The Use Of Rhodizonate In Enzymatic Histochemistry

A 0.5% solution of sodium rhodizonate stains lead precipitates, obtained in histochemical methods for enzymes, in a brown-black shade. The precipitate obtained in the method for acid phosphatase can be stained directly; those obtained in the methods for alkaline phosphatase and lipase, after conversion into the corresponding lead salts.     

Measurement of serum alkaline phosphatase

Alkaline phosphatase is a commonly requested enzyme test in clinical chemistry. However, the enzyme is not particularly substrate specific, which has led to a proliferation of methods for its analysis. It can exhibit a variable instability effect depending on the techniques required for its storage or analysis. Methods can also be highly dependent on sample isoenzyme distribution and reagent purity, leading to problems in the quality control of its analysis and in the comparison of results obtained from different methods. Alkaline phosphatase is not tissue specific and this may on occasion lead to uncertainty in the interpretation of its measured activity in blood serum. In recent years there has been a number of attempts to standardize methodology for this and other enzymes. Perhaps an alternative approach to the measurement of alkaline phosphatase activity will alleviate some of the problems encountered.     

Regio- and stereo-specific nitrile hydrolysis by the nitrile hydratase from Rhodococcus AJ270

Abstract Acid phosphatase activity was measured in individual cells by determining their optical densities through a scanning confocal laser microscope. The naphthol AS-TR (3-hydroxy-2-naphtoic acid 4'-chloro-2'-methylanilide) phosphate-hexazotized para-rosanilin method was used to visualise the acid phosphatase content in the light microscope. Evidence was obtained that the amount of enzyme varied in exponential growth phase cells as the fission age increased. By comparing the acid phosphatase activity with the rate of food vacuole formation, it appeared that the amount of enzyme inside the cells decreased in early clonal life, whereas the rate of food uptake increased. It was assumed that the reduction of acid phosphatase content could lead to a more extended life of vacuoles and to a decreased membrane recycling rate. In turn, the reduced supply of membrane available for food vacuole formation could partly be responsible for the decrease of the food uptake rate observed after the initial increase.     

Isolation and Characterization of Phosphatase from Thylakoid Membranes of Spinach Chloroplasts

A phosphatase from thylakoid membrane of spinach (Spinacia oleracea L. ) chloroplasts was isolated with the methods of extraction with n-ButanoL centrifugation at 100000 g for 30 min and chromatographic separation through DEAE-Cellulose (DE 52) column.The phosphatase catalyzed hydrolysis of phosphate monoesters (4-nitrophenyl phosphate). The optimal pH for enzyme catalysis was below 7. The peak rate of the enzyme reaction was obtained when it was incubated at 60℃ for 15 min. The phosphatase was inhibited by ATP and phosphate. The results from SDS-PAGE showed that the preparation of enzyme was composed of two proteins.     

Evaluation of cytochemical methods of studying the localization of alkaline phosphatase in B. subtilis]

A study was made of a possibility of using the "lead" and the "calcium" methods for the determination of localization of alkaline phosphatase in the bacterial cells. Cytochemical medium containing lead nitrate could not be used for determination of the true localization of the alkaline phosphatase in the microorganisms.     

False localization of acid phosphatase activity in the nuclear envelope and endoplasmic reticulum of peritoneal macrophages

Summary Acid phosphatase cytochemistry using lead salt methods was performed on rat peritoneal macrophages obtained by the intraperitoneal injection of dextran five days previously. Lead precipitate was present in the nuclear envelope, the rough endoplasmic reticulum, Golgi apparatus and lysosomes in about 50% of these cells. The formation of reaction product appeared to be substrate-specific and was sensitive to sodium fluoride in all these sites. However, only in the nuclear envelope, the rough endoplasmic reticulum and Golgi apparatus could lead salt precipitation be prevented by (a) omission of the washing procedure following the incubation step, (b) postincubation in a medium containing sodium fluoride, or (c) washing in buffer containing lead salt. It is concluded that precipitation of lead salt does not prove the presence of acid phosphatase activity in these organelles. The formation of precipitate in these sites is probably due to a local matrix effect, facilitated by the persistence of acid phosphatase activity in the lysosomes and a suboptimal trapping efficiency of phosphate ions during the washing procedure which follows in the incubation step.     

Mechanisms of metal-salt methods in enzyme cytochemistry with special reference to acid phosphatase

Summary This review is concerned with theoretical and experimental aspects of the factors governing the localizing potentialities of cytochemical enzyme reactions that are based on the metal-salt principle, that is, the precipitation of the primary product of the enzymatic reaction by a heavy-metal ion at the enzymatic site. Special attention is given to the lead phosphate precipitation process in acid phosphatase cytochemistry. The various model systems developed for the study of the factors involved in precipitation are described and their advantages and disadvantages discussed. Furthermore, the various cytochemical methods so far used for the demonstration of acid phosphatase activity are critically evaluated in the light of the results obtained with the model systems.     

The loss of enzyme reaction products from ultrathin sections during the staining for electron microscopy

Summary The effects of heavy metal salt staining procedures on the reaction products obtained in the demonstration of arylsulphatase and of acid phosphatase were studied.Lead citrate staining at pH 12 was found to cause a very marked dissolution of barium sulphate and a moderate dissolution of lead sulphate. The staining with uranyl acetate was found to dissolve moderately both barium and lead sulphate.Neither lead citrate nor uranyl acetate staining had any remarkable effect on lead phosphate.The mechanism of the dissolution and the possibilities to avoid it were discussed.     

Zinc and magnesium content of alkaline phosphatase from Escherichia coli.

Since alkaline phosphatase from Escherichia coli was first reported to contain 2.1 g-atoms of zinc and 0.8 g-aton of magnesium per molecular weight 80,000 (Plocke, D.J., Levinthal, D., and Vallee, B. L. (1962), Biochemistry 1, 373-378), the procedures for isolation and purification of the enzyme, as well as values for the protein molecular weight, specific absorptivity, and maximal activity, have changed repeatedly. Such variations have resulted in uncertainties concerning the molar metal content of this phosphatase. The present paper reviews the initial and recent results of metal analyses of alkaline phosphatase preparations in this laboratory and compares them with those obtained elsewhere, while simultaneously identifying some of the factors which have affected reports on the metal content of this enzyme. A purification procedure is described eliminating the features of all methods known to alter the metal content of phosphatase. In addition, the three isozymic forms, as well as preparations from four E. coli strains commonly employed for phosphatase isolation, were analyzed and compared.     

A comparison between osmiophilic reagents and the Gomori lead method for the electron cytochemical demonstration of two lysosomal enzymes in oral epithelium

Synopsis Osmiophilic reagents were used to study the histochemical localization of acid phosphatase and non-specific esterase in the keratinized oral mucosa of rat. The reaction product from both enzymes was found in the epithelium and in cells of the corium as discrete granules, suggestive of a lysosomal localization. Treatment with E-600 before incubation for non-specific esterase did not change this localization. The osmium black end-product, due to acid phosphatase activity, was examined with the electron microscope and compared with the localization obtained by the Gomori lead phosphate technique. Both methods produced a reaction product in membrane-bounded bodies resembling lysosomes, as described in other tissues. These organelles were present in the basal prickle and granular cell layers of the epithelium. In the keratinized layer the reaction product was localized between the cell membranes of the deeper cells and no deposits were present in the cells. It is suggested that the osmiophilic reagents provide a good alternative to the Gomori method for the localization of lysosomal acid phosphatase at both the light and electron microscope levels.     

Selective Extraction of Alkaline Phosphatase and 51 -Nucleotidase from Milk Fat Globule Membranes by a Single Phase n-Butanol Procedure

ABSTRACT

A single phase extraction procedure employing 8% (v/v) n-butanol at room temperature extracted over 90% of alkaline phosphatase activity and over 60% of 5'-nucleotidase activity from bovine milk fat globule membranes (MFGM). For 5'-nucleotidase, higher n-butanol concentrations lead to loss of activity, while lower concentrations were ineffective in extracting the enzyme. When extractions were performed at 0°C, similar yields were obtained for alkaline phosphatase extraction with 8% (v/v) n-butanol, but 5¹- nucleotidase extraction required 10% (v/v) n-butanol for similar yields. However, 5'-nucleotidase was less susceptible to denaturation during extraction at 0°C. The Km values and substrate specificities for both alkaline phosphatase and 5'-nucleotidase were unchanged by extraction with 8% (v/v) n-butanol. The 8% (v/v) n-butanol extraction procedure provides a 3-fold purification step, and an enzyme preparation suitable for further purification.     

Studies on an improved lead phosphate technique for the demonstration of non-specific acid phosphatase in non-deparaffinized organ and tissue sections

Summary The inconstant results and the aspecific reaction patterns obtained after use of the lead phosphate method for the demonstration of acid phosphatase cannot be attributed to destruction of the enzyme by acetone fixation and embedding in paraffin. The marked shrinking effect of pure acetone is partially avoided by fixation in 80% acetone at 0–4° C. The appearance of spotty gaps employing the lead phosphate method in non-deparaffinized sections is the result of the presence of by-products in the paraffin and not of an enzyme-inactivating action of the albumen-containing adhesive. Constant results in the localization of acid as well as of alkaline phosphatase can be obtained by pretreatment of non-deparaffinized sections with 80% acetone. Paraffin mixtures containing stearin and stearic acid are not suitable for this improved incubation technique. The conditions for the obtaining of reliable data as to the presence of non-specific acid phosphate activity are more favourable in non-deparaffinized sections of material fixed in cold acetone than in formaldehyde-fixed frozen sections. The effect of quantitative variations of the components of the substrate mixture on the result of the reaction is discussed. It has been clearly shown that the relative proportion of the concentrations of glycerophosphate ions and lead ions is of importance for optimal results. The distribution of non-specific acid phosphatase, histochemically demonstrable with the improved technique in a number of organs and tissues of the rabbit, rat and mouse is described.With 18 Figures in the Text     

A new direct lead technique for histochemical demonstration of alkaline phosphatase activity.

A lead method for demonstrating alkaline phosphatase is described. The method is based on direct precipitation of lead as lead phosphatase at pH 9.5, the pH optimum of the enzyme. Stable incubation medium was achieved by using tartrate, instead of maleate, as chelating for lead. The method was found to be suitable for visualization of alkaline phosphatase in different types of tissues.     

Acid phosphatase activity in intercellular spaces in roots of Nymphoides peltata (S. G. Gmel.) O. Kuntze

Abstract. Acid phosphatase was found cytochemically in intercellular spaces in the root of Nymphoides peltata. Different methods, using lead salts and azo-dyes, gave similar results. Reaction product appeared on material, possibly cytoplasmic, within the intercellular spaces and also against the outer walls of cells which formed the intercellular spaces. Possible functions of acid phosphatase in intercellular spaces are discussed.     

An examination of the age-related patterns of decay of acid phosphatase (ACP1) in human red cells from individuals of different phenotypes

A study has been made of the decay of acid phosphatase (ACP₁) in the human red cell using red cell fractions of different mean ages prepared by density gradient centrifugation. Red cells from acid phosphatase type A and type B individuals were used in the study. Acid phosphatase activity of the red cell fractions was determined by two different assay methods. The results obtained were comparable and have been combined. Acid phosphatase type A and type B showed a biphasic decay pattern with a rapid early loss of activity, followed by a more gradual rate of decline. Type A appeared to decay more rapidly than type B in both decay phases. It is proposed that differences in stability between type A and type B in vivo may explain the observed differences in activity between the enzyme types. There was no evidence for the generation of secondary isozymes by acid phosphatase type A or type B during red cell aging.     

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.     

Purification and characterization of two phosphorylase phosphatases from rabbit liver

Two phosphorylase phosphatase activities (I and III) have been purified from rabbit liver, with respective molecular weights of 117,000 and 230,000. Phosphatase III contained three different subunits of molecular weights 35,000, 67,000 and 80,000. Phosphatase I although majoritary in the preparation, was not homogeneous. Both phosphatases were dissociated by 2-mercaptoethanol treatment, releasing a catalytic subunit with a molecular weight of about 35,000. Phosphatases I and III activities responded very differently to incubation with trypsin and to ethanol precipitation. Phosphatase III was much more sensitive to inactivation by several ions and ATP than phosphatase I. On the basis of the obtained data, phosphatase I can be classified as a type-1 phosphatase and phosphatase III as a type-1 phosphatase.     

锌对铅染毒新生大鼠成骨细胞增殖分化的影响

目的:探讨铅锌联合染毒对乳鼠颅骨成骨细胞增殖分化的影响。方法:分离并培养原代成骨细胞,加入不同浓度铅、锌培养48h,检测其对成骨细胞增殖的作用;用碱性磷酸酶试剂盒检测ALP活力。结果:在染铅48h后,当铅浓度≥10μmol/L时,细胞增殖功能下降(P<0.05);加锌干预48h后,铅+锌组细胞增殖功能均高于各自单独染铅组,其中铅(1μmol/L、10μmol/L)+锌(50μmol/L)组、铅(10)+锌(100)组与对照组间的差异具有统计学意义(P<0.05)。铅干预48h后,100μmol/L铅组的ALP活力显著下(P<0.05),给予锌干预的铅锌联合染毒组,各组ALP活力均有增加,其中铅(1μmol/L、10μmol/L)+锌(50μmol/L)组ALP活力均高于对照组,而铅(100μmol/L)+锌(50μmol/L)组ALP活力低于对照组,差异均有统计学意义(P<0.05)。结论:铅对成骨细胞有毒性作用,影响其增殖和分化功能;50μmol/L锌在一定程度上可以拮抗铅对成骨细胞增殖和分化功能的损伤,且对ALP活力的作用更显著,为铅中毒骨病的防治提供一定的科学依据。     

Isolation of peroxisomes from the dog kidney cortex.

The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.     

Microwave oven and boiling waterbath extraction of hepatotoxins from cyanobacterial cells

Low-cost, straightforward methods for the extraction of microcystins and nodularins from cyanobacterial cells were developed using a microwave oven and boiling waterbath. The use of organic solvents, such as methanol, which can interfere with sensitive analytical procedures, e.g. immunoassays, can thus be avoided. Analysis by protein phosphatase inhibition assay and high performance liquid chromatography indicated that purified microcystin-LR was unaffected by the microwave oven and boiling waterbath treatments. Four microcystins of differing hydrophobicities were successfully extracted from Microcystis PCC 7813 by both treatments at yields equivalent to those obtained by longer protocols using methanol. Assessment of the microwave oven and boiling waterbath extraction methods with laboratory strains and environmental samples of cyanobacteria showed good correlation with results from lyophilisation and methanol extraction, when extracts were analysed by high performance liquid chromatography with diode array detection (R(2)>/=0.92). The microwave and boiling waterbath extraction methods also sterilised the environmental bloom samples, as evidenced by the abolition of heterotrophic bacterial growth.