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1.
Summary The Na+/K+ ionophore monensin is known to arrest the intracellular transport of newly synthesized proteins in the Golgi complex. In the present investigation the effect of monensin on the secretion of 3H-galactose-labeled and 3H-sialic acid-labeled thyroglobulin was studied in open thyroid follicles isolated from porcine thyroid tissue.Follicles were incubated with 3H-galactose at 20° C for 1 h; at this temperature the labeled thyroglobulin remains in the labeling compartment (Ring et al. 1987a). The follicles were then chased at 37° C for 1 h in the absence or presence of 1 M monensin. Without monensin substantial amounts of labeled thyroglobulin were secreted into the medium, whereas in the presence of the ionophore secretion was inhibited by 80%. Since we have previously shown (Ring et al. 1987 b) that monensin does not inhibit secretion of thyroglobulin present on the distal side of the monensin block we conclude that galactose is incorporated into thyroglobulin on the proximal side of this block.Secretion was also measured in follicles continuously incubated with 3H-galactose for 1 h at 37° C in the absence or presence of monensin. In these experiments secretion of labeled thyroglobulin was inhibited by about 85% in the presence of monensin. Identically designed experiments with 3H-N-acetylmannosamine, a precursor of sialic acid, gave similar results, i.e., almost complete inhibition of secretion of labeled thyroglobulin in the presence of monensin. The agreement between the results of the galactose and sialic acid experiments indicates that sialic acid, like galactose, is incorporated into thyroglobulin on the proximal side of the monensin block.Considering observations made in other cell systems the present results suggest that galactosylation and sialylation of thyroglobulin are completed within the Golgi complex. 相似文献
2.
Russell J. Howard Patricia M. Smith Graham F. Mitchell 《The Journal of membrane biology》1979,49(2):171-198
Summary The topography of the external surface of the Balb/c mouse erythrocyte has been investigated and compared to the human erythrocyte by using a series of protein radiolabeling probes. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the pattern of Coomassie Blue stained proteins was very similar for mouse and human erythrocyte ghosts, as was the distribution of radioactivity in protein bands after lactoperoxidase catalyzed radioiodination. The mouse erythrocyte glycoproteins identified by periodic-acid-Schiff and Stains-All reagents, sialic acid analysis of gel slices, binding of125I-wheat germ agglutinin and125I-concanavalin A to the gels, and glycoprotein radiolabeling techniques, differed markedly from the sets of proteins labeled by radioiodination, and also differed from the human erythrocyte glycoproteins. Instead of the PAS I to PAS IV series of sialoglycoproteins characteristic of human erythrocytes, the mouse erythrocyte possesses a broad band of sialoglycoproteins with several peaks ranging in mol wt from 65,000 to 32,000. The same group of sialoglycoproteins were labeled by the periodate/B3H4
– technique specific for terminal sialic acid, and the galactose oxidase/B3H4
– method (plus neuraminidase) specific for galactosyl/N-acetylgalactosaminyl residues penultimate to sialic acid. These results emphasize the necessity to employ a variety of protein radiolabeling probes based on different labeling specificities, to study the membrane topography of cells which are poorly understood compared to the human erythrocyte membrane. 相似文献
3.
Treatment of etiolated zucchini (Cucurbita pepo L.) hypocotyl tissue with sub-micromolar concentrations of the cationophore monensin rapidly (<20 min) inhibited the transport catalytic activity of the specific auxin-anion efflux carrier and reduced the inhibition of this carrier by the phytotropin N-1-naphthylphthalamic acid (NPA). Monensin inhibited the basipetal polar transport of indol-3yl-acetic acid (IAA) in long (30 mm) zucchini segments. At concentrations lower than 10–5 mol·dm–3 monensin did not affect uptake of the pH probe [2-14C]5,5-dimethyloxazolidine-2,4-dione (DMO) or that of the membrane-potential probe tetra[14C-phenyl]phosphonium bromide (TPP+), did not affect the response of IAA net uptake to external Ca2+ concentration and did not alter the metabolism of IAA. It was concluded that low concentrations of monensin inhibit transport through the Golgi apparatus of auxin efflux carrier protein and that the efflux carriers turn over very rapidly in the plasma membrane. Monensin pretreatment did not affect the saturable binding of [3H]NPA to microsomal membranes, indicating that the auxin-efflux catalytic sites and the NPA-binding sites are located on separate proteins. At higher concentrations (10–5 mol·dm–3) monensin inhibited both mediated uptake and mediated efflux components of IAA transport. This effect was at least in part attributable to perturbation by monensin of the driving forces for mediated uptake since high concentrations of monensin also reduced the uptake of DMO and TPP+.Abbreviations CH
cycloheximide
- DMO
5,5-dimethyloxazolidine-2,4-dione
- MDMP
2-(4-methyl-2,6-dinitroanlilino)N-methyl-propionamide
- NPA
N-1-naphthylphthalamic acid
- TPP+
tetraphenylphosphonium ion
We thank Mrs. R.P. Bell for technical assistance and Drs. G.F. Katekar and M.A. Venis for generous gifts of NPA. S.W. was supported by the U.K. Science and Engineering Research Council. 相似文献
4.
5.
A method for modifying and isotopic labeling the sialyl moiety of sialoglycoproteins is described. The basis of the procedure is the reductive amination of the exocyclic aldehyde group, generated on sialic acid by mild periodate oxidation, with a variety of amino compounds and sodium cyanoborohydride. Optimal conditions were selected to obtain maximum modification of sialic acid and minimal non-specific incorporation of the amino compound (glycine). The glycine modified model glycoproteins (α1-acid glycoprotein, fetuin) yielded single homogenous peaks upon gel filtration and on ion exchange chromatography. On gel electrophoresis a major band accounting for 92–98% of the modified glycoprotein and two minor bands consisting of dimers and trimers of the glycoprotein were observed. The modification did not alter the ability of the sialoglycoproteins to bind to wheat germ agglutinin-Sepharose or to interact with antibodies. The modified sialic acid was only partially released by mild acid hydrolysis suggesting that the introduction of an amino compound into the polyol chain of sialic acid has a stabilizing effect on the ketosidic linkage of the sugar. Interestingly, the modification rendered the sialic acid resistant to a variety of sialidases. The potential uses of this modification procedure include 1) the introduction of different isotopic labels (3H,14C,35S,125I) into the sialic acid moiety of glycoproteins; 2) the preparations of biologically active sialoglycoprotein (hormones, enzymes, co-factors) with increased circulating half-lives in animals; 3) preparation of substrates to search for endoglycosidases; 4) the direct comparison of sialoglycoprotein patterns obtained in small amounts from normal and pathological cells or tissues, and 5) the isolation and purification of cell surface sialoglycoproteins. 相似文献
6.
Kristina Oresic Caroline L. Ng Domenico Tortorella 《The Journal of biological chemistry》2009,284(9):5905-5914
The human cytomegalovirus proteins US2 and US11 have co-opted endoplasmic
reticulum (ER) quality control to facilitate the destruction of major
histocompatibility complex class I heavy chains. The class I heavy chains are
dislocated from the ER to the cytosol, where they are deglycosylated and
subsequently degraded by the proteasome. We examined the role of TRAM1
(translocating chain-associated membrane protein-1) in the dislocation of
class I molecules using US2- and US11-expressing cells. TRAM1 is an ER protein
initially characterized for its role in processing nascent polypeptides.
Co-immunoprecipitation studies demonstrated that TRAM1 can complex with the
wild type US2 and US11 proteins as well as deglycosylated and
polyubiquitinated class I degradation intermediates. In studies using US2- and
US11-TRAM1 knockdown cells, we observed an increase in levels of class I heavy
chains. Strikingly, increased levels of glycosylated heavy chains were
observed in TRAM1 knockdown cells when compared with control cells in a
pulse-chase experiment. In fact, US11-mediated class I dislocation was more
sensitive to the lack of TRAM1 than US2. These results provide further
evidence that these viral proteins may utilize distinct complexes to
facilitate class I dislocation. For example, US11-mediated class I heavy chain
degradation requires Derlin-1 and SEL1L, whereas signal peptide peptidase is
critical for US2-induced class I destabilization. In addition, TRAM1 can
complex with the dislocation factors Derlin-1 and signal peptide peptidase.
Collectively, the data support a model in which TRAM1 functions as a cofactor
to promote efficient US2- and US11-dependent dislocation of major
histocompatibility complex class I heavy chains.HCMV2 can
down-regulate cell surface expression of the immunologically important
molecule major histocompatibility complex class I to avoid immune detection by
cytotoxic T cells (1,
2). More specifically, the HCMV
US2 and US11 gene products alone can target the ER-localized major
histocompatibility complex class I heavy chains for extraction across the ER
membrane by a process referred to as dislocation or retrograde translocation.
The N-linked glycan is then removed upon exposure to the cytosol by
N-glycanase (3),
followed by proteasomal destruction
(4,
5). The HCMV US2 and US11
proteins utilize the ER quality control process to eliminate class I heavy
cells in a similar manner as misfolded or damaged ER proteins (e.g.
genetic mutants of α1-antitrypsin
(6) and the cystic fibrosis
transmembrane conductance regulator protein
(7)) are targeted for
degradation (8). Hence,
analysis of US2- and US11-mediated destruction of class I heavy chains
provides an excellent system to delineate viral protein function as well as
the ER quality control process.ER and cytosolic proteins are required for US2- and US11-mediated
dislocation/degradation of class I heavy chains. Some of these proteins have
also been identified in the processing of aberrant ER polypeptides. The ER
chaperones calnexin, calreticulin, and BiP have been implicated in
US2-mediated class I destruction
(9) as well as in the removal
of some misfolded ER proteins
(10). The ubiquitination
machinery also participates in the extraction of class I heavy chains as
ubiquitinated heavy chains are observed prior to dislocation
(11,
12). For misfolded ER
degradation substrates, ubiquitin conjugation enzymes (e.g. Ubc6p and
Ubc7p/Cue1p) and ubiquitin ligases Hrd1p/Der3p, Doa10p, and Ubc1p have been
implicated in the dislocation reaction
(8). Interestingly, the ER
membrane protein Derlin-1 along with SEL1L are involved in US11-mediated class
I heavy chain degradation
(13-15),
whereas SPP is critical for US2-induced class I destabilization
(16). The ubiquitinated
substrates are dislocated by the AAA-ATPase complex composed of p97-Ufd1-Npl4
(17) while docked to the ER
through its interaction with VIMP
(14) followed by proteasome
destruction. The inhibition of the proteasome causes the accumulation of
deglycosylated class I heavy chain intermediate in US2 and US11 cells,
allowing the dislocation and degradation reactions to be studied as separate
processes (4,
5).Despite the identification of some cellular proteins that assist US2- and
US11-mediated class I dislocation, the dislocation pore and accessory factors
that mediate the efficient extraction of class I through the bilayer have yet
to be completely defined. The current study explores the role of TRAM1
(translocating chain-associated membrane protein-1) in US2- and US11-mediated
class I dislocation. TRAM1 is an ER-resident multispanning membrane protein
that can mediate the lateral movement of select signal peptides and
transmembrane segments from the translocon into the membrane bilayer
(18), a property that makes it
uniquely qualified to participate in the dislocation of a membrane protein.
TRAM1 has been cross-linked to signal peptides as well as transmembrane
domains of nascent polypeptides during the early stages of protein processing
(19-25).
Interestingly, unlike the Sec61 complex and the signal recognition particle
receptor, TRAM1 is not essential for the translocation of all membrane
proteins into the ER (20,
21). Hence, TRAM1 may utilize
its ability to engage hydrophobic domains to assist in the efficient
dislocation of membrane proteins. In fact, association and TRAM1 knockdown
studies demonstrate that TRAM1 participates in US2- and US11-mediated
dislocation of class I heavy chains. Collectively, our data suggest for the
first time that TRAM1 plays a role in the dislocation of a membrane
glycoprotein. 相似文献
7.
K. Chandrasekhar A. Patricia Campbell Mei-Fen Jeng Arne Holmgren H. Jane Dyson 《Journal of biomolecular NMR》1994,4(3):411-432
Summary As a prelude to complete structure calculations of both the oxidized and reduced forms of Escherchia coli thioredoxin (Mr 11 700), we have analyzed the NMR data obtained for the two proteins under identical conditions. The complete aliphatic 13C assignments for both oxidized and reduced thioredoxin are reported. Correlations previously noted between 13C chemical shifts and secondary structure are confirmed in this work, and significant differences are observed in the C and C shifts between cis- and trans-proline, consistent with previous work that identifies this as a simple and unambiguous method of identifying cis-proline residues in proteins. Reduction of the disulfide bond in the active-site Cys32-Gly-Pro-Cys35 sequence causes changes in the 1H, 15N and 13C chemical shifts of residues close to the active site, some of them quite far distant in the amino acid sequence. Coupling constants, both backbone and side chain, show some differences between the two proteins, and the NOE connectivities and chemical shifts are consistent with small changes in the positions of several side chains, including the two tryptophan rings (Trp28 and Trp31). These results show that, consistent with the biochemical behavior of thioredoxin, there are minimal differences in backbone configuration between the oxidized and reduced forms of the protein. 相似文献
8.
Glycosylation of ion channel proteins dramatically impacts channel function. Here we characterize the asparagine (N)-linked
glycosylation of voltage-gated K+ channel α subunits in rat brain and transfected cells. We find that in brain Kv1.1, Kv1.2 and Kv1.4, which have a single
consensus glycosylation site in the first extracellular interhelical domain, are N-glycosylated with sialic acid-rich oligosaccharide
chains. Kv2.1, which has a consensus site in the second extracellular interhelical domain, is not N-glycosylated. This pattern
of glycosylation is consistent between brain and transfected cells, providing compelling support for recent models relating
oligosaccharide addition to the location of sites on polytopic membrane proteins. The extent of processing of N-linked chains
on Kv1.1 and Kv1.2 but not Kv1.4 channels expressed in transfected cells differs from that seen for native brain channels,
reflecting the different efficiencies of transport of K+ channel polypeptides from the endoplasmic reticulum to the Golgi apparatus. These data show that addition of sialic acid-rich
N-linked oligosaccharide chains differs among highly related K+ channel α subunits, and given the established role of sialic acid in modulating channel function, provide evidence for differential
glycosylation contributing to diversity of K+ channel function in mammalian brain.
Received: 17 December 1998/Accepted: 20 January 1999 相似文献
9.
The thermal fragmentation of human erythrocytes involves either surface wave growth and membrane externalization at the cell rim or membrane internalization at the cell dimple. In symmetrical monovalent electrolytes an increase in membrane internalization at the cell dimple correlates with the decrease in zeta potential arising from surface charge (sialic acid residue) depletion. The influence of divalent cations on thermal fragmentation is examined in this work. The erythrocyte zeta potential decreased when divalent cations replaced some Na+ in the cell-suspending phase. The incidence of membrane internalization increased in rank order Ca2+>Ba2+>Mg2+Sr2+. Calcium continued to influence the thermal fragmentation of cells highly depleted of sialic acid, suggesting that the ion also interacted with membrane sites other than sialic acid. The divalent cation influence on cell fragmentation was shown to be greater than that due to zeta potential decrease alone. This conclusion was supported by the observation that the divalent cation-induced changes in zeta potential showed much less cation specificity than did the changes induced in the thermal fragmentation pattern. The result implies that the specificity of the divalent cation effects was due to interactions within the erythrocyte shear layer. The possibility that the interaction is with membrane lipids is examined. 相似文献
10.
Skeletal myotubes responded to passive stretch by increased amino acid uptake (as measured with [3H]α-aminoisobutyric acid), increased incorporation of amino acids into total cellular protein and myosin heavy chains, and increased accumulation of total cellular protein and myosin heavy chains. These alterations were preceded by an increase in the uptake of ouabain-sensitive rubidium-86 (86Rb+), a potassium tracer used to measure membrane sodium pump activity (Na+K+ATPase). This stretch-induced stimulation of 86Rb+ uptake resulted from a 60-70% increase in the Vmax of the Na pump with little change in the Km. [3H] ouabain binding studies showed no stretch-induced change in the number of membrane Na pumps, indicating that stretch activates the Na pumps that are already present on the cell surface. Since the stretch-induced increases in amino acid transport and amino acid incorporation into proteins were inhibited by ouabain, Na pump activation may be involved in stretch-induced cell growth of skeletal muscle cells by hypertrophy. 相似文献
11.
Forebrains from day-old chicks were homogenized and fractionated by differential sedimentation and density gradient centrifugation to yield subcellular fractions. The synaptosomal plasma membrane fraction was further treated with Triton X-100 to yield subsynaptic membrane fractions including synaptic junctions. Glycoproteins from these subsynaptic membrane fractions were identified after separation by SDS-polyacrylamide gel electrophoresis by incubating the gel slabs with radioiodinated concanavalin A. Two lectin-binding proteins were discerned in the synaptic junction fraction while none were observed in the Triton-soluble portion of the synaptic plasma membrane. The carbohydrate content of the glycoproteins from each subcellular fraction was quantitated after methanolysis and derivatization aso-methyl-trifluoroacetyl analogs by gas-liquid chromatography. The lowest concentration of glycoprotein sugars was found in the synaptic junction, mitochondrial, and soluble fractions while the greatest concentration was found in the myelin, light-synaptic plasma membrane, and the Triton-soluble portion of the synaptic plasma membrane. Of the subcellular fractions, the synaptic junction contained the highest porportion of mannose and lowest proportion of sialic acid. Moreover, this fraction's content of galactose andN-acetylglucosamine, relative to mannose was the lowest while its content of fucose was low. The oligosaccharide chains extending into the synaptic cleft therefore are predominantly of the neutral, mannose-rich type and are attached to a limited number of high-molecular-weight glycoproteins. 相似文献
12.
Covalent modification ofSpiroplasma floricola membrane proteins by fatty acids was determined by in vivo labeling of the cells with radioactive fatty acids followed by separation on one-dimensional SDS-polyacrylamide gels and visualization by autoradiography. Approximately 25 different proteins were found to be labeled with [3H]-palmitate, whereas almost none were labeled with [3H]-oleate. The radioactivity could not be removed from the palmitoylated membrane proteins by boiling in SDS or by exhaustive extraction with chloroform-methanol (21). Nevertheless, treating the palmitoylated proteins with a 0.1N KOH solution removed approximately 70% of the bound [3H]-palmitate. The major protein-bound fatty acid species were identified, following their release from the protein by chemical cleavage, as palmitic acid and stearic acid (83% and 7.5%, respectively). 相似文献
13.
Summary A 4D HCCH-TOCSY experiment is described for correlating and assigning the1H and13C resonances of protein amino acid side chains that has several advantages over 3D versions of the experiment. In many cases, both the1H and13C chemical shifts can be obtained in the 4D experiment from a simple inspection of the13C(3),1H(4) planes extracted at the1H(1)/13C(2) chemical shifts. Together with the 3D and 4D triple resonance experiments, this allows sequence-specific assignments to be obtained. In addition, the increased resolution of the 4D experiment compared to its 3D counterpart allows. automation of resonance assignments. 相似文献
14.
The effect of monensin on the secretion of -amylase and other enzymes from the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya) was studied by electrophoresis followed by fluorography and by pulse-chase and organelle-isolation experiments. Monensin markedly inhibits the secretion, but not the synthesis, of -amylase, acid phosphatase, and at least four other proteins from the aleurone layer. Monensin treatment causes -amylase to accumulate within the protoplast, but its effect on the different -amylase isoenzymes is not equal. The accumulation of isoenzyme 2 is not influenced by monensin while isoenzymes 1, 3 and 4 are not secreted but rather accumulate in the cell when monensin is included in the incubation medium. The -amylase and acid-phosphatase activities which accumulate within the aleurone cells following treatment with monensin are localized in an organelle having a buoyant density greater than that of endoplasmic reticulum and less than that of mitochondria. In pulse-chase experiments with [35S]methionine, labelled proteins accumulate in this organelle in the presence of monensin and do not appear in the incubation medium. We conclude that monensin inhibits the secretion of proteins from the barley aleurone layer by influencing their intracellular transport.Abbreviations ER
endoplasmic reticulum
- GA3
gibberellic acid
- SDS-PAGE
sodium dodecyl-sulfate polyacrylamide-gel electrophoresis 相似文献
15.
Summary After treatment with the carboxylic ionophore monensin theChlorella containing perialgal vacuoles of the greenParamecium bursaria swell. TheParamecium cells remain motile at this concentration for at least one day. The swelling is only observed in illuminated cells and can be inhibited by DCMU. We assume that during photosynthesis the perialgal vacuoles are acidified and that monensin exchanges H+ ions against monovalent cations (here K+). In consequence the osmotic value of the vacuoles increases. The proton gradient is believed to drive the transport of maltose from the symbiont into the host. Another but light independent effect of the monensin treatment is the swelling of peripheral alveoles of the ciliates, likewise indicating that the alveolar membrane contains an active proton pump.Abbreviations HEPES
N-(2-hydroxyethyl)piperazine-N-2-ethane sulfonic acid
- DCMU
3-(3, 4-dichlorophenyl)-1,1-dimethylurea 相似文献
16.
Summary The effect of monensin on polysaccharide slime secretion by root tips of corn (Zea mays) was studied. Various treatment times and ionophore concentrations were tested: none resulted in inhibition of slime secretion. Because monensin changes the pH of the medium, its effect was also monitored in strongly buffered media and at different pH's. Even in such media, monensin did not inhibit slime secretion. We also measured the effect of the drug after a pulse with [3H]fucose or a pulse followed by a chase. The amount of labeled slimed secreted was not altered by the ionophore. However, 10M monensin affected the development of root tips and drastically reduced their growth. We showed that monensin inhibits the secretion of -amylase by the scutellum of the same plantlet. The importance of the nature of the secretory compound in relation to monensin inhibition of its secretion is discussed.Abbrevations Hepes
N-2-hydroxyethylpiperazine-N-2-ethane-sul-fonic acid
- Mes
2-(N-morpholino)ethane-sulfonic acid 相似文献
17.
Michael P. Williamson Tetsuo Asakura Eiji Nakamura Makoto Demura 《Journal of biomolecular NMR》1992,2(1):83-98
Summary The chemical shifts of CH protons have been calculated for 9 proteins, based on coordinates taken from high-resolution crystal structures. Chemical shifts were calculated using ring-current shifts, shifts arising from magnetic anisotropies of bonds, and shifts arising from the polarizing effect of polar atoms on the C-H bond. The parameters used were refined iteratively to give the best fit to (experimental — random coil) shifts over the set of 9 proteins. A further small correction was made to the averaged Gly CH shift. The calculated shifts match observed shifts with correlation coefficients varying between 0.45 and 0.86, with a standard deviation of about 0.3 ppm. The differences between calculated and observed shifts have been studied in detail, including an analysis of different crystal structures of the same protein, and indicate that most of the differences can be accounted for by small differences between the structure in solution and in the crystal. Calculations using NMR-derived structures give a poor fit. The calculations reproduce the experimentally observed differences between chemical shifts for CH in -helix and -sheet. Most of the differentiation in secondary structure-dependent shifts arises from electric field effects, although magnetic anisotropy also makes a large contribution to the net shift. Applications of the calculations to assignment (including stereospecific assignment) and structure determination are discussed. 相似文献
18.
Löhr F Pfeiffer S Lin YJ Hartleib J Klimmek O Rüterjans H 《Journal of biomolecular NMR》2000,18(4):337-346
A TROSY-based triple-resonance pulse scheme is described which correlates backbone 1H and 15N chemical shifts of an amino acid residue with the 15N chemical shifts of both the sequentially preceding and following residues. The sequence employs 1
J
NC and 2
J
NC couplings in two sequential magnetization transfer steps in an `out-and-back' manner. As a result, N,N connectivities are obtained irrespective of whether the neighbouring amide nitrogens are protonated or not, which makes the experiment suitable for the assignment of proline resonances. Two different three-dimensional variants of the pulse sequence are presented which differ in sensitivity and resolution to be achieved in one of the nitrogen dimensions. The new method is demonstrated with two uniformly 2H/13C/15N-labelled proteins in the 30-kDa range. 相似文献
19.
Molt cycle-associated changes in calcium-dependent proteinase activity that degrades actin and myosin in crustacean muscle 总被引:1,自引:0,他引:1
The role of calcium-dependent proteinase (CDP) in the proecdysial atrophy of crustacean claw muscle has been investigated. During atrophy the molar ratio of actin to myosin heavy chain decreased 31%, confirming earlier ultrastructural observations that the ratio of thin:thick myofilaments declined from 9:1 to 6:1 (D. L. Mykles and D. M. Skinner, 1981, J. Ultrastruct. Res., 75, 314–325). The release of TCA-soluble material in muscle homogenates at neutral pH was stimulated by Ca2+ and completely inhibited by EGTA. The specific degradation of the major myofibrillar proteins (actin, myosin heavy and light chains, paramyosin, tropomyosin, troponin-T, and troponin-I) was demonstrated by SDS-polyacrylamide gel electrophoresis. Proteolytic activity was more than twofold greater in proecdysial muscle homogenates. Degradation of myofibrillar proteins was inhibited by EGTA, and the two inhibitors of cysteine proteinases, leupeptin and antipain, but not pepstatin, an inhibitor of aspartic proteinases. Unlike CDPs from vertebrate muscle, the CDP(s) in crab claw muscle degrades actin and myosin in addition to other myofibrillar proteins. 相似文献
20.
C. S. Colwell D. Whitmore S. Michel G. D. Block 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1994,175(4):415-423
The eye of the marine mollusk Aplysia californica contains an oscillator that drives a circadian rhythm of spontaneous compound action potentials in the optic nerve. Both light and serotonin are known to influence the phase of this ocular rhythm. The aim of the present study was to evaluate the role of extracellular calcium in both light and serotonin-mediated phase shifts. Low calcium treatments were found to cause phase shifts which resembled those produced by the transmitter serotonin. However, unlike serotonin, low calcium neither increased ocular cAMP levels nor could these phase shifts be prevented by increasing extracellular potassium concentration. Low calcium-induced phase shifts were prevented by the simultaneous application of the translational inhibitor anisomycin and low calcium treatment resulted in changes in [35S]methionine incorporation into several proteins as measured by a two-dimensional electrophoresis gel analysis. Finally, light treatments failed to produce phase shifts in the presence of low calcium or the calcium channel antagonist nickel chloride. These results are consistent with a model in which serotonin phase shifts the ocular pacemaker by decreasing a transmembrane calcium flux through membrane hyperpolarization while light-induced phase shifts are mediated by an increase in calcium flux.Abbreviations ASW
artificial seawater
- EGTA
ethylene glycol-bis(-amino-ethyl ester) N,N,N N-tetraacetic acid
- CAP
compound action potential
- CT
circadian time 5-HT serotonin
- Ni++
nickel 相似文献