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At least 19 recordsLinked to original sources
PubMed1995

Design and expression of organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase.

Serine esterases and proteases are rapidly and irreversibly inhibited by organophosphorus (OP) nerve agents. To overcome this limitation, we selected several residues that were predicted to be within 3-10 A of both the active site Ser O gamma and the oxyanion hole of human butyrylcholinesterase for mutation to His (G115H, G117H, Q119H, and G121H). In remarkable contrast with wild-type (WT) and all other His mutants tested, G117H underwent spontaneous reactivation following OP inhibition to regain 100% of original esterase activity with maximum k3 values of approximately 6.8 x 10(-5) and 16 x 10(-5) s-1 for GB (sarin) and VX, respectively, in 0.1 M Bis-Tris, 25 degrees C. The free energy of activation for k3 was 19 kcal mol-1, and measurement of pH dependence suggested that reactivation resulted from an acidic group with pKa 6.2. To evaluate further the importance of His in achieving this result, we changed the same Gly to Lys (G117K) and compared its substrate and inhibitor kinetics with those of G117H. Both mutants retained esterase activity with Km values similar to those of WT for neutral ester hydrolysis, but G117K did not reactivate. Complete reactivation proves that G117H is not irreversibly inhibited but instead functions as a catalyst for OP hydrolysis. Dephosphonylation is the rate-limiting step, and G117H effects overall rate constant enhancements of approximately 100- and 2000-fold above the uncatalyzed hydrolysis of GB and VX, respectively, at pH 6.0, 25.0 degrees C. We conclude that an appropriately positioned imidazolium ion in the oxyanion hole catalyzes dephosphonylation and, thereby, confers a novel organophosphorus acid anhydride hydrolase activity upon butyrylcholinesterase.

Acid Anhydride Hydrolases↗
PubMed1998

Organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase: synergy results in a somanase.

Organophosphorus acid anhydride (OP) "nerve agents" are rapid, stoichiometric, and essentially irreversible inhibitors of serine hydrolases. By placing a His near the oxyanion hole of human butyrylcholinesterase (BChE), we made an esterase (G117H) that catalyzed the hydrolysis of several OP, including sarin and VX [Millard et al. (1995) Biochemistry 34, 15925-15930]. G117H was limited, however, because it was irreversibly inhibited by pinacolyl methylphosphonofluoridate (soman); soman is among the most toxic synthetic poisons known. This limitation of G117H has been overcome by a new BChE (G117H/E197Q) that combines two engineered features: spontaneous dephosphonylation and slow aging (dealkylation). G117H/E197Q was compared with the single mutants BChE G117H and E197Q. Each retained cholinesterase activity with butyrylthiocholine as substrate, although kcat/Km decreased 11-, 11- or 110-fold for purified G117H, E197Q, or G117H/E197Q, respectively, as compared with wild-type BChE. Only G117H/E197Q catalyzed soman hydrolysis; all four soman stereoisomers as well as sarin and VX were substrates. Phosphonylation and dephosphonylation reactions were stereospecific. Double mutant thermodynamic cycles suggested that the effects of the His and Gln substitutions on phosphonylation were additive for PSCR or PRCR soman, but were cooperative for the PSCS stereoisomer. Dephosphonylation limited overall OP hydrolysis with apparent rate constants of 0.006, 0.077, and 0.128 min-1 for the PR/SCR, PSCS, and PRCS soman stereoisomers, respectively, at pH 7.5, 25 degrees C. We conclude that synergistic protein design converted an archetypal "irreversible inhibitor" into a slow substrate for the target enzyme.

Amino Acid Substitution↗
PubMed1997

A single amino acid substitution, Gly117His, confers phosphotriesterase (organophosphorus acid anhydride hydrolase) activity on human butyrylcholinesterase.

The G117H mutant of human butyrylcholinesterase (EC 3.1.1.8) was expressed in Chinese hamster ovary cells. Substitution of Gly 117 with His to make the G117H mutant endowed butyrylcholinesterase with the ability to catalyze the hydrolysis of organophosphate esters. G117H was still able to hydrolyze butyrylthiocholine, benzoylcholine, and o-nitrophenyl butyrate, but in addition it had acquired the ability to hydrolyze the antiglaucoma drug echothiophate and the pesticide paraoxon. Wild-type butyrylcholinesterase was irreversibly inhibited by echothiophate and paraoxon, but G117H regained 100% activity within 2-3 min following reaction with these compounds. On a polyacrylamide gel, the same bands that stained for activity with butyrylthiocholine also stained for activity with echothiophate. G117H is the only enzyme known that hydrolyzes echothiophate. Diethoxyphosphorylated G117H aged with a half-time of 5.5 h, a rate 600 times slower than the rate of hydrolysis. Echothiophate and paraoxon were hydrolyzed with the same kcat of 0.75 min-1. This calculates to a rate acceleration of 100,000-fold for hydrolysis of echothiophate and paraoxon by the G117H mutant compared to the nonenzymatic rate.

Animals↗
PubMed1980

Cytochemical localization of acid phosphatase and trimetaphosphatase activities in exocrine acinar cells.

Acid phosphatase activity, a lysosomal marker, is commonly demonstrated using the Gomori technique with cytidine 5'-monophosphate or beta-glycerophosphate as substrate. Using this lead capture method on mouse and rat exorbital lacrimal, parotid, and pancreatic acinar cells, reaction product was localized in GERL, forming secretory granules, and secondary lysosomes. However, a different cytochemical localization was observed for inorganic trimetaphosphatase, another lysosomal enzyme. When the technique for trimetaphosphatase activity, a metal chelation method, was applied to exocrine acinar cells, reaction produce was conspicuously absent from GERL and forming secretory granules, but was present in secondary lysosomes, occasionally in Golgi saccules, and in previously unreported basal elongated lysosomes. The differences in the localization of the two enzymatic activities emphasizes the importance of employing more than one substrate where possible, and raises questions concerning the mechanism of delivery of acid hydrolases to secondary lysosomes.

Acid Anhydride Hydrolases↗
PubMed1999

Macrophage responses to Toxoplasma antigens in vitro: a possible role in inflammatory lesions in toxoplasmosis.

Toxoplasma antigen and Toxoplasma immune complex were shown to induce increased production and release of acid hydrolases from macrophage cell line P388D in a concentration-dependent manner. Antigen concentrations of 10-50 microg/ml gave a 2-4-fold increase in the activities of acid proteinase, acid phosphatase, and phospholipase A2 compared with control cells without antigen. Results were similar for immune complex concentrations of 30-80 microg/ml compared with controls. No significant lactate dehydrogenase activity was detected in the culture medium, indicating that enzyme release was selective and not due to cell death. These results suggest that increased release of acid hydrolases may play a role in the inflammatory lesions observed in Toxoplasma encephalitis.

Acid Anhydride Hydrolases↗
PubMed1984

The neuronal endoplasmic reticulum: its cytochemistry and contribution to the endomembrane system. II. Axons and terminals.

The morphology and cytochemistry of the endoplasmic reticulum (ER) in axons and terminals of a number of different types of neurons in brains from mice were investigated ultrastructurally. The neurohypophysis received particular attention because the morphology and enzyme cytochemical activities of many of the preterminal swellings of hypothalamo-neurohypophysial axons are altered by chronic salt-stress. Membrane contrast and enzyme cytochemical staining techniques were employed to characterize the axonal reticulum and to determine if organelles representing the lysosomal system in the axon and the tubular profiles participating in the anterograde axonal transport of native horseradish peroxidase (HRP) are associated with the ER. Potential enzyme cytochemical markers for the axonal ER included glucose-6-phosphatase (G6Pase), thiamine pyrophosphatase, nucleoside diphosphatase, and acid hydroxylase activities. The anterograde transport of HRP was analyzed in undamaged hypothalamo-neurohypophysial neurons and in facial and hypoglossal motoneurons of mice receiving the protein in the lateral cerebral ventricle. The ER pervaded the axon and appeared as parallel, 20-40-nm-wide tubules interconnected by oblique anastomoses. Membrane thickness of the axonal reticulum measured 60-100 A, which is similar to that of the perikaryal ER. Enzyme cytochemical activities associated with the ER or lysosomes were not conspicuous in axons and terminals under normal conditions but became prominent in some axons and preterminal swellings manifesting an autophagic appearance within neurohypophyses from salt-stressed mice. Only G6Pase activity was a marker for the ER in these axons and preterminals. Many ER profiles in non-incubated sections and in G6Pase cytochemical preparations of salt-stressed neurohypophyses were wrapped around or interspersed among secretory granules, multilamellar bodies, and vacuoles that may represent forms of lysosomes involved in autophagy and crinophagy. Acid hydrolase activities were localized within the vacuoles as well as within 80-130-nm-wide, blunt-ended tubules in pituitary stalk axons; similar reactive tubules were confluent with large secondary lysosomes in neurosecretory cell bodies and may be derived from these lysosomes. Morphologically identical tubules transporting HRP in the anterograde direction were observed only in the salt-stressed hypothalamo-neurohypophysial neuron. The HRP-positive tubules very likely are affiliated with the lysosomal system.

Acid Anhydride Hydrolases↗
PubMed2004

Earthworm leukocyte populations specifically harbor lysosomal enzymes that may respond to bacterial challenge.

Earthworm leukocytes (coelomocytes) are responsible for innate cellular immune functions such as phagocytosis and encapsulation against parasites and pathogens. Microbial killing results from the combined action of the phagocytic process with humoral immune factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), and various cytotoxic and antimicrobial molecules. There is also evidence of weak adaptive immune responses against foreign transplants. This study focused on aspects of the innate immune response. First, anti-human acid phosphatase (anti-AcP) polyclonal antibody characterized different acid hydrolase patterns in coelomocytes. Second, flow cytometry identified a strongly immunoreactive coelomocyte population. Third, ultrastructural and cytochemical analyses revealed acid phosphatase in discrete granules (lysosomes) of effector hyaline and granular coelomocytes but not in mature chloragocytes. Coelomocytes were exposed to bacteria to assess how phagocytosis influences: (a) the production of acid phosphatase using Western blot, and (b) release of acid phosphatase using ELISA from cell-free coelomic fluid. Fourth, after phagocytosis, acid phosphatase levels differed between controls and experimentals. Fifth, we found a 39-kDa molecule that reacted intensely with anti-AcP. Our results suggest that effector earthworm coelomocytes may not eliminate pathogens only by phagocytosis but also by extracellular lysis.

Acid Anhydride Hydrolases↗
PubMed1981

A new species of lysosome in rabbit polymorphonuclear leukocytes.

A species of lysosome containing an acid hydrolase activity, i.e., acid trimetaphosphatase (TMPase) was demonstrated in rabbit polymorphonuclear leukocytes (PMNs). The lysosome was less reactive near neutral pH and non-reactive at alkaline pH. The structure appeared typically ovoid, unlike the "tubular lysosomes" reported by Oliver (J Histochem Cytochem 28:78, 1980) in several exocrine acinar cells and also observed in monocytes in this study. Neither was there Golgi activity, although there are probably few, if any, Golgi elements present in the mature neutrophil. It is interesting that the TMPase-containing lysosome occurs in an inclusion the size of a specific granule at a greater frequency than the nonspecific acid phosphatase-containing lysosome.

Acid Anhydride Hydrolases↗
PubMed1987

Ultrastructural localization of lysosomal enzymes in the egg cortex of Brachydanio.

The localization of acid phosphatase (E.C. 3.1.3.2), inorganic trimetaphosphatase (E.C. 3.6.1.2), and aryl sulfatase (E.C. 3.1.6.1) in the cortex of unactivated and activated eggs of Brachydanio was examined by ultrastructural cytochemistry. Using a lead capture method, activity for all three acid hydrolases was demonstrated in organelles of the cortex before and after egg activation. Acid phosphatase (AcPase) reaction product was consistently present in primary lysosomes, secondary lysosomes, multivesicular bodies, and yolk bodies. AcPase activity was absent from mitochondria, profiles of the endoplasmic reticulum, coated pits of exocytosed cortical granules, and coated vesicles. Although most cortical granules of the mature, unactivated egg were unreactive for this enzyme, a few showed AcPase reaction product. It is not clear whether the AcPase-positive granules might be an immature form of cortical granules or a subpopulation of these organelles with lysosomal properties. Most cisternae of the Golgi apparatus did not stain for AcPase; however, reaction product was occasionally localized in a single cisterna as well as several small vesicles at the inner face of the Golgi. The intensity of the reaction product and the pattern of distribution of trimetaphosphatase (Tm-Pase) activity was very similar to that of AcPase. However, TmPase was never observed in cortical granules. Cortices of unactivated and activated eggs showed less overall aryl sulfatase (ArSase) activity when compared with AcPase and TmPase. The presence of ArSase reaction product in lysosomes and multivesicular bodies confirmed the acid hydrolytic nature of these organelles. AcPase and TmPase, and to a lesser extent ArSase, are adequate markers of a cortical lysosomal system in the danio egg.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Anhydride Hydrolases↗
PubMed1986

Ultrastructural and cytochemical studies of acid phosphatase and trimetaphosphatase in rat peritoneal mast cells developing in vivo.

The ultrastructural and cytochemical features of peritoneal mast cells of the rat were studied. Immature mast cells show specific cytoplasmic granules of different sizes, the smaller ones localized in the Golgi region. The rough endoplasmic reticulum and Golgi apparatus are well developed, and mitochondria are numerous. Nuclei show deep indentations. Acid phosphatase is present in the Golgi saccules, in GERL (Golgi apparatus-endoplasmic reticulum-lysosome) and in some small granules. It is not present in mature granules. Trimetaphosphatase is present in the Golgi saccules, in GERL, in most immature granules and in some mature granules. These enzymes appear to be transported and packaged into granules by the Golgi apparatus, suggesting that the specific mast cell granules may be a form of lysosome. The results of this study are consistent with the hypothesis that peritoneal mast cells may be derived from macrophage-like precursors.

Acid Anhydride Hydrolases↗
PubMed1994

Microglial and astroglial reactions to anterograde axonal degeneration: a histochemical and immunocytochemical study of the adult rat fascia dentata after entorhinal perforant path lesions.

The reaction of microglial and a stroglial cells to anterograde axonal degeneration was studied in the fascia dentata of adult rats at various timepoints after removal of the entorhinal perforant path projection. Microglial cells were identified by histochemical staining for nucleoside diphosphatase (NDPase) at light and electron microscopical levels. Astroglial cells were stained immunocytochemically for glial fibrillary acidic protein (GFAP). Activated astroglial cells and some microglial cells also stained immunocytochemically for the intermediate filament protein vimentin. Phagocytotic activity was detected by histochemical staining for acid phosphatase. The postlesional connective reorganization of the cholinergic septohippocampal projection was monitored by histochemical staining for acetylcholinesterase. Twenty-four hours after entorhinal cortex ablation, microglial cells in the perforant path zones of the fascia dentata and the adjacent neuropil reacted by shortening and coarsening of processes and an increase in NDPase reactivity. These changes occurred prior to a noticeable increase in GFAP immunoreactivity and hypertrophy of astroglial cells (first evident on postlesional day 2) or sprouting of cholinergic septohippocampal fibres (first evident on day 3). There was evidence of an early, local proliferation of microglial cells in the denervated perforant path zones and migration into these zones of microglial cells from adjacent intact areas. The specific accumulation of strongly stained microglial cells within the denervated parts of the dentate molecular layer persisted for at least 4 weeks, while the astroglial reaction subsided at 3 weeks. The results demonstrate an early activation of microglial cells by axonal degeneration, and indicate that these cells may play a pivotal, inductive role in the subsequent glial and neural events.

5'-Nucleotidase↗
PubMed1986

Ultrastructural ultracytochemical investigation of human gastric carcinomas.

We describe the ultrastructure of various types of gastric carcinoma cells as well as their histochemical properties as visualized at the electron-microscope level. The histochemical properties of tumour cells were compared with those of homologous normal epithelial cells. The localization and activity of ATPase, IDPase, acidic phosphatase and alkaline phosphatase as well as of the oxidoreductases (cytochrome oxidase, succinate dehydrogenase and NADH-dehydrogenase) were studied. Our findings demonstrated that, in tumour cells, a complicated process of structural-functional restructuring takes place. It seems that a number of ultracytochemical properties may be preserved or may disappear altogether; also, such properties may become enhanced or weaker. This heterogeneity of the histochemical properties of tumour cells is discussed with regard to the role of the stem (polypotent) cell in the process of the histogenesis (cytogenesis) of human gastric carcinomas.

Acid Anhydride Hydrolases↗
PubMed1987

Histochemical studies of the differentiation of microglial cells in the cerebral hemispheres of chick embryos and chicks.

Using histochemical procedures to reveal the presence of nucleoside diphosphatase (NDPase), thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase), we investigated the appearance, distribution and ultrastructure of amoeboid and microglial cells in the cerebral hemispheres of chick embryos and young chicks, in order to elucidate the relationship between these two cell populations. On day 6 of incubation, a few round cells exhibiting NDPase, TPPase and AcPase activity were first detected in the thin mantle layer of the cerebral hemisphere. In the corpus striatum, these round cells increased rapidly in abundance until day 13 of incubation, after which their numbers gradually decreased, so that, on day 19 of incubation, they had entirely disappeared. Between day 10 and day 17 or 18 of incubation, round cells were located mainly in the zone of the mantle layer closest to the lumen. On day 10 of incubation, NDPase-, TPPase- and AcPase-positive cells that had a few short cytoplasmic processes (poorly ramified cells) were detected in the intermediate and basal zones of mantle layer. They increased in abundance until day 17 or 18 of incubation and thereafter rapidly decreased in number. Round and poorly ramified cells exhibited NDPase activity on their plasma membranes and in their cytoplasmic vacuoles, with TPPase and AcPase activity being localized within their vacuoles. On day 19 of incubation, NDPase- and TPPase-positive cells with long, well-ramified cytoplasmic processes (well-ramified cells) were observed in the corpus striatum, these being mainly localized in the basal zone. After hatching, these cells increased rapidly in abundance and were distributed throughout the corpus striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Anhydride Hydrolases↗
PubMed1980

Studies on vinblastine-induced autophagocytosis in mouse liver. II. Origin of membranes and acquisition of acid phosphatase.

The origin of the membranes of autophagic vacuoles (AV) and acquisition of acid phosphatase into AV's were studied in vinblastine-induced autophagocytosis (VBL, 50 mg/kg, i.p.) in mouse hepatocytes. Using unbuffered OsO4, very intense staining was observed in the outer cisternae of the Golgi apparatus and also frequently in the cavity between the double membranes obviously destined to form AV's as well as in the cavity between the double membranes of newly formed AV's. There may occur a transformation process in the membranes limiting an AV analogous to that observed at the Golgi cisternae. The transformation of the outer AV membrane occurs independently of fusion with lysosomes. Inosine diphosphatase activity was localized within the cisternae and on the membranes of the endoplasmic recticulum and occasionally within the innermost cisterna of the Golgi apparatus. The results together with the unbuffered OsO4-staining pattern suggest that the membranes of most AV's are derived from the transformed smooth surfaced cisternae of the endoplasmic reticulum which do not have inosine diphosphatase activity. Acid phosphatase activity was localized in lysosomes, occasionally within the innermost cisternae of the Golgi apparatus, between the double membranes of a few newly formed AV's and within most older single membranes of a few newly formed AV's and within most older single membrane-limited AV's. VBL did not prevent the fusion of lysosomes with AV's.

Acid Anhydride Hydrolases↗
PubMed1986

Multivesicular bodies isolated from rat hepatocytes. Cytochemical evidence for transformation into secondary lysosomes by fusion with primary lysosomes.

Plasma lipoproteins (and other ligands) are endocytosed by hepatocytes and appear in multivesicular bodies (MVBs) in the Golgi-lysosome region of the cell prior to their degradation. We have isolated MVB fractions from livers of estradiol-treated rats, permitting studies of their properties (Hornick et al. 1985). Here we report our cytochemical studies of lysosomal enzyme activity in partially and highly purified MVB fractions and in MVBs in hepatocytes in situ. Only about 15% of partially or highly purified MVBs were positive for acid phosphatase and arylsulfatase, consistent with the prelysosomal nature of this compartment. Partially purified MVB fractions contained small round vesicles, 70-120 nm in diameter, which stained intensely for these enzymes; occasionally these vesicles appeared to fuse with MVBs, suggesting that these structures are primary lysosomes. Such stained vesicles were rarely seen in highly purified MVB preparations. Acid phosphatase reaction product with cerium as capture reagent appeared as uniform precipitates surrounding endocytosed plasma lipoproteins in positively stained MVBs. Arylsulfatase reaction product, however, appeared as distinctive arc or plaque-like deposits just inside the MVB-limiting membrane, often in continuity with intense reaction product contained in a fusing primary lysosome. Similar putative primary lysosomes were occasionally observed in isolated, "intact" Golgi fractions from the same livers. Similar histochemical reactivities of MVBs and putative primary lysosomes were observed in thin sections of hepatocytes in situ. These observations support the conclusion that, in hepatocytes, MVBs represent the immediate prelysosomal compartment in the endocytic pathway of macromolecular catabolism, and suggest that MVBs are converted to secondary lysosomes by direct fusion with primary lysosomes arising from closely adjacent Golgi compartments.

Acid Anhydride Hydrolases↗
PubMed1984

Cytochemical study on the effect of aluminum on neuronal Golgi apparatus and lysosomes.

Ultracytochemical reactions for enzymatic markers were applied to study the effect of aluminum on some cell organelles of neurons in the Ammon cortex and spinal cord in rabbits. The results showed that aluminum caused an appearance of secondary lysosomes and an increase in the number of lysosomes. The latter finding was endorsed by statistical analysis of Ammon neurons using a two-sided t-test. Concomitantly, decrease of the intensity of the reaction for thiamine pyrophosphatase (TPPase) and nucleoside diphosphatase (NDPase) in the Golgi apparatus was found. The reaction for NDPase in the endoplasmic reticulum remained unchanged. Cytochemical reactions for alkaline phosphatase and Mg2+-activated adenosine triphosphatase in the plasmalemma of neurons were negative both in control and in aluminum-treated animals. Our data point to the activation of the system of intracellular digestion and suppression of the enzymatic activities (NDPase, TPPase) of the Golgi apparatus in CNS neurons of rabbits treated with aluminum.

Acid Anhydride Hydrolases↗
PubMed1986

Effect of hyperthermia on morphology and histochemistry of spinal cord in the rat.

Studies were performed on mature Wistar strain rats, subjected to 43 degrees C environmental temperature for 4 h at a relative air humidity of 60-70%. Spinal cords of rats sacrificed 1, 24, 48, 72 h and one week after hyperthermia served as a material for the studies. Routinely stained preparations (H+E, Nissl) and enzymatic activity of some phosphatases and esterases was estimated as well in sections subjected to Feulgen reaction karyo- and cytophotometric measurements were performed on cell nuclei of anterior horns neurocytes, anterior columns oligodendrocytes and on anterior funicle astrocytes of the spinal cord lumbar segment. The hyperthermia resulted in rat spinal cords in several morphological and histochemical alterations. Signs of diffuse spinal cord lesion of vascular origin were present with degenerative alterations of neurocytes, oligodendroglia proliferation and astroglia hyperplasia. In histoenzymatic studies changes in enzymatic activity of NsE, AChE, ChE, AcP, ATPase and TPPase were noted. They were dependent upon the time which elapsed after hyperthermia. Karyo- and cytophotometric measurements demonstrated cell nuclei oedema in neurocytes, oligodendrocytes and astrocytes associated with a decrease in the relative DNA level and changes in density and concentration of nuclear chromatin. The observed morphological, histoenzymatic and cytophotometric changes were of a reversible type and majority of them vanished within a week after hyperthermia.

Acetylcholinesterase↗