The determination and localization of sialic acid in guinea-pig granulocytes.

When intact guinea-pig granulocytes (polymorphonuclear leucocytes) disrupted by sonication or with detergent were treated with neuraminidase from Vibrio cholerae, 3.1--3.2 nmol of sialic acid/10(7) cells was released. By using a chromatographic procedure for the specific determination of total cell sialic acid, this releasable portion was found to constitute 70% of the total sialate. All of the neuraminidase-releasable sialic acid of the cells could be removed by enzymic treatment of intact cells with neuraminidase. It thus seemed likely that the neuraminidase-releasable sialic acid is all on the cell surface. To make sure that the result was not due to entry of neuraminidase into the cells, the enzyme was bound covalently to Sepharose 6B, and intact polymorphonuclear leucocytes were treated with the bound enzyme. All of the neuraminidase-releasable sialic acid could still be removed, though more slowly. The cells remained intact and only 1.5--2% of the bound enzyme was released from the Sepharose during incubation. Freed enzyme could have been responsible, at the very most, for release of 18% of the sialic acid. Fractionation studies showed that the nucleus and cytoplasm contain low amounts of sialic acid and that the neuraminidase-releasable sialic acid distributes in a manner similar to the distribution of 5'-nucleotidase, an unambiguous marker for the plasma membrane in these cells. Thus neuraminidase-releasable sialate constitutes a clear marker for the membrane of polymorphonuclear leucocytes. Most of the neuraminidase-insensitive sialate was present in the granule fraction. Removal of sialic acid from intact polymorphonuclear leucocytes did not affect their ecto-AMPase, -ATPase and -p-nitrophenyl phosphatase activities.     

A comparison of the subcellular distribution of 5′-nucleotidase, (Na+K+)-ATPase and adenyl cyclase in beef thyroid gland

The validity of 5′-nucleotidase as a plasma membrane marker enzyme in beef thyroid has been tested by comparing the subcellular distribution of its activity to that of (Na⁺K⁺)-activated ATPase and adenyl cyclase. The specific activity and total activity of (Na⁺K⁺)-ATPase and adenyl cyclase were greatest in the 1000 × g (“nuclear”) and 33 000 × g (“mitochondrial and lysosomal”) fractions. In contrast, 5′-nucleotidase activity was concentrated in the 165 000 × g (“microsomal”) pellet and supernatant. Partially purified plasma membranes were separated from the 1000 (N₂), 30 000 (M₂) and 165 000 × g (P₂) pellets by discontinuous sucrose gradient centrifugation. Again a discordant distribution of these enzyme activities was observed. (Na⁺K⁺)-ATPase specific activity was increased approximately 30-fold over the homogenate in Fractions N₂ and M₂. Basal, thyroid-stimulating hormone-and fluoride-stimulated adenyl cyclase activities were concentrated in the same fractions. 5′-Nucleotidase activity was preferentially located in M₂ and P₂. These differences in distribution pattern suggest that 5′-nucleotidase activity is not uniquely located in the plasma membrane in the thyroid.     

Subcellular distribution of prostaglandin and gonadotrop in receptors in bovine corpora lutea.

The subcellular distribution of prostaglandin (PG) E₁, F₂α and gonadotropin receptors in bovine corpora lutea was critically examined by preparing various subcellular fractions, assaying for various marker enzymes to assess the purity and examining ³H-PGE₁, ³H-PGF₂α and ¹²⁵I-human lutropin (hLH) specific binding. The marker enzyme data suggested that subcellular fractions were relatively pure with little or no cross contamination. The binding of ³H-PGs and ¹²⁵I-hLH was markedly enriched in plasma membranes with respect to homogenate. The other subcellular fractions also exhibited binding despite very little or no detectable 5′-nucleotidase activity. If 5′-nucleotidase was assumed to lack sensitivity and reliability to detect minor contamination with plasma membranes and ³H-PGs or ¹²⁵I-hLH binding were used as sensitive plasma membrane markers, it was still difficult to explain binding in other fractions based on plasma membrane contamination. Therefore, these results lead to the inevitable conclusion that plasma membranes were primary (or one of the primary) but not exclusive sites for PGE₁, PGF₂α and gonadotropin receptors.     

Gonadotropin and prostaglandins binding sites in nuclei of bovine corpora lutea

Highly purified nuclei isolated from bovine corpora lutea showed marked enrichment of NAD pyrophosphorylase, a marker for this organelle. Rough endoplasmic reticulum and lysosomal markers were undetectable, whereas plasma membrane and Golgi markers were detectable but not enriched in nuclei. These highly purified nuclei exhibited specific binding with ¹²⁵I-labeled human choriogonadotropin, [³H]prostaglandin E₁ and [³H]prostaglandin F_2α. However, these bindings were only 15.4% (human choriogonadotropin), 7.9% (prostaglandin E₁) and 8.9% (prostaglandin F_2α) of the plasma membrane binding observed under the same conditions. Washing of nuclei and plasma membranes twice with buffer containing 0.1% Triton X-100 resulted in gonadotropin and prostaglandin F_2α binding site and 5′-nucleotidase (EC 3.1.3.5) losses from nuclei that were different from those observed for plasma membranes. More importantly, the washed nuclei exhibited 44% (human choriogonadotropin), 21–26% (prostaglandins) of original specific binding despite virtual disappearance of 5′-nucleotidase activity. The nuclear membranes isolated from nuclei, specifically bound ¹²⁵I-labeled human choriogonadotropin and [³H]prostaglandin F_2α to the same extent or significantly more ([³H]prostaglandin E₁, P < 0.05) than nuclei themselves, despite the marked losses of chromatin. In summary, our data suggest that gonadotropin and prostaglandins bind to nuclei and that this binding was intrinsic and was primarily associated with the nuclear membrane.     

Studies on the alkaline phosphatase and 5'-nucleotidase of Dictyostelium discoideum.

The alkaline phosphatase and 5′-nucleotidase activities of Dictyostelium discoideum are due to two distinct enzymes. Both enzymes are membrane bound, but over 90% of the 5′-nucleotidase activity is solubilized when the crude membrane fraction of the cell is treated with phospholipase C under conditions that release only 10% of the alkaline phosphatase.Part of the alkaline phosphatase activity can be detected in whole cells, suggesting that some of the enzyme molecules are located on the exterior surface of the plasma membrane. In contrast very low 5′-nucleotidase activity can be detected in whole cells. When membrane preparations, isolated from cells that had been surface labeled with ¹²⁵I, were subjected to sedimentation equilibrium on sucrose density gradients, the majority of the ¹²⁵I-radioactivity cosedimented with the alkaline phosphatase and 5′-nucleotidase activites, suggesting that both enzymes are plasma membrane components.The two enzymes have distinctly different pH optima, but otherwise their properties are remarkably similar. Both enzymes are inhibited by cyanide, sulfhydryl inhibitors and sulfhydryl reagents, although in each case the 5′-nucleotidase is slightly more susceptible. Both enzymes are inhibited by the levamisole analogue, R 8231, but the alkaline phosphatase is inhibited to a somewhat greater extent. Both enzymes are activated by incubation at 50 °C but inactivated by higher temperatures.The two enzymes increase in activity at identical times during differentiation, suggesting that they are under coordinate developmental control.     

Receptors for E type prostaglandins in the plasma membrane of nonpregnant human myometrium.

Binding sites for prostaglandin E₁ were present in the 1000g supernatant of nonpregnant human myometrium. When the 1000g supernatant was fractionated the distribution of prostaglandin E₁ binding sites followed that of the plasma membrane markers, phosphodiesterase-I and 5′-nucleotidase, but was different from that of the endoplasmic reticulum marker NADPH-cytochrome c reductase or the mitochondrial marker succinatecytochrome c reductase. It is concluded that a major portion of the prostaglandin E₁ receptors in the human myometrium is located at the plasma membrane. Scatchard analysis of prostaglandin E₁ binding to the plasma membrane-enriched fraction indicated the presence of both high and low affinity sites.     

Properties of 5′-nucleotidase in rat heart sarcolemma

The activity of 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) was examined in membrane fractions isolated by hypotonic shock-LiBr treatment (fraction HL) and sucrose gradient separation (fraction S) of rat ventricle homogenate. The enzyme activity in these two fractions differed significantly in several respects. In fraction HL, 5′-nucleotidase had a high affinity for AMP (K_m 35 μM), and ATP was a potent competitive inhibitor. In contrast, the 5′-nucleotidase displayed by fraction S showed a low substrate affinity (K_m 130 μM) and less sensitivity to ATP. Treatment of membranes with trypsin and neuraminidase markedly stimulated 5′-nucleotidase in fraction HL, whereas only a modest effect was observed in fraction S. Exposure of the membranes to Triton X-100 resulted in a 60% and 10% increase in the enzyme activity in fractions HL and S, respectively. The characteristic activity ratios of 5′-nucleotidase at 200 μM relative to 50 μM AMP in fractions HL and S were modified by alamethicin in an opposite way and became identical. Although concanavalin A almost completely inhibited the 5′-nucleotidase activity in both membrane preparations at a concentration of 2 μM, Hill plots of the data on concanavalin A inhibition revealed a coefficient of 2.2 for fraction S and 1.1 for fraction HL. The differences in 5′-nucleotidase activity of the two membrane fractions are considered to be due to differences in the orientation of the vesicles of the sarcolemmal preparations. These results suggest that two distinct catalytic sites for 5′-nucleotidase are present at the intra and extracellular surface of the rat heart sarcolemma.     

Separation of visual pigments on columns of controlled-pore glass beads

Plasma membranes have been isolated from 3T3 and SV-3T3 cells grown in culture. Cells were harvested mechanically and disrupted in simple isotonic buffered salt solutions without resorting to hypotonic swelling or chemical membrane “hardeners.” The method of storing collected cells, the cell concentration during disruption, and the method of mechanical disruption were found to be significant variables affecting the yield of plasma membranes. The plasma membranes were separated from mitochondria and other cellular organelles by a single centrifugation through a step sucrose gradient containing a viscosity barrier of Dextran T-500 (modified fromA. S. Sun and B. Poole (1975)Anal. Biochem.68, 260). The isolated plasma membranes were located by assay for the “marker” enzyme, alkaline phosphatase (EC 3.1.3.1). The isolated plasma membrane fraction was free of mitochondrial and essentially free of lysozymal and endoplasmic reticulum contamination, which were assayed by measuring cytochrome c reductase, arylsulfatase, and hydrolysis of α-naphthol acetate, respectively. Of the enzymes tested, the phosphodiesterase activity was found to be the most specific assay for the plasma membrane from culture mouse fibroblast cells. The 5′-nucleotidase (EC 3.1.3.5) activity, the other plasma membrane marker, was extremely low in activity and gave an additional peak of activity when 5′-adenilic acid was used as substrate as compared to the expected single peak obtained with 5′-cytidilic acid as substrate. Overall recovery of isolated plasma membranes was greater than 75% as measured by the final recovery of phosphodiesterase activity.     

Nicotinamide adenine dinucleotide splitting enzyme: a plasma membrane protein of murine macrophages.

The subcellular distribution of NADase in splenic and peritoneal macrophages of the mouse has been studied. Conventional procedures for fractionation and isolation of subcellular components demonstrated that the NADase of murine macrophages was localized in the microsomal fraction. By using the diazonium salt of sulfanilic acid, a nonpenetrating reagent known to inactivate ecto-enzymes in intact cells, purified plasma membrane preparations, and marker enzymes, 5′-nucleotidase for plasma membrane and glucose 6-phosphatase for the microsomal fraction, we have shown that: (i) NADase of murine macrophages is a plasma membrane ecto-enzyme and (ii) the microsomal fraction is a mixture of endoplasmic reticulum and plasma membrane elements. At 5 × 10^?4 M concentration, the diazonium salt of sulfanilic acid drastically decreased NADase in intact splenic and peritoneal macrophages of the mouse. 5′-Nucleotidase was similarly inhibited by this reagent, whereas the activity of glucose 6-phosphatase remained unaffected. There was a good recovery of NADase of high specific activity in plasma membrane preparations that were characterized by high 5′-nucleotidase and low glucose 6-phosphatase activity.     

Analytical subcellular fractionation of rat pituitary homogenates with special reference to the subcellular localization and properties of alkaline phosphatases

Alkaline phosphatase activities of the virgin rat anterior pituitary were studied with a highly sensitive fluorometric assay. Tissue whole homogenates were fractionated on sucrose density gradients in a Beaufay automatic zonal rotor and the gradient fractions assayed for alkaline phosphatase, prolactin and various organelle marker enzymes. Alkaline phosphatase was distributed between two peaks on the gradient. The low-density (1.10–1.15 g·cm^?3) alkaline phosphatase component co-sedimented with the plasma membrane marker, 5′-nucleotidase, had an apparent K_m for 4-methylumbelliferyl phosphate of approx. 59 μM, and was inhibited by levamisole. The high-density (1.20–1.25 g·cm^?3) peak was resistant to levamisole-inhibition, had an apparent K_m of approx. 30 μM and its distribution was distinct from plasma membrane, Golgi, lysosome, endoplasmic reticulum, mitochondria and prolactin granule markers on the isopycnic gradients.     

Cellular binding sites for insulin in rat liver

A study of the sites of insulin binding in subcellular fractions of rat liver is reported. A method for the isolation of liver plasma membranes, which permits one to follow quantitatively the distribution of all the parameters of interest, was modified and applied to the study of the cellular topography of insulin binding. The insulin-binding capacity did not follow closely the enzyme marker (5′-nucleotidase) for plasma membranes when differential centrifugation schemes were used, and the divergence from this marker was more prominent when separations were performed on discontinuous sucrose gradients. A significant amount of insulin binding capacity was always present in fractions with higher density than those containing the majority of 5′-nucleotidase. Results of studies on linear sucrose gradients have disclosed in some of the purified membrane fractions small but consistent differences in density of the insulin binding, and plasma membrane particles. It is suggested that there may be several types of intracellular membranes to which insulin can bind besides the plasma membranes.     

Highly enriched,minimally disrupted plasma membrane vesicles from aortic myocytes grown in primary culture

A plasma membrane-enriched fraction (fraction 1B) has been obtained from rat aortic myocytes grown in primary culture. Plasma membrane markers, 5′-nucleotidase and ouabain-sensitive (Na⁺ + K⁺)-ATPase, are enriched 4.1- and 8.7-fold, respectively, in this fraction. Although endoplasmic reticulum marker NADPH-cytochrome c reductase is the most enriched in mitochondrial and heavy sucrose density gradient fractions, substantial enrichment of this marker is also observed in membrane fraction 1. This membrane preparation therefore contains a certain quantity of endoplasmic reticulum. Cytochrome c oxidase is de-enriched by a factor of 0.04 in fraction 1, indicating that it is essentially clear of mitochondrial contamination. Homogenization of aortic media-intima layers using a whole-tissue technique induces greater disruption of mitochondria and subsequent contamination of membrane fractions than does the procedure for cell disruption. Analysis of electrophoretic gels, vesicle density distribution and electron micrographs of enriched membrane fractions provide evidence that plasma membrane enriched from cultured myocytes is less traumatized than comparable fractions obtained from intact tissue. The potential value of such a highly enriched, minimally disrupted plasma membrane preparation is discussed.     

Lymphocyte plasma membranes II. Cytochemical localization of 5′-nucleotidase in rat lymphocytes

Cytochemical studies of thymic and splenic lymphocytes from rats showed that 5′-nucleotidase was restricted to the plasma membranes. Isolated plasma membranes contained the highest specific activity of 5′-nucleotidase of any cellular fractions. The results indicate that this enzyme can be used as a plasma membrane marker for lymphocytes.     

Isolation of non-myelin plasma membranes unique to white matter

A procedure is described for isolating two membrane fractions from rabbit spina-cord white matter enriched with 5′-nucleotidase, a nonspecific plasma membrane marker, 2′, 3′-cyclic nucleotide phosphohydrolase, an oligodendroglial plasma membrane marker, and acetylcholinesterase, an axonal plasma membrane marker. While the two membrane fractions exhibited similar enrichments with respect to cyclic nucleotide phosphohydrolase, enrichments of 5′-nucleotidase and acetylcholinesterase were significantly greater in the heavier membranes were not detected. Moreover, gray matter did not yield homologous membrane fractions in the gradient when subjected to the identical procedure, indicating that the two membrane fractions were unique to white matter. While electronmicroscopic examination revealed that both membrane fractions were contaminated with myelin, the heavier fraction was least contaminated and exhibited a fair degree of homogeneity with respect to single membrane vesicular profiles. It was concluded that both membrane fractions were enriched with oligodendroglial and axonal plasma membranes, with the heavier fraction containing significantly more axolemma.     

Subfractionation of rat liver plasma membrane: Uneven distribution of plasma membrane-bound enzymes on the liver cell surface

Plasma membranes were islotaed from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5′-nucleotidase and (Na⁺+K⁺)-ATPase were used. The yield of plasma membrane was 0.6–0.9 mg protein per g wet weight of liver. The recovery of 5′-nucleotidase and (Na⁺+K⁺)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the acitvity of glucose-6 phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5′-nucleotidase, alkaline phosphatase, (Na⁺+K⁺)-ATPase and Mg²⁺-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na⁺+K⁺)-ATPase and Mg²⁺-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphatase was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.     

Studies on plasma membranes: XIX. Isolation and characterization of a plasma membrane fraction from calf thymocytes

A plasma membrane fraction was isolated from calf thymocytes by a modification of the method of Wallach and Kamat (Wallach, D. F. H. and Kamat, V. B. (1966) in Methods in Enzymology) (Colowick, S. P. and Kaplan, N. O., eds), Vol. 8, pp. 164–172, Academic Press, New York). Fractions were examined electron microscopically and subjected to chemical and enzymic assays.With respect to the cell homogenate and the final microsomal fraction, respectively, the plasma membrane fraction was enriched by a factor 23 and 5.1 in cholesterol, 11 and 2.4 in phospholipid, 5.1 and 4.2 in sialic acid, 20 and 5.2 in Mg²⁺-ATPase (EC 3.6.1.3), and 8 and 2.6 in 5′-nucleotidase (EC 3.1.3.5). Succinate:cytochrome $\text{c}$ oxidoreductase (EC 1.3.99.1) was lacking, and DNA was hardly if at all present in the plasma membrane fraction. The major part of the RNA found in this fraction (30 μg · mg^?1 protein) was concluded to be an authentic component of the plasma membrane.The concept of membrane “markers” was briefly considered and the conclusion was reached that by current criteria and electron microscopic evidence the plasma membrane fraction obtained from calf thymocytes consisted of reasonably clean plasma membranes.     

The synthesis and turnover of 5′-nucleotidase in primary cultured hepatocytes

The synthesis and degradation of 5′-nucleotidase has been studied in rat hepatocytes. Primary cultures of rat hepatocytes were established with the cells showing evidence of polarity after 24–36 h in culture. After a 30 h lag period 5′-nucleotidase activity increased to a plateau level similar to the activity found in whole liver. The half life of the enzyme after reaching the plateau of activity was 22.8 h. Pulse-chase biosynthetic labelling studies of 5′-nucleotidase in the cultured hepatocytes using [³⁵S]methionine showed that the 5′-nucleotidase monomer was synthesised as an M_r 67 000 form which was converted to the mature M_r 72 000 form. [³⁵S]Methionine labelling studies in the presence of tunicamycin showed that the unglycosylated protein monomer was an M_r 57 000 form. The immature M_r 67 000 form of 5′-nucleotidase was sensitive to endoglycosidase H, whereas the mature form was sensitive only to endoglycosidase F. The data presented are consistent with 5′-nucleotidase in a polarised cell being synthesised and processed like other membrane glycoproteins, in contrast to earlier reports.     

Fractionation of Isolated Rat CNS Myelinated Axons by Sucrose Density Gradient Centrifugation in a Zonal Rotor

Myelinated axons isolated from rat CNS brain stem by flotation in a buffered sucrose-salt medium were shocked by vigorous homogenization in hypotonie buffer and then fractionated on a 20-40% (wt/wt) linear sucrose gradient in a Beckman Ti-14 Zonal Rotor. After centrifu-gation to equilibrium, the gradient was fractionated on the basis of sucrose density into 13 individual fractions. The distributions of molecular markers related to myelin [(myelin basic protein, 2’3′-cyclic nucleotide 3′-phos-phodiesterase (EC 3.1.4.37), myelin-associated glycopro-tein (MAG)]; microsomes [CDP-choline:l,2 diglyceride cholinephosphotransferase (EC 2.7.8.2)]; mitochondria [cytochrome c oxidase (EC 1.9.3.1), monoamine oxidase (amine:oxygen oxidoreductase, deaminating, EC 1.4.3.4)], and axolemma [acetylcholinesterase (acetylcho-line hydrolase, EC 3.1.1.7), 5′-nucleotidase (5′-ribonu-cleotide phosphohydrolase, EC 3.1.3.5), Na⁺,K⁺-adeno-sine triphosphatase (EC 3.6.1.3), [³H]saxitoxin binding] were examined, as well as the protein composition and morphological appearance of the fractions. The myelin-related markers were most enriched in the 20-26% region of the gradient, although the MAG was broadly distributed throughout the entire gradient. The axolemma-related markers were most enriched in the 28-32% region of the gradient, whereas the microsomal and mitochondrial-related markers were enriched in the 35-40% region of the sucrose density gradient. Mixing experiments utilizing ¹²⁵I-labeled membrane preparations derived from cultured oligodendroglial and astroglial cells indicated that the constituents of the shocked myelinated axons were not significantly contaminated with glial membranes. The morphology of the fraction was consistent with the membrane molecular marker distribution: the light end of the gradient contained multilamellar myelin; fractions in the center of the gradient were enriched in un-ilamellar membrane fragments; the densest regions of the gradient were enriched in mitochondria. The myelin specific proteins were the prominent polypeptides in the 20-25% regions of the gradient, whereas polypeptides having a molecular weight of 50,000 or greater predominanted in the denser regions of the gradient. The significance of the distribution of these membrane markers and the utility of this fractionation procedure are discussed.     

Levels of membrane marker enzyme activity in normal and RNA and DNA virus-transformed fibroblasts

Activities of 5′-nucleotidase, a plasma membrane marker enzyme; esterase, a rough endoplasmic reticulum marker enzyme; and UDPase, a smooth endoplasmic reticulum marker enzyme, were measured in 3T3, RSV-3T3, MSV-3T3 and PY-3T3 cells. 5′-Nucleotidase and esterase activities were significantly elevated in the oncogenic virus-transformed cell lines compared to the 3T3 cells on a per mg protein basis. Activity of UDPase was slightly elevated in the oncogenic virus-transformed cells.     

Interactions of lectins with membrane glycoproteins effects of concanavalin A on 5′-nucleotidase

Concanavalin A causes a biphasic modification of the activity of the plasma membrane enzyme 5′-nucleotidase. The first stimulatory phase occurs from 0 to 0.05 μM concanavalin A, the second inhibitory phase at higher concentrations. The curve relating binding of ¹²⁵I-labelled concanavalin A and concentration of native lectin is similarly biphasie. The two phases likely result from occupation of distinct families of binding sites. When the enzyme is extracted from the membrane, the stimulatory phase disappears. Thus, the high affinity binding sites responsible for this phase depend upon the intact membrane structure while the others do not.