Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques: a combined histochemical and biochemical investigation 2. The biochemical investigation and comparison with the histochemical observations.

The reliability of enzyme histochemical semipermeable membrane techniques for the demonstration of acid hydrolases was investigated with a combined histochemical and biochemical study. In part 1 the histochemical findings were presented. In this communication the biochemical findings are reported and compared with the histochemical findings. In m. soleus, m. plantaris, m. gastrocnemius and diaphragm of vitamin E deficient rabbits the activity of the lysosomal acid hydrolases, cathepsin D, acid maltase, acid phosphatase and beta-glucuronidase is significantly increased. This increase in activity of the investigated acid hydrolases was equal for muscles with an aerobic or an anaerobic metabolism. By means of statistical calculations the activity of the enzymes demonstrated with histochemical techniques was compared with the enzyme activity determined with biochemical techniques. From the results of this investigation it can be concluded that the histochemical semipermeable membrane techniques for the demonstration of activity of acid hydrolases are very reliable. Considering the fact that these techniques are also tissue-saving, they are therefore extremely suitable for the study of catabolic wasting processes in skeletal muscle tissues of patients with inherited or acquired muscular diseases.     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques: A combined histochemical and biochemical investigation

Summary The reliability of enzyme histochemical observations of activities of acid hydrolases was investigated with a combined histochemical and biochemical study. Specimens of m. soleus, m. plantaris, m. gastrocnemius and diaphragm of normal and of vitamin E deficient rabbits were used. For the histochemical investigation, activity and localization of acid phosphatase, -glucuronidase, leucine aminopeptidase and E₆₀₀ resistant non-specific aryl-esterase were examined with semipermeable membrane techniques. For the biochemical investigation, activity of acid phosphatase, -glucuronidase, cathepsin D, acid maltase and neutral maltase was determined.By means of statistical calculations the enzyme activities demonstrated with histochemical techniques were compared with the enzyme activities determined with biochemical techniques.In the present communication the histochemical findings are reported and discussed. From the histochemical findings it appeared that activity of the acid hydrolases investigated is strongly increased in both a granular and a diffuse pattern in skeletal muscle of vitamin E deficient rabbits. The statistical calculations of the histochemical findings clearly reveal that the increased activity of one acid hydrolase was highly significantly paralleled by an increased activity of a second acid hydrolase. Moreover the probability that the activity of all other histochemically studied acid hydrolases was significantly increased was rather high.The increase in activity of the acid hydrolases studied was the same in muscles with an aerobic or an anaerobic metabolism. Moreover there was no difference in activity and localization of the acid hydrolases in aerobic type I and anaerobic type II fibres.The localization of acid phosphatase and -glucuronidase activity in muscle fibres mostly coincided. In cases where these enzymes were localized both centrally and in the subsarcolemnal areas of the muscle fibres, the activity of E₆₀₀ resistant naphtholesterase was usually, and the activity of leucine aminopeptidase was exclusively located in the subsarcolemnal areas. All of the examined acid hydrolases were found to be present in the inflammatory exudate and in the connective tissue.This study was partly extracted from the Ph. D. thesis of D.E. Israël (1977).     

Leishmania mexicana: a cytochemical and quantitative study of lysosomal enzymes in infected rat bone marrow-derived macrophages

The cellular localization and activity of the lysosomal enzymes acid phosphatase, trimetaphosphatase, and arylsulfatase were studied in rat bone marrow-derived macrophages infected with Leishmania mexicana amazonensis amastigotes. The specific activity of acid phosphatase normalized for protein content was similar in normal macrophages and in isolated amastigotes, whereas the latter were markedly deficient in trimetaphosphatase and arylsulfatase activities. It is thus likely that trimetaphosphatase and arylsulfatase activities detected in infected macrophages were of host cell origin. The activities of the three enzymes, assayed biochemically, varied independently in the infected macrophages. While arylsulfatase activity was unchanged after infection, the activity of acid phosphatase increased by 19, 40, and 94% at 6, 24, and 48 hr, respectively. Trimetaphosphatase activity rose only slightly during the first 24 hr after infection but increased by 74% at 48 hr. The rise in acid phosphatase activity could be accounted for only partially by multiplication of the amastigotes. Thus, as for trimetaphosphatase, these results suggest enhanced macrophage synthesis of acid phosphatase and/or reduced enzyme degradation by the infected macrophages. The reduction in host cell lysosomes previously described (Ryter et al. 1983; Barbieri et al. 1985) was confirmed but appearance of lysosomal enzyme activity in the parasitophorous vacuole is documented in the present report. Thus, Leishmania do not seem to reduce the amount and the activity of host lysosomal enzymes.     

Biochemical analysis of jejunal brush border membrane of golden hamster: pathogenic modulations due to ancylostomiasis.

The common hookworm (Ancylostoma ceylanicum) infection of humans was studied in golden hamsters model system. Significant biochemical modulations were observed in hamster jejunal brush border membrane (BBM), the primary site of infection. Analysis of BBM at the peak of infection (3-weeks) revealed a marked decrease in the activities of sucrase, lactase and maltase, while activities of alkaline phosphatase, (Ca2+ + Mg2+)-ATPase and gamma-glutamyl transpeptidase were increased. Kinetic studies conducted with maltase, a superficially localised enzyme of jejunal BBM, revealed loss of enzyme active site during the infection. Among other constituents, the levels of cholesterol and triglycerides were significantly decreased with slight increase in phospholipid content in the infected animals. The hookworm infection also caused a decline in total hexose content indicating an altered membrane glycocalyx. Conversely, there was significant enhancement of hydroxyproline and sialic acid contents. SDS-PAGE analysis showed an enhancement in both low and high molecular weight proteins in jejunal BBM preparations of the infected group. Gel electrophoresis of glycoproteins further revealed the appearance of two additional peaks in the low molecular weight region and concomitant disappearance of a peak in the high molecular weight region. These results strongly support the view that the hookworm infection causes severe damage not to the site of attachment alone but also to the entire cell lining of the jejunum and therefore could influence overall digestion and absorption.     

Effect of enterotoxigenic Escherichia coli heat stable toxin on intestinal lysosomal enzymes in the suckling rat

A preliminary study on 9 suckling Wistar rats, which received E. coli stable toxin, and on 12 sham-operated controls showed that acid phosphatase, the marker enzyme for lysosome, was significantly increased in the infected group whereas alkaline phosphatase, glucose 6-phosphatase, succinic dehydrogenase, and proteinase, the marker enzymes for brush border, microsome, mitochondria, and the soluble fraction, respectively, remained unaffected. The results suggest that lysosome, the subcellular organelle responsible for intracellular digestion could be modified by E. coli stable toxin. In another set of experiments, where 7 infected suckling rats and 7 sham-operated controls were used, the maximal activities of lysosomal enzymes (released by Triton X-100) were found to be increased in the infected group confirming the results obtained in the preliminary experiment. The values of the ratio between maximal and basal activity (an expression of the degree of retention of enzymes to lysosome) of acid phosphatase and cathepsin D were also significantly increased, indicating that lysosomal membrane may also be stabilized during the infection. The increased activities of lysosomal enzymes and the increased lysosomal membrane stability suggest that intracellular digestion by lysosome could be increased during E. coli stable toxin infection.     

Studies on the enzymology of purified preparations of brush border from rabbit kidney

1. A method for the preparation of brush border from rabbit kidneys is described. Contamination by other organelles was checked by electron microscopy and by the assay of marker enzymes and was low. 2. Seven enzymes, all hydrolases, were substantially enriched in the brush-border preparation and are considered to be primarily located in this structure. They are: alkaline phosphatase, maltase, trehalase, aminopeptidase A, aminopeptidase M, gamma-glutamyl transpeptidase and a neutral peptidase assayed by its ability to hydrolyse [(125)I]iodoinsulin B chain. 3. Adenosine triphosphatases were also present in the preparation, but showed lower enrichments. 4. Alkaline phosphatase was the most active phosphatase present in the preparation. The weak hydrolysis of AMP may well have been due to this enzyme rather than a specific 5'-nucleotidase. 5. The two disaccharidases in brush border were distinguished by the relative heat-stability of trehalase compared with that of maltase. 6. The individuality of the four peptidases was established by several means. The neutral peptidase and aminopeptidase M, both of which can attack insulin B chain, differed not only in response to inhibitors and activators but also in the inhibitory effect of a guinea-pig antiserum raised to rabbit aminopeptidase M. This antiserum inhibited both the purified and the brush-border activities of aminopeptidase M. The neutral peptidase and gamma-glutamyl transpeptidase were unaffected but aminopeptidase A was weakly inhibited. The characteristic responses to Ca(2+) and serine with borate served to distinguish aminopeptidase A and gamma-glutamyl transpeptidase from other peptidases. 7. No dipeptidases, tripeptidases or carboxypeptidases were identified as brush-border enzymes. 8. Incubation of brush border with papain released almost all the aminopeptidase M activity but only about half the activities of maltase, gamma-glutamyl transpeptidase and aminopeptidase A. No release of alkaline phosphatase, trehalase or the neutral peptidase was observed.     

In vitro study of the intestinal brush border enzyme activities in developing anuran amphibian: effects of thyroxine, cortisol, and insulin

The effects of several hormones on intestinal brush border membrane enzymatic activities have been investigated in intestinal explants taken from the amphibian midwife toad at different developmental stages. Explants were treated for at least 2 days with thyroxine (0.1 microgram/ml of culture medium) or for 2 days with cortisol (25 micrograms/ml) or insulin (6 mU/ml). The hydrolases examined were maltase, trehalase, glucoamylase, and alkaline phosphatase. In the explants from tadpoles in prometamorphosis, thyroxine had no effect on hydrolase activities; cortisol increased the activity of only glucoamylase, and insulin increased activity of maltase, glucoamylase, and alkaline phosphatase. When the explants were taken from tadpoles at the beginning of climax, cortisol and insulin generally stimulated the enzyme activities studied. When taken from tadpoles at the end of climax, at the moment when the embryonic cells under the degenerating epithelium divide, cortisol and insulin had little effect on these activities. When the animals terminate their metamorphosis, the intestinal epithelium of the explants is totally newly formed (secondary epithelium). At this time, cortisol stimulated the activities of maltase, glucoamylase, and alkaline phosphatase, while insulin decreased the activities of maltase and glucoamylase.     

Activities of brush border membrane bound enzymes of the small intestine in Metagonimus yokogawai infection in mice

The present study intended to evaluate the influences of Metagonimus yokogawai on the activities of brush border membrane bound enzymes of the small intestine. Mice were infected with 500 metacercariae respectively, and the worm recovery, morphological changes and enzyme activities were observed chronologically. A part of them were followed after the treatment. Recovered worms decreased in number continuously after the infection, and they were less than 10% after 2 weeks and almost zero after 28 weeks. Villous atrophy and stromal inflammation were found at two locations of the proximal jejunum from 2 weeks to 4 weeks after the infection. The enzymes, alkaline phosphatase, leucine aminopeptidase and disaccharidases (sucrase, lactase, maltase, and trehalase), showed lowered activities in the duodenum and proximal jejunum of the infected mice but they increased in the distal jejunum for the first two weeks. From three weeks after the infection, the activities were gradually recovered. In one week treated mice, they recovered the activities at 2 weeks from the treatment, but there found no differences of the activities between the 3 week treated group and infected controls. The present data reveal that M. yokogawai infection induces degenerative changes of the host's intestinal mucosa not only morphologically but functionally during the initial phase of infection. The lowered enzyme activities in acute metagonimiasis should be associated with malabsorption and diarrhea.     

Binuclear metal centers in plant purple acid phosphatases: Fe-Mn in sweet potato and Fe-Zn in soybean.

Purple acid phosphatases comprise a family of binuclear metal-containing acid hydrolases, representatives of which have been found in animals, plants, and fungi. The goal of this study was to characterize purple acid phosphatases from sweet potato tubers and soybean seeds and to establish their relationship with the only well-characterized plant purple acid phosphatase, the FeIII-ZnII-containing red kidney bean enzyme. Metal analysis indicated the presence in the purified sweet potato enzyme of 1.0 g-atom of iron, 0.6-0.7 g-atom of manganese, and small amounts of zinc and copper. The soybean enzyme contained 0.8-0.9 g-atom of iron, 0.7-0.8 g-atom of zinc per subunit, and small amounts of manganese, copper, and magnesium. Both enzymes exhibited visible absorption maxima at 550-560 nm, with molar absorption coefficients of 3200 and 3300 M(-1) cm(-1), respectively, very similar to the red kidney bean enzyme. Substrate specificities were markedly different from those of the red kidney bean enzyme. A cloning strategy was developed based on N-terminal sequences of the sweet potato and soybean enzymes and short sequences around the conserved metal ligands of the mammalian and red kidney bean enzymes. Three sequences were obtained, one from soybean and two from sweet potato. All three showed extensive sequence identity (>66%) with red kidney bean purple acid phosphatase, and all of the metal ligands were conserved. The combined results establish that these enzymes are binuclear metalloenzymes: Fe-Mn in the sweet potato enzyme and Fe-Zn in soybean. The sweet potato enzyme is the first well-defined example of an Fe-Mn binuclear center in a protein.     

Characteristics of lysosomes in the rat placental cells

Six acid hydrolases, cytochrome oxidase, and alkaline phosphatase were demonstrated in 0.25 m sucrose homogenates of rat chorioallantoic placenta. The acid hydrolases were: acid phosphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, β-galactosidase, and acid deoxyribonuclease, showing optimum activity near pH 4.5; cathepsin, with optimum activity near pH 3.6. The free acid hydrolases present in cytoplasmic extracts expressed 20–40% of their total activity. “Total” activity was defined as the enzyme activity observed in the presence of Triton X-100, while “free” activity denoted enzyme activity measured under similar assay conditions except in the presence of sucrose and absence of Triton X-100. The decreased activity or latency in the assays for the free activity of acid phosphatase, acid deoxyribonuclease, and cathepsin persisted after incubation at pH 5 and 37 ° up to an hour. In contrast, this latency did not persist after incubation of the β-glycosidases. Additionally, the free activity of all the designated enzymes of the cytoplasmic extract was in excess of the nonsedimentable activity observed.     

Age-related changes in enzyme activities, protein content and lipid composition of rat kidney brush-border membrane

Age-related changes in enzyme activities, protein electrophoretic patterns and lipid composition of kidney-brush-border membranes were studied in 10-20- and 30-month-old male and female Wistar rats. Polyacrylamide gel electrophoresis of membrane proteins revealed very little changes with increasing age, whether males or females were considered. The Km of three hydrolases - maltase, L-aminopeptidase and alkaline phosphatase - were not affected by age while the Vmax of maltase and alkaline phosphatase, but not of L-aminopeptidase, decreased from 10 to 30 months. The phospholipid to protein ratio which remained constant between 10 and 20 months, rose in both sexes from 20 to 30 months. In males, the cholesterol content of the membrane increased faster than that of phospholipid and the cholesterol over phospholipid ratio was then greater at 30 months than at 10 months, while in females this ratio remained unchanged in the course of aging. The fatty acid composition of the brush-border remained more or less constant with age in female rat whereas in the male, a 10% decrease in the proportion of arachidonic acid from 10 to 30 months was responsible for a lower unsaturation index.     

Histochemical and biochemical studies on the localization and activity of lysosomal enzymes in fracture callus

Summary Quantitative biochemical studies on the activities of four lysosomal hydrolases during different stages of fracture healing in the rat were performed, and the results obtained were integrated with those of histochemical observations relating to changes in the localization of acid phosphatase in the same tissue.The findings showed presence of all the four lysosomal enzymes assayed in the callus; during early callus formation the enzyme activities calculated on a DNA basis increased up to about 12 days after the fracture. The enzyme activities appeared to be roughly reflected histochemically by the acid phosphatase staining. The increasing activity during early callus formation seemed to depend on the presence of numerous macrophage-like cells in the tissue containing many large lysosomes. A decrease in enzyme activity was found after day 12. Comparison with the histochemical and ultrastructural findings suggested that this decrease was due to a reduction in the number of macrophage-like cells and a concomitant increase in osteogenic cells with a lower enzyme content.     

Characterization of Digestive Enzymes in the Intestine of the Pacific Halibut Hippoglossus stenolepis Schmidt, 1904 and the Starry Flounder Platichthys stellatus (Pallas, 1788)

The activity and temperature characteristics of hydrolases (maltase, α-amylase, total proteinase, alkaline phosphatase) in the intestine of Far Eastern flatfishes were studied. The character of the temperature dependence differed among the investigated enzymes. The activity of individual hydrolases in the two fish species varied. The results of this study suggest that flatfish intestinal hydrolases are efficient at low temperatures and that the digestive process in flatfishes is well adapted to the conditions in which it functions, thus ensuring optimal vital activity at low temperatures in the Far Eastern seas.Original Russian Text Copyright © 2005 by Biologiya Morya, Korostelev, Nevalenny, Levchenko.     

Binding of hydrolases from wheat aleurone to concanavalin A- and wheat-germ agglutinin-sepharose

We have studied the ability of hydrolases (acid phosphatase and glycosidases) from the aleurone layers of resting wheat grains to interact with Con A- and WGA-Sepharose as a way to examine their glycoprotein nature. Aliquots (6–85% depending on the enzyme) of all the enzymes interacted with Con A-Sepharose. The major part of α-mannosidase activity (85%) was present in this form. Aliquots (2–20% depending on the enzyme) of the following four enzymes, β-galactosidase, α-mannosidase, β-N-acetylglucosaminidase and acid phosphatase, interacted with WGA-Sepharose. All the enzymes were found in forms which were unable to interact with either lectin. No forms of hydrolases interacting with both lectins were found in the crude extract. The specific activities of most of the enzymes recovered from the lectin-Sepharose gels were greater than those measured in the crude extract. In particular, the highest specific activities were found for β-N-acetylglucosaminidase and β-galactosidase recovered from WGA-Sepharose. Different lectin-binding forms of hydrolases were compared with respect to pH optimum and stability under various conditions (heat and guanidine hydrochloride treatments). The lectin-binding pattern of the hydrolases released in the incubation medium by the aleurone layers was similar to that reported above for the enzymes extracted from these tissues, suggesting that none of the hydrolase forms found in the aleurone layers is selectively released during incubation of these tissues.     

Subcellular distribution of digestive enzymes in Ascaris suum intestine

Van den Bossche H. and Borgers M. 1973. Subcellular distribution of digestive enzymes in Ascaris intestine. International Journal for Parasitology3: 59–65. The microvilli of the intestinal cells of Ascaris suum resemble the microvilli of the mammalian intestine in respect to their morphologic structure; their behaviour to homogenization in the presence of a chelating agent; the presence of the disaccharide hydrolases, maltase, sucrase and trehalase and the presence of an enzyme which hydrolyses 5′-AMP at neutral pH. The microvilli of the Ascaris intestinal cells differ completely from those present in mammalian intestine in respect to the presence of non-specific phosphatases. The brush border fraction contains the bulk of acid phosphatase present in the intestinal cells. Although some pinocytotic vesicles have been observed only low endocytotic activity was found. We therefore suggest that the acid hydrolases found on the brush border membrane may be functionally related to extracellular digestion of macromolecules.     

The Histochemical Localization of Several Enzymes of Soybeans Infected with the Root-knot Nematode Meloidogyne incognita acrita

The sites of activity of alkaline phosphatase, acid phosphatase, esterase, peroxidase, adenosine triphosphatase, and cytochrome oxidase were demonstrated histochemically in fresh sections of ''Lee'' soybeans infected by the root-knot nematode Meloidogyne incognita acrita. Each of the six enzymes was more active at the sites of infection than in adjacent non-infected tissue. During the early stages of infection, an increase in enzyme activity was observed in several cells in the proximity of the lip region of the nematode. However, when definite syncytia were observed, increased enzyme activity was confined primarily within the limits of the syncytium. Increased activity paralleled syncytial development and nematode maturation.     

Changes in Two Acid Hydrolase Levels During Cyst Differentiation of a Ciliate,Histriculus muscorum1

Cellular levels of protein and two acid hydrolases, acid phosphatase (EC 3.1.3.2) and acid proteinase, were followed during cyst differentiation, arbitrarily divided into five stages, in the ciliate Histriculus muscorum Kahl. Extracellular enzyme activities were also measured. Protein content decreased gradually during cyst differentiation. In mature cysts the protein content was ca. 60% that of stationary phase organisms. The activities of both acid hydrolases remained unchanged during stage 1 and then decreased gradually; acid proteinase decreased more rapidly. Both enzymes remained slightly active in the mature cysts. The acid proteinase activity of stage 1 was reduced by cycloheximide treatment at time zero, whereas the enzyme was no longer sensitive to the inhibitor when treated at 1.5 h (late stage 1) after the first wash with encysting medium. Acid phosphatase activity was insensitive to the inhibitor. Extracellular release of acid phosphatase increased linearly at least until stage 5, although the extracellular release of acid proteinase was not detected. Cycloheximide blocked the extracellular release of acid phosphatase after stage 1. These results suggest that de novo synthesis of acid proteinase occurs during stage 1 and that lysosomes may play an important role during early stages of cyst differentiation.     

Enzymic solubilization of the human intestinal brush border membrane enzymes

The releases of proteins, maltase, lactase, sucrase, trehalase, alkaline phosphatase, γ-glutamyltransferase and leucylnaphthylamide-hydrolyzing activity from human intestinal brush border membrane vesicles by various enzymes (especially pancreatic proteases) have been studied.The brush border membrane enzymes are not solubilized by digestion with trypsin and chymotrypsin but are largely released after treatment with papain or elastase. Most of the enzymes are fully active after the proteolytic treatment. All proteins released by papain and elastase have been identified by electrophoresis to already known intestinal hydrolases.Electron microscopy of brush border membrane vesicles demonstrates “knob-like” structures (particles) attached to the external side of the membrane. During papain treatment, enzyme removal runs parallel with the disappearance of the particles. During elastase treatment it is not possible to correlate the release of th enzymic activities with the removal of the particles.The results indicate that most of the intestinal hydrolases are surface components attached to the external side of the membrane. They are in accord with the concept that the brush border membrane enzymes are organized within the membrane in a mosaic-like pattern.     

A comparative biochemical research in the Schistocephalus solidus (Cestoda)--three-spined stickleback Gasterosteus aculeatus L. system

The activity of five lysosomal hydrolases (acid phosphatase, DNAase, RNAase, beta-glucosidase, beta-galactosidase), alkaline phosphatase and aldolase have been examined in tissues of the cestode Schistocephalus solidus (Müller, 1776) and the three-spined stickleback Gasterosteus aculeatus (L.) forming a stable parasite-host system. As a rule, the activity of enzymes was higher in a cestode body than in fish tissues. The acid and alkaline phosphatases were the exception. The activity and variation of lysosomal nucleases and aldolase in the parasite differed notably from those in both infested and healthy hosts. The paper discusses the role of lysosomal and cytoplasmic enzymes in a cestode adaptation to parasitism, as well as in the mechanisms of the host's chemical and immunological response to infection.     

Enzymic solubilization of the human intestinal brush border membrane enzymes.

The releases of proteins, maltase, lactase, sucrase, trehalase, alkaline phosphatase, gamma-glutamyltransferase and leucylnaphthylamide-hydrolyzing activity from human intestinal brush bborder membrane vesicles by various enzymes (especially pancreatic proteases) have been studied. The brush border membrane enzymes are not solubilized by digestion with trypsin and chymotrypsin but are largely released after treatment with papain or elastase. Most of the enzymes are fully active after the proteolytic treatment. All proteins released by papain and elastase have been identified by electrophoresis to already known intestinal hydrolases. Electron microscopy of brush border membrane vesicles demonstrates "knob-like" structures (particles) attached to the external side of the membrane. During papain treatment, enzyme removal runs parallel with the disappearance of the particles. During elastase treatment it is not possible to correlate the release of the enzymic activities with the removal of the particles. The results indicate that most of the intestinal hydrolases are surface components attached to the external side of the membrane. They are in accord with the concept that the brush border membrane enzymes are organized within the membrane in a mosaic-like pattern.