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.     

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.     

Cytochemical studies of the types and localization of acid phosphatases in the various regions of the midgut epithelium of Carausius morosus

Synopsis Cytochemical studies on the localization and substrate specificities of acid phosphatase activities in the epithelial cells of the midgut ofCarausius morosus have revealed the presence of two distinct types of phosphatases. Acid naphthol AS-BI phosphatase activity was present at particulate (lysosomal) sites in all regions of the midgut and its activity was particularly high in the pear-shaped organs. Acid -glycerophosphatase of low activity was present in the mid and posterior midgut regions, but was absent from the pearshaped organs. In the anterior region of the midgut, acid -glycerophosphatase activity could only be found associated with the concentrically laminated vesicles.     

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.     

Histochemical study of a number of hydrolases,including a new acid phosphatase,in various tissues of men and mice

Summary Two acid phosphatases have been demonstrated histochemically in mouse ventral prostate, seminal vesicles, coagulating glands, and liver and in human prostate. The first is the lysosomal acid phosphatase demonstrable by the Gomori technique. The second differs from thisβ-glycerophosphatase in that it splits naphthol AS phosphates but notβ-glycerophosphate; it has a different histochemical pH optimum and it is not inhibited by MoO₄ or NaF. The enzyme does not represent the “tail” of alkaline phosphatase activity as it is not inhibited by inhibitors of alkaline phosphatase and it has a different localization in liver and in human prostate. The enzyme may be membrane-bound but a lysosomal localization has still to be confirmed.     

A histochemical study of lysosomal enzymes in the retina of the rat

Summary Sections of retinas from albino and pigmented rats were studied histochemically by the naphthol and lead methods for lysosomal enzymes (acid phosphatase, aryl sulfatase, glucosaminidase and -glucuronidase). Activity of the enzymes studied (except -glucuronidase) is demonstrable in granules which by their staining properties are identified as lysosomes. The matrix of the lysosome stains positively in PAS preparations and is diastaae resistant. The organelles are distributed mainly in the pigment epithelium, outer limiting membrane, inner nuclear layer, ganglion layer and inner limiting membrane of the retina.This investigation was supported by a generous grant from the Nuffield Foundation.     

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.     

Lysosomale Enzyme in der Oogenese und Follikulogenese des Meerschweinchens

Zusammenfassung Untersucht wurden die Ovarien von 75 Meerschweinchen (von 42 Embryonaltagen bis zum erwachsenen Tier) an Semidünnschnitten und lichtmikroskopisch enzym-histochemisch auf dad Vorkommen von saurer Phosphatase, -Glucuronidase und unspezifischer Esterase. Saure Phosphatase und -Glucuronidase können bereits im Primordialfollikel, unspezifische Esterase im Sekundärfollikel nachgewiesen werden. Alle drei Enzyme nehmen während des Oocytenwachstums an Aktivität zu, doch während Verteilung und Dichte der Hydrolasen sich im Zuge der Follikelreifung ändern, sind sie in denselben Follikelstadien in der Ontogenese der Tiere immer gleich. Im Follikelepithel fällt die Reaktion der unspezifischen Esterase, besonders in den sog. Call-Exner-bodies auf. Während der Liquor folliculi keine nennenswerte Aktivitäten aufweist, ist der Inhalt der Call-Exner-bodies und das Cytoplasma der sie umgebenden Follikelepithelzellen durch das Reaktionsprodukt stark gefärbt. Die Follikelatresie kann zumindest in fortgeschrittenen Stadien enzymhistochemisch und morphologisch erfaßt werden. Die Abgrenzung gegen intakte Follikel wird zuerst durhc den Vergleich des Follikelepithels möglich.

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Lysosomal enzymes in the oogenesis and folliculogenesis of the guinea pig

Summary In the ovaries of 75 guinea pigs from 42 days of gestational age to adult animals follicles of every developmental stage were investigated by means of histochemistry for the appearance of acid phosphatase, -glucuronidase and non-specific esterase in oocytes and follicle epithelial cells. For morphological control semi-thin sections were examined. Already in primordial follicles, oocytes exhibit a -glucuronidase and acid phosphatase reaction. Non-specific esterase can be detected in the oocytes and granulosa cells of secondary follicles. The three of them increase in activity during the growth of the follicles, but show no differences in the ontogenesis of the guinea pig. In the follicle epithelium the reaction of non-specific esterase is especially apparent in the cytoplasm of the cells surrounding the so called Call-Exner-Bodies. The highest activity is seen in the content of these formations whereas the liquor folliculi does not show a considerable activity. Atresia of follicles can be detected both by enzyme histochemistry and visually in semi-thin sections, at least in advanced stages. The boundaries with intact follicles can at first be recognized by comparison of the follicle epithelium.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft (SFB 105)

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Unter Leitung von Prof. Dr. Karl-Hermann Korfsmeier     

Zur Histochemie von Hydrolasen im Hoden des Hundes und der Katze

Zusammenfassung Wir untersuchten das Verteilungsmuster von unspezifischer Esterase, alkalischer Phosphatase, Adenosintriphosphatase, 5-Nucleotidase und -D-Glucuronidase im Hoden von Hund und Katze. Besonders hervorzuheben sind eine starke Aktivität der unspezifischen Esterase in den Sertolizellen der Katze, der Reichtum der Membrana propria aller Hodentubuli an alkalischer Phosphatase und Adenosintriphosphatase sowie die kräftige Reaktion auf -D-Glucuronidase in den Leydigzellen beider Tierarten.Die Befunde werden diskutiert.

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Summary The localization of unspecific esterase, alkaline phosphatase, adenosine triphosphatase, 5-nucleotidase, and -D-glucuronidase in the testes of cat and dog was demonstrated by histochemical means. We observed a strong esterase activity in the Sertoli cells of the cat and high amounts of alkaline phosphatase and adenosine triphosphatase in the membrana propria of all seminiferous tubules. In both species the principal site of -D-glucuronidase was in the Leydig cells. Our findings obtained being discussed.

     

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.     

Ultrastructural localization of esterase and acid phosphatase in digestive gland cells of fed and starvedCepaea nemoralis (L.) (Mollusca: Helicidae)

Summary The ultrastructural localizations of thiolacetic acid esterase, indoxyl acetate esterase and acid -glycerophosphatase have been studied in the digestive gland cells of fed and starvedCepaea nemoralis. In fed snails the major localization of all three enzymes was in the green granule vacuoles of digestive cells. In addition, the cytoplasm of calcium cells and the Golgi apparatus and GERL (?) of all cell types were acid phosphatase positive. Many digestive cells of starved snails showed a similar enzyme distribution to that found in fed snails but other digestive cells showed a very high cytoplasmic activity of all three enzymes. It is suggested that these cells are in the process of autolysis. New light is also thrown on the process by which food is transported from the digestive gland lumen to the phagosomes of digestive cells.     

Esterase

Zusammenfassung Mit den Essig- und Buttersäureestern von 8-Hydroxichinolin wurde eine Lipidtropfenesterase in der Leber und der Niere der Maus elektronenmikroskopisch dargestellt. Sie stellt in der fixierten Leber einen beträchtlichen Anteil der Esteraseaktivität dar und ist identisch mit der lichtmikroskopisch vorzugsweise im Läppchenzentrum tropfenförmig erscheinenden Esterase. Möglicherweise ist sie mit der tropfigen Esterase anderer Autoren nicht identisch. In der Niere ist die Lipidtropfenesterase weniger reichlich vertreten. Es werden Anhaltspunkte zur Herkunft der Lipidtropfenesterase gegeben. Die mögliche Bedeutung für den Lipidstoffwechsel wird diskutiert.

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EsteraseIX. On the esterase activity in lipid droplets within the mouse liver and kidney

Summary An esterase located within lipid droplets of mouse liver and kidney is demonstrated by electron microscopy employing acetic acid and butyric acid esters of 8-hydroxiquinoline. This enzyme accounts for a considerable amount of the esterase activity in the glutaraldehyde fixed liver and is identical with the droplet esterase found chiefly in the central area of the lobule. Possibly it is different from the droplet esterase described by other authors. In the kidney there is less of this lipid droplet esterase. Some aspects regarding its origin and its biological significance concerning lipid metabolism are discussed.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Metabolism of free and membrane-bound ribosomes during aging of Jerusalem artichoke tuber slices

Summary A biochemical and cytochemical study has been made of the distribution of -glycerophosphatase (EC 3.1.3.2) activity in mature and differentiating phloem cells of Nicotiana tabacum L. and the pH dependence and kinetics of -glycerophosphate hydrolysis of homogenates of fresh leaf midveins and midveins fixed in formaldehyde-gluteraldehyde. -glycerophosphatase showed two peaks of activity at pH 5.5 and 6.2. Enzyme saturation kinetics were exhibited by both fresh and fixed tissue homogenates. At a substrate concentration of 2 mM, 65% of the enzyme activity survived fixation. Specimens for cytochemical localization were incubated with 2 mM -glycerophosphate at pH 5.5 and 6.2. Specimens showed consistent patterns of reaction product deposition. Little or no reaction product was deposited in controls incubated without substrate or with substrate plus 0.01 M fluoride. -glycerophosphatase activity in the phloem and xylem is considerably higher than in surrounding tissue. Dense localization of reaction product was demonstrated on the vacuolar membranes, the inner membranes of mitochondria, and the dictysomes of phloem parenchyma and companion cells. The plasma membrane and endoplasmic reticulum cisternae of these cells were usually free of reaction products. Enzyme activity in mature sieve elements was associated with the parietal and stacked systems of endoplasmic reticulum and with the P-protein. There was inconsistency of staining of P-protein in mature sieve elements although the association of reaction products with the P-protein appeared to show a correlation with maturity and dispersal. The P-protein bodies of differentiating sieve elements showed no reaction product deposition. The distribution of -glycerophosphatase activity has been compared with that previously recorded for ATPase activity in the phloem of Nicotiana tabacum.     

Histochemical study of enzyme patterns in the human submaxillary gland

Summary The histochemical distribution of various enzymes, such as alkaline phosphatase, acid phosphatase, esterase, -glycosidase, aminopeptidase, succinic dehydrogenese and TPN diaphorase, in human submaxillary glands has been determined.Acini and ducts of human submaxillary gland were devoid of alkaline phosphatase activity, but this enzyme was observed in capillaries and somewhat in myoepithelium.Activities of acid phosphatase, esterase, -glucuronidase and -galactosidase were generally observed in the entire cytoplasm of serous acini; but the cytoplasm of mucous acini was either negative or showed only trace amounts.Aminopeptidase reaction of both acini and ducts was generally negative.Succinic dehydrogenase and TPN diaphorase activities were strongly active in intralobuler ducts. Serous acini exhibited less activity with these enzymes; and mucous cells showed still less and were almost negative. In serous acini, there was much greater activity of TPN diaphorase than of succinic dehydrogenase.With 7 Figures in the Text     

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.     

Metabolic variants of intestinal alkaline phosphatase in relation to fat absorption: in situ demonstration with the organ-specific inhibitors l-phenylalanine and l-homoarginine

Summary The localization of rat intestinal alkaline phosphatase has been studied in relation to fat absorption. The observations support a theory of conversion, within the intestinal mucosa, of intestinal type to liver type alkaline phosphatase when the criterion of differential sensitivity to two amino acid inhibitors, l-phenylalanine, and l-homoarginine, is applied.Following a three hour in vivo exposure to mixtures of oleic acid, sodium taurocholate, and lauric acid, the epithelium becomes depleted of its l-phenylalanine-sensitive, intestinal type alkaline phosphatase. At the same time, enriched activity is seen in the lamina propria; this activity is both particulate and diffuse, and is present both in the connective tissue matrix and in cells, including macrophages, eosinophils and lymphocytes. Most of this enzyme is inhibited by l-homoarginine, a property characteristic of liver type alkaline phosphatase.The localization of enzyme-positive particles 0.5 to 1.0 in diameter in both epithelium and lamina propria appears identical to that of particulate fat. A physical association between transport of absorbed fat and metabolic conversion of intestinal type alkaline phosphatase is postulated.This work was aided in part by grants-in-aid [CA-3332-01, K6-CA-18,453] from the National Cancer Institute, National Institutes of Health, U.S.P.H.S. and the John A. Hartford Foundation, Inc.Pre-doctoral trainee, U.S.P.H.S. grant GM01451.Holder of a Juan Marsh Foundation Fellowship, Lemuel Shattuck Hospital.     

Histochemical studies on the distribution of acid phosphatases in neurones of sensory ganglia; light and electron microscopy

Summary Acid phosphatase activity of rat sensory ganglia was studied by light and electron microscopic histochemistry. Neurones of trigeminal and spinal ganglia showed two characteristic patterns of -glycerophosphatase. Enzymic activity in small neurones appeared as granules, i.e. lysosomes, whereas activity in large neurones appeared as a network of filaments and scattered granules. The network pattern was due to localization of the reaction product in the Golgi apparatus and in association with the rough endoplasmic reticulum. The presence of at least two acid phosphatases was suggested on account of differences in sensitivity between the enzymes in the small and large cells toward several fixatives and inhibitors.     

Different tartrate sensitivity and pH optimum for two isoenzymes of acid phosphatase in osteoclasts. An electron-microscopic enzyme-cytochemical study

Summary By differentiation of substrate specificity, pH optimum range, and sensitivity to various inhibitors, 2 isoenzymes of acid phosphatase in bone cells have been studied at the electron-microscopic level. When p-nitrophenyl phosphate was used for the substrate, the demonstrable enzyme activity was affected by neither tartrate nor sodium fluoride. The reaction product, when incubated at pH 5–6, was detected in all sites along the pathway for the biosynthesis of acid phosphatase in the osteoclast, including the perinuclear space, cisternae of the endoplasmic reticulum, Golgi complex, various vesicles, and vacuoles. In the osteoclasts attached to bone, the enzymatic activity was demonstrated at the extracellular ruffled border and on the eroded bone surface. Reaction products became confined to lysosomes and extracellular ruffled border when incubated at pH 6–7. Unattached osteoclasts showed a similar intracytoplasmic localization of enzyme as the attached ones, except for the absence of the extracellular enzyme activity. The mononuclear, immature type of osteoclast also resembled the mature osteoclast in terms of enzymatic localization. Except for the osteoclasts, the acid p-nitrophenyl phosphatase activity was restricted to lysosomal vesicles in various bone cells, monocytes, and macrophages. Such activity was inhibited by adding 50 mM tartrate to the p-nitrophenyl phosphate medium. When -glycerophosphate or p-nitrocatechol sulfate was the substrate, most of the reaction product was localized intracellularly. Unlike the acid p-nitrophenyl phosphatase, the acid -glycerophosphatase or arylsulfatase activity in osteoclasts and other bone cells was inhibited completely by 10 mM tartrate or 10 mM sodium fluoride. Even preincubation of 100 mM tartrate in the buffer inhibited -glycerophosphatase activity completely, but p-nitrophenyl phosphatase activity was inhibited incompletely. Consequently, our results suggest that acid p-nitrophenyl phosphatase is a useful cytochemical marker for identification of the osteoclast family at electron-microscopic levels of resolution.     

A study of the biochemistry and cytochemical localization of β-glycerophosphatase activity in root tips of maize and pea

Summary A critical study has been made of the standard lead phosphate precipitation technique for the localization of-glycerophosphatase activity in young root tips. The effects of fixatives on enzyme activity and on the loss of activity during fixation and incubation have been determined. Cytochemical studies showed that the most prominent sites of-glycerophosphatase activity were at particulate sites in the cytoplasm and in the nucleus. Good correlation was obtained between frozen section and electron microscope studies although a number of problems were encountered. These particularly concerned the penetration of the staining medium, the loss of activity during incubation and the use of the acetic acid rinse. Recommendations are made for the reliable localization of the enzyme. The use of controls in this method were studied and their validity discussed.The study was supported in part by a National Science Foundation Grant GB 12371 to Dr. J.Cronshaw.     

The effects of lethal procedures on the histochemical localization of acid hydrolases

Summary The distribution and activities, as revealed by histochemical techniques, of acid phosphatase, -glucuronidase, arylsulphatase and -galactosidase in the kidney, liver and skeletal muscle of rats and dystrophic hamsters killed by each of the following methods were studied: overdose of ether, chloroform and nembutal anaesthesia, immersion in liquid air, stunning followed by decapitation, and carbon dioxide asphyxiation.In rats killed with overdoses of ether or chloroform, the activity of acid phosphatase in sections of formaldehyde-fixed kidney was, it was found, less than that in decapitated ones. Sometimes the activity was abolished completely. The intensity of colour, the particulate granularity and the distribution of final reaction product normally obtained for other acid hydrolases and tissues were also affected, e.g. the distribution was more diffuse. Often the minimum time of incubation for the final reaction product to appear was shortened. One example where this happened was the activity of arylsulphatase against 6-benzoyl-2-naphthyl sulphate. The activities of the three other enzymes studied were affected in the same way but to a different degree. However, killing animals with nembutal or liquid air or by decapitation seemed to have little effect on the activities of any of lysosomal enzymes investigated.In order to obtain a reproduceable localization of strong acid hydrolase activity in a particulate form (presumably indicative of lysosomes), it is necessary to bring experimental animals to a basal physiological condition by isolating them overnight away from other animals before killing them (with nembutal or by stunning and decapitation) after a substantial period of rest.