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At least 19 recordsLinked to original sources
PubMed1979

Differences between microsomal and mitochondrial-matrix palmitoyl-coenzyme A hydrolase, and palmitoyl-L-carnitine hydrolase from rat liver.

Palmitoyl-CoA hydrolase (EC 3.1.2.2) and palmitoyl-L-carnitine hydrolase (EC 3.1.1.28) activities from rat liver were investigated. 1. Microsomal and mitochondrial-matrix palmitoyl-CoA hydrolase activities had similar pH and temperature optima, although the activities showed different temperature stability. They were inhibited by Pb2+ and Zn2+. The palmitoyl-CoA hydrolase activities in microsomal fraction and mitochondrial matrix were differently affected by the addition of Mg2+, Ca2+, Co2+, K+ and Na+ to the reaction mixture. ATP, ADP and NAD+ stimulated the microsomal activity and inhibited the mitochondrial-matrix enzyme. The activity of both the microsomal and mitochondrial-matrix hydrolase enzymes was specific for long-chain fatty acyl-CoA esters (C12-C18), with the highest activity for palmitoyl-CoA. The apparent Km for palmitoyl-CoA was 47 microM for the microsomal enzyme and 17 microM for the mitochondrial-matrix enzyme. 2. The palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase activities of microsomal fraction had similar pH optima and were stimulated by dithiothreitol, but were affected differently by the addition of Pb2+, Mg2+, Ca2+, Mn2+ and cysteine. The two enzymes had different temperature-sensitivities. 3. The data strongly suggest that palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase are separate microsomal enzymes, and that the hydrolysis of palmitoyl-CoA in the microsomal fraction and mitochondria matrix was catalysed by two different enzymes.

Animals↗
PubMed1979

Intracellular localization of long-chain acyl-coenzyme A hydrolase and acyl-L-carnitine hydrolase in brown adipose tissue from guinea pigs.

The activities of long-chain acyl-CoA hydrolase (palmitoyl-CoA hydrolase, EC 3.1.2.2) and long-chain acyl-L-carnitine hydrolase, EC 3.1.1.28) in brown adipose tissue from cold-exposed and control guinea pigs were studied. Mitochondria from cold-exposed animals hydrolysed 21 nmol of palmitoyl-CoA/min per mg of protein and 1.3 nmol of palmitoyl-L-carnitine/min per mg of protein, and the specific activities were respectively 2 and 5 times as high in cold-exposed as in control animals. The subcellular-localization studies showed that both the long-chain acyl-CoA hydrolase and long-chain acyl-L-carnitine hydrolase were localized in the mitochondria. A location also in the soluble fraction cannot be excluded. The long-chain acyl-CoA hydrolase activity was doubled when the mitochondria were disrupted; this indicates that the enzyme is localized in the matrix compartment.

Adipose Tissue, Brown↗
PubMed1977

Identification of acylcholine acyl-hydrolase with carboxylic ester-hydrolase in human serum.

The relationship between pseudocholine esterase [acylcholine acyl-hydrolase, EC 3.1.1.8] and non-specific esterase [carboxylic ester-hydrolase, EC 3.1.1.1] in human serum was investigated. The purified preparation (purified 500-fold) which had both pseudocholine esterase and non-specific esterase activities, was found to give a single band with faint tailing on polyacrylamide gel electrophoresis. The ratio of the specific activity of pseudocholine esterase to that of non-specific esterase remained essentially the same during the purification procedures. Furthermore, the pseudocholine esterase was demonstrated to be identical with the non-specific esterase by immunochemical studies. All these results suggest that activities of pseudocholine esterase and non-specific esterase in human serum derive from the same enzyme molecule. Observation of Yoshida-cho in Ehime after the application of organophosphorus insecticide supported our results: the activity of pseudocholine esterase was found to be reduced with a concomitant decrease in the activity of non-specific esterase. Based on these results, the physiological significance of the esterase is discussed.

Butyrylcholinesterase↗
PubMed1975

Enzymatic hydrolysis of agar: purification and characterization of neoagarobiose hydrolase and p-nitrophenyl alpha-galactoside hydrolase.

The mixture of polysaccharides in the gelling component of agar (agarose) is hydrolyzed to D-galactose and 3,6-anhydro-L-galactose by a series of hydrolytic enzymes obtained from Pseudomonas atlantica. The final degradative step in the pathway of agarose decomposition is the hydrolysis of the alpha-linkage in the dissaccharide neoagarobiose yielding D-galactose and 3,6-anhydro-L-galactose. Pseudomonas atlantica when grown on agar produces two specific enzymes, p-nitrophenyl alpha-galactose hydrolase and neoagarobiose hydrolase. The purification and partial characterization of both enzymes are presented.

Agar↗
PubMed1977

Immunization with dextransucrases, levansucrases, and glycosidic hydrolases from oral streptococci. II. Immunization with glucosyltransferases, fructosyltransferases, and glycosidic hydrolases from oral streptococci in monkeys.

The feasibility of immunizing monkeys with enzymes from oral streptococci in an attempt to reduce dental caries was investigated. Forty rhesus monkeys, Macaca mulatta, were used. Cariogenic streptococci, S mutans, were implanted into all the monkeys' mouths. There was no pathological effect resulting from immunization. Of the 40 animals, 30 retained the implanted flora throughout the experiment; the remaining 10 were reimplanted until the streptococci remained. In six months, gross carious lesions were evident with plaque. Inhibitiors present in the monkey sera after immunization inhibited glucosyltransferase, fructosyltransferase, and neuraminidase activities. It was presumed the inhibitors were antibodies. There was a reduction of 68.6% in the total carious lesions in the animals immunized intraorally with glucosyltransferase, 62.4% reduction in those injected with fructosyltransferase, and 57.4% reduction in total lesions in those immunized with glycosidic hydrolases after 19 months, as compared to the control group. There were no gross lesions apparent in the group immunized with glycosidic hydrolases. It appears that immunization with enzymes significantly reduces carries and is feasible in a primate model.

Animals↗
PubMed1975

Lysosomal hydrolases of the epidermis. 2. Ester hydrolases.

Five distinct ester hydrolases (EC 3-1) have been characterized in guinea-pig epidermis. These are carboxylic esterase, acid phosphatase, pyrophosphatase, and arylsulphatase A and B. Their properties are consistent with those of lysosomal enzymes.

Acid Phosphatase↗
PubMed1975

Lysosomal hydrolases of the epidermis. 3. Peptide hydrolases.

Four distinct peptide hydrolases (EC 3-4) have been characterized in guinea-pig epidermis; these are cathepsin B1, cathepsin C, cathepsin D and arylamidase. Their properties are consistent with those of lysosomal enzymes. Cathepsin E was not detected.

Aminopeptidases↗
PubMed1978

Acid hydrolases in blister fluid. II. Characterization and quantification of glycoside hydrolases.

Seven glycoside hydrolases have been investigated in suction blister fluid, interstitial fluid and in serum. Six of these have been characterized; no differences could be demonstrated between the corresponding enzymes from the various sources. The remaining enzyme (beta-glucosidase) was not found. Quantitative data suggest that 2 enzymes (beta-acetylglucosaminidase and beta-glucuronidase) diffuse freely from the epidermis into blister fluid, whereas 4 (alpha-glucosidase, alpha- and beta-galactosidase and alpha-mannosidase) are almost entirely retained in the roof of the bulla.

Blister↗
PubMed1977

Purification and control of bovine adrenal cortical cholesterol ester hydrolase and evidence for the activation of the enzyme by a phosphorylation.

A procedure for the purification of cholesterol ester hydrolase from bovine adrenal cortical 105000 x g supernatant is described. Preincubation of a crude enzyme extract with [gamma-32P]ATP followed by purification resulted in the isolation of a phosphorylated preparation of cholesterol ester hydrolase. The phosphorylated cholesterol ester hydrolase appeared to be composed of 4 subunits, each having a molecular weight of 41000 +/- 280, only one of which may be phosphorylated. Preincubation of the crude enzyme preparation with [alpha-32P]ATP followed by purification did not produce a phosphorylated preparation of cholesterol ester hydrolase. Cyclic-AMP-dependent protein kinase, cyclic AMP, ATP and magnesium ions were required for activation of purified cholesterol ester hydrolase in vitro and the time course of activation closely paralleled the time course of phosphorylation of the enzyme. The addition of ATP, cyclic AMP and magnesium ions to the bovine adrenal cortical 105000 x g supernatant produced a 2.5-fold stimulation in cholesterol ester hydrolase activity. This stimulation was abolished if protein kinase inhibitor was added prior to the addition of ATP cyclic AMP and magensium ions. The addition of magnesium ions or calcium ions to a crude preparation of cholesterol ester hydrolase was found to inhibit activity; however the same additions made to a purified preparation of cholesterol ester hydrolase were not inhibitory. The decrease in cholesterol ester hydrolase activity on incubation with magnesium ion was accompanied by a loss of 32P radioactivity from the protein. Preincubation of a crude preparation of cholesterol ester hydrolase with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. It is suggested that bovine adrenal cortex cholesterol ester hydrolase is activated by a phosphorylation catalysed by a cyclic-AMP-dependent protein kinase. Deactivation of cholesterol ester hydrolase is accomplished by dephosphorylation catalysed by a phosphoprotein phosphatase, dependent on magnesium or calcium ions.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1976

Purification and properties of an N-formylmethionyl-tRNA hydrolase.

The isolation and properties of a novel N-formylmethionyl-tRNA hydrolase (hydrolase II) from Escherichia coli are described. This enzyme is difficult to detect in crude extracts; purification, however, unmasks the activity. Sedimentation and gel filtration parameters of this enzyme differ from those of the previously described peptidyl-tRNA hydrolase (hydrolase I), and preparations can be obtained where the two activities are free of each other. A mutant of hydrolase I has wild-type levels of hydrolase II. These data indicate that hydrolase II is a different enzyme, or an altogether different form of hydrolase I. The bulk of the enzymic activity occurs in the ribosome-free cytoplasm; the remainder is found on intact or dissociated 70-S ribosomes. Purified preparations of hydrolase II analyzed by two-dimensional gel electrophoresis contain 2 protein bands. These 2 proteins do not coincide in electrophoretic mobility with any known ribosomal proteins. Analysis after mixing experiments verifies this conclusion. The purified enzyme (hydrolase II) is inhibited by ribosomes bearing bound N-formylmethionyl-tRNA. The inhibition is potentiated by sparsomycin and other antibiotics that block specifically peptide-bond synthesis. The relationship of this enzyme to other hydrolytic activities, including a newly described ribosome-dependent hydrolase, are discussed.

Anti-Bacterial Agents↗
PubMed1976

Triglyceride, diglyceride, monoglyceride, and cholesterol ester hydrolases in chicken adipose tissue activated by adenosine 3':5'-Monophosphate-dependent protein kinase. Chromatographic resolution and immunochemical differentiation from lipoprotein lipase.

Hormone-sensitive lipase and cholesterol ester hydrolase of chicken adipose tissue were markedly activated by adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase (on the average, 235 to 275%; occasionally as much as 1000%). Diglyceride and monoglyceride hydrolases were also activated, but to a lesser extent (60 to 87%). The activation of all four hydrolases was inhibited by protein kinase inhibitor and reversed by the addition of exogenous protein kinase. Following activation by cAMP-dependent protein kinase, all four hydrolases were deactivated in a Mg2+-dependent reaction and then reactivated to or near initial levels on incubation with cAMP and Mg2+-ATP. The reversible deactivation is assumed to reflect activity of one or more protein phosphatases. The maximum activation obtainable for the four hydrolases decreased when the tissue had been previously exposed to glucagon, indicating that the glucagon-induced activation was probably similar to or identical with the activation demonstrated in cell-free preparations. The pH optima for the four hydrolase activities were similar (7.13 to 7.38). Although the absolute activities and relative degrees of kinase activation differed according to the particular emulsified substrates used, the results do not rule out the possibility that all four hydrolase activities are referable to a single hormone-sensitive hydrolase. Hormone-sensitive acyl hydrolases were separated from lipoprotein lipase by heparin-Sepharose affinity chromatography. Lipoprotein lipase was active against triolein, diolein, and monoolein, but not cholesterol oleate. Incubation of lipoprotein lipase with exogenous protein kinase, cAMP, and Mg2+ATP had no effect on any of the three hydrolase activities. Lipoprotein lipase was further purified to homogeneity and used to prepare antiserum in rabbits. The immunoglobin G fraction from these antisera completely inhibited lipoprotein lipase eluted from heparin-Sepharose columns. However, the hormone-sensitive hydrolase activities (not retained on heparin-Sepharose affinity chromatography) were not inhibited by anti-lipoprotein lipase immunoglobin G, and anti-lopoprotein lipase immunoglobin G did not affect the activation process in crude fractions. Thus, hormone-sensitive lipase and lipoprotein lipase, functionally distinct enzymes, have been physically resolved and immunochemically distinguished. Apparently lipoprotein lipase activity is not regulated, at least directly, by cAMP-dependent protein kinase.

Adenosine Triphosphate↗
PubMed1978

Extracellular hydrolases of the lung.

A pool of acid hydrolases exists within the acellular lining material of the alveoli and distal airways of the lungs. These extracellular hydrolases, obtained using pulmonary lavage procedures, appear to be of a selected variety insofar as some hydrolases (beta-N-acetylglucosaminidase and alpha-mannosidase) are highly active while others (beta-glucuronidase and arylsulfatase) are barely detectable. The origins of these hydrolases were investigated. Neither leakage of serum nor cell damage can account for the presence of the extracellular hydrolases in lavage effluents. Electrophoretic mobilities on acrylamide gels indicate that the extracellular hydrolases generally differ from those found in serum. Cytoplasmic soluble enzymes such as lactate dehydrogenase were used to monitor cell damage and show that the extracellular hydrolases did not originate from cell leakage during the lavage procedure. Hydrolases similar to those found extracellularly are associated with highly purified lysosome-free lamellar bodies isolated from homogenates of lung. The extracellular hydrolases are probably selected by the type 2 cells of the pulmonary alveolar epithelium during their selection of lamellar bodies.

Animals↗
PubMed1978

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques: a combined histochemical and biochemical investigation 1. The histochemical investigation.

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, beta-glucuronidase, leucine aminopeptidase and E600 resistant non-specific arylesterase were examined with semipermeable membrane techniques. For the biochemical investigation, activity of acid phosphatase, beta-glucuronidase, cathepsin D, acid maltase and neutral maltase was determined. By means of stastical 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 beta-glucuronidase activity 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.

Acid Phosphatase↗
PubMed1977

Immunological properties of medium-chain acyl-thioester hydrolase and fatty acid synthetase from lactating-rabbit mammary gland.

The cytosol from lactating-rabbit mammary gland contains a medium-chain acyl-thioester hydrolase. This hydrolase terminates chain lengthening of the fatty acids synthesised by fatty acid synthetase so as to release C8:0 and C10:0 fatty acids which are characteristic of rabbit milk. The medium-chain hydrolase and the fatty acid synthetase present in this cytosol have been shown to be immunologically distinct. When fatty acid synthetase was purified from this cytosol it showed unexpected immunological reactivity towards antiserum raised to the medium-chain hydrolase. The precipitate formed was not due to fatty acid synthetase, but to medium-chain hydrolase contaminating the synthetase. However, the proportion of this medium-chain hydrolase which was recovered with the purified synthetase was too small to be detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and was too small to elicit an antibody response in sheep. Immunological techniques have shown that the medium-chain hydrolase appears in rabbit mammary gland between days 17 and 22 of pregnancy. This coincides with the onset of milk-fat synthesis. The medium-chain hydrolase could not be detected in the cytosol from lactating-rabbit liver.

Animals↗
PubMed1975

Changes in electronegativity of lysosomal hydrolases during intracellular transport. An isoelectric-focusing study in subcellular fractions of rat kidney.

Isoelectric focusing was used to investigate the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase, beta-galactosidase and beta-N-acetylhexosaminidase in the following, previously characterized subcellular fractions from rat kidney: a special rough microsomal fraction, enriched up to 9-fold over the homogenate in acid hydrolases; a smooth microsomal fraction; a Golgi membrane fraction enriched about 2.5-fold in acid hydrolases and 10- to 20-fold in several glycosyl transferases; and a lysosomal fraction enriched up to 25-fold in acid hydrolases. The electro-focusing behavior of the hydrolases in these fractions was markedly sensitive to the autolytic changes that occur under acidic conditions, even at 4 degrees C. Autolysis was minimized by extracting fractions in an alkaline medium (0.2% Triton X-100, 0.1 M sodium glycinate buffer, pH 10, 0.1 % p-nitrophenyloxamic acid) and adding p-nitrophenyloxamic acid (0.1 %), AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND cathepsin D, to the pH gradient. The enzymes in the lysosomal fraction displayed a characteristic bimodal or trimodal distribution. Arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in an acidic form with an isoelectric point of 4.4, and a basic form with an isoelectric point of 6.2, 6.7 and 8.0, respectively. Acid phosphatase and beta-galactosidase occurred in an acidic, intermediate and basic form with isoelectric points of about 4. 1, 5.6 and 7.4, respectively. In the special rough microsomal fraction these enzymes were mostly in a basic form with isoelectric points between 7.5 and 9; these were 1-2 units higher than the corresponding basic forms in the lysosomal fraction. Treatment of extracts of the rough microsomal fraction with bacterial neuraminidase raised the isoelectric points of all five hydrolases by 1-2.5 units, indicating the presence of some N-acetylneuraminic acid residues in these basic glycoenzymes. The hydrolases in the Golgi fraction were largely in an acidic form with isoelectric points similar to or lower than those of the corresponding acidic components in the lysosomal fraction. The hydrolases in the smooth microsomal fraction showed isoelectric-focusing patterns intermediate between those in the rough microsomal and the Golgi fractions. These findings support the following scheme for the synthesis, transport and packaging of the lysosomal enzymes. Each hydrolase is synthesized in a restricted portion of the r

Acid Phosphatase↗