Demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver using a combined light- and electron-microscope procedure

Summary In the present study a technique was developed to demonstrate 5′-nucleotidase activity in unfixed cryostat sections of rat liver at the light- and electron-microscope level using a semipermeable membrane. In order to retain the ultrastructure of the unfixed material as much as possible, incubations were also performed at 4°C rather than at 37°C. The optimized incubation medium contained 300 mm Tris-maleate buffer, pH 7.2, 5 mm adenosine monophosphate as substrate, 30 mm cerium chloride as capturing agent for liberated phosphate, 10 mm magnesium chloride as activator and 1.5% agar. At the light-microscope level, similar localizations of 5′-nucleotidase activity were obtained when incubations were performed at 37°C and 4°C. Enzyme activity was present mainly at bile canalicular membranes and at sinusoidal membranes of hepatocytes; total activity was higher in pericentral than in periportal areas. Cytophotometric analyses revealed that specific formation of final reaction product (FRP) (test minus control reaction) at 37°C followed a hyperbolic curve with time. A linear relationship was found between specific amounts of FRP and section thickness up to 8μm. 5′-Nucleotidase activity was about three-fold higher after incubation for 30 min at 37°C than at 4°C. At the electron-microscope level, it was demonstrated that the ultrastructure of rat liver was rather well-preserved after incubating unfixed cryostat sections attached to a semipermeable membrane and electron-dense FRP was found at bile canalicular and sinusoidal plasma membrane of hepatocytes. The most distinct changes in ultrastructure after incubation at 37°C, in comparison with that at 4°C, were the appearance of multi-lamellar structures at bile canaliculi at 37°C. We conclude that the present method is valid for the demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver at both the light- and electron-microscope levels and that hypothermic incubations improve ultrastructural morphology substantially.     

Ultrahistochemical localization of adenylate cyclase activity in the electric organ ofTorpedo marmorata

Summary The lead pyrophosphate precipitation technique was used to visualize adenylate cyclase activity with the electron microscope in unfixed electric organ and synapto-somes ofTorpedo marmorata, with special attention to presynaptic membranes. Specificity of the deposition of reaction product was ensured by using 5′-adenylyl imidodiphosphate as substrate and 5′-guanylyl imidodiphosphate and sodium fluoride as activators. Under suitable conditions a reaction product was deposited on the Schwann cell, on presynaptic vesicles, on the inner side of membranes of cisternae and on glycogen granules of the presynaptic region of the endplate. In some cases, a precipitate was also found on postsynaptic membranes of the synaptic cleft and on mitochondria. In isolated synaptosomes localization of the reaction product was identical with that of minced tissue. However, most strikingly, on presynaptic membranes no precipitate was ever found, neither in pieces of electric organ nor in isolated synaptosomes. Furthermore, the extended membrane system of the postsynaptic region of the electroplax remained always free of leed pyrophosphate precipitate.     

Cytochemical demonstration of NPPase activity for detecting proton-translocating ATPase of Golgi complex in rat pancreatic acinar cells

Summary An attempt at cytochemical demonstration of acidification proton-translocating ATPase (H⁺-ATPase) of Golgi complex in rat pancreatic acinar cells has been made by using p-nitrophenylphosphatase (NPPase) cytochemistry which is used for detecting of Na⁺-K⁺-ATPase (Mayahara et al. 1980) and gastric H⁺-K⁺-ATPase (Fujimoto et al. 1986). K⁺-independent NPPase activity was observed on the membrane of the trans cisternae of Golgi complex, but not inside of cisternae. The localization of NPPase activity is different from that of acid phosphatase activity where reaction products were seen on the inside of the trans Golgi cisternae. Since this activity was insensitive to vanadate, ouabain and independent of potassium ions, it was distinct from plasma membranous ATPases such as Na⁺-K⁺-ATPase and Ca²⁺-ATPase. The K⁺-independent NPPase activity was diminished by the inhibitors of H⁺-ATPase such as N-ethylmaleimide (NEM) and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The NPPase reaction products were also seen on the membranes of other acidic organelles, i.e., lysosomes, endosomes, autophagosomes and coated vesicles. These results suggest that NPPase activity on the membrane of the Golgi complex and other acidic organelles corresponds with H⁺-ATPase which plays a role in acidification.     

Methodological dependence in the ultrastructural immunolocalization of laminin in tubular basement membranes of the mouse kidney

Summary The ultrastructural localization of the basement membrane glycoprotein laminin was investigated in basement membranes of proximal tubules of the mouse kidney. The localization of laminin was determined using two different immunoperoxidase and one immunogold preembedding technique and one immunogold postembedding technique on unfixed and formaldehyde fixed tissue. Strong differences in the immunolocalization for laminin were found in the lamina densa of the tubular basement membrane using different techniques.After preembedding immunostaining for laminin using JgG-PO as secondary antibody, a positive reaction for the lamina densa was found in the formaldehyde fixed as well as in the unfixed kidney. After preembedding immunostaining for laminin using Protein-A-PO, staining of the l. densa was seen in the unfixed, but not in the fixed kidney. It was striking that no clear immunoreaction in the l. densa of the tubular basement membrane was seen in either the fixed or unfixed tissue after preembedding immunostaining for laminin using protein A-gold. With a direct postembedding immunogold technique laminin was localized only in the l. fibroreticularis and the l. rara but not in the l. densa of basement membranes of proximal tubules of the unfixed and the fixed kidney.     

The endomembrane system and mechanism of membrane flow in the green alga,Gloeomonas kupfferi (Volvocales,Chlorophyta) II. A cytochemical analysis

Summary Cytochemical analysis of the endomembrane system of the chlamydomonad flagellate,Gloeomonas kupfferi (Chlorophyta), reveals distinct compartmentalization. Phosphatase localization shows that: IDPase is located throughout all cisternae of the dictyosome and vesicles associated with the contractile vacuole. Other alkaline phosphatases like TPPase, ATPase and ITPase were localized within the trans-face cisternae and vesicles of the contractile vacuole. IMPase was localized at the plasmamembrane and not within the endomembrane system. Acid phosphatases, incl. CMPase, NADPase and -glycerophosphatase, were localized in vesicles emerging from the central terminus of the trans-face of the dictyosome and in the peripheral vacuolar network. Silver proteinate labeling was noted in the dictyosome, contractile vacuole and on the anterior plasmamembrane. A summary of endomembrane compartmentalization and a putative interpretation of membrane flow and economy are presented.Abbreviations ER endoplasmic reticulum - IDPase inosine 5-diphosphatase - ITPase inosine 5-triphosphatase - ATPase adenosine 5-triphosphatase - TPPase thiamine pyrophosphatase - CMPase cytidine 5-monophosphatase - NADPase -nicotinamide adenine diphosphatase - AcPase acid phosphatase     

Phosphatase activities and osmium reduction in cell organelles ofMicrasterias americana

Summary Organelles in resting and growing cells ofMicrasterias americana were examined using electron microscopy after cytochemical procedures for four kinds of phosphatases, acid phosphatase (ACPase), alkaline phosphatase (ALPase), thiamine pyrophosphatase (TPPase), and inosine diphosphatase (IDPase), and osmium tetroxide reduction. Special attention was paid to activities in the Golgi apparatus.In resting cells, positive reactions for ACPase and TPPase were observed in all cisternae of the dictyosome, especially in the peripheral parts. A positive IDPase reaction was seen in one central cisterna and was frequent in the distal-most cisterna. Reduction of osmium tetroxide was seen in the proximal cisternae.In early growing cells, the dictyosomes gave positive reactions for ACPase in the proximal cisternae and the distal cisterna, while in late growing cells only in proximal cisternae. Both in early and late growing cells, the dictyosomes were positive for TPPase and IDPase in the distal cisternae and vesicles derived from the distal cisternae, and for the reduction of osmium tetroxide in the proximal cisternae. ALPase activity was detected in the growing cell wall but not in the dictyosome.     

Convergence of the endocytic and lysosomal pathways in soybean protoplasts

Ultrastructural analysis of endocytosis of cationized ferritin (CF) has been combined with ultrastructural localization of acid phosphatases (AcPase) in soybean (Glycine max (L.) Merr.) protoplasts. While CF is an electron-dense marker of organelles of the endocytic pathway, ultrastructural histochemistry of AcPase identifies the organelles involved in the synthesis, transport, and storage of lytic-compartment enzymes, i.e. the lysosomal pathway. Acid phosphatases have been localized using both lead- and cerium-precipitation techniques. Protoplasts have been exposed to CF for 5 min, 30 min, or 3 h and processed for AcPase localization. At 5 min, smooth vesicles contain both CF and AcPase. By 30 min, Golgi cisternae and multivesicular bodies contain both labels. By 3 h, vacuoles become labelled with both CF and AcPase. The large central vacuoles contain intraluminal membranes which are associated with both AcPase and CF. These observations extend the analogy between plant vacuoles and animal lysosomes and demonstrate the points at which the endocytic pathway of plants converges with the lysosomal pathway.Abbreviations AcPase acid phosphatase - CF cationized ferritin - ER endoplasmic reticulum - MVB multivesicular body - PCR partially coated reticulum - PM plasma membrane     

A postcoupling method for the demonstration of N-acetyl-beta-D-glucosaminidase in unfixed frozen tissue sections.

The localization and activity of the lysosomal enzyme, N-acetyl-beta-glucosaminidase, has been studied in unfixed frozen sections of tissues from normal and hemorrhaged rats with the use of a modified postcoupling technique. Discrete localization of the end product of the reaction was achieved in this method by the incorporation of 20% (w/v) polyvinyl alcohol (molecular weight 14,000) in the incubation medium. Advantages of the present method include the ability to overcome the inhibitory effects on enzyme activity of both tissue fixation and the presence of diazonium salts in the incubation medium. The staining reaction obtained with this technique demonstrates the enzyme activity at specific cytoplasmic sites within cells and has a wider applicability to the comparative study of N-acetyl-beta-glucosaminidase activity in normal and injured tissues.     

Association of inorganic-pyrophosphatase activity with human alkaline-phosphatase preparations

1. The inorganic-pyrophosphatase activity of alkaline phosphatases prepared from human liver and small intestine was investigated at different stages of purification. 2. Both liver and intestinal preparations possessed pyrophosphatase activity at all stages of purification, and the two types of activity were not separated by gel filtration or by anion-exchange or cation-exchange chromatography. 3. After starch-gel electrophoresis of the tissue extracts, the zones of pyrophosphatase activity coincided exactly with alkaline-phosphatase zones. 4. Hydrolysis of each type of substrate was inhibited by the presence of the other, and a constant ratio of alkaline-phosphatase activity to pyrophosphatase activity was maintained during inactivation of the enzymes by incubation at 55°. 5. These results are consistent with the view that alkaline phosphatases are also inorganic pyrophosphatases.     

Differences of adenylate cyclase localization in guinea-pig peritoneal macrophages under different physiological conditions: An ultracytochemical study

Summary The ultrastructural localization of adenylate cyclase activity has been investigated in unfixed guinea-pig peritoneal macrophages in different physiological states (such as suspension, adhesion and phagocytosis) using a medium containing 5-adenylyl-imidodiphosphate (AMP-PNP) as the substrate.Adenylate cyclase activity was observed in cytoplasmic vacuoles of macrophages in suspension; in the perinuclear space, endoplasmic reticulum, Golgi complex and pseudopods of adherent macrophages; and surrounding phagocytosed polystyrene particles. The activity was inhibited by Alloxan added to the incubation medium and no staining was observed when AMP-PNP was omitted from the medium.The segregation of this enzyme to phagocytic vacuoles and pseudopods may have significant implications in understanding cyclic nucleotide function in adhesion and phagocytosis.     

Cytochemical localization of enzymes in the spermatid and the spermatozoon of Culex quinquefasciatus say (Diptera : Culicidae)

Ultrastructural cytochemical techniques were used for the localization of phosphatases in spermatid and spermatozoon of the mosquito, Culex quinquefasciatus (Diptera : Culicidae). Acid phosphatase was found mainly in the trans-most portion of the Golgi complex. Thiamine pyrophosphotase was preferentially located in the cis-most portion of the Golgi complex. Glucose-6-phosphatase was located in the endoplasmic reticulum and cisternae of the transition zone between the endoplasmic reticulum and the Golgi complex. The complex membrane of the anterior acrosomal region and the axoneme showed acid phosphatase activity. Reaction products indicating the presence of acid phosphatase, thiamine pyrophosphatase, and glucose-6-phosphatase, were observed on the spermatozoon surface at the head and tail regions. These observations support the idea that various phosphatases may play some role in spermatid differentiation.     

Enzymatic synthesis of nucleoside analogues using immobilized 2′-deoxyribosyltransferase from <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis>

Covalent attachment of recombinant Lactobacillus reuteri 2′-deoxyribosyltransferase to Sepabeads EC-EP303 leads to the immobilized biocatalyst SLrNDT4, which displayed an enzymatic activity of 65.4 IU/g of wet biocatalyst in 2′-deoxyadenosine synthesis from 2′-deoxyuridine and adenine at 40°C and pH 6.5. Response surface methodology was employed for the optimization of SLrNDT4 activity. Optimal conditions for SLrNDT4 highest activity were observed at 40°C and pH 6.5. Immobilized biocatalyst retained 50% of its maximal activity after 17.9 h at 60°C, whereas 96% activity was observed after storage at 40°C for 110 h. This novel immobilized biocatalyst has been successfully employed in the enzymatic synthesis of different natural and therapeutic nucleosides effective against cancer and viral diseases. Among these last products, enzymatic synthesis of therapeutic nucleosides such as 5-ethyl-2′-deoxyuridine and 5-trifluorothymidine has been carried out for the first time. Importantly for its potential application, SLrNDT4 could be recycled for 26 consecutive batch reactions in the synthesis of 2,6-diaminopurine-2′-deoxyriboside with negligible loss of catalytic activity.     

Cytochemical localization of adenylate cyclase and of calcium ion, magnesium ion-activated ATPases in the dense tubular system of human blood platelets.

Cytochemical techniques have been employed to study the localization of adenylate cyclase and (Ca2+ + Mg2+)-stimulated ATPase activities in platelets after fixation. Biochemical analysis of adenylate cyclase demonstrated a 70% reduction in activity in homogenates from fixed cells, but the residual activity could be stimulated 10--20 times by prostaglandin E1 (1 micrometer) under the same incubation conditions as employed in the cytochemical studies (e.g. media containing 2 mM lead nitrate and 10 mM NaF). Adenylate cyclase activity employing 5'-adenylyl-imiodiphosphate (AMP-P(NH)P) as substrate was found to be associated with the dense tubular system (smooth endoplasmic reticulum) in intact fixed platelets, and was apparent only when the cells were incubated with prostaglandin E1. Less activity was found along the membranes of the surface connected open canalicular system and occasionally at the outer cell surface. Enzymatic activity was blocked by the adenylate cyclase inhibitor 9-(tetrahydro-2-furyl) adenine and was not due to AMP-P(NH)P phosphohydrolase activity. The low adenylate cyclase activity in the surface membranes may be due to enzyme inactivation as a result of fixation, since a surface membrane fraction obtained by the glycerol lysis technique from unfixed cells had an adenylate cyclase specific activity equivalent to that in the microsomal membrane fraction. (Ca2+ + Mg2+)-stimulated ATPase activity was found associated with the membranes of the surface connected open canalicular system in unfixed cells. After brief fixation (5--15 min) with glutaradehyde, strong (Ca2+ + Mg2+)ATPase activity became apparent in the dense tubular system. Longer periods of fixation inactivated enzymatic activity. Addition of Ca2+ (1.0 mM) to incubation medium with low Mg2+ (0.2 mM), or increasing Mg2+ to 4.0 mM, in both cases strongly stimulated enzyme activity. The ATPase activity in the platelet membranes was not inhibited by ouabain. It is suggested that the Ca2+-stimulated ATPase and adenylate cyclase activities in the dense tubules may possibly be involved in regulation of intracellular Ca2+ transport.     

Improved localization of thiamine pyrophosphatase activity in mounted cryostat sections using gel fixation and gel incubation media.

The presence of 1% agar in the fixation and substrate solutions for the histochemical demonstration of thiamine pyrophosphatase (4.4 mM TPP; 3.6 mM Pb2+; 0.025 Tris-maleate buffer, pH 7.2) clearly facilitates the localization of the enzyme in Golgi apparatus in cold microtome sections prepared from unfixed specimens.     

Ectonucleotidases in the kidney

Members of all four families of ectonucleotidases, namely ectonucleoside triphosphate diphosphohydrolases (NTPDases), ectonucleotide pyrophosphatase/phosphodiesterases (NPPs), ecto-5′-nucleotidase and alkaline phosphatases, have been identified in the renal vasculature and/or tubular structures. In rats and mice, NTPDase1, which hydrolyses ATP through to AMP, is prominent throughout most of the renal vasculature and is also present in the thin ascending limb of Henle and medullary collecting duct. NTPDase2 and NTPDase3, which both prefer ATP over ADP as a substrate, are found in most nephron segments beyond the proximal tubule. NPPs catalyse not only the hydrolysis of ATP and ADP, but also of diadenosine polyphosphates. NPP1 has been identified in proximal and distal tubules of the mouse, while NPP3 is expressed in the rat glomerulus and pars recta, but not in more distal segments. Ecto-5′-nucleotidase, which catalyses the conversion of AMP to adenosine, is found in apical membranes of rat proximal convoluted tubule and intercalated cells of the distal nephron, as well as in the peritubular space. Finally, an alkaline phosphatase, which can theoretically catalyse the entire hydrolysis chain from nucleoside triphosphate to nucleoside, has been identified in apical membranes of rat proximal tubules; however, this enzyme exhibits relatively high K _m values for adenine nucleotides. Although information on renal ectonucleotidases is still incomplete, the enzymes’ varied distribution in the vasculature and along the nephron suggests that they can profoundly influence purinoceptor activity through the hydrolysis, and generation, of agonists of the various purinoceptor subtypes. This review provides an update on renal ectonucleotidases and speculates on the functional significance of these enzymes in terms of glomerular and tubular physiology and pathophysiology.     

Improved localization of thiamine pyrophosphatase activity in mounted cryostat sections using gel fixation and gel incubation media

Summary The presence of 1% agar in the fixation and substrate solutions for the histochemical demonstration of thiamine pyrophosphatase (4.4 mM TPP; 3.6 mM Pb²⁺; 0.025 Tris-maleate buffer, pH 7.2) clearly facilitates the localization of the enzyme in Golgi apparatus in cold microtome sections prepared from unfixed specimens.     

Phosphorylation is Involved in the Regulation of the Taurine Influx Via the β-system in Ehrlich Ascites Tumor Cells

The role of 3′,5′-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), protein kinase C (PKC) and phosphatases in the regulation of the taurine influx via the β-system in Ehrlich ascites tumor cells has been investigated. The taurine uptake by the β-system in Ehrlich cells is inhibited when PKC is activated by phorbol 12-myristate 13-acetate (PMA) and when protein phosphatases are inhibited by calyculin A (CLA). On the other hand, taurine uptake by the β-system is stimulated by an increased level of cAMP or following addition of N⁶,2′-O-dibutyryl-3′,5′-cyclic adenosine monophosphate (dbcAMP). The effect of dbcAMP is partially blocked by addition of the protein kinase inhibitor H-89, and suppressed in the presence of CLA. It is proposed that the β-system in the Ehrlich cells exists in three states of activity: State I, where a PKC phosphorylation site on the transporter or on a regulator is phosphorylated and transport activity is low. State II, where the PKC phosphorylation site is dephosphorylated and transport activity is normal. State III, representing a state with high transport activity, induced by an elevated cellular cAMP level. Apparently, cAMP preferentially stimulates taurine transport when the β-system is in State II. Received: 8 September/Revised: 9 November 1995     

Enzyme Cytochemistry of the Alveolar Sacs and Golgian-Like Cisternae in the Ciliate Ichthyophthirius multifiliis

In the cell cortex of the parasitic ciliate Ichthyophthirius multifiliis different kinds of cisternae were observed: the alveolar sacs, thick membrane cisternae and the endoplasmic reticulum. The thick membrane cisternae possess coated dilated rims and sometimes could be observed close to the endoplasmic reticulum. Using cytochemical techniques acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in the thick membrane cisternae and in the alveolar sacs of trosphozoites. In the endoplasmic reticulum acid phosphatase activity was not detected and only very small amounts of thiamine pyrophosphatase and nucleoside diphosphatase reaction product were observed. After exit from the host, a reduction in acid phosphatase activity was evident in the alveolar sacs. At theront stage acid phosphatase activity is absent from these structures. However, high thiamine pyrophosphatase and nucleoside diphosphatase activities remain in the alveolar sacs during the whole life cycle. On the other hand, acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in thick membrane cisternae of theronts. Based on the morphological aspects and enzymatic content the thick membrane cisternae of the cell cortex are designated as golgian-like cisternae. The cytochemical results point out a relationship between the alveolar sacs and the Golgi complex.     

Ultrahistochemical localization of adenylate cyclase activity in the electric organ of Torpedo marmorata

The lead pyrophosphate precipitation technique was used to visualize adenylate cyclase activity with the electron microscope in unfixed electric organ and synaptosomes of Torpedo marmorata, with special attention to presynaptic membranes. Specificity of the deposition of reaction product was ensured by using 5'-adenylyl imidodiphosphate as substrate and 5'-guanylyl imidodiphosphate and sodium fluoride as activators. Under suitable conditions a reaction product was deposited on the Schwann cell, on presynaptic vesicles, on the inner side of membranes of cisternae and on glycogen granules of the presynaptic region of the endplate. In some cases, a precipitate was also found on postsynaptic membranes of the synaptic cleft and on mitochondria. In isolated synaptosomes localization of the reaction product was identical with that of minced tissue. However, most strikingly, on presynaptic membranes no precipitate was ever found, neither in pieces of electric organ nor in isolated synaptosomes. Furthermore, the extended membrane system of the postsynaptic region of the electroplax remained always free of lead pyrophosphate precipitate.     

In situ localization of transketolase activity in epithelial cells of different rat tissues and subcellularly in liver parenchymal cells.

Metabolic mapping of enzyme activities (enzyme histochemistry) is an important tool to understand (patho)physiological functions of enzymes. A new enzyme histochemical method has been developed to detect transketolase activity in situ in various rat tissues and its ultrastructural localization in individual cells. In situ detection of transketolase is important because this multifunctional enzyme has been related with diseases such as cancer, diabetes, Alzheimer's disease, and Wernicke-Korsakoff's syndrome. The proposed method is based on the tetrazolium salt method applied to unfixed cryostat sections in the presence of polyvinyl alcohol. The method appeared to be specific for transketolase activity when the proper control reaction is performed and showed a linear increase of the amount of final reaction product with incubation time. Transketolase activity was studied in liver, small intestine, trachea, tongue, kidney, adrenal gland, and eye. Activity was found in liver parenchyma, epithelium of small intestine, trachea, tongue, proximal tubules of kidney and cornea, and ganglion cells in medulla of adrenal gland. To demonstrate transketolase activity ultrastructurally in liver parenchymal cells, the cupper iron method was used. It was shown that transketolase activity was present in peroxisomes and at membranes of granular endoplasmic reticulum. This ultrastructural localization is similar to that of glucose-6-phosphate dehydrogenase activity, suggesting activity of the pentose phosphate pathway at these sites. It is concluded that the method developed for in situ localization of transketolase activity for light and electron microscopy is specific and allows further investigation of the role of transketolase in (proliferation of) cancer cells and other pathophysiological processes.