Esterases in histochemistry and ultrahistochemistry

Synopsis The histochemical identification of individual esterases is a problem that has not yet been overcome. Inhibitors and different substrates reveal different patterns of distribution. 8-hydroxyquinoline acetate is a useful substrate in ultrahistochemistry. There is evidence of a relationship between esterase distribution and function.ACTH adrenocorticotropic hormone - 5Bri–O-2 5-bromoindoxyl acetate - 5Br–4ClI–O-2 5-bromo-4-chloro indoxyl acetate - cAMP cyclic adenosine monophosphate - DFP di-isopropyl-fluorophosphate - hCG human chorion gonadotropin - HS-2/4 thiol acetate/butyrate - I-O-2/4 indoxyl acetate/butyrate - N-O-2/3/4 -naphthyl acetate/propionate/butyrate - N-O-2 -naphthyl acetate - N-S-2/9 -naphthyl thiolacetate/nonanoate - NAS-O-2 naphthol AS acetate - NASD-O-2 naphthol AS-D acetate - 4NP-O-2/3 p-nitrophenyl acetate/propionate - 4NP-S-2 p-nitrophenyl thiol acetate - P-O-2 phenyl acetate - Q-O-2/4 8-hydroxyquinoline acetate/butyrate - Q-S-2/4 8-mercaptoquinoline acetate/butyrate - TBA-S-2/9 -thiolbenzanilide acetate/nonanoate - TSH thyroid-stimulating hormone     

The distribution of acid phosphatases and esterases in differentiating roots of Vicia faba

Summary Lateral roots of Vicia faba have been examined cytochemically to determine the distribution of naphthol AS esterases, bromo-indoxyl esterases, acid -glycerophosphatases and acid naphthol AS phosphatases in the various tissues during differentiation. Attempts have been made to correlate the observed differences in the distribution of the hydrolases with respect to physiological and to structural differentiation processes in cells and tissues.     

Isoenzymes and histochemistry of non-specific esterases in normal and cancerous skin

Synopsis Non-specific esterases in normal and carcinomatous skin of the mouse have been investigated electrophoretically and histochemically. Three esterase bands were obtained on electrophoresis from homogenates of normal skin; homogenates of carcinomas showed an accumulation of esterase-Ia and esterase-Ib.^* However, using several ester substrates, substrate-specific patterns were demonstrated in the electrophoresis separations and histochemically in tissue sections. On the electrophoresis separations, -naphthyl acetate, -naphthyl acetate, 6-bromo-2-naphthyl acetate, naphthol AS acetate, naphthol AS-D acetate and naphthol AS-LC acetate gave rise to similar patterns, but with -naphthyl propionate as subsmate, more esterase-Ib was indicated and with 5-bromo-indoxyl acetate a distinctive preponderance. Peripheral or uniformly distributed staining was found histochemically in tumour epithelium using -naphthyl acetate, -naphthyl propionate and -naphthyl acetate, whereas with the substrates of naphthol AS acetate, naphthol AS-D acetate and indoxyl acetate an intermediate pattern of staining related to keratinization was obtained.     

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.     

Genetics of a tissue esterase polymorphism (Est-6) in the rabbit (Oryctolagus cuniculus)

Genetic analysis of a polymorphic tissue esterase revealed a new locus (Est-6) with two alleles (Est-6 ^a andEst-6 ^b) on linkage group VI of the rabbit.Est-6 is closely linked to theEst-1,2,4 cluster. Esterase ofEst-6 is found in many organs, particularly in liver and small intestine, but not in erythrocytes and serum.Est-6 esterase hydrolyzes -naphthyl acetate and butyrate, naphthol AS-D acetate, indoxyl acetate, and butyrate as well as 5-bromoindoxyl acetate,N-acetyl-l-alanine--naphthyl ester but not 4-methylumbelliferyl acetate and fluorescein diacetate. The enzyme is inhibited by bis-p-nitrophenyl phosphate and eserine but not byp-chloromercuribenzoate. It was classified as a carboxylesterase (EC 3.1.1.1). Based on chromosomal localization, tissue distribution, substrate specificity, inhibitor sensitivity, and range ofpI's, rabbitEst-6 is assumed to be homologous with mouseEs-7.The contribution of Dr. O. von Deimling (No. 59) was supported by the Deutsche Forschungsgemeinschaft (De 315/2-2).     

Fractions of non-specific esterase in root tip ofVicia faba L. revealed by disc electrophoresis in acrylamide gel

Fractions of non-specific esterase were studied in homogenates ofVicia faba L. root tips using disc electrophoresis in acrylamide gel. With α-naphthyl acetate, 7 bands were revealed in electrophoreograms, whereas only 5 bands appeared with naphthol AS acetate; the position of bands detected with naphthol AS acetate corresponds to 5 of 7 bands which appear when using α-naphthyl acetate. If incubated with α-naphthyl acetate, 1 band is totally blocked by the inhibitor E 600, whereas the other bands are weakened slightly and—more or less— proportionally, similarly, as if incubated with naphthol AS acetate. No bands appear after detection with α-naphthyl caprylate and with α-naphthyl myristate. Electrophoreograms developed with α-naphthyl propionate differ both from those treated with α-naphthyl acetate and from those treated with α-naphthyl butyrate. Remarkable, but only quantitative differences were revealed when comparing electrophoreograms from division, enlargement and maturation zones, detected with either α-naphthyl acetate, or α-naphthyl propionate. The non-specific esterase fractions revealed by disc electrophoresis are correlated with protein fractions distinguished in the same material by the same technique using xylene brilliant cyanine G staining and with fractions of the same enzyme found in sections of the same object treated histochemically.     

Acid phosphatase and esterase activity in orchid mycorrhiza

Summary A cytochemical study was made to examine the possibility that acid phosphatase may be specifically involved in the digestion of endophytic hyphae in orchid mycorrhiza. Esterase activity was studied for comparison. Frozen sections of unfixed or glutaraldehyde-fixed protocorms of Dactylorhiza purpurella infected by Thanatephorus cucumeris (Rhizoctonia solani) were reacted for acid naphthol AS BI phosphatase, acid -glycerophosphatase or naphthol AS D esterase.A marked increase in particulate acid naphthol AS BI phosphatase activity was observed during infection of host, central, parenchyma cells shortly before hyphae lysed; a diffuse reaction of high activity was localised on lysed fungus. Acid -glycerophosphatase was present at particulate sites only in fungal cytoplasm and as a diffuse reaction on lysed fungus.Naphthol AS D esterase showed highest activity at hyphal apices. Esterase seems to be associated with growth and differentiation of hyphae in orchid cells, rather than lysis of the fungus.     

Cathepsin C activity as related to some histochemical substrates

Summary Hog kidney homogenate was fractionated initially following the steps presented by De La Haba et al. (1959) for the purification of cathepsin C from beef spleen. The preparation was further fractionated by gel filtration using Sephadex G 200, starch gel and immunoelectrophoresis. The following enzymes were identified in the fractions obtained:Cathepsin C, which liberated ammonia from glycyl-phenylalanine amide and naphthylamine from glycyl-phenylalanyl--naphthylamide, pH optimum at 5.0, was activated by cysteine and inhibited by sulfhydryl reagents.An aminopeptidase, which liberated first of all glycine from glycyl-phenylalanine amide and glycyl-phenylalanyl--naphtylainide and after that ammonia and naphthylamine, respectively, hydrolysed numerous amino acid naphthylamides, pH optimum at 7.0–7.5, was activated by Co⁺⁺ and inhibited by EDTA.A peptidase, which liberated glycine from glycyl-phenylalanine amide and naphthylamide, did not hydrolyse amino acid naphthylamides, maximally active at neutral pH, was inhibited by EDTA.Several esterases, two in gel filtration, 5–6 in starch gel and immunoelectrophoresis, hydrolysing 5-bromoindoxyl acetate. The activities were sensitive to E 600.Both the studies on the characteristics of these activities as well as starch gel and immunoelectrophoretic studies support the view that none of the esterase activities is identical with cathepsin C. Cathepsin C, on the other hand, does not hydrolyse significantly 5-bromoindoxyl acetate and, consequently, this substrate can not be used to demonstrate cathepsin C histochemically.Glycyl-phenylalanyl--naphthylamide is recommended as a new sensitive chromogenic substrate for cathepsin C in biochemical studies in which the role of the aminopeptidase (s) can be adequately excluded but cannot be used in the histochemical demonstration of this enzyme.     

A cytochemical study of acid phosphatases and esterases during the induction of crown gall in the tomatoLycopersicon esculentum

Synopsis A cytochemical study has been made of acid -glycerophosphatase, acid naphthol-AS-BI-phosphatase and naphthol esterase activities during the induction of crown gall inLycopersicon esculentum byAgrobacterium tumefaciens. There was an increased activity of these enzymes with time after infection, being more marked for the phosphatases than the esterases. It is suggested that the esterase activities may play a part in cell wall development of the maturing gall.     

A method for the ultrastructural demonstration of non-specific esterase in human blood and lymphoid tissue

Summary 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 -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 histoocytes the granules appeared to the 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 cell lines at the ultrastructural level.     

Small enzymes with esterase activities from two thermophilic fungi, Emericella nidulans and Talaromyces emersonii

Extracellular esterase activities in Emericella nidulans and Talaromyces emersonii are attributed to small enzymes with molecular weights less than 10 kDa (microenzymes). A 1.6 kDa esterase accounted for most of the esterase activity observed in both organisms and one of them also contained a 4.1 kDa microenzyme with weaker esterase activity. These esterases were growth-associated and active towards fluorescein dibutyrate and -naphthyl acetate as well as tributyrin.     

Genetic variation as a tool for histochemical localization of a nonspecific esterase

Summary A prominent esterase activity was demonstrated histochemically in the straight portion of the proximal tubules in kidney of the mouse strain DBA/2J after inhibition with bis-p-nitrophenyl phosphate and subsequent staining, using 5-bromoindoxyl acetate as substrate. In the strain PUC/1Fre, the corresponding esterase was only weakly expressed. By comparing data from the literature (von Deimling et al. 1981) with the characteristic features of this kidney esterase including substrate preference, sensitivity to inhibitors, solubility, histochemical location, and strain differences, it was concluded that it was identical with the previously electrophoretically defined esterase-16.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Esterases of the ox adrenal: their histochemical and biochemical distribution

Summary Esterases were demonstrated in the ox adrenals histochemically. The substrates used were -naphthyl acetate, and naphthyl acetate AS on fresh and fixed tissue. The highest activity was in the zona glomerulosa, the adrenaline-storing cells and the ganglia. The other parenchymal cells showed a moderate activity. The cytological localization of the enzyme was both diffuse and particulate and highly polarized in the adrenaline-storing cells. The particulate form persisted after formaldehyde fixation. The following inhibitors were used to differentiate between the various esterases histochemically and biochemically: DFP, E600, eserine, p-chloromercuribenzoate and 62C47.The cortex, adrenaline storing- and noradrenaline-storing cells were separated by dissection. Suitable marker were used to assess contamination. Total esterase, carboxylesterase, arylesterase, acetylesterase, acetylcholinesterase and cholinesterase were assayed biochemically in conjunction with the inhibitors. Acetylesterase showed the highest activity and was fairly evenly distributed as was carboxylesterase, although at a lesser level of activity. Aryl esterase was more abundant in the cortex than the medulla. The reverse was found for acetylcholinesterase and at a lower level of activity cholinesterase. The results were compared with the histochemical findings.     

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.     

A study of acid phosphatase in mouse kidney using naphthol AS/BI phosphate as a substrate

Summary A kinetic study of mouse kidney acid phosphatase has been performed using an application of the histochemical method ofBurstone (1958a, b). The suitability of the use of naphthol AS/BI phosphate as a substrate for biochemical assays of acid phosphatase has been ascertained. However, the rate of inhibition of the enzyme by sodium molybdate and sodium fluoride suggests that naphthol AS/BI phosphate may represent a substrate for an acid phosphatase different from-glycerophosphatase.     

The use of 2-thionaphthyl acetate as a substrate for the localization and characterization of nonspecific esterase activity in rat alveolar and peritoneal macrophages

Summary A 2-thionaphthyl acetate substrate was utilized to assess the subcellular distribution of nonspecific esterases in rat pulmonary alveolar and peritoneal macrophages. The enzymatically liberated 2-thionaphthol was visualized at pH 7.1 by utilizing gold as a capture agent. Glutaraldehyde-fixed macrophages derived from healthy animals using standard lavage techniques exhibited a high affinity for the substrate and reaction times were thus relatively short (30–60 min). Alveolar macrophages had heavy reaction product on the external surface of the plasma membrane and membranes limiting cisternae of rough endoplasmic reticulum, Golgi complex and mitochondria. Only a thin layer of reaction density was observed associated with the limiting membranes of lysosomes and phagosomes. Peritoneal macrophages were similarly but much less intensely reactive, although they generally lacked or had very little plasma membrane-associated staining. The 2-thionaphthyl acetate esterase activities in both alveolar and peritoneal macrophages were sensitive to diisopropylfluorophosphate (DFP), while only the latter was inhibited by sodium fluoride. Polyacrylamide gel isoelectric focusing of whole cell homogenates indicated that the 2-thionaphthyl acetate esterase activity was the same as that for -naphthyl acetate in these cells. The data indicate that a significantly different distribution of nonspecific esterase activity results with use of a 2-thionaphthyl acetate substrate in the presence of gold ions than that previously reported with other methods. The rapid penetrability and sensitivity of this substrate make it a potentially useful tool for evaluating subcellular localization of esterase activity and probing characteristics of cellular organelles.     

Esterolytic and proteolytic enzymes of the rat adenohypophysis

Summary Characteristics of the hydrolysis of histochemical substrates 5-bromoindoxyl acetate, naphthyl acetate, proprionate, butyrate, caprylate, laurate, myristate, and palmitate, acetyl and butyryl thiocholhie, chloroacetyl and trifluoroacetyl -naphthylamide, benzoyl-arginine -naphthylamide and proteinase substrates human hemoglobin and glycyl-phenylalanine amide by the rat pituitary tissue homogenate and DEAE-cellulose chromatography fractions were determined.In DEAE-cellulose chromatography fractions four separate activities were found splitting short and long chain carboxylic esters. The activity hydrolysing most rapidly 5-bromoindoxyl acetate was resistant to E 600 and was identified as C esterase. Three of the remaining esterase activities were sensitive to E 600 and two of them hydrolysed more rapidly short-chain fatty acid esters while one preferred long-chain fatty acid esters as substrate.One peak of activity was identified as nonspecific cholinesterase on the basis of inhibition studies and hydrolysis of thiocholine substrates. Chloroacetyl -naphthylamide was hydrolysed minimally. Hydrolysis of trifluoroacetyl -naphthylamide was ascribed to E 600 resistant enzyme with pH-optimum at 8.3 hydrolysing also the thiocholine substrates and slowly long-chain fatty acid esters.Five different proteinases hydrolysing human hemoglobin were separated, three of them with pH-optima on the acid and two on the alkaline side of pH. The activities hydrolysing benzoyl-arginine naphthylamide were cysteine activated and had pH-optima around 5.3. None of the peaks of the proteinase activities appeared to coinside with the hydrolysis peaks of any of the histochemical ester substrates in the DEAE fractions.     

Genetics of two tissue esterase polymorphisms (Est-4 and Est-5) in the rabbit

Two polymorphic esterase systems were found after electrophoresis of rabbit tissue homogenates. Each of these systems is controlled by an autosomal locus with two alleles. Est-4 determines the absence (Est-4^a) or presence (Est-4^b) of two bands of esterase activity with intermediate anodal mobility and broad substrate specificity. This polymorphism was found to be present in liver, small intestine, and spleen but not in kidney, heart, and testis. Est-5 is coding for cathodally migrating esterases which differ in mobility (Est-5^a and Est-5^b). This polymorphism was found only in kidney and testis homogenates. Est-5 esterases are more active against -naphthyl acetate than against -naphthyl acetate and have no activity against -naphthyl butyrate. Linkage analysis indicated that Est-4 is localized on rabbit LG VI as part of a cluster of esterase loci, whereas Est-5 segregates independently. Rabbits from two inbred and nine partly inbred strains were tested for these polymorphisms.This investigation was supported in part by Public Health Service Research Grant RR-00251 from the Division of Research Resources and by funds from the University of Utrecht. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

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.     

Genetic variation as a tool for histochemical localization of a nonspecific esterase

A prominent esterase activity was demonstrated histochemically in the straight portion of the proximal tubules in kidney of the mouse strain DBA/2J after inhibition with bis-p-nitrophenyl phosphate and subsequent staining, using 5-bromoindoxyl acetate as substrate. In the strain PUC/1Fre, the corresponding esterase was only weakly expressed. By comparing data from the literature (von Deimling et al. 1981) with the characteristic features of this kidney esterase including substrate preference, sensitivity to inhibitors, solubility, histochemical location, and strain differences, it was concluded that it was identical with the previously electrophoretically defined esterase-16.