Cerium-based demonstration of phosphatase activity in plastic-embedded sections: a comparison with conventional methods

Cerium-based methods have been used for the demonstration of several phosphatases at alkaline, acid and neutral pH in low temperature acetone-fixed, plastic-embedded sections. At alkaline pH calcium is used as capturing agent and the precipitated calcium phosphate converted to cerium phosphate. At neutral and acid pH cerium is used directly as capturing agent. Cerium phosphate is subsequently visualized using the H2O2-DAB method. A comparison has been made with conventional calcium-cobalt and lead methods. It appeared that calcium-cobalt methods are more susceptible to improvement than lead methods.     

Cerium-Based Demonstration of Phosphatas Activity in Plastic-Embedded Sections: A Comparison with Conventional Methods

Cerium-based methods have been used for the demonstration of seve phatases at alkaline, acid and neutral pH in low temperature acetone-fixed, plastic-e sections. At alkaline pH calcium is used as capturing agent and the precipitated phosphate converted to cerium phosphate. At neutral and acid pH cerium is used d capturing agent. Cerium phosphate is subsequently visualized using the H₂C₂DAB n: comparison has been made with conventional calcium-cobalt and lead methods. It; that calcium-cobalt methods are more susceptible to improvement than lead methods     

Light microscopical demonstration of non-specific alkaline phosphatase activity with an incubation medium containing cerium and two calcium as the capturing agents. The cerium/calcium-hydrogen peroxide-P-phenylenediamine/pyrocatechol (Ce/Ca-H2O2-PPD/PC) double capture technique.

A new method for the light microscopical demonstration of alPase activity in cryotome sections by using simultaneously cerium and calcium as capturing agents (double capture technique) is described. This method has an increased sensitivity compared with the single cerium-based and the Gomori based-cerium (single calcium and cerium converted) with techniques described previously. Presuming that the enzymatic activity during incubation of sections in the presence of a defined capturing agent is constant, the increased sensitivity after employment of the double capture method could be attributed to a decrease of enzyme inhibition by cerium through the presence of calcium. Based on model experiments it is assumed that calcium phosphate and cerium phosphate are the primary reaction products, the former converting into cerium phosphate already during incubation. The remaining calcium phosphate is converted completely by treatment with cerium citrate solution (conversion reaction). After oxidation with H2O2 the cerium perhydroxyphosphate was visualized in a paraphenylenediamine/pyrocatechol (Hanker-Yates reagent) solution without H2O2 to give a black reaction product. This visualization procedure is superior to the DAB or DAB-Ni mode as published earlier. Some results concerning the mode of inhibition of the pseudoperoxidase activity of the hemoglobin are presented.     

New, improved lanthanide-based methods for the ultrastructural localization of acid and alkaline phosphatase activity

New, improved techniques for the ultrastructural localization of acid and alkaline phosphatase activity using lanthanide cations as the trapping agent were developed. Delayed penetration of the capture ions and the incubation constituents into cellular compartments was prevented by pretreating specimens with borohydride/saponin. Both the concentration of the capture agent in the incubation medium and the incubation time of the tissue specimens were optimized to achieve a satisfactory cytochemical reaction and to avoid precipitation artefacts caused by local matrix effects. The conversion of cerium phosphate into the almost insoluble cerium fluoride minimized losses of the reaction product during postincubation processing. Moreover, lanthanum itself as well as lanthanides other than cerium, e.g., gadolinium and didymium (praseodymium, neodymium), were successfully applied and can be recommended as capture agents for phosphatase cytochemistry.     

New,improved lanthanide-based methods for the ultrastructural localization of acid and alkaline phosphatase activity

Summary New, improved techniques for the ultrastructural localization of acid and alkaline phosphatase activity using lanthanide cations as the trapping agent were developed. Delayed penetration of the capture ions and the incubation constituents into cellular compartments was prevented by pretreating specimens with borohydride/saponin. Both the concentration of the capture agent in the incubation medium and the incubation time of the tissue specimens were optimized to achieve a satisfactory cytochemical reaction and to avoid precipitation artefacts caused by local matrix effects. The conversion of cerium phosphate into the almost insoluble cerium fluoride minimized losses of the reaction product during postincubation processing. Moreover, lanthanum itself as well as lanthanides other than cerium, e.g., gadolinium and didymium (praseodymium, neodymium), were successfully applied and can be recommended as capture agents for phosphatase cytochemistry.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Cytochemical demonstration of adenylate cyclase activity with cerium

Cerium was applied for the ultrastructural, cytochemical localization of adenylate cyclase (EC 4.6.1.1.). The enzyme activity was stimulated with norepinephrine, prenalterol and cholera toxin in the brown fat cells of newborn rats. The final reaction product was observed in the plasmalemmas of the stimulated adipocytes. The precipitate was finely crystalline, easily visible in the electron microscope and in the X-ray microprobe analysis it yielded cerium and phosphate peaks, respectively. The use of cerium offers a new tool valid for the cytochemical localization of adenylate cyclase enzyme related to the membrane receptors.     

Ultrastructural localization of 5'-nucleotidase in guinea pig neutrophils based upon the use of cerium as capturing agent

A cytochemical method for 5'-nucleotidase localization in which cerium serves as the capture agent in order to enzymatically detect liberated inorganic phosphate has been developed. The method has been established in cell-free model systems and in guinea pig neutrophils where 5'-nucleotidase is restricted to the plasmalemma as an ectoenzyme. This cerium-based method gives better results for ultrastructural localization of 5'-nucleotidase than conventional lead-based methods.     

Cytochemical demonstration of adenylate cyclase activity with cerium

Summary Cerium was applied for the ultrastructural, cytochemical localization of adenylate cyclase (EC 4.6.1.1.). The enzyme activity was stimulated with norepinephrine, prenalterol and choleratoxin in the brown fat cells of newborn rats. The final reaction product was observed in the plasmalemmas of the stimulated adipocytes. The precipitate was finely crystalline, easily visible in the electron microscope and in the X-ray microprobe analysis it yielded cerium and phosphate peaks, respectively. The use of cerium offers a new tool valid for the cytochemical localization of adenylate cyclase enzyme related to the membrane receptors.This study was supported by the grant from Reino Lahtikari Foundation     

A new cerium-based method for cytochemical localization of thiamine pyrophosphatase in the Golgi complex of rat hepatocytes. Comparison with the lead technique

The use of cerium chloride for the localization of thiamine-pyrophosphatase (TPPase) in rat liver parenchymal cells has been investigated and the results are compared with the classical lead capture method. A medium containing 3 mM cerium chloride gave the most uniform and consistent results with a homogeneous electron dense reaction product in the first trans lamella of the Golgi complex and a weak staining of endoplasmic reticulum. The fine deposits of cerium phosphate filled completely the first trans Golgi cisterna. In contrast the reaction product of the lead-based method appeared clumpy and aggregated with an irregular distribution over both Golgi complex and endoplasmic reticulum. Higher and lower concentrations of cerium chloride than 3 mM gave inconsistent results. The present study demonstrates that the cerium-based method is superior to the classical lead-technique for the localization of TPPase.     

A new cerium-based method for cytochemical localization of thiamine pyrophosphatase in the Golgi complex of rat hepatocytes

Summary The use of cerium chloride for the localization of thiamine-pyrophosphatase (TPPase) in rat liver parenchymal cells has been investigated and the results are compared with the classical lead capture method. A medium containing 3 mM cerium chloride gave the most uniform and consistent results with a homogenous electron dense reaction product in the first trans lamella of the Golgi complex and a weak staining of endoplasmic reticulum. The fine deposits of cerium phosphate filled completely the first trans Golgi cisterna. In contrast the reaction product of the lead-based method appeared clumpy and aggregated with an irregular distribution over both Golgi complex and endoplasmic reticulum. Higher and lower concentrations of cerium chloride than 3 mM gave inconsistent results. The present study demonstrates that the cerium-based method is superior to the classical lead-technique for the localization of TPPase.     

Localization of Hydrogen Peroxide Production in Pisum sativum L. Using Epi-Polarization Microscopy to Follow Cerium Perhydroxide Deposition

Cerium is becoming an increasingly popular reagent for histochemical localization of oxidases and phosphatases because it combines directly with reaction products to form fine precipitates of electron-dense materials that can be easily detected using transmission electron microscopy or laser confocal scanning microscopy. We used epi-polarization microscopy to detect cerium perhydroxide deposits formed when H2O2 was produced by diamine oxidase in pea (Pisum sativum L.) epicotyls exposed to exogenous putrescine. Diamine oxidase activity was abundant in cortical cell walls but showed little, if any, association with vascular tissues. Maps of cerium deposition generated using scanning electron microscopy/x-ray microanalysis verified these observations. This study demonstrates the use of epi-polarization microscopy to follow cerium deposition, and the ready accessibility of this microscopy technique should facilitate more widespread use of cerium for plant histochemistry and cytochemistry.     

Enzyme histochemical demonstration of alkaline phosphatase activity in plastic-embedded tissues using a Gomori-based cerium-DAB technique

We describe a new method for light microscopic demonstration of alkaline phosphatase (ALP) activity in plastic-embedded sections. Rat tissues were fixed in acetone (-20 degrees C), infiltrated in glycol methacrylate (GMA), and embedded at 0 degrees C. Sections were cut at 1 and 2 microns, dried at room temperature, and incubated in the conventional Gomori medium. Cerium chloride was used to convert calcium phosphate into cerium phosphate, which was subsequently converted into cerium perhydroxide. The slight yellow precipitate of cerium perhydroxide was amplified using 3,3'-diaminobenzidine tetrahydrochloride (DAB). For comparison, tissue sections were processed according to the calcium-cobalt method. The method described combines exact localization of ALP activity with optimal preservation of tissue morphology.     

Light-microscopic histochemistry of non-specific alkaline phosphatase using lanthanide-citrate complexes

Summary New lanthanide methods for the histochemical detection of non-specific alkaline phosphatase in the light microscope are described and compared with already existing techniques for the light microscopical demonstration of this enzyme. To avoid formation of insoluble lanthanide hydroxide at alkaline pH citrate complexes with the capture ions cerium, lanthanum and didymium were used. A molar ratio of 11 mM citrate/14 mM capture reagent is proposed. For preincubated sections, pretreatment in chloroform-acetone and fixation in glutaraldehyde, for non-preincubated sections fixation in glutaraldehyde yielded the best results. 4-Methylumbelliferyl and 5-Br-4-Cl-3-indoxyl phosphate were found to be the most suitable substrates. For routine purposes 4-nitrophenyl, 1-naphthyl, 2-naphthyl and 2-glycerophosphate were also sufficient; naphthol AS phosphates were inferior but still suitable. After incubation for 5–60 min at 37° C lanthanide phosphate was converted into lead phosphate which was visualized as lead sulfide. At pH 9.2–9.5 enzyme activity was demonstrated at many sites such as intestinal, uterine, placental, renal and epididymal microvillous zones, plasma membranes of arterial, sinus and capillary endothelial cells, vaginal and urethral epithelium, smooth muscle cells, myoepithelial cells as well as excretory duct cells of salivary and lacrimal glands and in secretory granules of laryngeal glands. In comparison with Gomori's calcium, Mayahara's lead, Burstone's and Pearse's azo-coupling, McGadey's tetrazolium salt and Gossrau's azoindoxyl coupling technique the lanthanide methods detected alkaline phosphatase activities at identical or additional sites depending on the respective procedure. However, in contrast to the other methods especially the cerium citrate procedure yielded a more precisely localized and more stable reaction product, can be used with all available alkaline phosphatase substrates including those up till now less suitable or unsuitable for light microscopic alkaline phosphatase histochemistry.     

Substrate specificity and kinetic mechanism of purine nucleoside phosphorylase from Mycobacterium tuberculosis

Purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP) is numbered among targets for persistence of the causative agent of tuberculosis. Here, it is shown that MtPNP is more specific to natural 6-oxopurine nucleosides and synthetic compounds, and does not catalyze the phosphorolysis of adenosine. Initial velocity, product inhibition and equilibrium binding data suggest that MtPNP catalyzes 2′-deoxyguanosine (2dGuo) phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate (P_i) binds first followed by 2dGuo, and ribose 1-phosphate dissociates first followed by guanine. pH-rate profiles indicated a general acid as being essential for both catalysis and 2dGuo binding, and that deprotonation of a group abolishes P_i binding. Proton inventory and solvent deuterium isotope effects indicate that a single solvent proton transfer makes a modest contribution to the rate-limiting step. Pre-steady-state kinetic data indicate that product release appears to contribute to the rate-limiting step for MtPNP-catalyzed reaction.     

Light microscopical visualization of phosphatase activities demonstrated with the cerium method in resin sections of the kidney with the microwave oven

The light microscopically invisible reaction product cerium phosphate in resin sections of rat kidney, that had been incubated for the demonstration of phosphatase activities before embedding, was converted into a visible reaction product by incubation for 10 min at 80 degrees C in alkaline lead citrate in a microwave oven. This method offers the possibility to study phosphatase activities with the cerium method in semithin Epon sections. Furthermore it is a suitable method to select areas with phosphatase activity to be studied with the electron microscope.     

Light microscopical visualization of phosphatase activities demonstrated with the cerium method in resin sections of the kidney with the microwave oven

Summary The light microscopically invisible reaction product cerium phosphate in resin sections of rat kidney, that had been incubated for the demonstration of phosphatase activities before embedding, was converted into a visible reaction product by incubation for 10 min at 80° C in alka-line lead citrate in a microwave oven. This method offers the possibility to study phosphatase activities with the cerium method in semithin Epon sections. Furthermore it is a suitable method to select areas with phosphatase activity to be studied with the electron microscope.     

Improved light microscopic demonstration of D-amino acid oxidase activity in cryotome sections using cerium ions as capturing and amplifying agent--the Ce/Ce-H2O2-DAB procedure.

The light microscopical demonstration of D-amino acid oxidase (AAOX) activity with cerium (Ce III) as the capturing agent was improved. The incubation medium was stabilized by the employment of triethanolamine and detrane complexed cerium. A considerable increase in intensity of the reaction was accomplished by treatment of the AAOX-incubated sections with Ce III which reacted with the primary reaction product Ce IV-perhydroxide to form Ce IV-hydroxide. In this way the primary reaction product was reduced and enlarged concomitantly. The Ce IV-hydroxide was converted into Ce IV-perhydroxide by H2O2, which was visualized by blue-black stained Ni-DAB complexes. Thus, Ce III is used as capturing agent as well as amplifier (Ce/Ce-H2O2-DAB method). The primary reaction product Ce III-phosphate formed by coreacting phosphatases was selectively extracted by citrate containing glycine-NaOH buffer while Ce IV-perhydroxide remained in the sections. In model experiments it was proven that the perhydroxide groups in the Ce IV-perhydroxide compound initiate predominantly the DAB polymerization while the contribution of Ce III and Ce IV is small.     

Quantitative histochemical analysis of glucose-6-phosphatase activity in rat liver using an optimized cerium-diaminobenzidine method

We have optimized a cerium-diaminobenzidine-based method for histochemical analysis of glucose-6-phosphatase (G6Pase) activity and have determined quantitative data on the zonal distribution pattern in the liver acinus of fasted male rats. In the cerium-diaminobenzidine technique, cerium instead of lead ions is used as capturing reagent for the enzymatically liberated phosphate. For light microscopy, the primary reaction product, cerium phosphate, is then visualized by conversion into cerium perhydroxide using hydrogen peroxide and subsequent oxidative polymerization of diaminobenzidine to diaminobenzidine brown as the final reaction product. Variation of the substrate (glucose-6-phosphate) concentration in the incubation medium yielded in periportal zones a KM value of 2.3 +/- 0.7 mM and a Vmax value of 0.96 +/- 0.18 (expressed as mean integrated absorbance). In perivenous zones a KM value of 1.1 +/- 0.4 mM and a Vmax value of 0.51 +/- 0.08 were calculated. The cytophotometric analysis performed in this study demonstrated for the first time that a functional difference of G6Pase, the key enzyme for gluconeogenesis, exists in the periportal and perivenous zones of the liver acinus. Periportal zones contain twice as many enzyme molecules (high Vmax) as perivenous zones, but the affinity for the substrate is twice as low. This may have important implications for the concept of metabolic zonation of the liver and also for glucose homeostasis in the blood.     

Cerium-based cytochemical method for detection of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity at physiological pH

We have developed a new cytochemical method for detecting the ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase (K-NPPase) activity of the sodium-potassium-activated adenosine triphosphatase (Na-K ATPase) complex. The incubation medium contains p-nitrophenylphosphate (p-NPP) as substrate, cerium chloride as capture agent, Tricine buffer, MgCl2, and KCl. Tricine buffer protected against the medium turbidity caused by non-enzymatic reaction at pH 7.5. Biochemically, the accumulation of p-nitrophenol and phosphate in the reaction precipitate was proportionally related to the enzyme concentration. Ultracytochemically, the reaction products of the K-NPPase activity were localized as fine and uniform electron-dense deposits in the cytoplasmic side of specialized basolateral plasma membranes of cells of kidney distal convoluted tubules, secretory cells of salt gland, and marginal cells of stria vascularis. This method has the advantage of being useful at physiological pH.     

Ultrastructural localization of coenzyme A phosphatase (CoA-Pase) activity to the GERL system in secretory ameloblasts of the rat incisor

Enzymatic hydrolysis of the monoester phosphate group from coenzyme A (CoA) was studied in rat incisor ameloblasts by incubating specimens from glutaraldehyde-fixed teeth in a cytochemical medium prepared with acetyl-CoA as substrate and lead ions as capture agent for phosphate. Ameloblasts incubated for 1 hr at 37 degrees C and at pH 5.0 in this medium showed reaction product localized almost exclusively along the trans (mature) aspect of the Golgi apparatus within a network of small granules and interconnecting tubular channels that comprise the GERL system in this cell. Reaction product was otherwise seen in trace amounts only within some Golgi saccules, a few lysosomal dense bodies and, in rare instances, within an occasional focal area of the endoplasmic reticulum. No selective staining of the GERL system was seen in control ameloblasts incubated at either pH 7.2 or pH 9.0 with acetyl-CoA as substrate, or incubated at pH 5.0 with dephospho-CoA as substrate. Control experiments at pH 5.0 also revealed that reaction product selectively stained the GERL system in ameloblasts when other molecules resembling CoA were used as substrate (e.g., crotonyl-CoA, 3'-NADP+), but not when adenosine 3'-monophosphate (3'-AMP) was used as substrate. That is, ameloblasts incubated at pH 5.0 with 3'-AMP showed heavy deposits of reaction product at many sites throughout the cell, including most lysosomal dense bodies, the Golgi saccules, the GERL system, most secretory granules, the nucleus, and extensively throughout the endoplasmic reticulum. These findings suggest that the GERL system of ameloblasts contains a CoA-specific phosphatase activity that may function to convert CoA to dephospho-CoA at acid pH. Biochemical studies included with this investigation further indicate that CoA-Pase activity saturates at exceptionally low concentrations of substrate (KM = 30 microM CoA) compared to other acid-dependent phosphatases.