Exposure of an antigen of chromaffin granules on cell surface during exocytosis

The synthesis rate of the membrane proteins of the catecholamine-storing vesicles (chromaffin granules) of the adrenal medulla is lower than that of the secretory proteins of the contents. Based on these results we proposed that after exocytosis the membranes of chromaffin granules are retrieved and are re-used for several secretion cycles (see also ref. 4). This concept of re-use of granule membranes has been further strengthened by the finding that exogenous markers which are taken up by secretory cells during stimulation can be traced to the Golgi region and to immature secretory organelles. However, one basic question remains: are the membranes of secretory organelles specifically and completely removed from the plasma membrane and if so, how fast is this process? By using an antiserum against a membrane glycoprotein of chromaffin granules we have now obtained quantitative data which demonstrate that during exocytosis this antigen becomes exposed on the cell surface and disappears again to a large degree within 30 min.     

Aggregation of chromaffin granules by calpactin at micromolar levels of calcium

Several cytosolic proteins bind to secretory granule membranes in a Ca2+-dependent manner and thus may be involved in the mediation of membrane interactions during exocytosis. One of these proteins, calpactin, is a tetramer consisting of two heavy chains of relative molecular mass (Mr) 36K (p36) and two light chains of 10K (p10). We report here that calpactin promotes the Ca2+-dependent aggregation and fatty acid-dependent fusion of chromaffin granule membranes at a level of Ca2+ that is lower than that reported for other granule-aggregating proteins, and which parallels the Ca2+ requirement for secretion from permeabilized chromaffin cells. We found subunits of calpactin to be inactive in promoting granule aggregation. Two distinct 33K proteolytic fragments of p36, differing at their N termini, also promote granule aggregation but with different Ca2+ sensitivities from calpactin. These differences suggest that the N-terminal portion of p36 modulates the Ca2+/lipid binding sites in the core portion of p36 (ref.5).     

Inhibition of exocytosis by intracellularly applied antibodies against a chromaffin granule-binding protein

Exocytotic secretion requires the interaction and fusion of secretory vesicles with the plasma membrane. This process could be mediated by specific recognition molecules acting as intracellular, membrane-bound receptors and ligands. One possible component of such a recognition site on the plasma membrane is a protein of relative molecular mass (Mr) 51,000 (51K) that has been isolated from bovine adrenal chromaffin cells. This protein binds strongly to chromaffin granules, the secretory vesicles of these cells. To determine the function of this membrane-anchored chromaffin granule-binding protein in exocytosis, we tested the effect of intracellularly injected antibodies on secretion. Here we show, by two independent techniques in two different cell types, that antibodies against this protein inhibit exocytosis. In rat pheochromocytoma cell cultures, monospecific antibodies, applied by erythrocyte ghost fusion, impair the release of 3H-noradrenaline. The same antibodies, introduced into individual chromaffin cells through a patch pipette, block exocytosis, as revealed by the measurement of membrane capacitance. These results demonstrate the functional involvement in exocytosis of a plasma membrane protein with high affinity for secretory vesicles.     

Roles of G protein subunits in transmembrane signalling

A family of proteins called G proteins couples cell surface receptors to a variety of enzymes and ion channels. Since many cells contain several very similar G proteins, an important question is how signals remain specific as they cross the cell membrane.     

Dynamics of ATP-dependent chromatin assembly by ACF

The assembly of DNA into chromatin is a critical step in the replication and repair of the eukaryotic genome. It has been known for nearly 20 years that chromatin assembly is an ATP-dependent process. ATP-dependent chromatin-assembly factor (ACF) uses the energy of ATP hydrolysis for the deposition of histones into periodic nucleosome arrays, and the ISWI subunit of ACF is an ATPase that is related to helicases. Here we show that ACF becomes committed to the DNA template upon initiation of chromatin assembly. We also observed that ACF assembles nucleosomes in localized arrays, rather than randomly distributing them. By using a purified ACF-dependent system for chromatin assembly, we found that ACF hydrolyses about 2#150;4 molecules of ATP per base pair in the assembly of nucleosomes. This level of ATP hydrolysis is similar to that used by DNA helicases for the unwinding of DNA. These results suggest that a tracking mechanism exists in which ACF assembles chromatin as an ATP-driven DNA-translocating motor. Moreover, this proposed mechanism for ACF may be relevant to the function of other chromatin-remodelling factors that contain ISWI subunits.     

RP-HPLC法测定心肌安颗粒中绿原酸和芍药苷

建立RP-HPLC同时测定心肌安颗粒剂中含绿原酸、芍药苷两种有效成分的方法.采用Dia-monsil C18(4.6 mm×250 mm,5μm)色谱柱,以乙腈-0.2%磷酸水为流动相梯度洗脱,流速1.0mL/min,柱温35℃,检测波长230 nm.绿原酸和芍药苷质量浓度分别在20～120μg/mL(r=0.999 9)和30～180μg/mL(r=0.999 8)范围内与峰面积线性关系良好,绿原酸和芍药苷平均加样回收率(n=6)分别为99.38%(RSD=0.96%)和99.91%(RSD=0.82%).三批制剂测定结果显示,含绿原酸量为10.36 mg/g(RSD=0.73%),含芍药苷量为8.18 mg/g(RSD=0.58%).本方法快速、准确,重复性好,可用于同时测定心肌安颗粒剂中绿原酸和含芍药苷的量.     

Channel properties of an insect neuronal acetylcholine receptor protein reconstituted in planar lipid bilayers

A pentameric membrane protein composed of four types of polypeptide has been identified as the minimal structural unit responsible for the electrogenic action of acetylcholine on electrocytes and muscle cells. Because many populations of central and peripheral neurons also have nicotinic acetylcholine receptors (AChRs), considerable effort has recently gone into identifying the neuronal receptor. The central nervous tissue of insects contains very high concentrations of nicotinic AChRs, and we have recently purified an alpha-toxin binding protein, a putative AChR, from neuronal membranes of locusts. It is a component of high relative molecular mass, clearly composed of identical subunits, a structure predicted for an ancestral AChR protein. To verify that the purified polypeptides not only represent ligand binding sites but that they are indeed functional receptors, we have now reconstituted the isolated protein in a planar lipid bilayer. We show that in this system cholinergic agonists activate functional ion channels, that have properties comparable to those exhibited by the peripheral AChRs in vertebrates; thus, for the first time a functional acetylcholine receptor channel has been identified in nerve cells.     

A transmembrane protein required for acetylcholine receptor clustering in Caenorhabditis elegans

Clustering neurotransmitter receptors at the synapse is crucial for efficient neurotransmission. Here we identify a Caenorhabditis elegans locus, lev-10, required for postsynaptic aggregation of ionotropic acetylcholine receptors (AChRs). lev-10 mutants were identified on the basis of weak resistance to the anthelminthic drug levamisole, a nematode-specific cholinergic agonist that activates AChRs present at neuromuscular junctions (NMJs) resulting in muscle hypercontraction and death at high concentrations. In lev-10 mutants, the density of levamisole-sensitive AChRs at NMJs is markedly reduced, yet the number of functional AChRs present at the muscle cell surface remains unchanged. LEV-10 is a transmembrane protein localized to cholinergic NMJs and required in body-wall muscles for AChR clustering. We also show that the LEV-10 extracellular region, containing five predicted CUB domains and one LDLa domain, is sufficient to rescue AChR aggregation in lev-10 mutants. This suggests a mechanism for AChR clustering that relies on extracellular protein-protein interactions. Such a mechanism is likely to be evolutionarily conserved because CUB/LDL transmembrane proteins similar to LEV-10, but lacking any assigned function, are expressed in the mammalian nervous system and might be used to cluster ionotropic receptors in vertebrates.     

Modulation of an RNA-binding protein by abscisic-acid-activated protein kinase

Protein kinases are involved in stress signalling in both plant and animal systems. The hormone abscisic acid mediates the responses of plants to stresses such as drought, salinity and cold. Abscisic-acid-activated protein kinase (AAPK -- found in guard cells, which control stomatal pores -- has been shown to regulate plasma membrane ion channels. Here we show that AAPK-interacting protein 1 (AKIP1), with sequence homology to heterogeneous nuclear RNA-binding protein A/B, is a substrate of AAPK. AAPK-dependent phosphorylation is required for the interaction of AKIP1 with messenger RNA that encodes dehydrin, a protein implicated in cell protection under stress conditions. AAPK and AKIP1 are present in the guard-cell nucleus, and in vivo treatment of such cells with abscisic acid enhances the partitioning of AKIP1 into subnuclear foci which are reminiscent of nuclear speckles. These results show that phosphorylation-regulated RNA target discrimination by heterogeneous nuclear RNA-binding proteins may be a general phenomenon in eukaryotes, and implicate a plant hormone in the regulation of protein dynamics during rapid subnuclear reorganization.     

Activation of facilitation calcium channels in chromaffin cells by D1 dopamine receptors through a cAMP/protein kinase A-dependent mechanism

Facilitation calcium channels in unstimulated bovine chromaffin cells are normally quiescent but are activated by large pre-depolarizations or by repetitive depolarization in the physiological range. The activation of these 27-pS dihydropyridine-sensitive channels by repetitive stimulation, such as by increased splanchnic nerve activity, can lead to an almost twofold increase in Ca2+ current in these cells. This increase in Ca2+ current is of probable physiological importance in stimulating rapid catecholamine secretion in response to danger or stress. We have identified D1 dopaminergic receptors on bovine chromaffin cells by fluorescence microscopy. Here we show that stimulation of the D1 receptors activates the facilitation Ca2+ currents in the absence of pre-depolarizations or repetitive activity, and that activation by D1 agonists is mediated by cyclic AMP and protein kinase A. The recruitment of facilitation Ca2+ channels by dopamine may form the basis of a positive feedback loop mechanism for catecholamine secretion.     

ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin

Proteins contain thiol-bearing cysteine residues that are sensitive to oxidation, and this may interfere with biological function either as 'damage' or in the context of oxidant-dependent signal transduction. Cysteine thiols oxidized to sulphenic acid are generally unstable, either forming a disulphide with a nearby thiol or being further oxidized to a stable sulphinic acid. Cysteine-sulphenic acids and disulphides are known to be reduced by glutathione or thioredoxin in biological systems, but cysteine-sulphinic acid derivatives have been viewed as irreversible protein modifications. Here we identify a yeast protein of relative molecular mass M(r) = 13,000, which we have named sulphiredoxin (identified by the US spelling 'sulfiredoxin', in the Saccharomyces Genome Database), that is conserved in higher eukaryotes and reduces cysteine-sulphinic acid in the yeast peroxiredoxin Tsa1. Peroxiredoxins are ubiquitous thiol-containing antioxidants that reduce hydroperoxides and control hydroperoxide-mediated signalling in mammals. The reduction reaction catalysed by sulphiredoxin requires ATP hydrolysis and magnesium, involving a conserved active-site cysteine residue which forms a transient disulphide linkage with Tsa1. We propose that reduction of cysteine-sulphinic acids by sulphiredoxin involves activation by phosphorylation followed by a thiol-mediated reduction step. Sulphiredoxin is important for the antioxidant function of peroxiredoxins, and is likely to be involved in the repair of proteins containing cysteine-sulphinic acid modifications, and in signalling pathways involving protein oxidation.     

Primary structure and transmembrane orientation of the murine anion exchange protein

The amino-acid sequence of murine band 3, deduced from the nucleotide sequence of a complementary DNA clone, confirms that this integral membrane glycoprotein is composed of two major structural domains which correlate with its dual functions as the anchor for the erythrocyte cytoskeleton and as a plasma membrane anion antiporter. This latter activity resides within a highly hydrophobic domain that crosses the plasma membrane at least 12 times.     

Biological function of H1V-1 transmembrane protein gp41

Since 1992, the study of biological functions of HIV-1 gp41 has made great progress. Experimental evidence from several research groups demonstrated that gp41 has a putative cellular receptor. A recombinant soluble gp41 (aa539–684) and gp41 immunosuppressive peptide (aa583–599) could bind to human B lymphocytes and monocytes, but weakly bind to T lymphocytes. It was found that gp41 contains two cellular binding sites (aa583–599 and 641–675). GP41 could selectively inhibit cell proliferation of human T, B lymphocytes and monocytes, enhance human MHC class I, II and ICAM-1 molecule expression on cell surface. Gp41 binding proteins and a monoclonal antibody against the first binding site could inhibit this modulation effect. Amino acid sequence homology exists between gp41 and human type I interferons, and the homologous region is located in the first binding site on gp41 and in the receptor binding site on type I interferons. Studies in other groups indicate that both binding sites in gp41 may be associated with HIV infection of cells. Peptides containing two binding sites could respectively inhibit HIV infection of cells. A monoclonal antibody recognizing the second binding site could neutralize lab-strains and recently separated strains of HIV-1. Besides, antibodies against two regions (homologous with gp41 binding sites) of SIV transmembrane protein gp32 could protect macaques from SIV infection. These results suggest that the study of gp41 binding sites and cellular receptor could contribute to understanding the mechanism of HIV infection and to developing HIV vaccine and anti-HIV drugs.     

Synchronised transmembrane insertion and glycosylation of a nascent membrane protein.

Studies of the synthesis and incorporation of the vesicular stomatitis virus glycoprotein into membranes in a synchronised cell-free system demonstrate a tight coupling between polypeptide synthesis and membrane insertion, as a result of which the nascent chain crosses the membrane. The studies reveal a surprisingly precise sequence by which the nascent chain of this membrane glycoprotein is glycosylated in two steps. These findings have important implications for the mechanisms of membrane assembly.