Acid hydrolases in HeLa cells: comparison of methods for light microscopy

To distinguish lysosome populations of HeLa cells, acid phosphatase, beta-glucuronidase, 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 beta-glucuronidase, naphthol AS-BI beta-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 beta-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 beta-glucuronidase, esterase or arylsulfatase, but rarely acid phosphatase.     

QUANTITATIVE ESTIMATION OF LYO- AND DESMOENZYMES IN TISSUE SECTIONS WITH AND WITHOUT FIXATION

1. Tissue sections eight microns thick were exposed to various experimental conditions used in histochemistry, and the effect upon the activities of esterase, the phosphatases, leucine aminopeptidase, β-glucuronidase, and arylsulfatase was determined colorimetrically. 2. Significant differences were found in the amounts of the lyo and desmo fractions of these enzymes. The desmo components were found to be for esterase, alkaline phosphatase, leucine aminopeptidase, acid phosphatase, β-glucuronidase, and arylsulfatase, ⅓, 2/3, 2/3, ½, ⅛, and ⅛ of the total enzymatic activity respectively. 3. Variations in the time and in the temperature at which diffusion was studied and of the pH and salt concentration of the solution into which the sections were placed, resulted in differences in the amount of enzymatic activity which remained in the tissue section. Some enzyme loss by diffusion was noted even after fixation of the tissue section. 4. The significance of the findings with respect to some of the concepts of localization of enzymes in tissue sections was discussed.     

Effect of Formalin Fixation on the Activity of Five Enzymes of Rat Liver

The effect of formalin fixation on the enzymatic activity of five enzymes in rat liver was studied as a function of time and temperature, β-glucuronidase was found to be most resistant to the inactivating effects of formalin. At a time when fixation was satisfactory for preparing frozen sections (24 hours), considerable enzymatic activity was present in decreasing order; β-glucuronidase, sulfatase, acid phosphatase, esterase, and alkaline phosphatase.     

A new method for the preparation of rat liver lysosomes. Separation of cell organelles of rat liver by carrier-free continuous electrophoresis

A combination of differential centrifugation and carrier-free continuous electrophoresis is introduced as a new method for the isolation of animal cell organelles. Various buffers were systematically checked in order to find the system which preserves the organelles and gives as well a good separation in the free-flow electrophoresis apparatus. Triethanolamine-acetate buffer (10 mM), pH 7.4 was used. The isolated lysosomes were pure according to marker enzymes and electron micrographs. A heterogeneity of the lysosomes in electrophoretic mobility was demonstrated with respect to the marker enzymes arylsulfatase and β-glucuronidase. The lysosomes with higher mobility showed a maximum enrichment of 240-fold with respect to arylsulfatase. The lysosomes with lower electrophoretic mobility showed a 65-fold enrichment with respect to β-glucuronidase. The ratio of β-glucuronidase to arylsulfatase varied from 2:1 to 1:2 in lysosomes of different mobility. The yield amounted to approximately 1 mg of lysosomal protein per gram of liver protein. 5–8 mg of lysosomes can be obtained in one experiment. The electrophoretic separation proves to be an effective tool in obtaining pure and well preserved lysosomes.     

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.     

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.     

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.     

The increase in activity of acid hydrolases in muscles of rats after subcutaneous administration of dimethyl-para-phenylene diamine. A combined histochemical and biochemical investigation

Summary The increase in activity of acid hydrolases in skeletal muscles of rats after subcutaneous administration of dimethyl-para-phenylene diamine (DPPD) was studied with a combined histochemical and biochemical investigation. In part I the histochemical findings were presented. In this communication the biochemical findings are reported and compared with the histochemical findings.In homogenates of m. biceps femoris, m. gastrocnemius and m. rectus femoris of DPPD-treated rats, the activity of the lysosomal acid hydrolases, cathepsin D, acid maltase, acid phosphatase, and -glucuronidase was increased. This increase in activity was maximal after 7 to 9 days of DPPD treatment and ran parallel to the severity of the pathological changes. Statistical calculations clearly reveal that the increased activity of one acid hydrolase was significantly paralleled by an increased activity of a second acid hydrolase. Moreover these calculations reveal that the biochemical activity findings correlated with the histochemical activity findings. However it was remarkable that in the histochemical study, the estimated increase in acid phosphatase activity was much more than the increase in acid phosphatase activity found biochemically, whilst on the other hand the histochemically estimated increase in -glucuronidase activity corresponded with the biochemical observations. The results of gel filtration techniques have shown that this discrepancy of acid phosphatase activity was caused by different substrate specificity of the different isoenzymes of acid phosphatase and that as a result of the DPPD treatment the isoenzyme pattern had been altered. The elution patterns showed three distinct isoenzymes of acid phosphatase of normal and of DPPD treated rats. These isoenzymes, termed I, II and III, have molecular weights of: 200,000 or more, 83,500–104,500 and 14,500–18,100. Isoenzymes I and II split the substrates 4-methylumbelliferyl phosphate and naphthol AS-BI phosphate and the activity is strongly increased in the muscles of the DPPD treated rats. Isoenzyme III does not split naphthol AS-BI phosphate and the activity is not increased in the muscles of the DPPD treated rats. Considering the fact that it has been shown that the activity of isoenzyme III is high compared with that of the isoenzymes I and II, it is important to realise that by using naphthol AS-BI phosphate not all acid phosphatase can be demonstrated in sections of skeletal muscle.This study was partly supported by a grant from the Prinses Beatrix Fonds, 's Gravenhage, The Netherlands, and was mainly extracted from the Ph. D. thesis of D.E. Israël (1977).     

Immunochemical quantitation of enzymes in human diploid cell line WI-38

A quantitative immunochemical study was carried out of four enzymes, cathodal esterase, acid phosphatase, glucosaminidase and β-glucuronidase. In homogenates of the human diploid cell line WI-38, the relative amounts of the enzymes increased with the passage number of the culture, although great variation was found in later passages just before death of the culture.     

Lysosomes in skeletal muscle tissue. Zonal centrifugation evidence for multiple cellular sources

Postnuclear supernates from homogenates of skeletal muscle from rats subjected to starvation, injections of Triton WR-1339, dextran-500, and dextran + corticosterone were fractionated by means of rate and isopycnic zonal centrifugation in sucrose—0.02 M KCl gradients. Zonal fractions were analyzed for protein, RNA, cytochrome oxidase, and up to six acid hydrolases. The results indicate the presence of two groups of lysosome-like particles. One group contributes approximately 95% of the cathepsin D and acid phosphatase activity and 75% of the acid ribonuclease, β-glucuronidase, and arylsulfatase activity in muscle. It is characterized by a modal equilibrium density of 1.18 that is decreased by starvation, but is not shifted by dextran-500 or Triton WR-1339. The second group has a higher proportion of acid ribonuclease, β-glucuronidase, and arylsulftase; the equilibrium density can be shifted by dextran-500 and Triton WR-1339. It is suggested that this group of lysosomes is derived from macrophages and other connective tissue cells, whereas the former group represents lysosome-like particles from muscle cells.     

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 α-naphthylphosphatase, with optimum pH at ? 6.0; α-galactosidase, β-galactosidase, β-glucosidase, N-acetyl-β-glucosaminidase, cathepsin A and peptidase I and III, with optimum pH between 5.0 and 6.0: and arylsulfatase cathepsin D, α-arabinase and α-mannosidase with optimum pH at ? 4.0 α-Glucosidase, gluccse-6-phosphatase and peptidase II had optimum pH at ? 7.0. β-Glycerophcsphatase 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 α-fucosidase, β-xylosidase, β-glucuronidase, elaidate esterase. acid lipase, and alkaline phospho-diesterase.     

DIFFERENCES IN ENZYME CONTENT OF AZUROPHIL AND SPECIFIC GRANULES OF POLYMORPHONUCLEAR LEUKOCYTES : I. Histochemical Staining of Bone Marrow Smears

Histochemical procedures for PMN granule enzymes were carried out on smears prepared from normal rabbit bone marrow, and the smears were examined by light microscopy. For each of the enzymes tested, azo dye and heavy metal techniques were utilized when possible. The distribution and intensity of each reaction were compared to the distribution of azurophil and specific granules in developing PMN. The distribution of peroxidase and six lysosomal enzymes (acid phosphatase, arylsulfatase, β-galactosidase, β-glucuronidase, esterase, and 5'-nucleotidase) corresponded to that of azurophil granules. Progranulocytes contained numerous reactive granules, and later stages contained only a few. The distribution of one enzyme, alkaline phosphatase, corresponded to that of specific granules. Reaction product first appeared in myelocytes, and later stages contained numerous reactive granules. The results of tests for lipase and thiolacetic acid esterase were negative at all developmental stages. Both types of granules stained for basic protein and arginine. It is concluded that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appears to be restricted to one of the granules. The findings further indicate that azurophil granules are primary lysosomes, since they contain numerous lysosomal, hydrolytic enzymes, but the nature of specific granules is uncertain since, except for alkaline phosphatase, their contents remain unknown.     

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     

Granulation staining and cytochemistry of peripheral blood leucocytes in healthy carp (Cyprinus carpio L.) I. Granulocytes

The cytochemistry and staining of granula in peripheral blood granulocytes in healthy carp ( Cyprinus carpio L.) are described. Blood smears were stained for periodic acid-Schiff (PAS), peroxidase, oxidase, alkaline and acid phosphatase, α-naphthyl-acetate esterase, α-naphthyl-butyrate esterase, naphthol-AS-chloroacetate esterase (AS-D), naphthol-AS-acetate esterase and β-glucuronidase. Different granula types were shown by triazid-staining (eosinophil and neutrophil granula) and methylenblue-staining for basophil granulation. Toluidinblue-staining was used for basophil granulocytes. Lipids were shown by the Sudan-black-reaction. Four granulocyte subpopulations are described: neutrophil, heterophil, basophil and eosinophil granulocytes. Neutrophils possess all tested granula types, whereas heterophil and basophil granulocytes show only basophil granula. Neutrophils and heterophils show no activity of the tested esterases with the exception of AS-D. Only neutrophils were peroxidase-positive. Alkaline phosphatase and β-glucuronidase were not detected in granulocytes. Basophils and especially eosinophils were rarely found in peripheral blood.     

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.     

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.     

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.     

Ultrastructural localisation of acid phosphatase,non-specific esterase and β-glucuronidase in the midgut epithelium of Tenebrio molitor,Schistocerca gregaria and Carausius morosus

Summary Acid phosphatase, non-specific esterase and -glucuronidase have been localised in the midgut epithelium of three species of insect using naphthol esters as substrates and triphenyl-p-amino-phenethyl lead as coupling salt. In all three species acid phosphatase and -glucuronidase appear to be confined to primary and secondary lysosomes. Non-specific esterase activity was demonstrated within membrane-enclosing bodies in all three species, associated with lipid droplets in T. molitor and C. morosus and with an unidentified intranuclear structure in C. morosus.     

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.     

CHARACTERIZATION AND LOCALIZATION OF ACID HYDROLASE ACTIVITY IN THE SYNAPTOSOMAL FRACTION FROM RAT CEREBRAL CORTEX

Synaptosomes were prepared from the cerebral cortex of adult rats by a rapid technique of centrifugation in a Ficoll-sucrose discontinuous gradient. The synaptosomal fraction contained 40 per cent of the total gradient activity of acid α-naphthyl phosphatase (EC 3.1.3.2). Quantitative electron microscopy of this fraction revealed rare, typical, extrasynaptosomal dense body lysosomes. pH-activity profiles of free and Triton X-100 (total) activities were prepared for α-naphthyl phosphatase, β-glucuronidase (EC 3.2.1.31), β-galactosidase (EC 3.2.1.23), arylsulfatase (EC 3.1.6.1) and N-acetylglucosaminidase (EC 3.2.1.30). The ratios of total to free activity varied in the order: arylsulfatase > β-galactosidase > β-glucuronidase > N-acetylglucosaminidase > acid phosphohydrolase. Incubation of synaptosomal fractions at pH 5 and 37°C produced significant activation of β-galactosidase and N-acetylglucosaminidase but no activation of cryptic lactate dehydrogenase (EC 1.1.1.27). Hyposmotic suspension and subfractionation of the synaptosomal fraction produced considerable solubilization of lactate dehydrogenase, arylsulfatase and β-galactosidase but only partial liberation of α-naphthyl phosphatase, the remainder being associated with synaptosomal membrane fragments. Incomplete equilibrium sedimentation of synaptosomes in a continuous sucrose gradient (0·55-1·5 M) provided a broad lactate dehydrogenase and Na + K ATPase (EC 3.6.1.4) peak (peak I) at low sucrose densities. β-Glucuronidase, β-glucosidase and α-naphthyl phosphatase were significantly present in peak I. Conversely, N-acetylglucosaminidase, arylsulphatase and β-galactosidase were predominantly located in denser particles sedimenting through 1·2 M sucrose (peak II). Electron microscopy confirmed the heterogeneity of this second peak and the presence of numerous extrasynapto-somal dense body lysosomes.