Electron-cytochemical localization of alkaline phosphatase to G cells of Necturus maculosus antrum

Summary Electron-cytochemical localization of alkaline phosphatase activity was performed on G cells of Necturus maculosus antral mucosa. Alkaline phosphatase activity was localized to the nuclear membrane, the Golgi/endoplasmic reticulum, and the limiting membranes of G cell peptide-secretion vesicles. There was no specific localization of alkaline phosphatase activity to the plasma membrane. Treatment of the tissues with levamisole (an alkaline phosphatase inhibitor) did not markedly reduce the specific alkaline phosphatase activity. Specific lead deposition was reduced by removal of the substrate from the reaction mixture. The results from this study on N. maculosus G cells demonstrate that alkaline phosphatase activity can be found in a non-mammalian gastric endocrine cell and that specific activity was localized primarily to those intracellular structures involved with protein biosynthesis.     

The ultrastructural localization of human neutrophil alkaline phosphatase in normal individuals during pregnancy and in patients with chronic granulocytic leukaemia

Summary The intracellular localization of alkaline phosphatase was determined in human neutrophils by electron microscope cytochemistry. In normal individuals, the largest and most intense deposits of reaction product were seen in a unique cytoplasmic granule population termed phosphasomes. Lighter deposits were seen on nuclear membranes, some intracytoplasmic membranes lining vacuoles and granules and occasionally in focal patches on the internal surface of the plasma membrane.In cell isolated from women in the third trimester of pregnancy, activity was found in the same intracellular sites but there were, on average, more alkaline phosphatase-containing granules per cell than in the cells from non-pregnant individuals. Neutrophils from pregnant women were also characterized by the presence of large deposits of reaction product on the external surface of the plasma membrane (extramembranous). This activity had properties characteristic of the placental isoenzyme of alkaline phosphatase, found in serum during pregnancy.Neutrophils from patients with chronic granulocytic leukaemia, showing normal mature morphology, contained significant amounts of granule reaction product but there were fewer phosphasomes per cell than in normal individuals. In morphologically immature cells, reaction product was present in nuclear membrane, endoplasmic reticulum and large granules. These results were in agreement with previous biochemical data confirming a quantitative lack of alkaline phosphatase in chronic granulocytic leukaemia.     

Cytochemistry of Ca(++)-dependent adenosine triphosphatase (Ca-ATPase) in rat anterior pituitary cells

In the present study, we demonstrate the localization of Ca(++)-ATPase in the anterior pituitary of the male rat. Ca(++)-ATPase was mainly distributed on the membrane system of the granular cells, which included the plasma membrane, the outer mitochondrial membrane, the enveloping membrane of secretory granules, the smooth endoplasmic reticulum and some components of the Golgi complex. No reaction product was detected on the membrane of the rough endoplasmic reticulum or that surrounding the lysosomes. A positive reaction was clearly observed on the membranes surrounding 'large' secretory granules, while that present on the membranes of the 'small' granules was comparatively weak. The cells which contained the 'large' granules were interpreted as growth hormone-secreting cells and those in which the 'small' granules were located as gonadotrophs. There were either no reaction or one that was barely detectable on the plasma membrane of the folliculo-stellate cells. These data along with our previous findings (Soji, 1982, 1984) suggest that the membranous enzymes are not uniformly distributed over all pituitary cells but rather are specific for a given cell population(s).     

Alkaline phosphatase biosynthesis in the endoplasmic reticulum and its transport through the Golgi apparatus to the plasma membrane: cytochemical evidence

Enzyme induction of HeLa cell placental alkaline phosphatase with various agents such as prednisolone, sodium butyrate, hyperosmolality (NaCl), or combination of these inducers resulted in the appearance of enzyme activity in the rough endoplasmic reticulum, nuclear envelope, Golgi apparatus, and plasma membrane. In the Golgi apparatus, intense reaction product deposits tended to be concentrated on its trans side, with small vesicles and granules also being positively stained. Inhibition of protein synthesis with cycloheximide was followed by the disappearance of enzyme activity from these cytoplasmic organelles but not from the plasma membrane. Treatment with monensin, a secretory protein transport inhibitor, uniformly increased activity in the rough endoplasmic reticulum while causing marked dilatation of the intensely positive Golgi cisternae. These results suggest that intracellular alkaline phosphatase is newly synthesized in the endoplasmic reticulum and then passes en route through the Golgi apparatus to the plasma membrane. Accordingly, the present system could represent the biosynthesis, transport, and incorporation of the model cell surface enzyme protein to add to the vesicular stomatitus virus glyco-1 (VSV-G) protein and acetylcholine receptor model systems for studying the dynamics of cell surface protein genesis, transport, and membrane integration.     

Acid phosphatase in the guinea-pig oocytes

In guinea-pig oocytes, at every developmental stage, acid phosphatase is found histochemically in cytoplasmic granules. Ultracytochemically the reaction product is located in lysosomes and is some cisternae of the rough endoplasmic reticulum, but not in cortical granules or in vesicles with a rough endoplasmic reticulum membrane which are filled with a moderately dense homogeneous substance. It is discussed whether the acid phosphatase transforms reserve material into a storable form as has been proposed for the deposition of vitellogenin in the oocytes of lower vertebrates.     

Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes

beta-Hexosaminidase B purified from human fibroblast secretions was used as a ligand to study phosphomannosyl-enzyme receptors in membranes from rat tissues. Enzyme binding to rat liver membranes was saturable, competitively inhibited by mannose 6-phosphate, not dependent on calcium, and destroyed by prior treatment of the hexosaminidase with either alkaline phosphatase or endoglycosidase H. Most (90%) of the phosphomannosyl-enzyme receptors were found in endoplasmic reticulum, Golgi apparatus, and lysosomes; 9.5% in the plasma membrane, and less than 1% in nuclei and mitochondria. Receptors were vesicle-enclosed in all fractions except plasma membrane. Receptors in the endoplasmic reticulum apparently were occupied by endogenous ligands, but most receptors in lysosomes and plasma membrane were unoccupied. Most of the endogenous beta-hexosaminidase was in lysosomes and was released from vesicles by detergent treatment. Displacement of the residual receptor-bound endogenous beta-hexosaminidase (mostly in endoplasmic reticulum and Golgi apparatus) from detergent-treated membranes by mannose 6-phosphate released high uptake enzyme with properties expected for phosphomannosyl-enzymes. Mannose 6-phosphate-inhibitable enzyme receptor activity was found in nine rat organs and correlated roughly with their lysosomal enzyme content. These data support a general model for lysosomal enzyme transport in which the phosphomannosyl-enzyme receptor acts as a vehicle for delivery of newly synthesized acid hydrolases from the endoplasmic reticulum to lysosomes.     

Separation of the microvillous (maternal) from the basal (fetal) plasma membrane of human term placenta: methods and physiological significance of marker enzyme distribution.

Plasma membranes from normal, full-term human placental trophoblast have been isolated by a new procedure. The method depends upon isopycnic zonal centrifugation using linear sucrose/Ficoll density gradients. Enrichment of plasma membrane marker enzymes with respect to trophoblast homogenate is found in two distinct peaks (designated B and D) of the fractionated effluent recovered from the rotor. Fraction B is enriched with membrane-bound alkaline phosphatase and 5'-nucleotidase, but not with (Na+, K+)-ATPase of F(-)-stimulated adenylate cyclase. It is suggested that this material is derived from the maternal-facing microvillous plasma membrane. Fraction D, enriched with (Na+, K+)-ATPase, F(-)-stimulated adenylate cyclase and, to a smaller extent, with 5'-nucleotidase and alkaline phosphatase is, by exclusion, proposed to be derived from the fetal-facing basal plasma membrane. Both plasma membrane fractions are shown to be free of appreciable contamination, using specific markers for endoplasmic reticulum, mitochondria, nuclei and lysosomes. The separation of the two membrane fractions is shown to depend both upon these membranes forming closed vesicles during homogenization and upon the buoyant densities of such vesicles differing in such a way that microvillous plasma membranes band at a lower density than basal plasma membranes. No separation of the membranes is achieved in gradients in which the vesicles are collapsed.     

Acid phosphatase in the guinea-pig oocytes

Summary In guinea-pig oocytes, at every developmental stage, acid phosphatase is found histochemically in cytoplasmic granules, Ultracytochemically the reaction product is located in lysosomes and in some cisternae of the rough endoplasmic reticulum, but not in cortical granules or in vesicles with a rough endoplasmic reticulum membrane which are filled with a moderately dense homogenous substance. It is discussed whether the acid phosphatase transforms reserve material into a storable form as has been proposed for the deposition of vitellogenin in the oocytes of lower vertebrates.This investigation was supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

The isolation and subfractionation of plasma membrane from the cellular slime mould Dictyostelium discoideum

A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N(2) and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [(125)I]iodide. 5'-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH-cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH-cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants.     

A cytochemical study of the calcium-activated adenosinetriphosphatase in hamster adrenal medulla: its occurrence in the golgi region of chromaffin cells

Summary Several different fixation procedures and incubation media were used in order to demonstrate the ultrastructural localisation of Ca²⁺-activated adenosinetriphosphatase (ATPase) in the hamster adrenal medulla. Fixation by perfusion with 2.5% glutaraldehyde gave the best preservation of fine structure without markedly inhibiting the enzymic activity. The localisation of Ca²⁺-activated ATPase was different from that of Mg²⁺-activated ATPase: the Mg²⁺-dependent enzyme was confined to plasma membranes. Ca²⁺-dependent ATPase also occurred on the plasma membranes of neurons and of some chromaffin cells, but the most prominent site of this enzyme was in the Golgi apparatus of chromaffin cells. Most of the reaction product was localised between Golgi lamellae, but some was found in Golgi vesicles and in prosecretory granules. The nucleus, mature chromaffin granules, roughsurfaced endoplasmic reticulum and mitochondria were usually free of reaction product. Rarely, some precipitate was found in the matrix of mitochondria and in lysosomes.Wellcome Research Fellow.J. H. Burn Research Scholar.This work was supported by a grant from the Medical Research Council.     

Studies on isolated subcellular components of cat pancreas. I. Isolation and enzymatic characterization.

Pancreas of the cat was fractionated into its subcellular components by centrifugation through an exponential ficoll-sucrose density gradient in a zonal rotor. This enables a preparation of four fractions enriched in plasma membranes, endoplasmic reticulum, mitochondria and zymogen granules, respectively. The first fraction, enriched by 9- to 15-fold in the plasma membrane marker enzymes, hormone-stimulated adenylate cyclase, (Na+K+)-ATPase, and 5'-nucleotidase, is contaminated by membranes derived from endoplasmic reticulum but is virtually free from mitochondrial and zymogen-granule contamination. The second fraction from the zonal gradient shows only moderate enrichment of the above marker enzymes but contains a considerable quantity of plasma membrane marker enzymes and represents mostly rough endoplasmic reticulum. The third fraction contains the bulk of mitochondria and the fourth mainly zymogen granules as assessed by electron microscopy and marker enzymes for both mitochondria and zymogen granules, namely succinic dehydrogenase, trypsin and amylase. Further purification of the plasma membrane fractions by differential and sucrose step-gradient centrifugation yields plasma membranes enriched 40-fold in basal and hormone-stimulated adenylate cyclase and (Na+K+)-ATPase.     

Subcellular localization of phospholipids and enzymes involved in PAF-acether metabolism

The biosynthesis of platelet-activating factor (PAF-acether or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) through the remodeling pathway was investigated at the subcellular level in two different cell lines. In human neutrophils, plasma membrane was isolated not only from granules, but also from internal membranes related to endoplasmic reticulum. Interestingly, the latter exhibited enhanced acetyltransferase upon neutrophil stimulation with ionophore A23187. A similar study was undertaken on the tumor strain Krebs-II cells. The enzyme acetyltransferase was found to be located only on an endoplasmic reticulum subfraction, whereas most alkylacyl-GPC, the source of PAF-precursor alkyl-lyso-GPC, was located in the plasma membrane inner leaflet. The topographical separation of enzyme and precursor emphasizes the central role of the intracellular phospholipase A2 in providing lyso-PAF to the acetyltransferase to form PAF-acether.     

Demonstration of lysosomal and extralysosomal sites for acid phosphatase in mouse kidney tubule cells with p-nitrophenylphosphate lead-salt technique.

Dual localization of acid phosphatase in lysosomal and extralysosomal sites of the tubule epithelial cells of normal mouse kidney was observed at the light and electron microscope level using a modified Gomori lead-salt method with p-nitrophenylphosphate (pNPP) as substrate. Based on previous biochemical and cytochemical findings, we developed optimal conditions for the enzyme activity in extralysosomal sites. The conditions used for the light microscopic level consisted of 1.5 mM PNPP, 2.0 MM Pb(NO3)2 and 0.05 M acetate buffer (pH 5.8). Those for the electron microscopic study required 3.0 mM PNPP, 3.6 MM Pb(NO3)2 and 0.1 M acetate buffer (pH 5.8). This modified lead-salt technique was highly specific and provided a suitable method for the demonstration of nonlysosomal as well as lysosomal sites of acid phosphatase activity in the tubule epithelial cells of normal mouse kidney. As expected, the enzyme activity appeared in the lysosomes, but the prominent reaction in the brush border, the rough endoplasmic reticulum and basal infolding plasma membranes was not anticipated. We were able to demonstrate in situ organelle precursors of microsomal acid phosphatase such as endoplasmic reticulum, plasma membrane and basal infolding membranes showing the same substrate preference, which had been observed previously in biochemical studies in our laboratory. Since the possible participation of alkaline phosphatases, K+-pNPPase or Na+-K+-adenosine triphosphatase was ruled out by use of appropriate inhibitors, the enzyme-reactive sites can be interpreted as reflecting nonspecific acid phosphatase.     

Localization of filipin-sterol complexes in cell membranes of eosinophils

The polyene antibiotic filipin was used as a probe for the detection of cholesterol in the cell membranes of eosinophils isolated from the peritoneal exudate of rats. A homogenous distribution of filipin-sterol complexes was observed, both in thin sections and freeze-fracture replicas throughout the whole plasma membrane but not in the membrane of pynocytic vesicles, Golgi complex, endoplasmic reticulum, mitochondria and the nucleus. Few complexes were seen in freeze-fracture replicas showing the membrane of the specific granules. Treatment of living cells with filipin induced aggregation of filipin-sterol complexes at some points of the plasma membrane.     

Localization of filipin-sterol complexes in cell membranes of eosinophils

Summary The polyene antibiotic filipin was used as a probe for the detection of cholesterol in the cell membranes of eosinophils isolated from the peritoneal exudate of rats. A homogenous distribution of filipin-sterol complexes was observed, both in thin sections and freeze-fracture replicas throughout the whole plasma membrane but not in the membrane of pynocytic vesicles, Golgi complex, endoplasmic reticulum, mitochondria and the nucleus. Few complexes were seen in freeze-fracture replicas showing the membrane of the specific granules. Treatment of living cells with filipin induced aggregation of filipin-sterol complexes at some points of the plasma membrane.     

Fine structural localization of alkaline phosphatase in rat ovum during cleavage

On a submicroscopic level alkaline phosphatase activity was demonstrated by cytochemical methods in all stages of segmenting rat ova under survey, i.e. in the unfertilized and fertilized ovum, in the two-, four- and eight-cell stages and in the blastocyst. The reaction product was present in some cytoplasmic organelles as well as on cell membranes. A considerable number of cytoplasmic organelles with alkaline phosphatase activity was found in all stages from the one-cell up to the eight-cell stage. The reaction product was deposited in the tubules and vesicles of the smooth endoplasmic reticulum, in the nuclear envelope and in the Golgi complex as well. Some multivesicular bodies, autophagic vacuoles and majority of residual bodies out of the secondary lysosomes showed enzymatic activity. In the multicellular stages no significant differences were observed between the individual blastomeres in the incidence and distribution of the alkaline phosphatase activity. On the blastocyst-stage was found a low incidence of enzymatically active cytoplasmic organelles. Alkaline phosphatase activity was demonstrated in some minute vesicles below the cell membrane and in some secondary lysosomes. No essential differences were found between the cells of the embryoblast and the cells of the trophoblast in the incidence of enzymatically active structures. In the one-cell stage the activity of alkaline phosphatase was present on the cell membrane only sporadically, in the two- and four-cell stages enzymatic activity was found in this localization in a third of all specimen. In the eight-cell stage alkaline phosphatase activity was demonstrated on the cell membranes of all blastomeres. In the blastocyst the reaction product was deposited regularly on the membranes of the trophoblastic cells turned towards the zone pellucida, frequently on membranes of mutual tactile cells of the trophoblast and the embryoblast and only sporadically on cell membranes limiting the blastocyst cavity.     

Dephosphorylation of phosphoproteins of human liver plasma membranes by endogenous and purified liver alkaline phosphatases

Purified alkaline phosphatase and plasma membranes from human liver were shown to dephosphorylate phosphohistones and plasma membrane phosphoproteins. The protein phosphatase activity of the liver plasma membranes was inhibited by levamisole, a specific inhibitor of alkaline phosphatase, and by phenyl phosphonate and orthovanadate, but was relatively insensitive to fluoride (50 mM). Endogenous membrane protein phosphatase activity was optimal at pH 8.0, compared to pH 7.8 for purified liver alkaline phosphatase. Plasma membranes also exhibited protein kinase activity using exogenous histone or endogenous membrane proteins (autophosphorylation) as substrates; this activity was cAMP-dependent. Autophosphorylation of plasma membrane proteins was apparently enhanced by phenyl phosphonate, levamisole, or orthovanadate. The dephosphorylation of phosphohistones by protein phosphatase 1 was not inhibited by levamisole but was inhibited by fluoride. Inhibition of endogenous protein phosphatase activity by orthovanadate during autophosphorylation of plasma membranes could be reversed by complexation of the inhibitor with (R)-(-)-epinephrine, and the dephosphorylation that followed was levamisole-sensitive. Neither plasma membranes nor purified liver alkaline phosphatase dephosphorylated glycogen phosphorylase a. These results suggest that the increased [32P]phosphate incorporation by endogenous protein kinases into the membrane proteins is due to inhibition of alkaline phosphatase and that the major protein phosphatase of these plasma membranes is alkaline phosphatase.     

Enzymatic characterization of subcellular samples from gypsy moth larval midgut following differential centrifugation and fractionation on Percoll-sucrose gradient

A method is described for isolation of an enriched fraction of plasma membranes from gypsy moth (Lymantria dispar) larval midgut tissue. Following differential centrifugation of tissue homogenate, a microsomal sample is obtained and fractionated on a Percoll®-sucrose gradient that yields 2 distinct regions of high protein concentration: one enriched in plasma membranes, the other in mitochondrial membranes. The procedure is relatively rapid, being completed within approximately 5 h. Protein yields and accompanying specific activities are reported for marker enzymes used to indicate the presence of plasma membranes (leucine aminopeptidase and alkaline phosphatase), endoplasmic reticulum (NADPH-cytochrome c reductase), and mitochondria (succinate dehydrogenase). The apparent differences between the plasma membrane enriched fraction vs. brush border membrane vesicles prepared from insect midguts are discussed, as is the suitability of the plasma membrane enriched fraction for ATP-dependent calcium ion transport studies. © 1992 Wiley-Liss, Inc.     

Plasma membranes from rat Sertoli cells: purification and properties

A method is reported for preparing surface (plasma) membranes from rat Sertoli cells. The procedure is based upon homogenization in hypotonic buffer, extraction in a two-phase system, and sedimentation through two sucrose density gradients. The purified membranes consist of large sheets of membrane. The identity and purity of the membranes was demonstrated by electron microscopy, enzyme markers, and functional activities associated with the membranes (binding of follicle-stimulating hormone [FSH] and production of cyclic adenosine 5'-monophosphate [cAMP]. Electron microscopy showed membranes with small fragments of cytoplasm attached to the inside of the membrane sheets. Marker enzymes for plasma membrane (5'-nucleotidase and alkaline phosphatase) showed more than 16- and 6-fold enrichment, respectively, and other enzymes showed that contamination by nuclei, mitochondria, endoplasmic reticulum, or cytosol was negligible. Binding of FSH was found to be specific, with KD 1.2 nM and the equivalent of 7500 sites per cell. This binding was enriched 20-fold compared to whole homogenate. Production of cAMP by membranes was increased by addition of FSH and by forskolin to the purified membranes in vitro.     

Studies on isolated subcellular components of cat pancreas

Summary Pancreas of the cat was fractionated into its subcellular components by centrifugation through an exponential ficoll-sucrose density gradient in a zonal rotor. This enables a preparation of four fractions enriched in plasma membranes, endoplasmic reticulum, mitochondria and zymogen granules, respectively. The first fraction, enriched by 9- to 15-fold in the plasma membrane marker enzymes, hormone-stimulated adenylate cyclase, (Na⁺K⁺)-ATPase, and 5-nucleotidase, is contaminated by membranes derived from endoplasmic reticulum but is virtually free from mitochondrial and zymogen-granule contamination. The second fraction from the zonal gradient shows only moderate enrichment of the above marker enzymes but contains a considerable quantity of plasma membrane marker enzymes and represents mostly rough endoplasmic reticulum. The third fraction contains the bulk of mitochondria and the fourth mainly zymogen granules as assessed by electron microscopy and marker enzymes for both mitochondria and zymogen granules, namely succinic dehydrogenase, trypsin and amylase. Further purification of the plasma membrane fractions by differential and sucrose step-gradient centrifugation yields plasma membrane enriched 40-fold in basal and hormone-stimulated adenylate cyclase and (Na⁺K⁺)-ATPase.