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A simple and convenient colorimetric assay for the quantification of non-specific esterase is described. The assay takes advantage of the microtitration plate format, and the high capacity of repetitive dispensers and the microplate reader for this format. The assay is well suited for the quantification changes in the esterase activity in homogeneous cell populations like monocytes maturing into macrophages.

The histochemical distributions of 14 enzymes in the human amnion at term are described. Adenosine triphosphatase, 5′-nucleotidase, acid phosphatase and nonspecific esterase show continuous gradations of enzyme activity. Cells with intense activity are prominent in acid phosphatase and nonspecific esterase preparations. A distinct subpopulation of amniotic cells can be defined by their alkaline phosphatase activity. The possible correlations with morphological studies are discussed.

A Wang model 2200 computer has been interfaced with the Bio/Physics Systems, Inc. model 6300 Cytograf and model 2100 Distribution Analyzer. Using a custom designed software program, in conjunction with an azo-dye technic for staining monocytes for nonspecific esterase activity, it has been possible to obtain rapid and reliable data concerning relative values for intracellular monocyte esterase activity. The method is based on measuring the axial light-loss voltage signal for each of one thousand stained monocytes. Individual stained monocytes were assigned to one of four groups (A, B, C, D), dependent upon the magnitude of the signal and were given different rating values (1, 2, 3, 4) according to their group designation. A "score" was derived for each blood sample by multiplying the percentage of cells (monocytes) in each group category by the appropriate factor and summing these values. The technic permits rapid objective assessment of intracellular nonspecific esterase activity in monocytes suspended in a mixed cell population. Both Gaussian and bi-modal patterns for monocyte esterase were observed. The latter suggests a dual monocyte population.

Nonspecific esterase activity using alpha-naphthyl butyrate as substrate was found to be present in hairy cells from patients with hairy cell leukemia. Activity of this enzyme was markedly diminished and in some instances obliterated by sodium fluoride. Since alpha-naphthyl butyrate is believed to be a more specific substrate for monocytic-type nonspecific esterase than alpha-naphthyl acetate, its presence in hairy cells combined with fluoride inhibition which is also characteristic of monocytic nonspecific esterase provides additional evidence of monocytic properties of hairy cells.

The results of a histochemical study of changes in the activity of nonspecific esterases and α-glycerophosphate dehydrogenase (α-GPD) and the fat content in the regenerating liver of domestic chickens are given. Accumulation of fat in the hepatocytes in the early states of regeneration (1st–5th days of the experiment) is due partly to a decrease in nonspecific esterase activity. Maximal steatosis in the liver is also accompanied by an increase in α-GPD activity, evidence of intensification of glycolysis and of primary synthesis of triacylglycerides — the reserve lipids of the liver — in the period of regeneration.

Enzyme histochemistry has revealed that encapsulation reactions surrounding larvae of the nematode Angiostrongylus cantonensis 4 wk after infection of the gastropod Biomphalaria glabrata are the sites of highly localized acid phosphatase and nonspecific esterase activities. Lesser amounts of alkaline phosphatase and β-glucuronidase activities also occur within such capsules, but aminopeptidase activity cannot be demonstrated. In addition, it has been ascertained that acid phosphatase activity gradually increases as the encapsulation reaction progresses during the first to the fourth week postinfection.

Using the substrate 2-naphthylthiol acetate (NTA), we developed a reproducible method of demonstrating a non-specific esterase while retaining nuclear and cytoplasmic details at the ultrastructural level. The NTA esterase had a distribution and pattern of staining similar to those of esterases demonstrable at the light microscopic level by the alpha-naphthyl acetate or naphthol AS-D acetate esterase reaction. The NTA esterase appeared as intensely electron-dense granules of varying size and shape in the cytoplasm. The granules were most abundant in the cells of the histiomonocytic series. The large number of diffusely scattered granules in the cytoplasm of the histiocytes and monocytes made it possible to separate these cells from other haematopoietic elements. There was usually no direct relationship between the NTA esterase positivity and the amount or the location of lysosomes or mitochondria, although in some histiocytes the granules appeared to be associated with lysosomes. The NTA esterase-positive granules were usually more numerous than lysosomes and were located outside the lysosomal granules. Some of the lymphocytes outside the germinal centres and most of the lymphocytes in the blood showed a punctate positivity in the form of 1--4 electron-dense dots. Plasma cells were usually negative but, in rare cases, contained an occasional single dot-like reaction product similar to that in some of the lymphocytes. Granulocytes were always negative. The method described in this paper can be used effectively for identification and study of human haematopoietic cells lines at the ultrastructural level.

Localization and activity of five hydrolases (alkaline phosphatase, adenosine triphosphatase, acid phosphatase, nonspecific esterase and leucylamino-peptidase) were evaluated histochemically in the epididymides of mature dogs. In the ductuli efferentes, cilia and apical parts of the epithelial cells displayed high activity of alkaline phosphatase and adenosine triphosphatase. Strong activity of acid phosphatase, nonspecific esterase and leucylaminopeptidase was present in the basal and supranuclear zones of the epithelium of the ductuli efferentes. Stereocilia of all three segments of the ductus epididymidis showed a high activity of alkaline phosphatase. Positive adenosine triphosphatase reaction was confined to the stereocilia of the initial segment. A complex pattern of acid phosphatase activity was observed in the middle segment. The subdivision of the middle segment in four subsegments was therefore suggested. In the epithelium of the initial segment only a few nonspecific esterase-positive cells were seen. The infranuclear and basal areas of the epithelium in the middle segment and the supranuclear zone of the terminal segment displayed distinct nonspecific esterase activity. The possible contribution of the hydrolases to the function of the epididymis is discussed.

The liver and spleen of gnotobiotic Wistar rats were studied by histochemical methods and the liver electron-microscopically. Under germfree conditions of existence of the animal the succinate dehydrogenase and nonspecific esterase activity in the liver decreased, fatty infiltration of the cytoplasm of the hepatocytes and Kupffer cells increased, and some of the cells developed fatty degeneration. Meanwhile acid phosphatase activity and the number of lysosomes increased in the biliary poles of the hepatocytes, whereas in the spleen destruction of erythrocytes and the liberation of free iron and pigments, which stimulate the excretion of bile in germfree animals, were increased.

Polyacrylamide gel electrophoresis was used to separate Sancassania berlesei (Michael) and Tetranychus urticae (Koch) extracts into 10 and 7 non-specific esterases respectively. These non-specific esterases could not be classified according to common esterase classification schemes from their substrate and inhibition characteristics. Hence a specific staining technique was used to identify the acetylcholinesterase isozymes; 3 were separated from S. berlesei and 2 from T. urticae extracts. Major differences in the structure and membrane binding properties of the acetylcholinesterase isozymes from these two mites were indicated by their different abilities to penetrate into gels, and their different electrophoretic mobilities and solubilisation characteristics. S. berlesei non-specific esterases and acetylcholinesterase isozymes were much less sensitive to organophosphorus inhibitors than T. urticae enzymes, a finding which implicates strongly these enzymes as being responsible, in part, for the or...

Summary The human promyelocytic leukemia cell line HL-60 can be differentiated to cells resembling either neutrophils or mononuclear phagocytes by a diverse group of stimuli. However, the underlying mechanisms remain unknown. We report that 1- O -hexadecyl-2-acetyl- sn -glycerol inhibits the growth of HL-60 cells and induces differentiation to cells resembling mononuclear phagocytes. HL-60 cultures incubated for 6 days with 1- O -hexadecyl-2-acetyl- sn -glycerol (5 μg/ml) demonstrated a ten-fold increase in nonspecific esterase activity, and produced cells with morphological features similar to those of monocytes and macrophages. Higher concentrations of 1- O -hexadecyl-2-acetyl- sn -glycerol significantly inhibited the growth of HL-60 cells and resulted in the virtual absence of cells resembling the original HL-60 line. 1- O -Oleoyl-2-acetyl- rac -glycerol added under the same conditions did not induce cell differentiation or inhibit cell growth.

The effects of various parameters on the nonspecific esterase electropherograms of the Knights Landing strain of the mosquito Culex tarsalis were determined by isolelectric focusing electrophoresis in acrylamide gels. The parameters examined were substrate preference, isolation/incubation media composition, tissue distribution, aging, deoxycholate stimulation and acetylthiocholine hydrolysis. The results corroborate the identity of two major acetylcholinesterases and document a single lipase in this mosquito. The nonspecific esterases of C. tarsalis can now be classified as: one acetylesterase, one arylesterase, one lipase, two acetylcholinesterases and thirteen carboxylesterases.

A microtiter plate spectrophotometric system has been used together with the Bradford, Ellman, and van Asperen assays to measure protein concentration (to 0.5 microgram) and the activities of acetylcholinesterase (to 10(-3) units) and carboxylesterase (to 30 micrograms beta-naphthol reaction product) in small samples such as high-performance liquid chromatographic eluate fractions. For 100-microliter samples, at least 300 Ellman acetylcholinesterase or Bradford protein assays can be conducted and read in less than 30 min, and a like number of van Asperen nonspecific esterase assays (including 1-h incubation) run in less than 90 min. The eluate from a single 20-min high-performance liquid chromatographic run of a biological sample can be collected as up to 300 fractions directly into microtiter plate wells, and the three assays run on all fractions in less than 90 min.

Examination of seed samples of Gossypium thurberi by means of acrylamide gel electrophoresis was undertaken to detect the presence of molecular polymorphism in nonspecific esterases. Although little morphological variation is observed within and between populations of this species, six different non-specific seed esterase zymograms were demonstrated suggesting that high molecular polymorphism is present. Studies using urea indicated that the specific isozyme patterns did not result from secondary alterations in structures of polypeptide species during extraction and therefore are not in vitro artifacts.

Electrophoreograms of protein extracts from leaves of the variety Lord Lambourne, detached at the flowering period toward the end of April, differed quantitatively from electrophoreograms of protein extracts from leaves detached from shoots at the end of May and June. Electrophoreograms of protein extracts of leaves from juvenile seedlings, detached at the end of May and June, did not differ from electrophoreograms of protein extracts of leaves from shoots of the variety Lord Lambourne detached at the same period. Electrophoretic pattern of protein fractions and the fractions of non-specific esterase from leaves differed from the corresponding electrophoretic pattern of flowers quantitatively and in some areas even qualitatively; the pattern of acid phosphatase differed quantitatively. The results obtained are discussed in relation to organogenesis.

Flat be(l gradient acrylamide gel electrophoresis at constant pulsed power was used to resolve muiltiple forms of nonspecific esterases amid acetylchsolinesterases of ejaculated bull spermatozoa cytoplasmic droplets, seminal plasma and isolated spermatozoan heads, midpieces arid tail fragments. Several substrates and inhibitors were ussed to (histiniguish the esterases. Eleven distinct zones of spermatozoan non specific esterase activity not fousnd in droplets or seminal plasma were detected with a-niaphithiyl acetate as sumbstrate. All 11 zones were present in sperm heads arid tails. A single nonspecific esterase was demonstrated inn sperm heads, midpieces and tails arid in seminal plasmna with a-naphthyl hutyrate or a-naphthyl caproate. Two oilier seminal plasma nonspecific esterases were detected: one with a-naphthyl acetate arid one with a-naphthyl butyrate or a-naphthyl caproate. Three seminal plasma, two cytoplasmic droplet and one spermatozoan acetylchiolirsesterases were demonstrated with acetyithiocholine. The electrophoretic stusdies, together with previous cytochemical studies, suggested thiat certain bull seminal plasma nonspecific esterases are adsorbed by spermatozoa.

Zymogramic analysis of esterase in the mouse, rat and guinea-pig salivary glands was undertaken and demonstrated species and organ specificities of esterase with electrophoretic method. Salivary glands esterase was classified into A, B and C types based on the electrophoretic mobility. Mouse submandibular gland had the most complicated pattern, while guinea-pig showed the simpliest patterns which was devoid of B type of esterase. Rat salivary glands exhibited rather regular patterns. Similar zymogram patterns were obtained with many kinds of ester compounds, that is simple and substituted naphthol esters and indoxyl derivatives. The tests of inhibition and activation for esterase activity was obtained. Histochemical properties applied to inhibitor test in the esterase zymogram patterns showed no marked differences between ducts and acini.

Azo-coupling methods were used for demonstrating non-specific esterase in the wheat root in all parts of a transverse section, usually with the exception of the woody parts of the vascular bundle. The central cylinder gave a more intense reaction than the primary cortex and the rhizoderm. The reaction was not inhibited by dodecyl sulphate. A weakening of the reaction intensity was observed after application of AgNO3. The Tween method did not yield reliable results.

1. 1. Three lysosomal enzymes: Acid phosphatase, nonspecific esterase, and E-600 resistant esterase, have been investigated histochemically in the human endometrium through the menstrual cycle. 2. 2. Cyclic variations of these enzymes were observed in the glandular epithelium, endometrial stroma, and macrophages, in relation to the various stages of the menstrual cycle. 3. 3. The relationships of these enzymes to the lysosomes and their regulation by ovarian sex steroid hormones is briefly discussed. 4. 4. The nonspecific esterase activity of the endometrial epithelium appears to be due the action of a lysosomal cathepsin or cathepsins. 5. 5. A more active role than hitherto believed is postulated for the endometrial macrophages, especially in relation to spermatocyte survival in the uterus.

The activity and the isozyme pattern of nonspecific esterase in HeLa cells (variant 77) were found to depend on the type and concentration of serum present in the culture medium.

The localization of lipids and the activities of nonspecific esterase, aryl sulfatase and acid phosphatase were studied in different stages of spermatogenesis in rats. In addition, the distribution of acid phosphatase activity was demonstrated electron histochemically. The spermatogenetic cycle was divided into two phases—corresponding to the first and the last four stages of Roosen-Runge-Giesel (RG) classification. Spermatids in the first phase contained abundant endoplasmic reticulum with rosette formation and well developed Golgi apparatus with numerous vesicles. They displayed high activity of hydrolytic enzymes but contained no appreciable amount of lipids. The Sertoli cells contained large lipid granules but showed minimal enzyme activity. During the second phase reduction of the cytoplasm of spermatids with fragmentation of the endoplasmic reticulum and Golgi lamellae, accumulation of lipids, aggregation of ribonucleo-protein particles, formation of residual bodies and marked decrease of enzyme activity were seen. The Sertoli cells contained large mitochondria, well developed endoplasmic reticulum and numerous dense bodies and revealed high activities of hydrolytic enzymes and rapid depletion of lipids. These ultrastructural and histochemical findings suggested an interaction between the Sertoli cells and the developing spermatids which probably contributed to the regulation of spermatogenesis.

1. 1. Isoelectric focusing on polyacrylamide gels was used to detect malate dehydrogenase and non-specific esterase isoenzymes in diploid honey bee ( Apis mellifera L.) eggs of several known ages. 2. 2. It was determined that the number of MDH and EST isoenzymes increased as development proceeded with an apparent increase in the quantity of some of the isoenzymes.

A method for the demonstration of nonspecific esterase activity in plastic-embedded tissues using Meldola Blue is described. Although Meldola Blue does not function as an electron carrier in this method, it may account for the short incubation time and excellent localization of the reaction product. Use of Meldola Blue is an advancement in the demonstration of enzyme activity in plastic sections.

Although larval walleyes (Stizostedion vitreum) readily ingest formulated feeds, survival is low. The present study was conducted to determine if the gut of larval walleyes has digestive capability. Preliminary data indicate that the gut exhibits considerable enzyme activity for four intracellular enzymes—acid phosphatase, alkaline phosphatase, nonspecific esterase, and aminopeptidase—that catalyze the breakdown of a wide variety of organic substances and are capable of digesting many breakdown products of proteins, fats, and carbohydrates. If the digestive system of walleyes becomes functional before the yolk sac is absorbed, as seems likely from the data, problems encountered in feeding formulated feeds to larvae probably do not result from lack of digestive capability.

Rabbit antiserums were prepared against three submicrosomal fractions from liver of normal or phenobarbital-treated rats. The two membrane fractions originating from the rough- and smooth-surfaced endoplasmic reticulum were characterized by the same soluble antigens, with the exception of a highly basic coinponent present only in extracts of rough membranes. The third fraction, whose subcellular origin is unknown, was different. It contained at least two typical marker antigens not present in the other fractions. Of eight tissue antigens common for the endoplasmic reticulum, five displayed nonspecific esterase activity. Some of these esterases were also found in other organs, but none was seen in rat serum. Phenobarbital treatment of the rats led to a rise in activity and characteristic changes in the esterase patterns of all these submicrosomal liver fractions.

Sebaceous gland non-specific esterase activity was suppressed following application to mouse skin of 2 electrophilic alpha beta-unsaturated compounds, o-chlorobenzylidenemalononitrile and beta-nitrostyrene, but not by three others.

SUMMARY The localization of the enzymatic activity of non-specific esterase and endogenous peroxidase of the Langerhans’ cells in murine epidermal sheets separated with EDTA was observed by means of light and electron microscopy. The Langerhans’ cells exhibited non-specific esterase activity in the outer portion of the plasma membrane and endogenous peroxidase activity in the nuclear envelope and the endoplasmic reticulum.

Separation of blood serum prealbumin from ten strains of inbred mice was accomplished using acrylamide electrophoresis. Nine of these strains demonstrated the same five prealbumin bands; however, the C57BL/6JWg strain showed a sixth band. The use of appropriate crosses of C57BL/6JWg and DBA/2fWg showed this unique band to be the product of a single autosomal dominant gene. We have named the gene for this prealbumin band Pre-1 and have shown a map distance between Pre-1 and b of 2.25 cM. Only one of the five prealbumins present in all strains of mice tested showed nonspecific esterase activity.

A new cytochemical method is described for the simultaneous demonstration of nonspecific esterase in monocytes and chloracetate esterase in granulocytes. The procedure uses both alpha-naphthyl butyrate and naphthol AS-D chloroacetate as substrates and hexazotized pararosaniline as the coupler. The enzyme reaction products are highly chromogenic and their localization is precise. This method is potentially useful for the accurate diagnosis of the acute monocytic leukemias. Its advantages and limitations are also discussed.

Using high-resolution technics of isoelectric focusing in polyacrylamide gel, isoenzymatic components of nonspecific esterases extracted from various cytologic types of leukemic blasts were visualized using alpha-naphthyl acetate and alpha-naphthyl butyrate as substrates. For each cytologic type of leukemic blast, consistent and distinctive patterns of nonspecific esterase activity were visualized. These studies provide further evidence for heterogeneity of nonspecific esterases, and for elaboration of isoenzymatic components as a reflection of cellular differentiation.

A simple, cold-embedding method (on cracked ice at 2 C) is presented for the demonstration of acid phosphatase, beta-galactosidase and nonspecific esterase in glycol methacrylate tissue sections.

Publisher Summary Esterase activity has, over the years, been used in conjunction with activities such as adherence and phagocytic tests to identify and quantitate mononuclear phagocytes. Esterase activity is generally demonstrated by using α-naphthyl acetate or butyrate as substrates. The naphthol liberated by the enzyme is coupled with a diazonium salt to give an insoluble, brightly colored, azo dye. Most esterase-staining procedures have employed cells fixed as a smear on glass slides or as cytocentrifuged preparations. Nonspecific esterase-staining mononuclear phagocytes are easily distinguished because of the red or reddish brown staining pattern produced in esterase-positive cells. The cells that do not have nonspecific esterase activity appear unstained. When investigators from Technicon Corporation tested peripheral blood of various species for esterase activity according to the method used for human blood, they concluded that only human and rabbit monocytes stained well, while staining of mouse, rat, guinea pig, and bovine monocytes was less reliable.

The present investigations reveal that the non specific esterase from mouse liver nuclei is exclusively located in the perinuclear cistern and that the nuclear chromatin does not contain any esterase as a rule. However, esterase is associated with lipid droplets which are seldomly found within nuclei. Two different esterases are demonstrated with the employed substrates in the nuclear envelope. It is exemplified that the esterase, found in isolated nuclei, can neither be understood as qualitative nor as quantitative equivalent of the esterase demonstrated ultrahistochemically at nuclear membranes.

Subcellular fractions of the human lymphoid cell line PGLC-33H were obtained by N2 cavitation and differential centrifugation. The purity of the fractions was assessed by the use of the following marker enzymes: β-glucuronidase for the lysosomal-intermediate fraction; a nonspecific esterase for the microsomal fraction; and LDH for the supernatant fraction. These subcellular fractions were studied for MIF activity utilizing human lymphoid cells from established lines as target cells. MIF activtiy was most consistently found in the microsomal fraction. Also, MIF activity was closely associated with the relative specific activity of esterase. No such correlation with MIF activity could be demonstrated for β-glucuronidase or LDH.

The purpose of this study is to determine if inframammalian vertebrate (amphibian) lung contains certain nonspecific esterases that have been identified as enzyme markers for mammalian (rat and mouse) pulmonary surfactant. Density gradient centrifugation procedures were utilized to concentrate any surface-active material in frog lung homogenates. Lipid and protein analyses of one of the derived fractions and of pulmonary lavage fluid were consistent with other techniques indicating that these preparations were surface active. A comparison of the nonspecific esterases in the derived fractions and the pulmonary lavage fluid allowed the identification of a nonspecific esterase that has an electrophoretic mobility comparable to one of the nonspecific esterases already identified as an enzyme marker for mammalian (rat and mouse) pulmonary surfactant. These results indicate that these enzyme markers may be useful in the further investigation of the surfactant systems of other inframammalian vertebrates.

— Isolated bovine central nerve myelin sheath preparations showed non-specific esterase activity towards naphthyl ester substrates of increasing chain length from acetate to palmitate. Short chain esters were hydrolysed much faster than long chain substrates by myelin, the specific activity for the hydrolysis of β-naphthyl acetate being the highest. Micro-somal fractions from brain white matter were much higher in esterase activity to all naphthyl ester substrates. NADPH-cytochrome c reductase activity was absent from isolated myelin samples. Distilled water and salt and buffer solutions of different ionic strengths and pH were ineffective in releasing non-specific esterase activity from myelin although tri-potassium citrate caused marked inhibition of the membrane-bound esterase activity. The detergent Triton X-100 released esterase activity from the myelin preparations but at a concentration of 0.1 per cent was also inhibitory.