Discontinuous Sucrose Density Gradient Centrifugation Research Articles

Separation of Single Core and Multicore Lytic Granules by Subcellular Fractionation and Immunoisolation.

Subcellular fractionation is an important tool used to separate intracellular organelles, structures or proteins. Here, we describe a stepwise protocol to isolate two types of lytic granules, multicore (MCG), and single core (SCG), from primary murine CTLs. We used cell disruption by nitrogen cavitation followed by separation of organelles via discontinuous sucrose density gradient centrifugation. Immunoisolation with a Synaptobrevin 2 antibody attached to magnetic beads was then used to harvest Synaptobrevin 2 positive granules for immunoblotting, mass spectrometry, electron, and light microscopy.

A2E Distribution in RPE Granules in Human Eyes.

A2E (N-retinylidene-N-retinylethanolamine) is a major fluorophore in the RPE (retinal pigment epithelium). To identify and characterize A2E-rich RPE lipofuscin, we fractionated RPE granules from human donor eyes into five fractions (F1–F5 in ascending order of density) by discontinuous sucrose density gradient centrifugation. The dry weight of each fraction was measured and A2E was quantified by liquid chromatography/mass spectrometry (LC/MS) using a synthetic A2E homolog as a standard. Autofluorescence emission was characterized by a customer-built spectro-fluorometer system. A significant A2E level was detected in every fraction, and the highest level was found in F1, a low-density fraction that makes up half of the total weight of all RPE granules, contains 67% of all A2E, and emits 75% of projected autofluorescence by all RPE granules. This group of RPE granules, not described previously, is therefore the most abundant RPE lipofuscin granule population. A progressive decrease in autofluorescence was observed from F2 to F4, whereas no autofluorescence emission was detected from the heavily pigmented F5. The identification of a novel and major RPE lipofuscin population could have significant implications in our understanding of A2E and lipofuscin in human RPE.

Development of a Japanese encephalitis virus-like particle vaccine in silkworms using codon-optimised prM and envelope genes.

We have successfully prepared a Japanese encephalitis virus (JEV) − Nakayama virus like particle (NVLP) vaccine using synthetic codon-optimized prM and E genes. The expression of the recombinant JEV Nakayama-BmNPV (JEV-NNPV) virus was determined in infected silkworm Bm-N cells by fluorescence and Western blot analysis. The recombinant was inoculated into silkworm pupae and the yield of Nakayama VLP (NVLP) reached a peak in the homogenates after 3 days. Additionally, in the peptide analysis of infected pupae homogenate, it appeared approximately 300–500 μg E protein/pupa were produced. When purified the above eluates on the discontinuous sucrose density gradient centrifugation, NVLP showed a strong hemagglutination (HA) activity by using chicken red blood cell in phosphate-buffered saline (PBS) free from Mg++ and Ca++ ions. The immune antisera against NVLP strain could efficiently neutralize the plaque formation of Nakayama, Beijing-1 and Muar strains, showing tendency of much higher reaction with heterologous Muar strain than homologous Nakayama strain. Our findings suggest that the JEV-NVLP may be useful for JEV epidemic control in many endemic areas of Asian countries as a widely effective and less expensive JE vaccine.

Comparison of Chloroplast Proteomes Extracted from Florets of Physiological and Genic Male Sterile Lines and Their Maintainer Line in Wheat

The objective of this study was to explain the male sterile mechanism of wheat (Triticum aestivum L.) based on chloroplast proteome. A method for isolating intact chloroplast proteome from wheat floret was established. Using this method, the chloroplast proteomes were extracted from florets of the genic and physiological male sterile lines and their maintainer line, and separated in 2-dimensional electrophoresis (2-DE) gels. The cytoplasmic-nuclear sterile line, ms(S)-1376, had identical nuclear background with the maintainer line, (A)-1376, and the physiological male sterile line, ms(A)-1376, was derived from (A)-1376 after induction of chemical hybridizing agent SQ-1. The extraction method was effective to obtain high purity of intact chloroplast using discontinuous sucrose density gradient centrifugation with 3-step gradient densities of 30, 45, and 60%. The 2-DE result showed that the floret chloroplast protein profiles were different among the 3 lines at uninucleate anther stage, and 239 protein spots were visible on each gel (pH 4-7, molecular weight 14.4-66.2 kD). Six differentially expressed proteins were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) and indexed in bioinformation database. They were identified as acyl-CoA dehydrogenase domain protein, calmodulin-binding protein phosphatase, multiple catalytic peptidase, heat-shock protein 60, light receptor protein 2, and a protein of unknown function. These proteins are involved in series of physiological reactions such as metabolism of energy substances, chloroplast defendance, chloroplasts signal transduction, and plant growth. The differential expressions of these proteins among the 3 lines are likely related to the male sterility in wheat.

Detergent-free isolation and characterization of cholesterol-rich membrane domains from trans-Golgi network vesicles

Cholesterol is an abundant lipid of the trans-Golgi network (TGN) and of certain endosomal membranes where cholesterol-rich microdomains are important in the organization and compartmentalization of vesicular trafficking. Here we describe the development of a rapid method to isolate a cholesterol-rich endomembrane fraction. We show that widely used subcellular fractionation techniques incompletely separate cholesterol-rich membranes, such as the TGN, from organelles, such as late endosomes and lysosomes. To address this issue, we devised a new subcellular fractionation scheme involving two rounds of velocity centrifugation, membrane sonication, and discontinuous sucrose density gradient centrifugation. This strategy resulted in the isolation of a cholesterol and GM1 glycosphingolipid-enriched membrane fraction that was completely cleared of plasma membrane, endoplasmic reticulum, and mitochondria. This buoyant fraction was enriched for the TGN and recycling endosome proteins Rab11 and syntaxin-6, and it was well resolved from cis-Golgi and early and late endosomal membranes. We demonstrate that this technique can give useful insights into the compartmentation of phosphoinositide synthesis, and it facilitates the isolation of cholesterol-rich membranes from a population of TGN-trafficking vesicles.

Effect of calmodulin-like protein from buffalo (Bubalus bubalis) seminal plasma on Ca2+, Mg(2+)-ATPase of purified plasma membrane of buffalo spermatozoa.

Buffalo sperm heads and tails were cleaved by sonication and isolated in relatively pure proportions i.e. 95% and 98% respectively, by discontinuous sucrose density-gradient centrifugation. Purified plasma membranes from the isolated sperm heads and tails were obtained by hypotonic treatment and brief sonication followed by discontinuous sucrose density-gradient centrifugation. Ca2+, Mg(2+)-ATPase activity was evident in plasma membrane from sperm heads and tails, although activity was greater in the latter. A calmodulin-like protein isolated from buffalo seminal plasma increased the Ca2+, Mg(2+)-ATPase of plasma membrane from the sperm heads and tails by 128 and 136% respectively. Based upon the data obtained here and elsewhere (Sidhu & Guraya, 1989a) a model is proposed which explains regulation of Ca2+ in buffalo spermatozoa and implicates calmodulin-like protein and Ca2+, Mg(2+)-ATPase in sperm acrosome reaction.

Selective targeting of cAMP signaling components and adhesion molecules to caveolin‐enriched fractions of pulmonary microvascular endothelial cells (PMVECs)

Strongly adherent PMVECs interact to form the pulmonary capillary barrier, necessary to maintain efficient gas exchange in the lung. Tight cell‐cell junctions between adjacent pulmonary capillary endothelial cells and cytoskeletal elements control barrier integrity and are dynamically regulated by cAMP. Within PMVECs, calcium sensitive adenylyl cyclase (AC) activity has been resolved in a phosphodiesterase 4 regulated membrane fraction. However, compartmentation of AC and other signaling molecules involved in barrier regulation within PMVEC membranes has not been described. Utilizing detergent free discontinuous sucrose density gradient centrifugation to separate caveolae/lipid raft fractions from the bulk plasma membrane we detect AC5/6 in the caveolin rich light density fractions. TRPC4 a putative molecular component of the store operated calcium entry channel also resides in caveolin fractions. In addition, filamin, an actin binding protein that when PKA phosphorylated stabilizes the actin cytoskeleton, also localizes to caveolae with cell adhesion molecules N‐ and E‐cadherin, and ALCAM. In contrast, VE‐cadherin is found outside of lipid rafts in the bulk plasma membrane compartment. Therefore, it appears that AC6 compartmentalizes to caveolae with other signaling components that dynamically regulate endothelial barrier. Supported by the Welcome Trust, HL66299 and F32HL091651

Bacillus thuringiensis BT-1, BT-2가 생산하는 δ-endotoxin의 특성 규명

Bacillus thuringiensis is a well-known species of entomophathogenic bacteria that is widely used as a biopesticide against many insect pests. It produces parasporal crystals (δ-endotoxin) and endospores during sporulation. In this report, the δ-endotoxin produced by Bacillus thuringiensis BT-1 and BT-2 were characterized by Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM), SDS-PAGE, and solubilization activity by alkaline solution. BT-1, BT-2 were cultured in the GBY medium, and the δ-endotoxin of them was purified with discontinuous sucrose density gradient centrifugation. Their δ-endotoxin was observed by SEM and TEM. Morphologically, the δ-endotoxin of BT-1 was a square and flat type, whose size was 1.73 ㎛ × 0.7 ㎛, and the δ-endotoxin of the BT-2 was spherical form whose size was 1.1 ㎛ × 0.9 ㎛ determined by SEM and TEM. The δ-endotoxin of the BT-1 was composed of 28 kDa and 21 kDa, however, it of the BT-2 was composed of 50 kDa, 35 kDa, and 22 kDa bands determined by SDS-PAGE. The purified crystals of BT-1 and BT-2 were dissolved gradually in alkaline solution as time goes by, and it was perfectly dissolved after 3 hours. It is supposed that the δ-endotoxin of crystal was converted to a state of activation in the course of time in the intestines of insect.

Adenylyl cyclase 5/6 colocalizes with TRPC4 and cell adhesion molecules to caveolin‐enriched fractions of pulmonary microvascular endothelial cells (PMVECs)

Strongly adherent PMVECs interact to form the pulmonary capillary barrier, necessary to maintain efficient gas exchange in the lung. Tight cell-cell junctions between adjacent pulmonary capillary endothelial cells contribute to the integrity of this barrier and are dynamically regulated by transitions in cAMP. Within PMVECs calcium sensitive adenylyl cyclase activity has been resolved in a phosphodiesterase 4 regulated membrane fraction. However, compartmentation of adenylyl cyclase and other signaling molecules involved in barrier regulation within the PMVEC membrane has not been described. Here we utilize a detergent free discontinuous sucrose density gradient centrifugation technique to separate caveolae/lipid raft fractions from the bulk plasma membrane. AC5/6 immunoreactivity was detected in the caveolin rich light-density fractions while beta adaptin immunoreactivity was detected in the high-density fraction of non-caveolae regions within the plasma membrane. TRPC4 a putative molecular component of the store operated calcium entry channel also resides in the caveolin rich fraction. In addition, cell adhesion molecules N-cadherin and ALCAM and the scaffolding molecule Dlg localize to caveolae. Therefore, it appears that AC6 compartmentalizes to caveolae with other signaling components that dynamically regulate endothelial barrier. Supported by the Welcome Trust, HL66299 and HL60024

Fas-, Caspase 8-, and Caspase 3-dependent Signaling Regulates the Activity of the Aminophospholipid Translocase and Phosphatidylserine Externalization in Human Erythrocytes

Apoptosis and erythrocyte senescence share the common feature of exposure of phosphatidylserine (PS) in the outer leaflet of the cells. Western analysis showed that mature red cells contain Fas, FasL, Fas-associated death domain (FADD), caspase 8, and caspase 3. Circulating, aged cells showed colocalization of Fas with the raft marker proteins Galpha(s) and CD59; the existence of Fas-associated FasL, FADD and caspase 8; and caspase 8 and caspase 3 activity. Aged red cells had significantly lower aminophospholipid translocase activity and higher levels of PS externalization in comparison with young cells. In support of our contention that caspases play a functional role in the mature red cell, the oxidatively stressed red cell recapitulated apoptotic events, including translocation of Fas into rafts, formation of a Fas-associated complex, and activation of caspases 8 and 3. These events were independent of calpain but dependent on reactive oxygen species (ROS) as evident from the effects of the ROS scavenger N-acetylcysteine. Caspase activation was associated with loss of aminophospholipid translocase activity and with PS externalization. ROS was not generated by treatment of cells with t-butyl hydroperoxide at 10 degrees C, and Fas did not translocate into rafts. Concomitantly, neither formation of a Fas-associated signaling complex nor caspase activation could be observed, supporting the view that translocation of Fas into rafts was the trigger for the chain of events leading to caspase 3 activation. Our data demonstrate for the first time the novel involvement of Fas/caspase 8/caspase 3-dependent signaling in an enucleated cell leading to PS externalization, a central feature of erythrophagocytosis and erythrocyte biology.

Mitochondrial DNA integrity is not dependent on DNA polymerase-β activity

Mutations in mitochondrial DNA (mtDNA) are involved in a variety of pathologies, including cancer and neurodegenerative diseases, as well as in aging. mtDNA mutations result predominantly from damage by reactive oxygen species (ROS) that is not repaired prior to replication. Repair of ROS-damaged bases occurs mainly via base excision repair (BER) in mitochondria and nuclei. In nuclear BER, the two penultimate steps are carried out by DNA polymerase-β (Polβ), which exhibits both 5′-deoxyribose-5-phosphate (5′-dRP) lyase and DNA polymerase activities. In mitochondria, DNA polymerase-γ (Polγ) is believed to be the sole polymerase and is therefore assumed to function in mitochondrial BER. However, a recent report suggested the presence of Polβ or a “Polβ-like” enzyme in bovine mitochondria. Consequently, in the present work, we tested the hypothesis that Polβ is present and functions in mammalian mitochondria. Initially we identified two DNA polymerase activities, one corresponding to Polγ and the other to Polβ, in mitochondrial preparations obtained by differential centrifugation and discontinuous sucrose density gradient centrifugation. However, upon further fractionation in linear Percoll gradients, we were able to separate Polβ from mitochondria and to show that intact mitochondria, identified by electron microscopy, lacked Polβ activity. In a functional test for the presence of Polβ function in mitochondria, we used a new assay for detection of random (i.e., non-clonal) mutations in single mtDNA molecules. We did not detect enhanced mutation frequency in mtDNA from Polβ null cells. In contrast, mtDNA from cells harboring mutations in the Polγ exonuclease domain that abolish proofreading displayed a ≥17-fold increase in mutation frequency. We conclude that Polβ is not an essential component of the machinery that maintains mtDNA integrity.

Characteristics of the Polyamine Transporter TPO1 and Regulation of Its Activity and Cellular Localization by Phosphorylation

The subcellular localization of the polyamine transporter TPO1 of Saccharomyces cerevisiae was determined by sucrose gradient centrifugation and indirect immunofluorescence microscopy. When expressed from a multi-copy vector, TPO1 was located mainly on the plasma membrane, but with some localization on the vacuolar membrane. Polyamine transport by TPO1 was dependent on pH. Uptake of spermidine and spermine occurred at alkaline pH (pH 8.0), whereas inhibition of spermidine uptake, but not spermine uptake, was observed at acidic pH (pH 5.0). This suggests that TPO1 catalyzes polyamine excretion at acidic pH, similar to the PotE transporter in Escherichia coli. Paraquat, a polyamine analogue, was excreted by TPO1 at a rate comparable with the excretion of spermidine (deduced from the inhibition of spermidine uptake) at pH 5.0. However, excretion of preloaded radiolabeled spermidine and spermine was not observed in intact cells, suggesting that preloaded spermidine (or spermine) exists mainly as spermidine (or spermine)-ribosome complex in cells. The transport activity of TPO1 was enhanced through phosphorylation at Ser19 by protein kinase C and at Thr52 by casein kinase 1. Sorting of TPO1 from the endoplasmic reticulum to the plasma membrane was enhanced through phosphorylation at Ser342 by cAMP-dependent protein kinases 1 and 2.

Regulated Internalization and Phosphorylation of the Native Norepinephrine Transporter in Response to Phorbol Esters: EVIDENCE FOR LOCALIZATION IN LIPID RAFTS AND LIPID RAFT-MEDIATED INTERNALIZATION

The effects of norepinephrine in the brain and periphery are terminated primarily by active reuptake of the catecholamine via cocaine- and amphetamine-sensitive norepinephrine transporters (NETs). Activation of protein kinase C (PKC) down-regulates NET by sequestering it from the plasma membrane, although the underlying mechanism is not yet known. Previously, we showed robust expression of endogenous NETs in rat placental trophoblasts (Jayanthi, L. D., Vargas, G., and DeFelice, L. J. (2002) Br. J. Pharmacol. 135, 1927-1934). Here we report a significant reduction in native NET function and surface expression in these cells following phorbol ester (beta-PMA) treatment. The beta-PMA-mediated down-regulation of NET occurs by a rapid sequestration of NETs from the plasma membrane and is calcium-independent. Reversible biotinylation experiments revealed a significant enhancement of NET endocytosis following beta-PMA treatment. Chemical treatments and expression of dominant negative mutants of dynamin 1 and 2 failed to prevent the beta-PMA effect, suggesting a clathrin-independent pathway. In contrast, treatment with the cholesterol-disrupting agent filipin, which blocks caveolae/lipid raft-mediated internalization, completely blocked the beta-PMA-mediated NET sequestration. Discontinuous sucrose density gradient centrifugation revealed NET in the lipid raft fractions. Following beta-PMA treatment, there was reduced NET levels in the lipid raft fractions suggesting that cholesterol-rich lipid rafts mediate PKC-triggered NET internalization. Metabolic labeling and immunoprecipitation studies revealed that NET phosphorylation is stimulated severalfold by PKC activation and protein phosphatase 1/2A inhibition. Together, these findings demonstrate for the first time that in trophoblasts (i) PKC activation regulates NET function and surface expression by an enhanced internalization process that is lipid raft-mediated and (ii) PKC and protein phosphatase(s) modulation regulates NET phosphorylation.

Isolation and partial characterisation of the plasma and outer acrosomal membranes of goat spermatozoa

In order to achieve successful fertilization, the essential steps include maturation, species-specific binding and fusion of gametes followed by activation of the ovum. Implicit in this are a variety of molecular events, the most important among them being the receptor–ligand interaction. A protocol is described to isolate highly enriched fractions of goat sperm plasma and the outer acrosomal membranes and present data on the ultrastructure and enzyme assays of these membrane domains. The sperm, washed with 1.3 M sucrose sedimentation, was sonicated and subjected to discontinuous sucrose density gradient centrifugation, which allowed the separation of isolated plasma and the outer acrosomal membranes in a single step. While the isolated plasma membranes formed vesicles, the cap shaped acrosomal segments retained its characteristic morphology, as was seen in the ultrastructure. The plasma membrane fraction was relatively pure and free of any contamination of other cell organelles. The matrix associated with the isolated acrosomal membranes gave it a relatively higher density and a crescent shape. Enzyme assays showed high alkaline phosphatase activity in the plasma membrane fraction, with low activity of the acrosome bound enzymes. The acrosomal fraction had a higher specific activity of acrosin and hyaluronidase and a low alkaline phosphatase activity. Suggesting that both the membranes were pure. Membranes, thus purified could be used as initial material for elucidating the lipid as well as the antigenic composition and in understanding the mechanism involved in capacitation, gametic interaction and acrosome reaction, which eventually lead to fertilization.

Separation of distinct compartments of rice Golgi complex by sucrose density gradient centrifugation

We studied the separation of distinct compartments of the Golgi complex in rice and tobacco employing the discontinuous sucrose density gradient centrifugation technique with EDTA or MgCl 2. In the presence of EDTA, rice Golgi marker enzymes, nucleoside diphosphatase and N-acetylglucosaminyltransferase were fractionated in the 28 and 33% sucrose fractions, respectively. Peanut lectin-recognized glycoproteins and reversibly glycosylated polypeptide-1 that are shown to be located at Golgi complex were broadly distributed around a 31.5% sucrose density. Under a MgCl 2-supplemented condition, the peak of nucleoside diphosphatase was shifted to 29 and 37% sucrose and N-acetylglucosaminyltransferase was distributed in the 37% sucrose fraction. The distribution of peanut lectin-recognized glycoproteins and reversibly glycosylated polypeptide-1 were also shifted to the 34.5% sucrose fraction. Furthermore, glucan synthase-I and UDP-glucose pyrophosphorylase were fractionated in the 26.5% sucrose fraction, regardless of the presence or absence of MgCl 2. These results indicate that there exist at least four distinct compartments of the rice Golgi complex. The tobacco Golgi marker enzymes and Golgi-localized proteins were distributed in the 30–31% sucrose fractions in the presence of EDTA and slightly shifted to the 32–33% sucrose fractions under a MgCl 2-supplemented condition. These results suggest that the sedimentation behavior of compartments of the rice Golgi complex in the gradients is specific for rice cells.

Low-density caveolae-like membrane from Xenopus laevis oocytes is enriched in Ras.

Detergent-free discontinuous sucrose density gradient centrifugation was used to resolve low- and high-density membrane fractions from Xenopus laevis oocytes. Compared to high-density membrane, low-density oocyte membrane is enriched two-fold in cholesterol and highly enriched in ganglioside GM1. Protein immunoblotting of membrane fractions from whole cells with polyclonal anti-human caveolin antibody detected multiple bands, including a distinctive triad with apparent molecular weights of 21, 33, and 48 kDa. To more clearly determine which of these caveolin-like protein(s) is associated with the oocyte plasma membrane, microdissection was used to separate external membrane (cortical preparations containing plasma membrane) from intracellular membrane. Cortical membrane preparations displayed a single 21-kDa caveolin-like protein in low-density membrane. Internal oocyte membrane displayed the higher molecular weight bands of 33 and 48 kDa and a lesser amount of the 21-kDa protein in low-density membrane fractions. Monoclonal anti-human Ras antibody detected a single 23-kDa immunoblot band that is enriched an average of eight-fold in low-density membrane fractions prepared from whole cells. This is the first report of caveolin-associated, low-density membrane in amphibian oocytes, and is consistent with a role for caveolin and caveolae-like microdomains in oocyte signal transduction.

Characterization of caveolae from rat heart: Localization of postreceptor signal transduction molecules and their rearrangement after norepinephrine stimulation

Caveolae are plasma membrane subcompartments that have been implicated in signal transduction. In many cellular systems, caveolae are rich in signal transduction molecules such as G proteins and receptor-associated tyrosine kinases. An important structural component of the caveolae is caveolin. Recent evidence show that among the caveolin gene family, caveolin-3 is expressed in skeletal and cardiac muscle and caveolae are present in cardiac myocyte cells. Both the ANP receptor as well as the muscarinic receptor have been localized to the caveolae of cardiac myocytes in culture. These findings prompted us to conduct a further analysis of cardiac caveolae. In order to improve our understanding of the mechanisms of signal transduction regulation in cardiac myocytes, we isolated cardiac caveolae by discontinuous sucrose density gradient centrifugation from rat ventricles and rat neonatal cardiocytes. Our analysis of caveolar content demonstrates that heterotrimeric G proteins, p21ras and receptor-associated tyrosine kinases are concentrated within these structures. We also show that adrenergic stimulation induces an increase in the amount of diverse a- and b-subunits of G proteins, as well as p21ras, in both in vivo and in vitro experimental settings. Our data show that cardiac caveolae are an important site of signal transduction regulation. This finding suggests a potential role for these structures in physiological and pathological states. J. Cell. Biochem. 77:529-539, 2000. © 2000 Wiley-Liss, Inc.

Characterization of caveolae from rat heart: Localization of postreceptor signal transduction molecules and their rearrangement after norepinephrine stimulation

Caveolae are plasma membrane subcompartments that have been implicated in signal transduction. In many cellular systems, caveolae are rich in signal transduction molecules such as G proteins and receptor-associated tyrosine kinases. An important structural component of the caveolae is caveolin. Recent evidence show that among the caveolin gene family, caveolin-3 is expressed in skeletal and cardiac muscle and caveolae are present in cardiac myocyte cells. Both the ANP receptor as well as the muscarinic receptor have been localized to the caveolae of cardiac myocytes in culture. These findings prompted us to conduct a further analysis of cardiac caveolae. In order to improve our understanding of the mechanisms of signal transduction regulation in cardiac myocytes, we isolated cardiac caveolae by discontinuous sucrose density gradient centrifugation from rat ventricles and rat neonatal cardiocytes. Our analysis of caveolar content demonstrates that heterotrimeric G proteins, p21ras and receptor-associated tyrosine kinases are concentrated within these structures. We also show that adrenergic stimulation induces an increase in the amount of diverse alpha- and beta-subunits of G proteins, as well as p21ras, in both in vivo and in vitro experimental settings. Our data show that cardiac caveolae are an important site of signal transduction regulation. This finding suggests a potential role for these structures in physiological and pathological states.

32] Identification of phosphorylation sites within vertebrate and invertebrate rhodopsin

Publisher Summary Studies of vertebrate and invertebrate rhodopsin have been applicable to our understanding of amplification and desensitization processes of Gprotein-coupled receptors in general. In vertebrate rod outer segments, photoisomerization of rhodopsin (Rho*) initiates activation of hundreds of G-protein (G t ) molecules, which in turn stimulate cGMP phosphodiesterase, resulting in closure of cGMP gated channels in response to the decrease in cytosolic cGMP concentrations. To study the enzymatic and functional roles of Rho phosphorylation, it was necessary to develop protocols for the identification of phosphorylation sites within vertebrate and invertebrate Rho. This chapter describes a method that employs mass spectrometry in conjunction with specific proteolysis, and should be applicable to the study of phosphorylation in other systems. Rho phosphorylation is carried out on rod outer segment (ROS) membranes isolated from fresh bovine retinas by discontinuous sucrose density gradient centrifugation in 10 mM Bis-Tris propane (BTP) buffer, pH 7.5, containing 5 mM MgCl2 and 1 mM ATP at 30° for 0–45 minutes under illumination from a 150-W lamp at a distance of 10 cm. Phosphorylated forms of the peptide could, in principle, be analyzed by tandem mass spectrometry (MS/MS) to identify the specific residues phosphorylated.

Effects of Exogenous Fatty Acids on H +-ATPase Activity and Lipid Composition of Plasma Membrane Vesicles Isolated from Roots of Barley Seedlings under Salt Stress

Summary The effects of exogenous fatty acids of different saturaturation degrees on the H+-ATPase activity and lipid composition of the plasma membrane (PM) vesicles isolated from the roots of two barley cultivars differing in salt-sensitivity (Jian 4, salt-tolerant; Kepin 7, salt-sensitive) were investigated under 100 mmol L−1 NaCl stress for 2 days. The PM vesicles with high purity were prepared by discontinuous sucrose density gradient centrifugation. The H+-ATPase activities, phospholipid contents and the index of unsaturated fatty acids (IUFA) increased in the PM vesicles isolated from the roots of seedlings of two barley cultivars under NaCl stress. However, there was no obvious change in the IUFA value with exogenous fatty acids of different saturation degrees. Exogenous stearic acid and linoleic acid increased the H+-ATPase activities and phospholipid contents, but their effects on galactolipid contents of the PM of Kepin 7 were different. For Jian 4, treated with linoleic acid, the galactolipid content increased by 115.85 %; the PM H+-ATPase activity and phospholipid contents increased slightly. This is consistent with the lower Na+ content and Na+/K+ ratio in leaves of salt-tolerant Jian 4 under salt stress.