Analysis of Antigens from Membrane and Soluble Fractions of Entamoeba histolytica

ABSTRACT. Axenic cultures of Entamoeba histolytica strains HK-9, HM-1, and Rahman were fractionated to provide plasma membranes, internal, vesiculated membranes, and a soluble cytosol. Each particulate fraction was solubilized and all fractions were analyzed by techniques designed to demonstrate molecular complexity and serologic reactivity. The cytosol contained more antigenic moieties than either membrane fraction; however, the antigens associated with the membranes had very high reactivity and lower nonspecific activity than the cytosol.     

Effects of the cytosol fraction and ATP on the kinetics of cAMP binding to human erythrocyte membranes

The cytosol fraction of human erythrocytes as well as 1 mM ATP decreased the rate constant (Kl) of association of [3H]cAMP with human erythrocyte membranes. However, the effect of 1 mM ATP on the association kinetics was much greater than that of the cytosol fraction. Accordingly, the cytosol fraction and 1 mM ATP increased the rate constant (k-l) of dissociation of bound [3H]cAMP from the human erythrocyte membranes. However, the cytosol fraction affected the dissociation kinetics to a far greater extent than 1 mM ATP. Thus, although both cytosol and 1 mM ATP decreased the affinity of [3H]cAMP for binding sites on human erythrocyte membranes at equilibrium, the detailed mode of their action on the membrane [3H]cAMP binding sites appears to differ.     

Alteration in protein kinase C activity and subcellular distribution in sickle erythrocytes

In agreement with previous data, membrane protein phosphorylation was found to be altered in intact sickle cells (SS) relative to intact normal erythrocytes (AA). Similar changes were observed in their isolated membranes. The involvement of protein kinase C (PKC) in this process was investigated. The membrane PKC content in SS cells, measured by [3H]phorbol ester binding, was about 6-times higher than in AA cells. In addition, the activity of the enzyme, measured by histone phosphorylation was also found to be increased in SS cell membranes but decreased in their cytosol compared to the activity in AA cell membranes and cytosol. The increase in membrane PKC activity was observed mostly in the light fraction of SS cells, fractionated by density gradient, whereas the decrease in cytosolic activity was only observed in the dense fraction. PKC activity, measured in cells from the blood of reticulocyte-rich patients, exhibited an increase in both membranes and cytosol, thus explaining some of the effects observed in the SS cell light fraction, which is enriched in reticulocytes. The increase in PKC activity in the membranes of SS cells is partly explained by their young age but the loss of PKC activity in their cytosol, particularly in that of the dense fraction, seems to be specific to SS erythrocytes. The relative decrease in membrane PKC activity between the dense and the light fractions of SS cells might be related to oxidative inactivation of the enzyme.     

Interaction of fatty acid binding protein with microsomes: removal of palmitic acid and retinyl esters.

[14C] palmitic acid or [3H] retinyl esters incorporated in microsomal membranes were removed by a cytosolic fraction enriched in fatty acid binding protein. When mouse liver cytosol was fractionated by 70% ammonium sulphate, a precipitate and a soluble fraction were obtained. The soluble fraction containing the fatty acid binding protein was able to remove from microsomal membranes, [14C] palmitic acid or [3H] retinyl esters, whereas the precipitate fraction had no removal capacity. Retinoid analysis indicated that 70% ammonium sulphate soluble fraction was enriched in endogenous retinyl esters with regard to cytosol or 70% ammonium sulphate precipitate fraction.     

Subcellular localization and some properties of lipoxygenase activity in human blood platelets.

Lipoxygenase activity was measured in human platelet subcellular fractions. From a sonicated platelet preparation, a granule fraction, mixed membranes (surface and intracellular) and cytosol fractions were separated by differential centrifugation. With respect to activities in the sonicated preparation, the lipoxygenase was slightly enriched in both the cytosol and mixed-membrane fractions and consistently de-enriched in the granule fractions. Approx. 65% and 20% of the total cell enzyme activity were found in the cytosol and mixed membranes respectively, with only 8% present in the granule fraction. Additionally we measured the lipoxygenase activity in purified surface- and intracellular-membrane subfractions prepared from the mixed membranes by free-flow electrophoresis. There was a slight enrichment in activity in the intracellular membrane fraction compared with that in the mixed membranes, and a depletion of activity in the surface membranes. Characterization of the enzyme activity, i.e. time course, pH-dependence, Ca2+-dependence, Vmax. and Km for arachidonic acid, and the carbon-position specificity for this acid, failed to reveal any significant differences between the membrane-bound and soluble forms of the lipoxygenase. These findings suggest that in human platelets the same lipoxygenase is associated with the membranes as in the cytosol and that the membrane-bound activity predominates in intracellular membrane elements.     

Increase in ornithine decarboxylase activity and polyamine levels of the rat liver during an early period of hepatocarcinogenesis

The ornithine decarboxylase activity and the polyamine content in the fraction of plasma membranes and cytosol of the rat liver are studied in the early period (1-4 weeks) of nitrosodiethyl amine-induced hepatocarcinogenesis. The enzyme activity and polyamine levels in the cytosol of the rat liver cells are found to increase sharply during the first month of the disease, the maximum being observed the first-second week. By the end of the fourth week these indices become lower but they remain significantly higher than the normal levels. The polyamine level increases considerably in the fraction of plasma membranes with the maximum observed the second week.     

Phospholipid/Ca2+-dependent protein kinase activity in human parathyroid adenoma

We have examined the activities of phospholipid/Ca2+-dependent and cyclic AMP-dependent protein kinases of the parathyroid adenomas and the atrophic glands which were resected from three patients with primary hyperparathyroidism. Phospholipid/Ca2+-dependent protein kinase activity of atrophic parathyroid gland was exclusively present in cytosol fraction (90.7 +/- 12.3%). On the other hand, phospholipid/Ca2+-dependent protein kinase activity of parathyroid adenomas was 66.9 +/- 6.4% in cytosol and 33.1 +/- 6.4% in membrane fraction, suggesting a translocation of the enzyme from the cytosol to the membranes. Cyclic AMP-dependent protein kinase activity appeared to be higher in parathyroid adenoma than in atrophic parathyroid gland in both cytosol and membrane fractions.     

Evidence of the presence of extraperoxisomal catalase in chloragogen cells of the earthworm, Lumbricus terrestris L

The DAB reactivity of the midintestine of the earthworm, consisting of epithelial layer, muscle layer, and chloragogen tissue, was examined electron microscopically. Besides the mitochondrial membranes of the examined cell types and the hemoglobin content of the blood vessels and chloragogen cells, a considerable DAB reactivity was found in the whole cytosol of the chloragocytes. The DAB reaction of the cytosol was more intensive when incubation medium for catalase, less intensive when incubation medium for peroxidase, was used and did not occur when H2O2 was omitted. Cytosol of the chloragogen cells was isolated and preliminary assay of catalase and peroxidase activities was made. Cytosol samples showed moderate peroxidase activity, but catalase activity measured by the decomposition of hydrogen peroxide showed a very high rate. Catalase and peroxidase activities of the cytosol were heat-sensitive and might have been inhibited by azide and cyanide, respectively. Results prove the assumption that the intensive DAB reactivity of the chloragocyte cytosol is caused by its extraperoxisomal catalase content.     

Involvement of pp60c-src in platelet-activating factor-stimulated platelets. Evidence for translocation from cytosol to membrane.

We have investigated the characteristics of platelet-activating factor (PAF)-stimulated protein tyrosine phosphorylation in rabbit platelets and its relationship to pp60c-src. 32P-Labeled platelets were challenged with PAF (10(-7) M) for 15 s, the reaction was killed by lysis at 4 degrees C, and samples were loaded onto a phosphotyrosine monoclonal antibody (Tyr(P)-mAb)-agarose column. The column was eluted with 10 mM phenyl phosphate, and the fractions were collected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography of the column fractions, showed that PAF increased the radioactivity of about a dozen protein bands with predominant ones of approximate molecular masses of 50, 60, 71, 82, and 300 kDa. When Tyr(P)-mAb-agarose column fractions were subjected to immunoblotting with pp60v-src mAb, it was observed that PAF treatment increased the reactivity of 50- and 60-kDa protein species. Immunoprecipitation with pp60v-src mAb further confirmed that PAF treatment increased phosphorylation of the 60- and 50-kDa proteins. Polyclonal antibody to G-protein (alpha-subunit) did not exhibit any reactivity to the column fractions and thus ruled out this protein as substrate for the tyrosine kinase. We next attempted to localize the pp60c-src. Platelet membrane particulate and cytosol fractions were separated from control and PAF-treated platelets, and it was observed that the immunoreactivity to pp60v-src mAb dramatically increased in the particulate membrane fraction from PAF-treated platelets. A concomitant decrease in the immunoreactivity in the cytosol fraction of PAF-treated platelets was also noted. It is concluded that PAF stimulates phosphorylation of pp60c-src tyrosine kinase and causes its rapid translocation from cytosol to membranes in rabbit platelets.     

Evidence of the presence of extraperoxisomal catalase in chloragogen cells of the earthworm,Lumbricus terrestris L.

Summary The DAB reactivity of the midintestine of the earthworm, consisting of epithelial layer, muscle layer, and chloragogen tissue, was examined electron microscopically. Besides the mitochondrial membranes of the examined cell types and the hemoglobin content of the blood vessels and chloragogen cells, a considerable DAB reactivity was found in the whole cytosol of the chloragocytes. The DAB reaction of the cytosol was more intensive when incubation medium for catalase, less intensive when incubation medium for peroxidase, was used and did not occur when H₂O₂ was omitted.Cytosol of the chloragogen cells was isolated and preliminary assay of catalase and peroxidase activities was made. Cytosol samples showed moderate peroxidase activity, but catalase activity measured by the decomposition of hydrogen peroxide showed a very high rate. Catalase and peroxidase activities of the cytosol were heat-sensitive and might have been inhibited by azide and cyanide, respectively. Results prove the assumption that the intensive DAB reactivity of the chloragocyte cytosol is caused by its extraperoxisomal catalase content.     

Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation

Formation of the nuclear pore is an intricate process involving membrane fusion and the ordered assembly of up to 1,000 pore proteins. As such, the study of pore assembly is not a simple one. Interestingly, annulate lamellae, a cytoplasmic organelle consisting of stacks of flattened membrane cisternae perforated by numerous pore complexes, have been found to form spontaneously in a reconstitution system derived from Xenopus egg extracts, as determined by electron microscopy (Dabauvalle et al., 1991). In this work, a biochemical assay for annulate lamellae (AL) formation was developed and used to study the mechanism of AL assembly in general and the assembly of individual nucleoporins into pore complexes in particular. Upon incubation of Xenopus egg cytosol and membrane vesicles, the nucleoporins nup58, nup60, nup97, nup153, and nup200 initially present in a disassembled form in the cytosol became associated with membranes and were pelletable. The association was time and temperature dependent and could be measured by immunoblotting. Thin-section electron microscopy as well as negative staining confirmed that annulate lamellae were forming coincident with the incorporation of pore proteins into membranes. Homogenization and subsequent flotation of the membrane fraction allowed us to separate a population of dense membranes, containing the integral membrane pore protein gp210 and all other nucleoporins tested, from the bulk of cellular membranes. Electron microscopy indicated that annulate lamellae were enriched in this dense, pore protein-containing fraction. GTP gamma S prevented incorporation of the soluble pore proteins into membranes. To address whether AL form in the absence of N-acetylglucosaminylated pore proteins, AL assembly was carried out in WGA-sepharose-depleted cytosol. Under these conditions, annulate lamellae formed but were altered in appearance. When the membrane fraction containing this altered AL was homogenized and subjected to flotation, the pore protein- containing membranes still sedimented in a distinct peak but were less dense than control annulate lamellae.     

Biogenesis of microsomal membrane glycoproteins in rat liver. I. Presence of glycoproteins in microsomes and cytosol

The glycoproteins of microsomes and cytosol were studied. Various washing procedures did not release the proteins from the microsomes, and immunological tests demonstrated that the sialoproteins are not serum components. Low concentrations of deoxycholate and incubation in 0.25 M sucrose solution liberated a small amount of microsomal sialoprotein and this fraction exhibited a high degree of labeling of protein-bound N-acetylneuraminic acid. A part of the glycoprotein fraction could not be solubilized, even with a high concentration of the detergent. Thoroughly perfused rat liver contained sialoproteins in the particle-free supernate. The level of sialoprotein present could not be due to contamination with serum or broken organelles. The high in vivo incorporation of [3H]glucosamine into protein-bound sialic acid of Golgi membranes and cytosol was paralleled by a delayed and lesser rate of incorporation into the rough and smooth microsomal membranes. This incorporation pattern suggests the possibility that the glycoproteins of cytosol and Golgi may later be incorporated into the membrane of the endoplasmic reticulum.     

Intestinal caveolin-1 is important for dietary fatty acid absorption

How dietary fatty acids are absorbed into the enterocyte and transported to the ER is not established. We tested the possibility that caveolin-1 containing lipid rafts and endocytic vesicles were involved. Apical brush border membranes took up 15% of albumin bound ³H-oleate whereas brush border membranes from caveolin-1 KO mice took up only 1%. In brush border membranes, the ³H-oleate was in the detergent resistant fraction of an OptiPrep gradient. On OptiPrep gradients of intestinal cytosol, we also found the ³H-oleate in the detergent resistant fraction, separate from OptiPrep gradients spiked with ³H-oleate or ³H-triacylglycerol. Caveolin-1 immuno-depletion of cytosol removed 91% of absorbed ³H-oleate whereas immuno-depletion using IgG, or anti-caveolin-2 or -3 or anti-clathrin antibodies removed 20%. Electron microscopy showed the presence of caveolin-1 containing vesicles in WT mouse cytosol that were 4 fold increased by feeding intestinal sacs 1 mM oleate. No vesicles were seen in caveolin-1 KO mouse cytosol. Caveolin-1 KO mice gained less weight on a 23% fat diet and had increased fat in their stool compared to WT mice. We conclude that dietary fatty acids are absorbed by caveolae in enterocyte brush border membranes, are endocytosed, and transported in cytosol in caveolin-1 containing endocytic vesicles.     

Differential Activities of Protein Phosphatase Types 1 and 2A in Cytosolic and Participate Fractions from Rat Forebrain

Abstract: The activities and concentrations of protein phosphates type 1 (PP1) and type 2A (PP2A) were compared in cytosol and particulate fractions of rat forebrain. Although the activity of PP2A was highest in the cytosol, immunoblot analysis with a PP2A-specific antibody showed that there were significant levels of the enzyme in the particulate fraction. There was no significant difference between the concentration of PP2A in the cytosol and particulate fractions such that the low activity of PP2A in the particulate fraction represents an inactivation of this form of the enzyme. Similar analysis in skeletal muscle, heart, and liver showed this finding was unique to the brain. Similarly, the majority of PP1 activity was recovered in the cytosol, but most PP1 enzyme was associated with the particulate fraction. Comparison with other tissues showed that the activities of PP1 in the particulate fractions were similar but that the forebrain contained significantly more enzyme than the other tissues. Thus, like PP2A it appears that the specific activity of PP1 in the particulate fraction of rat forebrain is much lower than that of the cytosol and of the particulate fractions of other tissues. Elution of PP1 and PP2A from membranes with 0.5 M NaCl plus 0.3% Triton X-100 resulted in severalfold activation of both enzymes. That the majority of PP1 and PP2A in rat forebrain are associated with membrane structures but in a low activity state suggests that novel regulatory mechanisms exist that have considerable and unique potential for activation of protein dephosphorylation.     

Phosphorylation of low molecular weight proteins in purified preparations of rat heart sarcolemma and sarcoplasmic reticulum

A rat heart sarcolemmal preparation could be obtained in which both 5'-nucleotidase and adenylate cyclase were enriched approx. 9-fold by subjecting a homogenate to a discontinuous sucrose gradient, without the use of a high salt extraction. After incubation of this fraction with Mg[gamma-32P]ATP, the majority of 32P incorporated was present in 24 000- and 9000-dalton protein components. Only when a heart cytosol fraction or a purified cyclic AMP-dependent protein kinase was added, was enhancement of 32P-incorporaton found by addition of cyclic AMP. The 9000- and 24 000-dalton proteins appeared to be interconvertible. The degree of conversion could be affected by changing the temperature during solubilizaion of the membranes in SDS prior to electrophoresis. This suggested that the 24 000-dalton protein does not correspond to phospholamban, first identified by others in canine heart sarcoplasmic reticulum. Moreover, it could be excluded that the 24 000-dalton protein was derived from contaminating myofibrillar troponin I. When the sarcolemmal fraction was preincubated with Ca2+, Mg2+, ATP and oxalate, contaminating sarcoplasmic reticulum vesicles, loaded with calcium oxalate, settled to a greater density in the sucrose gradient. Membrane constituents other than those with enzymatic activity were monitored to confirm the separation between sarcolemmal and sarcoplasmic reticulum membranes: Coomassie blue staining material, sialic acid, cholesterol and phospholipid. The 24 000- and 9000-dalton proteins were equally distributed among the sarolemmal and sarcoplasmic reticulum fractions present in the sucrose gradient. However, the rate of 32P-incorporation in the presence of heart cytosol fraction was much slowr in the sarcoplasmic reticulum than in the sarcolemmal fraction.     

Catechol-O-methyl transferase in mouse liver plasma membranes.

Catechol-o-methyl transferase is usually localized predominantly in the cytosol fraction of cells, but fractionation of mouse liver showed plasma membranes contain ～ 70% of the total enzyme activity and have a specific activity ～ 10x greater than the cytosol fraction. Treatment of the membrane fraction with Lubrol-PX solubilized 47% of the membrane protein and 95% of the enzyme activity. A comparison of Lubrol-solubilized enzyme and [³H]norepinephrine binding activities in a variety of experimental conditions suggest binding is not related to interaction with the active site of catechol-o-methyl transferase. Isoelectric focusing of solubilized membrane proteins showed the enzyme has an isoelectric pH of 4.5-4.8.     

Multiple small molecular weight GTP-binding proteins in bovine brain cytosol. Purification and characterization of a 24KDa protein

We have separated multiple small Mr GTP-binding proteins (G-proteins) from bovine brain crude membranes, purified a novel 24KDa G protein (smg p25A) to near homogeneity and characterized it. In this paper, we have studied these small Mr G proteins in the cytosol fraction of bovine brain. [35S]GTP gamma S-binding activity is detected in the cytosol fraction but this activity is one-sixth to one-eighth of that of the crude membrane fraction. When G proteins in the cytosol fraction are purified by successive chromatographies on DEAE-cellulose, Ultrogel AcA-44, hydroxyapatite and Mono Q HR5/5 columns, multiple small Mr G proteins are separated. One of these G proteins shows a Mr of about 24KDa. Its physical, immunological and kinetic properties are indistinguishable from smg p25A. These results indicate that there are also multiple small Mr G proteins in the cytosol fraction of bovine brain, and suggest that one of the cytosol G proteins is the soluble form of smg p25A.     

Characterization of estrogen and antiestrogen binding to the cytosol and microsomes of breast tumors

The binding of [3H]estradiol and [3H]hydroxytamoxifen to the cytosol and microsomal fractions of several human breast tumors was investigated. By washing microsomal membranes with a KCl-free or a KCl-containing medium we could distinguish between intrinsic, extrinsic and contaminant estradiol binding sites in these membranes. We observed that treatment of the microsomes with low salt medium removes about 80% of the total estradiol binding sites, whereas 20% are not extractable. The concentration of unextractable [3H]estradiol binding sites in the microsomes varies in proportion to the level of cytosolic estrogen receptors (ER). About 10% of the total extranuclear specific estrogen binding sites was consistently found tightly associated to the microsomal fraction, which displays an affinity for estradiol (Kd = 0.1-0.6 nM) similar to that of the cytosolic ER. The displacement of [3H]estradiol with unlabeled hormone or with the antiestrogens, nafoxidine, enclomiphene and tamoxifen (TAM) exhibits identical IC50 values either in the cytosol or in the microsomal membranes. On the other hand, the microsomal fraction of breast tumors also binds [3H]hydroxyTAM, but with higher capacity and lower affinity than those of the cytosolic fraction. Furthermore, we did not observe correlation between the concentrations of ER and of antiestrogen binding sites (AEBS) in the tumors. These results indicate that microsomal membranes of human breast tumors contain estrogen binding sites which may be related to the cytosol ER recycling and that specific AEBS are predominantly localized in this membrane system. Furthermore, it is shown that the magnitude of estradiol binding to microsomes depends on the ER positive degree of the tumors, whereas the magnitude of the antiestrogen binding to the microsomes is independent of the ER status of the tumors.     

The synthesis of reduced metabolites of aldosterone by subcellular fractions of rat kidney: effects of antimineralocorticoids

Subcellular fractionation of male rat kidney revealed that the nuclear and plasma membrane fractions isolated from the 1,000 g pellet retained a significant proportion of the aldosterone ring-A reducing activity. Improved HPLC solvent systems separated all six possible ring-A reduced metabolites of aldosterone and revealed that 80-90% of the reduced metabolites synthesized by purified nuclei and plasma membranes were 5 alpha-reduced compounds consisting of 5 alpha-DHA and 3 alpha,5 alpha-THA in ratios of 1:2 (nuclei) and 1:1 (membranes). The 105,000 g cytosol also synthesized significant quantities of reduced, hydroxylated, and conjugated metabolites of aldosterone. In contrast, the majority of the reduced metabolites of aldosterone synthesized by kidney cytosol were 5 beta-products, consisting principally of 5 beta-DHA and smaller quantities of 3 alpha,5 beta-THA and 3 beta,5 beta-THA. The synthesis of reduced aldosterone metabolites in the cytosol, nuclear, and plasma membrane fraction was inhibited by both 5 and 50 microM concentrations of the antimineralocorticoids, progesterone, K+-canrenoate, and corticosterone. Progesterone was the strongest inhibitor of the synthesis of 5 alpha-DHA and 3 alpha,5 alpha-THA in both nuclei and plasma membranes. The overall order of inhibition of the synthesis of ring-A reduced metabolites in the kidney subcellular fractions was progesterone greater than K+-canrenoate greater than corticosterone; both progesterone and K+-canrenoate inhibited 5 alpha-reduction more than 5 beta-reduction.     

Similarities between a dipeptide hydrolase from brush-border and cytosol fractions of guinea-pig intestinal mucosa.

A dipeptide hydrolase from the brush border of guinea-pig intestinal mucosa was purified. The enzyme resembles another dipeptide hydrolase isolated from the cytosol fraction of intestinal mucosa. Studies on the binding of cytosol peptide hydrolase to brush-border membranes indicate that the enzyme found in the brush border may be a cytoplasmic contaminant.