-363-->Leu mutant was refractory to L-threo-chloramphenicol. Studies of chloramphenicol covalent binding and metabolism by the Val-367-->Ala mutant showed that its resistance to inactivation is largely attributable to an inability to bioactivate the inhibitor. The expression of P450 2B1 wild-type and mutants in E. coli provides an excellent opportunity to study structure/function relationships by site-directed mutagenesis.

Activation of a Gi protein in digitonin/cholate-solubilized membrane preparations of mouse sperm by the zona pellucida, an egg-specific extracellular matrix.

Mammalian sperm possess guanine nucleotide-binding regulatory proteins (G proteins) that are involved in signal transduction pathways leading to zona pellucida (ZP)-mediated acrosomal exocytosis. We have previously examined ZP-G protein dynamics in mouse sperm homogenates, as well as cell-free membrane preparations, and our data support the existence of ZP receptor-G protein complexes in sperm membranes. However, the composition of this complex has not been identified due to experimental limitations of the membrane preparations. In the present study, a detergent-solubilized preparation from mouse sperm membranes that retained the signaling properties of cell homogenates and cell-free membrane preparations was developed using buffers containing digitonin and cholate. GTP gamma S, a poorly hydrolyzable analogue of GTP, bound to these solubilized preparations in a specific and concentration-dependent fashion that reached saturation at 100 nM. Incubation of this solubilized membrane preparation with heat-solubilized ZP resulted in an increase in specific GTP gamma S binding in a concentration-dependent manner, with a maximal response at 4-6 ZP/microliters. Mastoparan (50 microM) increased GTP gamma S binding to levels similar to that seen with solubilized ZP. Mastoparan plus ZP stimulated GTP gamma S binding to the same extent as mastoparan or ZP alone. Pertussis toxin completely inhibited ZP-stimulated GTP gamma S binding and decreased mastoparan-stimulated GTP gamma S binding by 50-60%. Purified ZP3, the ZP component that possesses quantitatively all of the sperm binding and acrosomal exocytosis-inducing activities of the intact ZP, stimulated GTP gamma S binding to an extent similar to that of solubilized ZP.(ABSTRACT TRUNCATED AT 250 WORDS)

Solubilization and partial purification from mouse sperm membranes of the specific binding activity for 3‐quinuclidinyl benzilate, a potent inhibitor of the zona pellucida‐induced acrosome reaction

3-Quinuclidinyl benzilate (QNB), a potent antagonist of muscarinic acetylcholine receptors, has been demonstrated to inhibit specifically the zona pellucida (ZP)-induced acrosome reaction (AR) in mouse sperm (Florman and Storey, 1982; Dev Biol 91:121-130). In this study we describe the solubilization and partial purification of the mouse sperm QNB binding activity which may represent a component of the putative receptor complex for ZP on the sperm plasma membrane. Sperm membranes were isolated from cell homogenates of washed, capacitated, epididymal mouse sperm. Scatchard plots of QNB binding to these membranes indicated a single class of binding sites with KD = 7.2 nM and Bmax = 8700 sites/cell. These binding characteristics are similar to those seen with QNB binding to whole cells (Florman and Storey, 1982, J Androl 3:157-164). Sperm membranes were solubilized using 1% digitonin/0.2% cholate, and the resultant detergent-soluble fraction possessed QNB binding activity similar to that of intact membranes. The detergent-soluble fraction maintained intact ZP receptor(s)-G protein coupling in that treatment of this fraction with either ZP or mastoparan resulted in a 35% or 65% increase in specific GTP gamma S binding, respectively. The solubilized membrane preparation was fractionated by gel permeation HPLC. A majority of specific QNB binding activity was confined to one HPLC fraction. Analysis of this fraction by SDS-PAGE revealed a complex of approximately 5 proteins unique to this fraction. The most prominent protein had a M(r) of 72 kDa, which is within the M(r) range for muscarinic receptors. A protein with M(r) = 41 kDa was also present within this fraction. Subsequent pertussis toxin (PTX)-catalyzed ADP-ribosylation of this fraction revealed this protein to be the alpha subunit of the G(i) class of G proteins. Although the QNB binding activity could not be positively identified, we propose that it is contained in one or more of the proteins unique to this fraction and that these proteins, including G(i), may act as part of a sperm receptor complex for the ZP.

Modulation of guanylate cyclase-coupled atrial natriuretic factor receptor activity by mastoparan and ANF in murine Leydig tumor cells: Role of G-proteins

Mastoparan potently stimulated catalytic activity of guanylate cyclase-coupled atrial natriuretic factor receptor (GC-A/ANF-R), both in the plasma membranes and intact Leydig tumor (MA-10) cells. In plasma membrane preparations, a maximum of 5-fold GC catalytic activity was stimulated by 100 μM mastoparan and the half maximum stimulation (EC 50) was achieved at 40 μM concentration. Mastoparan potentiated GC activity by more than 40%, above the level, stimulated by ANF. Mas 7, an active analog of mastoparan, stimulated the GC activity in a similar manner to mastoparan whereas Mas 17, an inactive analog, did not enhance GC activity. In membranes prepared from mastoparan-treated intact MA-10 cells, GC catalytic activity was enhanced by more than 4-fold as compared with untreated control cells. Pretreatment of membranes with either anti-Gsα or anti-Giα antibodies had no effect on mastoparan-stimulated GC activity, however, anti-Goα antibodies inhibited the stimulatory effect of mastoparan by almost 50%. Agents known to modulate the effect of mastoparan such as EGTA (Ca 2+ chelator), W7 (calmodulin inhibitor) and staurosporine (protein kinase C inhibitor) had no effect on the mastoparan-stimulated GC activity. Mastoparan enhanced the ANF-stimulated GC activity in detergent solubilized membrane preparations without a significant change in ANF-binding capacity. The data establish a role for mastoparan in the ANF-dependent stimulation of GC-A/ANF-R catalytic activity, both in the plasma membrane preparations and intact Leydig tumor (MA-10) cells. Furthermore, these findings provide new evidence that mastoparan (isolated from wasp venom) potently stimulates guanylate cyclase activity of GC-A/ANF-R by activating G-proteins.

Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H2. The enzyme showed a maximal activity of 120±40 μmol H2 oxidized · min−1 · min−1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 μmol H2 · min−1 · mg−1 with methyl viologen as electron donor, and an apparent K m for hydrogen oxidation of 5.6±1.7 μM. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S2−, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F420 or ferredoxin, nor with FAD, FMN, or NAD(P)+. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.