Leucine aminopeptidase as an ecto-enzyme of polymorphonuclear neutrophils

Intact polymorphonuclear neutrophils were modified chemically by a poorly permeable reagent, diazotized sulfanilic acid, and the changes in the activity of 5′-nucleotidase, alkaline phosphodiesterase, and leucine aminopeptidase were examined. Among three plasma membrane enzymes, 5′-nucleotidase activity was hardly detected in the human neutrophils. The activity of alkaline phosphodiesterase was observed in all the neutrophils examined, but was not inhibited by diazotized sulfanilic acid in the guinea-pig neutrophils. On the other hand, the activity of leucine aminopeptidase was not only found but also inhibited by diazotized sulfanilic acid without the inhibition of lactate dehydrogenase, a cytosol enzyme, in all the neutrophils, suggesting that leucine aminopeptidase is located generally on the plasma membrane as an ecto-enzyme in the neutrophils.     

Inactivation of phagocytosis-stimulating activity of tuftsin by polymorphonuclear neutrophils: A possible role of leucine aminopeptidase as an ecto-enzyme

The subcellular localization of the tuftsin-inactivating activity was studied using guinea-pig polymorphonuclear neutrophils and the following results were obtained. 1. The tuftsin-inactivating activity was present in the membrane function but not in the cytosol and the granular fractions. 2. Intact neutrophils inactivated tuftsin rapidly. However, when neutrophils were modified chemically by a poorly permeant reagent, diazotized sulfanilic acid, the tuftsin-inactivating activity decreased sifnificantly without any inhibition of marker enzymes of cytosol, microsome, granulesa and mitochondria, suggesting that the tuftsin-inactivating activity is located on the plasma membrane as an ecto-enzyme. 3. When neutrophils were modified by diazotized sulfanilic acid at different concentrations, the tuftsin-inactivating activity of neutrophils was inhibited in proportion to the degree of inhibition of the activity of leucine aminopeptidase, an ecto-enzyme. 4, Hydrolysis of L-leucyl-β-napthylamide, a synthetic substrate of leucine aminopeptidase, was inhibited competitively by tuftsin. 5. Treatmetn of neutrophils with serine protease inhibitors affected neither tuftsin-inactivating nor leucine aminopeptidase activity at all, indicating no involvement of serine proteases, which is said to be located on the cell surface membrane, in the tuftsin-inactivating activity of neutrophils. The possibility was deduced from the above results that leucine aminopeptidase may act as a tuftsin-inactivating enzyme.     

Possible exposure of leucine aminopeptidase on the cell surface of rabbit blood neutrophils by digitonin treatment

The sensitivity of leucine aminopeptidase to diazotized sulfanilic acid (DSA) was compared between neutrophils from blood and peritoneal exudates of rabbit. The leucine aminopeptidase activity of peritoneal neutrophils was inhibited about 40% by DSA, whereas that of blood neutrophils was not inhibited at all by the reagent. However, pretreatment of blood neutrophils with digitonin in the presence and in the absence of divalent cations rendered leucine aminopeptidase sensitive to DSA to the same extent as peritoneal neutrophils, without affecting the cell viability and lactate dehydrogenase activity. These findings seem to indicate that the leucine aminopeptidase of blood neutrophils, which is normally inaccessible to DSA, was exposed on the cell surface by digitonin treatment.     

Release of alkaline phosphodiesterase I from rat kidney plasma membrane produced by the phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis

The release of plasma-membrane-bound enzymes by phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis was investigated. Among the ectoenzymes of plasma membrane tested, alkaline phosphodiesterase I was released markedly from rat kidney cortex slices, in addition to alkaline phosphatase and 5'-nucleotidase. Other membrane-bound enzymes; alanine aminopeptidase, leucine aminopeptidase, dipeptidyl peptidase, leucine aminopeptidase, dipeptidyl peptidase IV, esterase and gamma-glutamyl transpeptidase could not be liberated from the treated slices. Alkaline phosphodiesterase I was released linearly from rat kidney slices with the concentration of phosphatidylinositol-specific phospholipase C, but little enzyme was released from rat liver slices. Alkaline phosphodiesterase I separated from kidney tissue with n-butanol still retained phosphatidylinositol and was transformed into a lower molecular weight form by phosphatidylinositol-specific phospholipase C. This suggests an important function for phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membrane of rat kidney cells. The alkaline phosphodiesterase I released from rat kidney had a molecular weight of about 240,000 and an isoelectric point (pI) of 5.4. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9 and had a Km value of 0.3 mM. The enzyme was activated by Mg2+ and Ca2+, but was inhibited by EDTA. Strong inhibition took place on the addition of adenosine 5'-phosphosulfate or the nucleotide pyrophosphates, i.e., UDP-galactose and alpha, beta-methylene ATP.     

Possible involvement of aminopeptidase, an ecto-enzyme, in the inactivation of bradykinin by intact neutrophils

The subcellular localization of the bradykinin-inactivating activity was studied using guinea-pig neutrophils and the following results were obtained. The bradykinin-inactivating activities were found to be present in the cytosol and membrane fractions but not in the granular and nuclear fractions. The bradykinin-inactivating activity of the cytosol fraction was inhibited by N-carbobenzoxy-Gly-Pro, an inhibitor of prolyl endopeptidase, whereas that of the membrane fraction was inhibited by bestatin, an inhibitor of aminopeptidase. Prolyl endopeptidase and aminopeptidase activities were located predominantly in the cytosol and membrane fractions, respectively, and their activities were inhibited by their respective inhibitors. Prolyl endopeptidase and aminopeptidase activities measured with synthetic substrates were competitively inhibited by bradykinin, suggesting that bradykinin is a possible substrate for prolyl endopeptidase and aminopeptidase. Intact neutrophils inactivated bradykinin rapidly. However, when neutrophils were modified chemically by diazotized sulfanilic acid, a poorly permeant reagent which inactivates ecto-enzymes selectively, both the bradykinin-inactivating activity and aminopeptidase activity of neutrophils decreased significantly without any inhibition of cytosol prolyl endopeptidase. The possibility that aminopeptidase, an ecto-enzyme, would be responsible for the inactivation of bradykinin by intact neutrophils was deduced from the results above, although both cytosol prolyl endopeptidase and membrane aminopeptidase could inactivate bradykinin.     

Inhibition of platelet aggregation by synthetic phosphatidylcholines: possible involvement of vesiculation of platelet plasma membranes

The purpose of this investigation was to determine which enzyme activities are true canine neutrophil plasma membrane markers. Three enzymes thought to be present on plasma membranes were chosen for study: 5'-nucleotidase, magnesium-dependent adenosine triphosphatase (Mg2+-ATPase), and leucine aminopeptidase. Both 5'-nucleotidase and Mg2+-ATPase were found to be ectoenzymes in the canine neutrophil but additional Mg2+-ATPase activity was located intracellularly. An endogenous inhibitor of 5'-nucleotidase was found in the cytosol of canine neutrophils. The specific 5'-nucleotidase inhibitor, adenosine 5'-[alpha, beta-methylene] diphosphate also inhibited the canine enzyme in intact cells. Leucine aminopeptidase was located solely in the myeloperoxidase-containing granules of the canine neutrophil. Plasma membrane, as identified by the presence of Mg2+-ATPase and 5'-nucleotidase activities, was separated from other cell organelles by Percoll-density gradient centrifugation of a 10 000 X g supernatant of nitrogen cavitated neutrophils.     

Inactivation during phagocytosis of leucine aminopeptidase, an ecto-enzyme of polymorphonuclear neutrophils

Changes in enzyme activities of the plasma membrane makers were examined during phagocytosis using guinea-pig polymorphonuclear neutrophils. Incubation of neutrophils with fresh serum-opsonized zymosan particles showed a significant reduction in leucine aminopeptidase activity, whereas 5′-nucleotidase and alkaline phosphodieterase activities remained unchanged. Inactivation of leucine aminopeptidase activity was also observed by exposure of neutrophils to complement-opsonized zymosan particles, but not to non-opsonized zymosan, IgG-coated zymosan or polysterene latex particles. Pretreatment of neutrophils with cytochalasin B, which prevents phagocytosis but not surface binding of particles, provoked inactivation to the same degree as when the cells were allowed to phagocytose the particles. However, the inactivation during phagocytosis was protected by serine protease inhibitors. These findings suggest that loss of leucine aminopeptidase activity from phagocytosing cells may be mediated by certain serine protease inhibitor-sensitive factor(s) which are probably activated by the attachment of an opsonized zymosan particle to a specific membrane receptor, probably the C3b receptor.     

Studies on the leukotriene D4-metabolizing enzyme of rat leukocytes, which catalyzes the conversion of leukotriene D4 to leukotriene E4

Leukotriene D4-metabolizing enzyme was studied using rat neutrophils, lymphocytes and macrophages. These leukocyte sonicates converted leukotriene D4 to leukotriene E4. However, the leukotriene D4-metabolizing activity varied with cell type, and macrophages showed the highest activity among these leukocytes. The subcellular localization of the leukotriene D4-metabolizing enzyme of macrophages was examined, and the leukotriene D4-metabolizing activity was found to be present in the membrane fraction, but not in the nuclear, granular and cytosol fractions. When macrophages were modified chemically with diazotized sulfanilic acid, a poorly permeant reagent which inactivates cell-surface enzymes selectively, the leukotriene D4-metabolizing activity of macrophages decreased significantly (about 95%) without any inhibition of marker enzymes of microsome, cytosol, lysosome and mitochondria. When neutrophils and lymphocytes were modified with diazotized sulfanilic acid, the leukotriene D4-metabolizing activity was also inhibited about 90% by the modification. Among various enzyme inhibitors used, o-phenanthroline, a metal chelator, remarkably inhibited the leukotriene D4-metabolizing activity of leukocytes, and the o-phenanthroline-inactivated enzyme activity was fully reactivated by Co2+ and Zn2+. These findings seem to indicate that rat neutrophils, lymphocytes and macrophages possess the leukotriene D4-metabolizing metalloenzyme which converts leukotriene D4 to leukotriene E4, on the cell surface, although macrophages have a higher enzyme activity than the other two.     

Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. III. Changes of enzyme activities on cell membranes during culture

When liver cells were dispersed with collagenase, their 5'-nucleotidase activity decreased to half the initial level, but it increased to the original level again on culture of the cells for a few days. The activity of another membrane enzyme, alkaline phosphatase, did not decrease on dispersion of the cells, but it increased about 10-fold on culture of the cells. These inductions did not require any hormone, but the effects were greater at a high cell density. These enzymes are located in both the plasma membranes and the cytoplasm, but the enzymes in these two locations can be distinguished by differences in their pH optima, substrate specificities, and susceptibilities to inhibitors. The increases were found to be due to increases in the activity of only the enzymes in the plasma membranes. The increases in enzyme activities were inhibited by actinomycin D, cycloheximide, and puromycin. The activities of leucine aminopeptidase and aminopeptidase B, other membrane enzymes, remained constant during dispersion and culture of the cells. These results show that enzymes in the cell membranes are affected in different ways by cell dispersion with collagenase and subsequent culture of the cells.     

Subcellular localization of a membrane-associated transglutaminase activity in rat liver

Fractionation of rat liver by homogenization and differential centrifugation revealed that only about 83% of the transglutaminase activity in the tissue is in a soluble form, and that the remainder is associated with the particulate fraction. This latter activity remained with the membranes even after they were extensively washed to remove 99% of such soluble enzymes as lactate dehydrogenase and aldolase. Subsequent fractionation of the membranes by isopycnic density gradient centrifugation in sucrose resulted in a single band of transglutaminase activity at a density of 1.194 g/cm3. This activity was coincident with the major band of plasma membranes, which was identified by its content of 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and leucine aminopeptidase activities. After treatment with digitonin and fractionation on sucrose gradients, the transglutaminase activity and the plasma membrane marker enzyme activities were found at a new density of 1.210 g/cm3, while the enzyme markers for the other membrane fractions remained unchanged. From these data, we conclude that approximately 17% of the transglutaminase activity in rat liver is specifically associated with the plasma membranes.     

Dependence of histochemical staining of leukocyte aminopeptidase upon its biochemical enzyme activity

The relationship between histochemical staining and biochemical activity of the enzyme was investigated using leukocytes with different aminopeptidase activities. In guinea-pig neutrophils and macrophages which have a relatively high enzyme activity, the histochemical staining correlated with the biochemical enzyme activity. On the other hand, guinea-pig lymphocytes and mouse neutrophils whose enzyme activities were 8.25 +/- 0.27 mU/10(7) cells and 6.18 +/- 0.87 mU/10(7) cells, respectively, were not stained by histochemical techniques. When guinea-pig neutrophils were modified chemically by diazotized sulfanilic acid at different concentrations, the histochemical staining of neutrophils decreased in proportion to the degree of inhibition of their biochemical enzyme activity and hardly became detectable below 10 mU/10(7) cells. However, guinea-pig neutrophils contained the soluble enzyme, corresponding to 5 mU/10(7) cells, which leaked out rapidly from cells during staining procedure, suggesting that the limit of visualization of the membrane-bound aminopeptidase activity by the histochemical techniques is about 5 mU/10(7) cells. The membrane-bound enzyme activities in guinea-pig lymphocytes and mouse neutrophils were 5 mU and 3 mU per 10(7) cells, respectively, and so it is possible that these leukocytes hardly stained histochemically.     

Independent secretion of proteoglycans and collagens in chick chondrocyte cultures during acute ascorbic acid treatment.

1. Liver plasma membranes originating from the sinusoidal, lateral and canalicular surface domains of hepatocytes were covalently labelled with sulpho-N-hydroxysuccinamide-biotin. After solubilization in Triton X-114, treatment with a phosphatidylinositol-specific phospholipase C (PI-PLC), two-phase partitioning and 125I-streptavidin labelling of the proteins resolved by PAGE, six major polypeptides (molecular masses 110, 85, 70, 55, 38 and 35 kDa) were shown to be anchored in bile canalicular membrane vesicles by a glycosyl-phosphatidylinositol (G-PI) 'tail'. 2. Permeabilized 'early' and 'late' endocytic vesicles isolated from liver were also examined. Two polypeptides (110 and 35 kDa) were shown to be anchored by a G-PI tail in 'late' endocytic vesicles. 3. Analysis of marker enzymes in bile-canalicular vesicles treated with PI-PLC showed that 5'-nucleotidase and alkaline phosphatase, but not leucine aminopeptidase and ecto-Ca2(+)-ATPase activities were released from the membrane. A low release and recovery of alkaline phosphodiesterase activity was noted. The cleavage from the membrane of 5'-nucleotidase as a 70 kDa polypeptide was confirmed by Western blotting using an antibody to this enzyme. 4. Antibodies raised to proteins released from bile-canalicular vesicles by PI-PLC treatment, and purified by partitioning in aqueous and Triton X-114 phases, localized to the bile canaliculi in thin liver sections. Antibodies to proteins not hydrolysed by this treatment stained by immunofluorescence the sinusoidal and canalicular surface regions of hepatocytes. 5. Antibodies generated to proteins cleaved by PI-PLC treatment of canalicular vesicles were shown to identify, by Western blotting, a major 110 kDa polypeptide in these vesicles. Two polypeptides (55 and 38 kDa) were detected in MDCK and HepG-2 cultured cells. 6. Since two of the six G-PI-anchored proteins targeted to the bile-canalicular plasma membrane were also detected in 'late' endocytic vesicles, the results suggest that a junction where exocytic and endocytic traffic routes meet occurs in a 'late' endocytic compartment.     

Localization of leucine aminopeptidase and vitamin B-12 binding protein in rabbit peripheral blood polymorphonuclear leukocytes

Polymorphonuclear leukocytes were isolated from the peripheral blood of rabbits by Ficoll-Hypaque centrifugation followed by dextran sedimentation. The granulocytes were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. Leucine aminopeptidase, when assayed with L-leucine-7-amido-4-methyl-coumarin as substrate, showed a similar distribution to N-acetyl-ß-glucosaminidase and thus is localized to the tertiary granules. There was no leucine aminopeptidase associated with the plasma membrane of these cells. Further experiments with purified plasma membranes and inhibitor studies using diazotized sulphanilic acid further confirmed that leucine aminopeptidase had a purely intracellular localization. Vitamin B-12 binding protein showed a similar localization to alkaline phosphatase indicating that, as in human polymorphonuclear leukocytes, vitamin B-12 binding protein is located to the specific granules.     

Mg2+-ATPase as a membrane ecto-enzyme of human granulocytes. Inhibitors, activators and response to phagocytosis.

(1) The Mg2+-ATPase of purified human granulocytes is located at the plasma membrane. Thus, no additional enzyme activity was detected when the cells were disrupted. Moreover, the Mg2+-ATPase activity of intact cells was inhibited by such poorly permeant reagents as diazotized sulfanilic acid and suramin. Finally, the enzyme activity of cell homogenates was recovered in particulate fractions. (2)The surface Mg2+-ATPase of human granulocytes had an apparent Km of 50 microns for ATP and displayed substrate inhibition. (3) The enzyme was not affected by ouabain, but was inhibited by N-ethyl malemide, sodium meta-periodate, suramin and diazotized sulfanilic acid. The enzyme was activated by cytochalasins B and D and by UDP. Activation by UDP was characterized by changes in the enzyme's apparent Km and V and by belief of substrate inhibition. (4)Internalization of surface membranes subsequent to phagocytosis of suitable particles did not result in depletion of Mg2+-ATPase from the cell surface. The enzyme activity did not decrease after exposure to several varieties of paraffin oil emulsion particles, even if the challenged cells had been pretreated with colchicine of cytochalasin B. (5) Since suramin, which inhibited Mg2+-ATPase, had no effect upon other granulocyte functions such as chemotaxis, superoxide anion generation, or phagocytosis, it is unlikely that the enzyme plays a major role in these functions.     

Localization of leukotriene D4-metabolizing metalloenzyme on the cell surface of human neutrophils

The localization of leukotriene D4-metabolizing enzyme on the cell surface was examined using human neutrophils. Intact neutrophils rapidly converted leukotriene D4 to leukotriene E4. However, when neutrophils were modified chemically by diazotized sulfanilic acid, a poorly permeant reagent which inactivates cell surface enzymes selectively, the leukotriene D4-metabolizing activity of neutrophils decreased significantly without any inhibition of the cell viability or marker enzymes of cytosol, granules, microsome and mitochondria. The leukotriene D4-metabolizing enzyme activity of the membrane fraction was inhibited by modification to the same extent as that of Mg2+-dependent ATPase, a cell-surface marker enzyme. Among various enzyme inhibitors examined, a metal chelator, o-phenanthroline, strongly suppressed the leukotriene D4-metabolizing activity of intact neutrophils and the o-phenanthroline-inactivated enzyme activity was fully reactivated by Co2+, Mn2+ and Zn2+. These results would suggest that some metalloenzyme located on the cell surface is involved in the conversion of leukotriene D4 to leukotriene E4 by neutrophils.     

Changes in plasma membrane enzyme activities during liver regeneration in the rat.

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na+ + K+)-ATPase, leucine aminopeptidase, 5'-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na+ + K+)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48--108 h and in 5'-nucleotidase activity at 12--24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.     

Glycerylphosphorylcholine phosphodiesterase in rat liver. Subcellular distribution and localization in plasma membranes

1. A simple new assay for glycerylphosphorylcholine phosphodiesterase is described, in which radioactive glycerylphosphorylcholine is used as substrate and the reaction products are separated by adsorption on an anion-exchange resin. 2. Rat liver subcellular fractions contained both particulate (58%) and soluble (42%) glycerylphosphorylcholine phosphodiesterase. Both activities released free choline from glycerylphosphorylcholine. 3. The particulate glycerylphosphorylcholine phosphodiesterase was recovered mainly in the nuclear and microsomal fractions and showed a distribution similar to those of 5'-nucleotidase and alkaline phosphodiesterase I, both of which are constituents of the liver plasma membrane. 4. During purification of plasma membranes glycerylphosphorylcholine phosphodiesterase, 5'-nucleotidase and alkaline phosphodiesterase I showed largely similar behaviour, indicating that glycerylphosphorylcholine phosphodiesterase is also localized in liver plasma membranes. Slight differences in the distributions of these three enzymes in density-gradient separations are discussed in relation to the possibility that they are unevenly distributed on different areas of the cell surface. 5. The differences between glycerylphosphorylcholine phosphodiesterase and alkaline phosphodiesterase I indicate that these two activities are not functions of a single enzyme. 6. The glycerylphosphorylcholine phosphodiesterase of liver plasma membranes has a pH optimum of 8.5 and a K(m) for glycerylphosphorylcholine of 0.95mm. It is inhibited by EDTA and fully reactivated by a variety of bivalent cations (and Fe(3+)).     

Relations between plasma membrane and lysosomal membrane. 2. Quantitative evaluation of plasma membrane marker enzymes in the lysosomes

We have quantified, in cultured rat fibroblasts, the association to the lysosomal membrane of two classical plasma membrane markers, 5'-nucleotidase and alkaline phosphodiesterase I. To isolate highly purified lysosomal preparations, lysosomes were loaded with horseradish peroxidase (2-h cell uptake, 16-h chase) and isolated by isopycnic centrifugation in linear Percoll gradients, followed by a 3,3'-diaminobenzidine-induced density shift in sucrose gradients. Purified lysosomal preparations contained up to 50% of N-acetyl-beta-glucosaminidase of the homogenate. This lysosomal enzyme was enriched 33-fold in the most purified preparations. In the electron microscope, these preparations appeared to be highly purified and only contained organelles filled with diaminobenzidine reaction products. Analysis of purified preparations indicates that 0.5-0.8% of 5'-nucleotidase, but as much as 10.9-14.3% of alkaline phosphodiesterase I activities of the homogenate, are associated with lysosomes. After freezing-thawing, these activities remained essentially membrane-associated. The larger value obtained for alkaline phosphodiesterase I could not be ascribed to other lysosomal enzymes, as no such activity was detected at acidic pH. These two plasma membrane markers are thus unevenly distributed in the lysosomal compartment.     

Dependence of histochemical staining of leukocyte aminopeptidase upon its biochemical enzyme activity

Summary The relationship between histochemical staining and biochemical activity of the enzyme was investigated using leukocytes with different aminopeptidase activities. In guinea-pig neutrophils and macrophages which have a relatively high enzyme activity, the histochemical staining correlated with the biochemical enzyme activity. On the other hand, guinea-pig lymphocytes and mouse neutrophils whose enzyme activities were 8.25±0.27 mU/10⁷ cells and 6.18±0.87 mU/10⁷ cells, respectively, were not stained by histochemical techniques. When guinea-pig neutrophils were modified chemically by diazotized sulfanilic acid at different concentrations, the histochemical staining of neutrophils decreased in proportion to the degree of inhibition of their biochemical enzyme activity and hardly became detectable below 10 mU/10⁷ cells. However, guinea-pig neutrophils contained the soluble enzyme, corresponding to 5 mU/10⁷ cells, which leaked out rapidly from cells during staining procedure, suggesting that the limit of visualization of the membrane-bound aminopeptidase activity by the histochemical techniques is about 5 mU/10⁷ cells. The membrane-bound enzyme activities in guinea-pig lymphocytes and mouse neutrophils were 5 mU and 3 mU per 10⁷ cells, respectively, and so it is possible that these leukocytes hardly stained histochemically.     

Enzymatic histochemistry of mouse kidney in plastic

Two-micrometer sections of methacrylate-embedded kidney were used to investigate the enzymatic activities of mouse kidney where the proximal tubule and Bowman's capsule from the same corpuscle were viewed in the same section. Alkaline phosphatase, acid phosphatase, 5'-nucleotidase, gamma-glutamyl transpeptidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, alpha-naphthyl butyrate esterase, and adenosine triphosphatase activities were observed in the proximal tubule, but only 5'-nucleotidase, alpha-naphthyl butyrate esterase, and alkaline phosphatase were observed in the squamous portion of the parietal epithelium of Bowman's capsule. The use of methacrylate-embedded tissue allowed more precise localization of enzymatic activity than is possible with most frozen sections. This may provide interesting applications not only for characterization of kidney diseases but also for characterization of other normal and abnormal tissues.