Concanavalin A and phorbol ester cause opposite subcellular redistribution of protein kinase C

Concanavalin A and phorbol ester induce human blood monocytes to produce superoxide. We tested whether activation of human monocytes by these agents is accompanied by a subcellular redistribution of protein kinase C. Phorbol ester predictably caused a profound shift of the enzyme from the cytosol to the particulate fraction. In contrast concanavalin A induced a shift of the enzyme from the particulate fraction to the cytosol. The opposite effect of these agents on kinase C translocation was observed also by analysis of the phosphorylation of cytosolic proteins. Kinase C is either not involved in monocyte activation or does so by distinct pathways determined by the activating agent.     

Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70

We reconstituted prepro-alpha-factor translocation and signal peptide processing using a yeast microsomal detergent soluble fraction formed into vesicles with soybean phospholipids. Reconstituted translocation required ATP, and was deficient when sec63 and kar2 (BiP) mutant cells were used as a source of membranes. Normal translocation was observed with vesicles reconstituted from a mixture of pure wild-type yeast BiP and a soluble fraction of kar2 mutant membranes. Two other heat-shock cognate (hsc) 70 homologs, yeast cytosolic hsc70 (Ssalp) and E. coli dnaK protein did not replace BiP. Conversely, BiP was not active under conditions where translocation into native ER vesicles required cytosolic hsc70. We conclude that cytosolic hsc70 and BiP serve noninterchangeable roles in polypeptide translocation, possibly because distinct, asymmetrically oriented membrane proteins are required to recruit each protein to opposing surfaces of the ER membrane.     

Proteomic and bioinformatic analysis of outer membrane proteins of the protobacterium Bartonella henselae (Bartonellaceae)

Bartonella henselae, an infectious agent causing cat-scratch disease and vasculoproliferative disorders in humans, is a fastidious facultative intracellular pathogen. The outer membrane proteins of B. henselae are key molecules that play a primary role in host-cell interactions. We isolated B. henselae outer membrane proteins, using the ionic detergent N-lauroyl sarcosine sodium salt and sodium carbonate, purification by two-dimensional (2-D) gel electrophoresis, and protein identification using mass spectrometry. Treatment with buffers containing ASB-14 and ZWITTERGENT 3-10 increased solubilization of B. henselae proteins, particularly proteins with basic pI. Three hundred and sixty-eight spots were detected from the sarcosine-insoluble outer membrane fraction; 94 distinct protein species were identified from 176 spots. In the outer membrane fraction from carbonate incubation, 471 spots were calculated and 259 spots were identified, which included 139 protein entries. There were six outer membrane proteins in the sarcosine-insoluble outer membrane fraction compared with nine outer membrane proteins from samples subjected to carbonate incubation. We used bioinformatic analysis to identify 44 outer membrane proteins by prediction of their domains and tertiary structures and documented the potential virulence factors. We established the 2-D reference maps of the outer membrane subproteome of B. henselae using the two different extraction methods, which were partly complementary to each other. Sodium carbonate extraction isolated low-abundance and basic proteins better than the lauroyl sarcosine sodium salt extraction, which enriched high-abundance porins.     

Alterations in the synthesis of proteins in hepatocytes of rainbow trout fed cyclopropenoid fatty acids

Hepatocytes from rainbow trout reared on a diet containing cyclopropenoid fatty acids were analyzed for alterations in protein composition and synthesis by double label experiments. Both cytosolic and microsomal hepatocyte fractions were investigated. In the cytosolic fraction, the synthesis of proteins in the range of 68,000 to 74,000 daltons were significantly decreased. The identity of these proteins remains uncertain. A pronounced depression in both the mass and apparent synthesis of a 200,000 to 240,000 dalton microsomal protein was also observed. Immunoblotting with antibodies raised against goose acetyl-CoA carboxylase and avidin-peroxidase staining suggest that this protein is acetyl-CoA carboxylase. Moreover, synthesis of this protein as well as mass of the protein in cyclopropenoid fatty acid-fed fish were less than 20% of that found in control fish.     

Distribution of Calretinin, Calbindin D28k, and Parvalbumin in Subcellular Fractions of Rat Cerebellum: Effects of Calcium

Abstract: The distribution of calretinin, calbindin D28k, and parvalbumin was examined in subcellular fractions prepared from rat cerebellum and analyzed by immunoblot. Calretinin was also quantified by radioimmunoassay. As expected, all three soluble, EF-hand calcium-binding proteins were predominantly localized in the cytosolic fraction. Calretinin and calbindin D28k were also detected in membrane fractions. Calretinin was more abundant in synaptic membrane than in microsomal fractions. The cerebellar microsomal fraction contained the greatest concentration of membrane-associated calbindin D28k. The association of calretinin and calbindin D28k with membrane fractions was decreased in samples prepared or incubated in low calcium. Quantification of calretinin in subcellular fractions of rat cerebellum revealed a greater amount of calretinin in cytosolic fractions prepared or incubated in low calcium and reduced amounts of calretinin in all membrane fractions incubated in low calcium with the exception of the mitochondrial fraction. These results imply that calretinin and calbindin D28k might have physiological target molecules that are associated with, or are components of, brain membranes.     

Regulation of microsomal stearoyl-coenzyme A desaturase. Purification of a non-substrate-binding protein that stimulates activity.

Crude cytosolic fraction from rat liver was examined for proteins that may be involved in regulation of microsomal stearoyl-CoA desaturase activity. Gel filtration revealed the presence of several components that either stimulate or inhibit this enzyme. In addition, other components bind the acyl-CoA substrate, thus affecting observed activities in vitro. A protein that stimulates stearoyl-CoA desaturase but does not bind substrate was purified approx. 1100-fold. The purified protein had no visible absorption spectrum and an approximate mol.wt. of 26500. Maximal stimulation of desaturase activity occurred with less than 2 micrometer purified protein. The protein was heat-labile and exhibited neither catalase nor glutathione peroxidase activity. Addition of the cytosolic protein produced no effect on the desaturase reaction stoicheiometry; the proportions O2 consumed/NADH oxidized/stearoyl-CoA desaturated remained 1:1:1. Because the Km' for stearoyl-CoA was also unchanged by addition of the cytosolic protein, no change in substrate affinity was suggested. Furthermore addition of the cytosolic protein also produced no effect on desaturase inhibition by oleoyl-CoA, which suggested that the protein does not simply relieve apparent product inhibition. These results indicate that, in analogy to other cytosolic proteins that stimulate microsomal oxidase activities, the protein may have a regulatory function, perhaps related to activity modulation via organization of the multienzymic desaturase in the membrane.     

Regulation of cytosolic COX-2 and prostaglandin E2 production by nitric oxide in activated murine macrophages

Murine macrophages (RAW 264.7) when stimulated with LPS show 90% distribution of cyclooxygenase-2 (COX-2) in the nuclear fraction and approximately 10% in the cytosolic fraction. Further analysis of this cytosolic fraction at 100,000 x g indicates that the COX-2 is distributed both in the 100,000 x g soluble fraction and membrane fraction. Stimulation of RAW 264.7 cells with LPS in the presence of inducible nitric oxide synthase inhibitor L-NMMA at concentrations that inhibit nitrite accumulation by /=85% with higher concentrations of L-NMMA shows 1) up-regulation of PGE2 production, 2) accumulation of COX-2 protein in the 100,000 x g soluble and membrane fractions of the cytosolic fraction, and 3) with no significant effects on the accumulation of COX-2 mRNA. These experiments suggest that low concentrations of nitric oxide (10-15% of the total) attenuate PGE2 production in response to LPS in RAW 264.7 cells. This inhibition is, in part, due to decreased expression of cytosolic COX-2 protein.     

The in vivo turnover of rat liver microsomal epoxide hydrolase and both the apoprotein and heme moieties of specific cytochrome P-450 isozymes

The in vivo turnover rates of liver microsomal epoxide hydrolase and both the heme and apoprotein moieties of cytochromes P-450a, P-450b + P-450e, and P-450c have been determined by following the decay in specific radioactivity from 2 to 96 h after simultaneous injections of NaH14CO3 and 3H-labeled delta-aminolevulinic acid to Aroclor 1254-treated rats. Total liver microsomal protein was characterized by an apparent biphasic exponential decay in specific radioactivity, with half-lives of 5-9 and 82 h for the fast- and slow-phase components, respectively. Most (approximately 90%) of the rapidly turning over microsomal protein fraction was immunologically distinct from membrane-associated serum protein, and thus appeared to represent integral membrane proteins. The existence of two distinct populations of cytochrome P-450a was suggested by the apparent biphasic turnover of both the heme and apoprotein moieties of the holoenzyme. The half-lives of the apoprotein were estimated to be 12 and 52 h for the fast- and slow-phase components, respectively, and 7 and 34 h for the heme moiety. The turnover of cytochromes P-450b + P-450e was identical to that of cytochrome P-450c, with half-lives of 37 and 28 h for the apoprotein and heme moieties, respectively. In all cases, the shorter half-lives of the heme component compared to the protein component were statistically significant. In contrast to the cytochrome P-450 isozymes, epoxide hydrolase (t1/2 = 132 h) turned over slower than the "average" microsomal protein (t1/2 = 82 h). The differential rates of degradation of these major integral membrane proteins during both the rapid and slow phases of total microsomal protein turnover argue against the concepts of unit membrane degradation and unidirectional membrane flow of liver endoplasmic reticulum.     

The process of promutagen biotransformation studied by the Ames test. II. The extent of the manifestation of the mutagenic action of nitrosomorpholine, diethylnitrosamine and cyclophosphane using various subfractions of rat liver homogenate

The data are presented on involvement of components of microsomal and cytosolic subfractions composing the S-9 fraction of rat liver homogenate in processes leading to formation of active metabolites of nitrosomorpholine (NM), diethyl nitrosoamine (DENA) and cyclophosphane (CP) promutagens and their detoxication resulting from the reaction with glutathione (G-SH) added to the system. It is established that the process of metabolic activation is only connected with microsomal subfraction, while reactions of the first phase of CP and DENA metabolism take place when both microsomal and cytosolic subfractions are added. Decrease in the effect of all promutagens studied under the action of G-SH was observed after microsomal and cytosolic subfractions of the S-9 fraction were introduced into the activating mixture. Various values of dependence of the metabolic activation level and the extent of decrease in the mutagenic action, upon addition of G-SH, on the protein content in microsomal and cytosolic subfractions were obtained.     

Phosphatidylinositol kinase,phosphatidylinositol-4-phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions

Subcellular fractions isolated and purified from rat brain cerebral cortices were assayed for phosphatidylinositol (PI-), phosphatidylinositol-4-phosphate (PIP-), and diacylglycerol (DG-) kinase activities in the presence of endogenous or exogenously added lipid substrates and [γ-³²P]ATP. Measurable amounts of all three kinase activities were observed in each subcellular fraction, including the cytosol. However, their subcellular profiles were uniquely distinct. In the absence of exogenous lipid substrates, PI-kinase specific activity was greatest in the microsomal and non-synaptic plasma membrane fractions (150–200 pmol/min per mg protein), whereas PIP-kinase was predominantly active in the synaptosomal fraction (136 pmol/min per mg protein). Based on percentage of total protein, total recovered PI-kinase activity was most abundant in the cytosolic, synaptosomal, microsomal and mitochondrial fractions (4–11 nmol/min). With the exception of the microsomal fraction, a similar profile was observed for PIP-kinase activity when assayed in the presence of exogenous PIP (4 nmol/20 mg protein in a final assay volume of 0.1 ml). Exogenous PIP (4 nmol/20 mg protein) inhibited PI-kinase activity in most fractions by 40–70%, while enhancing PIP-kinase activity. PI- and PIP-kinase activities were observed in the cytosolic fraction when assayed in the presence of exogenously added PI or PIP, respectively, but not in heat-inactivated membranes containing these substrates. When subcellular fractions were assayed for DG-kinase activity using heat-inactivated DG-enriched membranes as substrate, DG-kinase specific activity was predominantly present in the cytosol. However, incubation of subcellular fractions in the presence of deoxycholate resulted in a striking enhancement of DG-kinase activities in all membrane fractions. These findings demonstrate a bimodal distribution between particulate and soluble fractions of all three lipid kinases, with each exhibiting its own unique subcellular topography. The preferential expression of PIP-kinase specific activity in the synaptic membranes is suggestive of the involvement of PIP₂ in synaptic function, while the expression of PI-kinase specific activity in the microsomal fraction suggests additional, yet unknown, functions for PIP in these membranes.     

rap1B, a cAMP-dependent protein kinase substrate, associates with the platelet cytoskeleton

rap1B is a member of the ras superfamily of low molecular weight GTP binding proteins which constitutes a focal point of GTP and cAMP signal transduction systems. Like other members of this superfamily, rap1B is membrane-associated in resting platelets, presumably through polyisoprenylation. The studies presented here were undertaken to determine the subcellular changes in rap1B localization during cell activation. Activated and unactivated platelets were fractionated by Triton X-100 lysis followed by differential centrifugation to obtain a 10,000 x g cytoskeleton fraction, a 100,000 x g membrane skeleton fraction, and a 100,000 x g supernatant fraction containing solubilized proteins. In unactivated platelets, rap1B was present in the 100,000 x g supernatant fraction. In contrast, in platelets activated with 1 unit/ml alpha-thrombin or with the calcium ionophore, A23187, rap1B was quantitatively recovered in the 10,000 x g cytoskeleton fraction. rap1B was absent from the 100,000 x g fraction containing the membrane skeleton and could not be detected in the 100,000 x g supernatant containing cytosolic proteins and solubilized membrane components. These results indicate that rap1B associates with the cytoskeleton during cell activation.     

Subcellular localization of ubiquitin and ubiquitinated proteins in Arabidopsis thaliana.

Ubiquitin is a highly conserved, 76-amino acid, eukaryotic protein. Its widely accepted role as a proteolytic cofactor depends on its unique ability to covalently ligate to other cellular proteins. While there is good evidence for the existence of such ubiquitinated proteins in the cytosolic and nuclear compartments, relatively little is known about the presence of free ubiquitin and ubiquitinated proteins in other subcellular compartments. This is especially true of higher plants, which have not previously been the subject of extensive biochemical subcellular localizations of ubiquitinated proteins. We extracted cell wall proteins and purified nuclei, vacuoles, chloroplasts, and microsomes from chlorophyllous tissues of Arabidopsis. Immunoblot analyses were used to compare the profiles of ubiquitinated proteins from purified subcellular fractions to those from unfractionated extracts. Purified nuclei contained, in addition to a complex mixture of high molecular mass ubiquitinated proteins, a strongly immunoreactive 28-kDa protein. In the apoplastic extract, we did not detect any ubiquitinated proteins enriched above the background level of those due to cytosolic contamination. Vacuoles appeared to contribute significantly to the ubiquitinated proteins present in the whole protoplast extract. At least three high molecular mass ubiquitinated proteins were unique to the vacuolar extract. Chloroplast stromal proteins did not react specifically with anti-ubiquitin antibodies. When microsomal ubiquitinated proteins were compared to those found in a whole protoplast extract, a distinct pattern was evident. Microsomal ubiquitinated proteins were not visible in the 10,000 x g supernatant used to prepare the 100,000 x g pellet, indicating that they were probably low abundance proteins in the protoplast extract.     

Biogenesis of microsomal membrane glycoproteins in rat liver. II. Purification of soluble glycoproteins and their incorporation into microsomal membranes

Sialoproteins isolated from the soluble fraction of rat liver could be incorporated into microsomal membranes. This incorporation was dependent on protein concentration, time, and temperature. Sodium dodecyl sulfate gel electrophoresis of membrane proteins after in vitro incorporation showed four major sugar-containing peaks and was similar to that found after in vivo labeling. Most of the incorporated protein was tightly bound to the microsomal membrane. Gel filtration and ion-exchange chromatography revealed the presence of several cytosolic glycoproteins that could be incorporated into microsomes. During prolonged centrifugation in a KBr solution with a density of 1.21 a highly labeled ([3H]glucosamine) protein (mole wt approximately to 70,000) that was actively incorporated into microsomes could be recovered in the upper region of the tube. These results demonstrate that several cytoplasmic glycoproteins of rat liver are transferred into microsomal membranes and that one of these is a lipoprotein.     

Reproducibility,sensitivity and compatibility of the ProteoExtract® subcellular fractionation kit with saturation labeling of laser microdissected tissues

Laser microdissection (LMD), a method of isolating specific microscopic regions of interest from a tissue that has been sectioned, is increasingly being applied to study proteomics. LMD generally requires tissues to be fixed and histologically stained, which can interfere with protein recovery and subsequent analysis. We evaluated the compatibility and reproducibility of protein extractions from laser microdissected human colon mucosa using a subcellular fractionation kit (ProteoExtract^®, Calbiochem). Four protein fractions corresponding to cytosol (fraction 1), membrane/organelle (fraction 2), nucleus (fraction 3) and cytoskeleton (fraction 4) were extracted, saturation labeled with Cy5 and 5 μg separated by both acidic (pH 4–7) and basic (pH 6–11) 2‐DE. The histological stains and fixation required for LMD did not interfere with the accurate subcellular fractionation of proteins into their predicted fraction. The combination of subcellular fractionation and saturation CyDye labeling produced very well resolved, distinct protein spot maps by 2‐DE for each of the subcellular fractions, and the total number of protein spots consistently resolved between three independent extractions for each fraction was 893, 1128, 1245 and 1577 for fractions 1, 2, 3 and 4, respectively. Although significant carryover of protein did occur between fractions, this carryover was consistent between experiments, and very low inter‐experimental variation was observed. In summary, subcellular fractionation kits are very compatible with saturation labeling DIGE of LMD tissues and provide greater coverage of proteins from very small amounts of microdissected material.     

Characterization of microsomal and cytosolic alpha-1,2-mannosidases from mung bean hypocotyls

Microsomal and cytosolic alpha-mannosidase activities, which hydrolyze alpha-1,2-mannosyl-mannose linkages in the Man5GlcNAc2 oligosaccharide, have been isolated from homogenates of mung bean hypocotyls. The alpha-1,2-mannosidase activities were readily distinguished from previously described aryl alpha-mannosidases by several criteria. They were optimally active in the presence of Ca2+ between pH 5.5 and 6, they were inhibited by Zn2+, and they had essentially no activity with p-nitrophenyl-alpha-mannoside. The microsomal and cytosolic alpha-1,2-mannosidases demonstrated specificity for oligosaccharides with terminal nonreducing alpha-1,2-mannosyl linkages, and they were inhibited by mannosyl-mannose disaccharides, with the inhibition decreasing in the order of alpha-1,2-greater than alpha-1,3-greater than alpha-1,6-mannosyl-mannose. The cytosolic alpha-1,2-mannosidase activity, which was present in the 100,000 g supernatant, was separated from the aryl alpha-mannosidase by ammonium sulfate precipitation. The microsomal alpha-1,2-mannosidase, which was tightly associated with the particulate fraction, was solubilized with Triton X-100 and 0.2 M KCl. The two alpha-1,2-mannosidase activities were readily differentiated by gel-filtration chromatography. The solubilized microsomal enzyme chromatographed in approximately the same position as a Mr 460,000 globular protein whereas the cytosolic enzyme was eluted in a retarded position, indicating a much smaller protein.     

Localization of the estrogen receptor in uterine cells by affinity labeling with [3H]tamoxifen aziridine

The possibility that estrogen receptors may exist in uterine plasma membranes was investigated by covalent labeling of estrogen receptors in mouse uterine cells with [3H]tamoxifen aziridine (TA). Isolated epithelial and stromal cells of immature mice were incubated with [3H]TA in the presence or absence of unlabeled tamoxifen, homogenized and separated into nuclear, cytosolic and microsomal fractions by differential centrifugation. These fractions were subjected to SDS-polyacrylamide gel electrophoresis and the proteins labeled covalently with TA were visualized by autoradiography. Proteins labeled specifically with [3H]TA were observed almost exclusively in the nuclear fraction of both epithelial and stromal cells. In contrast, very little labeled protein was detected in the cytosolic or microsomal fraction. Although these data do not preclude the possibility that estrogen binding sites are present in plasma membranes of uterine cells, this cellular fraction is definitely not labeled to a significant extent by [3H]TA. Thus, if membrane estrogen binding sites exist, their structural conformations may be different from that of nuclear estrogen receptors.     

Effect of microsome-liposome fusion on the rotational mobility of cytochrome P450IIB4 in rabbit liver microsomes

Membrane fusion of microsomes with soybean phospholipid vesicles was performed at pH 6.5 to investigate the effect of lipid-enrichment in the membrane on the rotational mobility of cytochrome P450. Rotational diffusion of cytochrome P450 in the microsomal membrane of phenobarbital-induced rabbit liver was measured by detecting the decay of absorption anisotropy after photolysis of the heme CO complex by a vertically polarized laser flash. The fusion procedures yielded three separate fractions upon sucrose density gradient centrifugation with lipid-to-protein ratio in weight (L/P) as follows: 1.5 in the bottom fraction, 2.2 in the middle fraction, and 3.9 in the top fraction. In each fraction, co-existence of mobile and immobile cytochrome P450 was observed. The percentage of rotationally mobile P450 (with the mean rotational relaxation time of phi=505-828 micros) in each of the different bands was found to be 59% in the bottom fraction, 61% in the middle fraction, and 68% in the top fraction. This increase in mobile population of P450 due to lipid-enrichment indicates that aggregated proteins in microsomal membranes dissociate with increasing L/P which is inversely proportional to the protein concentration in the membrane. With freeze-fracture electron microscopy, it was shown that the average distance increased between intramembrane particles by lipid-enrichment. Thus, the significant immobile population (32%) of P450 in microsomal membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P of 0.8. The decrease in the mobile population in the bottom fraction compared with intact microsomes was shown to be due to the pH 6.5 incubation used for fusion.     

High density lipoprotein binding protein of eel (Anguilla japonica) liver with specificity of binding to apoAI as a ligand

High-density lipoprotein (HDL) binding protein (HBP) was isolated from the microsomal fraction of eel liver homogenate by affinity chromatography with a HDL-column. After SDS-PAGE and blotting, HBP on the PVDF membrane was detected by FITC-labeled HDL and apolipoprotein AI (apoAI) as a ligand. HBP in the microsomal fraction was most abundant among microsomal, mitochondrial and cytosolic fractions. The HBP isolated by a HDL-column consisted of at least three proteins with low molecular weights of 18.5, 14.5 and 13.5 kDa; the main component was 14.5 kDa. These proteins are not products of protease digestion, as the procedure was carried out in the presence of protease inhibitors including (p-aminophenyl) methansulfonyl fluoride, 4-(2-aminoethyl)-benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, aprotinin and EDTA. The HBP specifically bound to FITC-apoAI and faintly bound or did not bind to FITC-apoAII. Furthermore, binding of HDL labeled with lipophilic fluorescence to isolated eel hepatocytes was inhibited by the antibody to apoAI, but not inhibited by the antibody to apolipoprotein AII (apoAII). These results strongly suggest that the HBP isolated from the microsomal fraction is present on the plasma membrane of eel liver and plays important roles for the lipid transport through the interaction with HDL.