Acid Hydrolases in Hela Cells: Comparison of Methods for Light Microscopy

TO distinguish lysosome populations of HeLa cells, acid phosphatase, /8-glu-curonidase, arylsulfatase and esterase were demonstrated using various substrates and couplers with different fixations, pHs and inhibitors. The substrates chosen were for acid phosphatase, naphthol AS-BI phosphate with fast red violet LB at pH 4.6; for β-glucuronidase, naphthol AS-BI β-D-glucuronide with fast red violet LB at pH 4.4; for arylsulfatase, p-nitrocatechol sulfate, with lead as the capturing ion, at pH 4.8 and 5.6; and for esterase, naphthol AS-D acetate with fast blue BB at pH 6.5. In the azo-dye methods, the coupling was always simultaneous and results were satisfactory with unfixed cells. For optimal demonstration of arylsulfatase, cells were fixed in glutaraldehyde in 0.1 M cacodylate buffer pH 7.2, 2% for 24 hr or 6.25% for 2 hr, and washed for 1-9 days in 0.1 M veronal acetate buffer pH 7.2, 7.5% with respect to sucrose. Two groups of lysosomes were distinguished. One comprised small bodies, probably primary lysosomes, which lay in a cluster near the nucleus. They had quite stable membranes and were mostly acid phosphatase-positive. They sometimes contained β-glucuronidase or esterase, but rarely arylsulfatase. The other group included all the acid hydrolase-positive bodies scattered throughout the rest of the cytoplasm. They were mostly larger, with more labile membranes, and contained β-glucuronidase, esterase or arylsulfatase, but rarely acid phosphatase.     

Isoelectric-focusing behavior of acid hydrolases in rat kidney lysosomes. Effects of the pH gradient, autolysis and neuraminidase.

Isoelectric focusing was used to study the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase in lysosomes isolated from rat kidney. The isoelectric points of the main protein and hydrolase peaks were 1-1.5 units lower when electrofocusing was done in a pH 3-10 gradient than in a pH 10-3 gradient, apparently because the lysosomal constituents aggregated strongly at their isoelectric points and tended to settle somewhat in the gradient due to gravity. In the extended pH gradient the acidic form of each hydrolase occurred as asingle, relatively discrete peak. However, when pooled acidic fractions were refocused in a restricted pH gradient (pH 6-3 or 3-5) multiple acidic enzyme and protein components were resolved with isoelectric points between 2.7 and 5.1. When autolysis was minimized by extracting lysosomal fractions at alkaline pH (0.2% Triton X-100, 0.1%p-nitrophenyloxamic acid, 0.1 M glycine buffer, pH9) and including 0.1%p-NITROPHENYLOXAMIC ACID, AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND CATHEPSIN D, in the pH gradient, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in two forms, an acidic form with an isoelectric point of about 4.4, and a basic form with an isoelectric point close to 6.2, 6.7 and 8.0, respectively. Acid phosphatase occurred in three forms with isoelectric points of 4.1, 5.6 and 7.4. When some autolytic digestion was permitted by extracting lysosomal fractions in an acidic medium (0.2% Triton X-100, 0.1 M sodium acetate buffer, pH 5.2) AT 0-4DEGREES C and omitting p-nitrophenyloxamic acid from the gradient, the acidic form of beta-glucuronidase and the intermediate form of acid phosphatase were lost, the isoelectric points of the acidic forms of acid phosphatase, arylsulfatase and beta-N-acetylhexosaminidase were increased 0.6-1.2 units, and the isoelectric point of the basic forms of acid phosphatase, arylsulfatase and beta-glucuronidase was increased 0.5 unit. When lysosomal extracts were incubated with bacterial neuraminidase before electrofocusing, the acidic forms of acid phosphatase, arylsulfatase and beta-glucuronidase were largely lost, the isoelectric point of the acidic form of beta-N-acetylhexosaminidase was increased from 4.5 to 6.4, and the isoelectric points of the basic forms of all four hydrolases were increased 0.5-1.5 units. Autoincubation of lysosomal extracts in vitro at pH 5.2 PRODUCED SIMILAR, THOUGH LESS MARKED, effects. cont'd     

Simultaneous Formalin Fixation and Edta Decalcification, With Carbowax Embedding for Preservation of Acid Phosphatase

Carbowax serial sections from pubic symphyses of female mice, fixed and decalcified in a 10% formalin-5% Versenate solution for 18 hr at 4 C, pH 5.2, were incubated for 30 min with Burstone's simultaneous coupling reagent (pH 5.2); substrate: naphthol AS-TR and the diazonium salt, fast red violet L.B. All sections were counterstained with 1% methyl green at pH 4.0 in a phospho-citrate buffer. Inhibition by 0.01 M NaF, 0.0002 M CuCl₂, 10% tartaric acid and 0.01 M NaCN, as well as substrate-deficient and heat-inactivated controls, demonstrated conclusively that acid phosphatase was functionally preserved. Strong enzymatic activity was exhibited by osteoclasts, chondroclasts and free multinucleated giant cells. In addition, megakaryocytes, histiocytes, plasma cells, and monocytes exhibited moderate activity. The results demonstrated the technique to be consistently reproducible.     

Ultrastructural cytochemistry of chronic T-cell leukaemias. A study with four acid hydrolases

The ultrastructural localization of four acid hydrolases (acid phosphatase, beta-glucuronidase, beta-glucosaminidase and alpha-naphthylacetate esterase) has been studied in lymphocytes from 16 patients with three types of chronic T-cell leukaemia, namely, T-prolymphocytic leukaemia (T-PLL), T-chronic lymphocytic leukaemia (T-CLL) and adult T-cell lymphoma leukaemia (ATLL). Different patterns of enzyme distribution were observed in the leukaemic T-cells from these disorders. In T-PLL, reactivity for the four acid hydrolases was confined to single or a few large granules. Gall bodies were reactive for beta-glucuronidase, b-glucosaminidase and alpha-naphthylacetate esterase but apparently unreactive for acid phosphatase. In T-CLL, scattered small- to medium-size cytoplasmic granules and many parallel tubular arrays were strongly reactive for acid phosphatase, beta-glucuronidase and beta-glucosidase but showed no reactivity for alpha-naphthylacetate esterase. Intermediate features were observed in ATLL. The observed differences in enzyme reactivity reflect a different content of lysosomal granules in the various types of leukaemic T-cells. They also suggest that similar differences may be found in normal T-lymphocyte subsets.     

Localization of Acid Hydrolase Activity in Zea mays L. Root Tips

Naphthol AS-BI phosphatase, esterase, aryl sulphatase, glucuronidase,and ß-glycerophosphatase have been studied in frozensections of maize root tips. In general these enzymes showedhighest activities at the root surface and at particulate sitesin the cytoplasm although the indigogenic method for esteraseshowed no particulate activity and the naphthol AS-D acetatereaction gave no pronounced surface activity. With electronmicroscopy highest activity for ß-glycerophosphatasewas observed in the cell walls and associated with the vacuoles.The significance of these observations are discussed in relationto the function of surface hydrolytic activity and to the presenceof lysosome-like bodies in higher plant cells.     

Effects of Triton X-100 on acid phosphatases with different substrate specificities

Summary The effect of Triton X-100 on the activities of acid phosphatases from wheat germ, potato and human prostate was tested using -glycerophosphate, p-nitro-phenyl phosphate and naphthol AS BI phosphate as substrates. There was little effect on -glycerophosphatase activity at the concentrations of Triton X-100 tested. However at low concen trations of the detergent there was a stimulation of the activities of p-nitrophenyl phosphatase and naphthol phosphatase which were inhibited with the higher concentrations. Triton X-100 was found to enhance colour production between naphthol AS BI and fast red violet LB.Further evidence is presented confirming the presence of more than one acid phosphatase from each of the sources employed.     

Acid and neutral hydrolases in Trypanosoma cruzi. Characterization and assay.

Twelve acid hydrolases, 4 near-neutral hydrolases, and alkaline phosphatase were demonstrated in 0.34 M sucrose homogenates of Trypanosoma cruzi strain Y: p-nitrophenylphosphatase and alpha-naphthylphosphatase, with optimum pH at approximately 6.0; alpha=ga;actpsodase. beta=ga;actpsodase. beta=g;icpsodase, N-acetyl-beta-glucosaminidase, cathepsin A and peptidase I and III, with optimum pH between 5.0 and 6.0; and arylsulfatase, cathepsin D, alpha-arabinase and alpha-mannosidase with optimum pH at approximately 4.0. alpha-Glucosidase, glucose-6-phosphatase and peptidase II had optimum pH at approximately 7.0. beta-Glycerophosphatase had a broad pH-activity curve from 4,0 to 7.4, with maximum activity at pH 7.0. The main kinetic characteristics of these enzymes and their quantitative assay methods were studied. No activity was detected for alpha-fucosidase, beta-xylosidase, beta-glucuronidase, elaidate esterase, acid lipase, and alkaline phosphodiesterase.     

Electron cytochemical demonstration of four hydrolytic enzymes in the rat corpora lutea at second half of gestation

Summary Corpora lutea from rat ovaries at mid pregnancy were fixed by perfusion and studied by electron cytochemistry for localisation of four hydrolytic enzymes. Using the metal-salt methods for acid phosphatase and aryl sulphatases activity was localised in small and large lysosomes, multivesicular bodies, Golgi complex and within cisternae of endoplasmic reticulum. The azo-dye coupling method for Beta-glucuronidase was less satisfactory and gave positive results in lysosomes, lipids and in the globules within the mitochondrial matrix. The latter two localisation were probably associated with affinity of the naphthol AS-BI for lipid material. In addition to plasma membranes, the reaction product for alkaline phosphatase with the lead-salt method was seen in lysosomelike bodies, in smooth endoplasmic reticulum and in occasional Golgi elements of granulosa lutein and endothelial cells.Increased activity of lysosomal acid hydrolases occurs when regressive changes of lutein cells start at the end of gestation and this might probably reflect the initiation of lytic processes.     

Electron cytochemical demonstration of four hydrolytic enzymes in the rat corpora lutea at second half of gestation

Corpora lutea from rat ovaries at mid pregnancy were fixed by perfusion and studied by electron cytochemistry for localisation of four hydrolytic enzymes. Using the metal-salt methods for acid phosphatase and aryl sulphatases activity was localised in small and large lysosomes, multivesicular bodies, Golgi complex and within cisternae of endoplasmic reticulum. The azo-dye coupling method for Beta-glucuronidase was less satisfactory and gave positive results in lysosomes, lipids and in the globules within the mitochondrial matrix. The latter two localisation were probably associated with affinity of the naphthol AS-BI for lipid material. In addition to plasma membranes, the reaction product for alkaline phosphatase with the lead-salt method was seen in lysosomelike bodies, in smooth endoplasmic reticulum and in occasional Golgi elements of granulosa lutein and endothelial cells. Increased activity of lysosomal acid hydrolases occurs when regressive changes of lutein cells start at the end of gestation and this might probably reflect the initiation of lytic processes.     

Histochemical identification of osteoclasts. Review of current methods and reappraisal of a simple procedure for routine diagnosis on undecalcified human iliac bone biopsies

Osteoclasts are known to have a high acid phosphatase content. We have adapted the simple simultaneous mono-coupling azo-dye method of Grogg and Pearse to undecalcified bone sections. A cold embedding in a mixture of glycol and methyl methacrylate was shown to well preserve the enzyme activity. Sodium alpha-naphtyl phosphate (1 mg/ml) and fast violet B (2 mg/ml) are used in 0.1 M acetate buffer, pH 5.0. The addition of 1 mM L(+) sodium tartrate selectively inhibits the acid phosphoprotein phosphatase ("osteoblastic acid phosphatase") but not osteoclastic lysosomal acid phosphatase. Counterstaining with phosphomolybdic aniline blue WS leads to well contrasted sections, providing accurate measurements of osteoclast number.     

Histochemical studies on the adrenal glands of the marmosets (Callithrix jacchus and Callithrix penicillata).

The histrochemistry of the adrenal glands was studied in four adult male marmosets (two Callithrix jacchus and two Callithrix penicillata). It was impossible to demonstrate any reactivity to UDPG-GT, ADH, alanyl aminopeptidase, leucine aminopeptidase, xilitol (NAD-dependent) dehydrogenase, beta-glucuronidase and aryl-sulfatase in these glands. Total phosphorylase was found in scattered cells of the glomerulosa and adjacent outer fasciculata of one C. penicillata. The dehydrogenases (LDH, G-6-PDH,6-PGDH, NADPH2-TR,ICDH,SDH,NADH2-TR, alpha-GPDH, beta-OHBDH) as well as the hydrolases (except alkaline phosphatase, ATPase, and acetylcholinesterase) showed a stonger reactivity in the cortical part. Some hydrolases (naphthol acetate esterase, acid phosphatase) and cytochrome oxidase were less reactive in the zona glomerulosa, where the dehydrogenases were more abundant. The outer fasciculata and the reticularis also showed a strong dehydrogenase reactivity.     

Cytochemical localization of acid phosphatase and trimetaphosphatase activities in Kurloff cells

After stimulation of guinea pigs with estradiol to increase their Kurloff cell number, spleen imprints were prepared in order to detect non-specific acid phosphatase (AcPase) activity by light microscopic cytochemistry using naphthol AS-BI phosphate as substrate and pararosanilin or fast garnet GBC as coupler. For ultracytochemistry, Kurloff cells were prepared from spleens by filtration through a homogeneizer screen followed by repeated centrifugation. AcPase and trimetaphosphatase activities were tested using beta-glycerophosphate, cytidine-5'-monophosphate and inorganic trimetaphosphate as substrates. Significant enzymatic activities were demonstrated with all the substrates used in the cytoplasmic inclusion body of the Kurloff cells.     

A biochemical study of non-specific esterases from plant cells, employing the histochemical substrate, naphthol AS-D acetate

Synopsis The reagents routinely employed in the histochemical detection of nonspecific esterases, namely naphthol AS-D acetate and Fast Red Violet LB salt, have been used to study these enzymes biochemically with spectrophotometric procedures. A range of parameters that affect the coupling of the hydrolyzed substrate and diazonium salt were examined and their relevance to future histochemical procedures is noted.TheK _m of broad-bean root-tip esterases was estimated to be approximately 0.07 mM, but other kinetic data suggest that the true value is lower. From an analysis of the kinetics of the hydrolytic reaction it appears that they are of a mixed nature over the time course and substrate concentrations used.The effect of pH on root tip esterase activity has been examined and the recorded optimum of 5.5 is similar to that reported by other workers for plant cells. Non-enzymic hydrolysis of the substrate at alkaline pH levels prevented measurement of enzyme activity beyond pH 7.2.Magnesium ions at a final concentration of 5 mM are important in retaining esterases in their natured state during enzyme extraction, although the addition of increasing amounts of the ion to the assay system caused a corresponding increase in esterase inhibition.     

Nature of human spleen red pulp cells with special reference to sinus lining cells

Summary A histological and cytological as well as enzyme histo- and cytochemical analysis (alpha-naphthyl acetate esterases, naphthol AS acetate esterases, naphthol AS-D chloroacetate esterases, acid and alkaline phosphatases) of human spleen cells in sections and imprints was carried out with special reference to sinus lining cells. These cells show strong naphthol AS esterase activity and no or only little alpha-naphthyl acetate esterase and acid phosphatase activity. Thus they can be distinguished from reticular cells in pulp cords and from other macrophages in cords and sinuses. From the morphological and enzyme histochemical aspect it can be deduced that the sinus lining cells are a distinct cell type of the human spleen. The comparison of these enzyme cytochemical findings with the results of biochemical and electron microscopical investigations suggests that reticular cells of pulp cords and littoral cells of sinuses also have different functions: reticular cells seem to have a high phagocytotic activity while littoral cells seem to be only facultatively phagocytic.This work was supported by the Deutsche Forschungsgemeinschaft.     

Histochemical studies on the albino rat pancreas in different periods following vagotomy and simultaneous pyloromyotomy.

The effect of complete subphrenic vagotomy and simultaneous pyloromyotomy on the morphological state and the activities of some intracellular enzymes of the albino rat was studied histochemically. Within the first weeks after vagotomy, the pancreatic acini were found to diminish in size, and the beta-cells in the islets of Langerhans became oedematous. In the acini, the activities of succinate dehydrogenase, cytochrome oxidase, AS naphthol acetate esterase, and glucose-6-phosphatase were observed to decline, but the reactions for beta-glucuronidase and aryl sulphatase showed intensifications and polymorphic behaviour both in acinar and in islet cells. The latter also and particularly the beta-cells simultaneously revealed enhanced activities of succinate dehydrogenase, cytochrome oxidase, beta-glucuronidase, and aryl sulphatase, and an entire disappearance of the reaction for glucose-6-phosphatase. The alpha-cells increased their AS naphthol acetate esterase activity. After 5 weeks following vagotomy, morphological and enzymatic changes in the acini and islets were negligible, and after 5 and 9 months no differences were noted between the vagotomized rats and the control animals.     

The presence of a low molecular weight acid phosphatase in liver tissue that cannot be demonstrated with the histochemical substrate naphthol AS-BI phosphate

Summary Three distinct isoenzymes of acid phosphatase have been separated from extracts of liver tissue of rats by gel filtration. These isoenzymes have molecular weights of 180,000±35,000; 74,000±11,000 and 13,000±2,500. High molecular weight isoenzymes and a low molecular weight isoenzyme of acid phosphatase (molecular weight 13,000±2,100) were also present in extracts of normal human and mouse liver tissue, and of pathologically altered liver tissue of mice in which the activity of acid phosphatase was strongly increased as a result of intraperitoneal injections of dextran solutions. Activity of acid phosphatase was determined with three substrates. The isoenzymes showed different conversion rates for the three substrates. The high molecular weight isoenzymes split the substrates 4-methylumbelliferyl phosphate, p-nitrophenyl phosphate and naphthol AS-BI phosphate. The activity was sensitive to the inhibitors fluoride and L(+)tartrate. In the pathologically altered liver tissue, which had stored dextran, the activity of these isoenzymes was strongly increased. The low molecular weight isoenzyme split 4-methylumbelliferyl phosphate and p-nitrophenyl phosphate but not naphthol AS-BI phosphate. Therefore this isoenzyme cannot be demonstrated with histochemical techniques using the substrate naphthol AS-BI phosphate. In contrast to the activity of the high molecular isoenzymes the activity of the low molecular isoenzyme was not changed in the pathologically altered liver tissue of mice and was not sensitive to the inhibitors fluoride and L(+)tartrate.This study was supported by a grant from the Prinses Beatrix Fonds, s'Gravenhage     

Dynamic assay of enzyme activities in single cells by flow cytometry.

Three enzymes in single cells were assayed dynamically by flow cytometry using four fluorogenic substrates. Acid phosphatase was determined with 7-bromo-3-hydroxy-2-naphtho-o-anisidine (naphthol AS-BI) phosphate and 4-methylumbelliferone (MU) phosphate, neutral esterase with fluorescein diacetate, and lactic dehydrogenase with NAD-sodium lactate. Fluorescence measurements obtained with the flow cytometer were converted into relative specific enzyme activities for single cells with molar fluorescence coefficients determined with a spectrofluorometer. Specific activities obtained from spectrofluorometric data were compared with activities calculated from flow cytometeric data. Flow cytometric assays gave lower specific single cell activities for 4-methylumbelliferone phosphate hydrolysis and for lactic dehydrogenase than did similar assays by standard spectrofluorometry. Product diffusion may be the greatest cause for this discrepancy.     

The enzyme cytochemistry and composition of elasmobranch granulocytes

Peroxidase, alkaline phosphatase, acid phosphatase, β-glucuronidase, α-naphthyl acetate esterase (ANAE), α-naphthyl butyrate esterase, naphthol AS-D chloroacetate esterase, acetyl-L-tyrosine-α-naphthyl esterase (ATNE), tosyl-L-lysine-α-naphthyl esterase (TLNE) and periodic acid-Schiff (PAS) were studied in 17 species of elasmobranchs in which granulocytes had previously been identified at the ultrastructural level.
Eosinophils, eosinophilic and neutrophilic granulocytes contained variable acid phosphatase, esterases and PAS, but they were strongest in neutrophilic granulocytes; particularly ANAE. Esterases were released into surrounding plasma and therefore probably function as ectoenzymes. In eosinophils and some neutrophilic granulocytes there were indications of weak peroxidase, but this could not be conclusively demonstrated. Alkaline phosphatase was diffuse between granules in some eosinophils of Pavoraja , and (β-glucuronidase was diffuse in neutrophilic granulocytes of Etmopterus baxteri , otherwise granulocytes lacked these enzymes. Neutrophilic granulocytes stained moderately to strongly for ATNE and weakly and inconsistently for TLNE in Squalus acanthias and Dalatias licha . with a similar reaction in granular lymphocytoid and thrombocytoid cells of Galeorhinus ausiralis and Raja nasuta . The enzyme composition of these granulocytes is discussed.     

Enzyme Histochemistry on Paraffin Embedded Tissue Sections

To obtain diagnostic enzyme reactions in paraffin embedded tissue sections, we compared four fixatives (buffered formol sucrose, Baker's formol calcium, periodate lysin paraformaldehyde, and buffered formalin acetone) and subsequent acetone dehydration with or without graded concentrations of Triton X-100. Four spleens and 14 lymph nodes were tested for peroxidase, naphthol ASD chloroacetate esterase, acid phosphatase, alpha naphthyl acetate esterase, and alpha naphthyl butyrate. Best results were obtained by a processing method using buffered formalin acetone, Holt's gum sucrose, dehydration in acetone with 0.03% Triton X-100, and paraffin for embedding.     

Combined immunohistochemical staining for surface IgD and T-lymphocyte subsets with monoclonal antibodies in human tonsils

The aim of the present paper is to detect two different antigens simultaneously in a single slide. In cryostat sections of human tonsils, B-lymphocytes of follicle mantle-bearing surface IgD were immunostained with the alkaline phosphatase method using monoclonal anti IgD. The subsequent staining for T-lymphocyte subsets (T-helper and T-suppressor lymphocytes) was performed again with the alkaline phosphatase method using one of the monoclonal antibodies OKT 4, OKT 8, Leu 3a, Leu 2a. The best results with the alkaline phosphatase method were achieved using naphthol AS phosphate and Fast Blue BB for the revelation of the first antigen and naphthol AS-BI phosphate and diazotized New Fuchsin for the second.