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PubMed1979

Effect of ethanol on cholesteryl ester hydrolases in adrenal.

An assay of cholesteryl ester hydrolase activity of microsomes from guinea pig adrenal indicated maximal activity at pH at pH 4.0 and 5.5. When ethanol was added to the microsomes, these enzymes showed differences in response with increasing ethanol concentration. Rats administered 15% ethanol as drink for 4--5 months showed a small (20%) increase in cholesteryl ester hydrolase (pH 4.0) activity in adrenal as compared to controls. However, a 50% decrease in cholesteryl ester hydrolase activity resulted when rats were administered ethanol by intragastric tubing technique (15 days) as compared to controls treated similarly with saline. The decrease in cholesteryl ester hydrolase activity was correlated with an increase in cholesteryl ester level in serum. In a different study, rats intubated with an acute dose of ethanol showed an increase in adrenal cholesteryl ester hydrolase activity as early as 1 hr after administration. This effect seemed to last for about 24 hr before activity returned to control level. It is concluded that different modes of ethanol administration to rats may elicit a change in cholesteryl ester hydrolase activity in the adrenal. An increase in cholesteryl ester hydrolase activity may be related to an increase in steroidogenic activity and activation of adrenocortical function.

Adrenal Glands↗
PubMed1984

Characterization of multiple forms of cholesteryl ester hydrolase in the rat testis.

Cholesterol ester hydrolase (EC 3.1.1.13) activity from the 104,000 X g supernatant of rat testis was fractionated into 28-kDa, 72-kDa, and 420-kDa molecular mass forms by high performance size exclusion chromatography. The 72-kDa and 420-kDa forms (temperature-labile) were completely inactivated by elevation of temperature from 32 to 37 degrees C. Apparent disaggregation of the 420-kDa form suggested that the 72-kDa and 420-kDa enzymes are monomeric and multimeric forms of the same enzyme. The 28-kDa form was shown to be a different enzyme (temperature-stable) which retained activity at 37 degrees C. In contrast, cholesteryl ester hydrolase activities from 104,000 X g supernatants of liver or adrenal gland were unaffected and increased 4-fold, respectively, by elevation of temperature from 32 to 37 degrees C. Both testicular enzymes exhibited pH optima at about 7.3, and were activated by sodium cholate at concentrations near the critical micellar concentration (0.03-0.07%), but inhibited by higher concentrations. The temperature-labile cholesteryl ester hydrolase exhibited a high specificity for cholesteryl esters of monoenoic fatty acids of 18-24 carbons, especially nervonate (24:1), whereas the temperature-stable cholesteryl ester hydrolase exhibited highest specificity for cholesteryl oleate and arachidonate. Neither enzyme hydrolyzed cholesteryl acetate, myristate, palmitate, linoleate, or docosahexaenoate . Both enzymes reached maximum rates of hydrolysis at 150 microM substrates, with each substrate and at both reaction temperatures. Substrate inhibition was observed at higher concentrations (200 microM). The temperature-labile cholesteryl ester hydrolase was induced 20-fold in hypophysectomized rats by injection of follicle-stimulating hormone (FSH) and was localized in Sertoli cells, the target cells for FSH, but was not induced by luteinizing hormone. The temperature-stable cholesteryl ester hydrolase was induced by both FSH and LH and was found in both Sertoli cells and Leydig cells, the respective target cells for FSH and luteinizing hormone. Neither form of the enzyme was present at detectable levels in the germinal cells. The unique properties, localization, and hormonal regulation of both temperature-labile and temperature-stable cholesterol ester hydrolases suggest important roles for these enzymes in the testis.

Animals↗
PubMed1983

Identification and characterization of a new epoxide hydrolase from mouse liver microsomes.

A new microsomal epoxide hydrolase (mEH2) has been identified and characterized. This enzyme has properties which distinguish it from previously described cytosolic (cEH) or membrane-bound (mEH1) epoxide hydrolases. The enzyme is an integral microsomal protein which is not dissociated from the membrane by repeated washing, high ionic strength salt, or chaotropic agent solutions, or by sonication. It is very different from the normally described microsomal epoxide hydrolase (mEH1) as shown by its different substrate specificity and kinetic properties and by immunological criteria. In contrast to the hitherto described microsomal epoxide hydrolase, mEH1, the new enzyme effectively catalyzes the hydration of transdisubstituted oxiranes such as trans-stilbene oxide and trans-beta-ethyl styrene oxide and has no appreciable activity toward benzo(a)pyrene 4,5-oxide. It is also structurally distinct, in that it does not cross-react with antibodies raised against the normally described microsomal epoxide hydrolase mEH1. This newly described microsomal epoxide hydrolase probably represents an important factor in the control of reactive epoxides; its location in the membrane ensures access to lipophilic epoxides generated by membrane-bound monooxygenases, and its substrate specificity is such that it can hydrolyze epoxides poorly metabolized by the previously described microsomal epoxide hydrolase.

Animals↗
PubMedDate not supplied

Epoxide hydrolase: a marker for experimental hepatocarcinogenesis.

Rat liver chemical hepatocarcinogenesis, induced by interrupted feeding of 2-acetylaminofluorene, results in various cellular preneoplastic stages and finally in a hepatoma in about 70 to 90 percent of the rats. The putative precursors of hepatomas, called hyperplastic nodules, appear after 12 weeks of feeding and, after 16 weeks of feeding carcinogen, most of them are persistent. Epoxide hydrolase is a tightly bound endoplasmic reticulum enzyme which is strongly induced in hyperplastic nodules and hepatomas. This enzyme has been purified, high-titre rabbit antiserum prepared to it, and this antiserum used to search for epoxide hydrolase immunodeterminants in the sera from chemically induced nodule or hepatoma bearing rats. An enzyme linked immunosorbent assay (ELISA) assay using this antiserum showed significant titres of circulating microsomal epoxide hydrolase antigen, range 0.01 to 2.50 (mean 1.18 +/- 0.30) micrograms per ml, in all 24 hepatoma bearing rats tested. Eight sera from animals with large hyperplastic nodules were also significantly positive for this antigen, while sera from six normal controls were negative (less than 0.004 microgram per ml serum). A passive hemagglutination inhibition assay with sheep or normal rat red blood cells sensitized with pure rat microsomal epoxide hydrolase was capable of detecting 0.2 microgram hydrolase per ml serum. With this assay, sera from four rats with hepatomas were found to contain 0.8 to 1.6 micrograms epoxide hydrolase immunodeterminants per ml. Control rat sera had no detectable immunodeterminants. Thus epoxide hydrolase, a marker induced during experimental chemical hepatocarcinogenesis and called the preneoplastic antigen, has been shown to be circulating in the tumor bearing host.

2-Acetylaminofluorene↗
PubMed1980

Repetitions in the NH2-terminal amino acid sequence of beta-ketoadipate enol-lactone hydrolase from Pseudomonas putida.

Muconolactone delta-isomerase (EC 5.3.3.4) and beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24) mediate consecutive reactions in the beta-ketoadipate pathway of bacteria. An earlier investigation (Yeh, W.K., Davis, G., Fletcher, P., and Ornston, L.N. (1978) J. Biol. Chem. 253, 4920-4923) revealed that the respective NH2-terminal amino acid sequences of Pseudomonas putida muconolactone isomerase and Acinetobacter calcoaceticus beta-ketoadipate enol-lactone hydrolase II are evolutionarily homologous. In this report, we describe the purification of Pseudomonas beta-ketoadipate enol-lactone hydrolase and present evidence indicating that the protein is a trimer composed of identical 11,000-dalton subunits. The NH2-terminal amino acid sequences of Pseudomonas muconolactone isomerase and Pseudomonas enol-lactone hydrolase have diverged widely from each other, yet the two sequences contain different fragments of an ancestral sequence which is represented in Acinetobacter enol-lactone hydrolase II. The widely divergent Pseudomonas muconolactone isomerase and Pseudomonas enol-lactone hydrolase sequences each contain unique sets of repeated peptides. In principle, the repetitive sequences might have been introduced by elongation mutations which occurred early in the evolution of the proteins. However, the divergence of Pseudomonas muconolactone isomerase and Pseudomonas enol-lactone hydrolase is so extreme that the observed sequence repetitions cannot have been conserved from ancestral duplication mutations. Rather, the data favor the interpretation that copies of DNA were substituted into structural genes for the enzymes as they diverged.

Adipates↗
PubMed1980

Evolutionary divergence of co-selected beta-ketoadipate enol-lactone hydrolases in Acinetobacter calcoaceticus.

Muconolactone isomerase (EC 5.3.3.4) and beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24) mediate consecutive catabolic steps in bacteria. Separately inducible beta-ketoadipate enol-lactone hydrolases I and II are formed in representatives of Acinetobacter calcoaceticus. When subjected to DEAE-cellulose chromatography, Acinetobacter enol-lactone hydrolase I displays heterogeneous behavior which, in whole or in part, appears to be due to modifications of sulfhydryl groups in the protein; the enzyme is unusual in that its NH2-terminal amino acid is cysteine. Comparison of the NH2-terminal amino acid sequence of Acinetobacter enollactone hydrolase I, reported here, with the corresponding amino acid sequences of Acinetobacter enollactone hydrolase II and Pseudomonas enol-lactone hydrolase indicates that all three proteins have diverged widely from a common evolutionary origin. Sequence comparisons suggest that divergence of the Acinetobacter enol-lactone hydrolase structural genes was achieved by substitution with DNA derived from an ancestral muconolactone isomerase structural gene.

Acinetobacter↗
PubMed2004

Enzymatic hydrolysis of alpha- and beta-oligo(L-aspartic acid)s by poly(aspartic acid) hydrolases-1 and 2 from Sphingomonas sp. KT-1.

The enzymatic hydrolysis of alpha- and beta-oligo(L-aspartic acid)s by PAA hydrolase-1 and PAA hydrolase-2 (purified from Sphingomonas sp. KT-1) was performed to elucidate the mechanism of the microbial degradation by Sphingomonas sp. KT-1 of the thermally synthesized alpha,beta-poly(D,L-aspartic acid) (tPAA). GPC analysis of the hydrolyzed products of alpha- and beta-tetra(L-aspartic acid)s by PAA hydrolase-1 has showed that PAA hydrolase-1 is capable of hydrolyzing only the specific amide bonds between beta-aspartic acid units. The RP-HPLC analysis of the enzymatic hydrolysis of beta-oligo(L-aspartic acid)s (4 and 5 mers) by PAA hydrolase-1 has suggested that the enzymatic hydrolysis of beta-oligo(L-aspartic acid)s occurs via an endo-mode cleavage. In contrast, PAA hydrolase-2 hydrolyzed both alpha- and beta-oligo(L-aspartic acid)s via an exo-mode cleavage to yield L-aspartic acid as a final product. A kinetic study on the enzymatic hydrolysis of alpha-oligo(L-aspartic acid)s (3 to 7 mers) by PAA hydrolase-2 has indicated that Km values are almost independent of the number of monomer units in oligomers of 4 to 7 mers, while that Vmax values are markedly dependent on the chain length and show a maximum value at 5 mer.

Aspartic Acid↗
PubMed1986

Stimulation of a neutral triacylglycerol hydrolase from rat heart by phosphatidylethanolamine and lysophosphatidylethanolamine.

Triacylglycerol hydrolase activity measured at pH 7.5 in a pH 5.2 precipitate fraction from rat heart was increased two- to three-fold by the presence of phosphatidylethanolamine (PE) or lysophosphatidylethanolamine (LPE). This stimulatory effect also could be obtained in assays with particulate and soluble subcellular fractions and was observed with two different methods of preparing triolein substrate emulsions. Ethanolamine and glycerophosphorylethanolamine had no effect on hydrolase activity, whereas phosphatidylcholine (PC) and acidic phospholipids such as cardiolipin were inhibitory. Palmitic acid, palmityl CoA and palmityl carnitine inhibited PE-stimulated hydrolase activity, but ethyl esters of palmitate had no effect. The preparation of acetone-ether powders resulted in a marked reduction of triacylglycerol hydrolase activity, but PE and LPE now stimulated hydrolase activity by ten-fold or greater, suggesting that these phospholipids may have an obligatory role in modulating triacylglycerol hydrolase activity. Triton X-100 also stimulated hydrolase activity in acetone-ether powders.

Animals↗
PubMed2026

Acyloxyacyl Hydrolase-Mediated Lipopolysaccharide Inactivation Limits Macrophage Endotoxin Tolerance and Promotes Inflammation and Fibrosis in Metabolic Dysfunction-Associated Steatohepatitis.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatohepatitis, a chronic liver disease, is characterized by persistent low-grade inflammation, partially driven by gut-derived lipopolysaccharide. Although repeated lipopolysaccharide exposure can induce endotoxin tolerance in innate immune cells, its role in chronic liver diseases remains unclear. Acyloxyacyl hydrolase is an endogenous enzyme that inactivates lipopolysaccharide, potentially modulating this process. We aimed to investigate how acyloxyacyl hydrolase regulates endotoxin tolerance in Kupffer cells and how this affects hepatic inflammation and fibrosis during metabolic dysfunction-associated steatohepatitis progression. METHODS: Acyloxyacyl hydrolase-deficient mice and wild-type controls were subjected to multiple dietary metabolic dysfunction-associated steatohepatitis models. Inflammatory responses, fibrosis, and transcriptomic changes in liver tissues and isolated Kupffer cells were analyzed. Endotoxin tolerance was modulated through β-glucan administration or lipopolysaccharide preconditioning. Lipopolysaccharide bioactivity was assessed using Toll-like receptor 4-reporter cell assays. RESULTS: Lipopolysaccharide-preconditioned Kupffer cells exhibited reduced proinflammatory cytokine production and transcriptional suppression of inflammatory pathways, indicating tolerance. Despite slight elevation of plasma lipopolysaccharide levels in metabolic dysfunction-associated steatohepatitis, upregulation of hepatic acyloxyacyl hydrolase positively correlated with disease severity, suggesting enhanced lipopolysaccharide inactivation but impaired establishment of tolerance. In contrast, acyloxyacyl hydrolase-deficient Kupffer cells displayed reinforced endotoxin tolerance, leading to diminished hepatic inflammation and fibrosis. Reversal of tolerance using β-glucan reactivated inflammatory and fibrogenic responses in acyloxyacyl hydrolase-deficient mice, whereas tolerance induction by low-dose lipopolysaccharide preconditioning mitigated metabolic dysfunction-associated steatohepatitis pathology, supporting the protective role of macrophage tolerance in chronic liver injury. CONCLUSIONS: Endotoxin tolerance in Kupffer cells represents a protective mechanism against chronic liver inflammation and fibrosis. Acyloxyacyl hydrolase regulates this state by limiting bioactive lipopolysaccharide, thereby modulating the establishment of endotoxin tolerance and downstream inflammatory and fibrotic responses. Enhancing macrophage tolerance by utilizing lipopolysaccharide may offer a novel therapeutic avenue to control the progression of metabolic dysfunction-associated steatohepatitis.

AOAH↗
PubMed2004

Sucrose hydrolases from the midgut of the sugarcane stalk borer Diatraea saccharalis.

A beta-fructosidase (EC 3.2.1.26) was isolated from the midgut of larval sugar cane stalk borer Diatraea saccharalis by mild-denaturing electrophoresis and further purified to near homogeneity by gel filtration. beta-Fructosidase hydrolysed sucrose, raffinose and the fructosyl-trisaccharide isokestose, but it had no activity against maltose, melibiose and synthetic substrates for alpha-glucosidases. Two other sucrose hydrolases, one resembling a alpha-glucosidase (EC 3.2.1.20) and the other one active specifically against sucrose (sucrase) were detected in the larval midgut of D. saccharalis. All three sucrose hydrolases were associated with the midgut epithelium of larval D. saccharalis. Relative molecular mass (M(r)) of the beta-fructosidase was estimated around 45,000 (by gel filtration). The other two sucrose hydrolases had M(r) of 54,000 (alpha-glucosidase) and 59,000 (sucrase). The pH optima of the sucrose hydrolases were 5-10 for both alpha-glucosidase and sucrase and 7-8 for beta-fructosidase. Considering V(max)/K(m) ratios, beta-fructosidase preferentially cleaves isokestose rather than raffinose and sucrose. In order to evaluate the possible contribution of microorganisms isolated from the midgut to the pool of sucrose hydrolases, washed midgut epithelia were homogenised and plated onto appropriate media. Seven bacterial and one yeast species were isolated. None of the sucrose hydrolases extracted from the microorganisms corresponded to the enzymes isolated from midgut tissue homogenates. This result suggests that the major sucrose hydrolases found in the midgut of larval D. saccharalis were probably produced by the insect themselves not by the gut microflora.

Animals↗
PubMed2001

Profiling serine hydrolase activities in complex proteomes.

Serine hydrolases represent one of the largest and most diverse families of enzymes in higher eukaryotes, comprising numerous proteases, lipases, esterases, and amidases. The activities of many serine hydrolases are tightly regulated by posttranslational mechanisms, limiting the suitability of standard genomics and proteomics methods for the functional characterization of these enzymes. To facilitate the global analysis of serine hydrolase activities in complex proteomes, a biotinylated fluorophosphonate (FP-biotin) was recently synthesized and shown to serve as an activity-based probe for several members of this enzyme family. However, the extent to which FP-biotin reacts with the complete repertoire of active serine hydrolases present in a given proteome remains largely unexplored. Herein, we describe the synthesis and utility of a variant of FP-biotin in which the agent's hydrophobic alkyl chain linker was replaced by a more hydrophilic poly(ethylene glycol) moiety (FP-peg-biotin). When incubated with both soluble and membrane proteomes for extended reaction times, FP-biotin and FP-peg-biotin generated similar "maximal coverage" serine hydrolase activity profiles. However, kinetic analyses revealed that several serine hydrolases reacted at different rates with each FP agent. These rate differences were exploited in studies that used the biotinylated FPs to examine the target selectivity of reversible serine hydrolase inhibitors directly in complex proteomes. Finally, a general method for the avidin-based affinity isolation of FP-biotinylated proteins was developed, permitting the rapid and simultaneous identification of multiple serine peptidases, lipases, and esterases. Collectively, these studies demonstrate that chemical probes such as the biotinylated FPs can greatly accelerate both the functional characterization and molecular identification of active enzymes in complex proteomes.

Animals↗
PubMed1999

Aspartate 142 is involved in both hydrolase and dehydrogenase catalytic centers of 10-formyltetrahydrofolate dehydrogenase.

The enzyme 10-formyltetrahydrofolate dehydrogenase (FDH) catalyzes conversion of 10-formyltetrahydrofolate to tetrahydrofolate in either a dehydrogenase or hydrolase reaction. The hydrolase reaction occurs in a 310-residue amino-terminal domain of FDH (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. N(t)-FDH shares some sequence identity with several 10-formyltetrahydrofolate-utilizing enzymes. All these enzymes have a strictly conserved aspartate, which is Asp(142) in the case of N(t)-FDH. Replacement of the aspartate with alanine, asparagine, glutamate, or glutamine in N(t)-FDH resulted in complete loss of hydrolase activity. All the mutants, however, were able to bind folate, although with lower affinity than wild-type N(t)-FDH. Six other aspartate residues located near the conserved Asp(142) were substituted with an alanine, and these substitutions did not result in any significant changes in the hydrolase activity. The expressed D142A mutant of the full-length enzyme completely lost both hydrolase and dehydrogenase activities. This study shows that Asp(142) is an essential residue in the enzyme mechanism for both the hydrolase and dehydrogenase reactions of FDH, suggesting that either the two catalytic centers of FDH are overlapped or the dehydrogenase reaction occurs within the hydrolase catalytic center.

Amidohydrolases↗
PubMed1982

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution.

By in vitro translation of mRNA's isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, beta-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA's for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.

Animals↗
PubMed1980

Role of lipids in functions of photosynthetic membranes revealed by treatment with lipolytic acyl hydrolase.

1. Thylakoid membranes and subchloroplast particles I and II enriched in photosystem I and photosystem II, respectively, were treated with a potato lipolytic acyl hydrolase. 2. In the thylakoid membrane fraction, this treatment inhibited electron flows involving both photosystems and the associated photophosphorylations. However, electron flows involving either photosystem I or photosystem II were still preserved. The treatment of thylakoid membranes by lipolytic acyl hydrolase brought about a temporal convergence of different events such as maximal activity of reduced dichloroindophenol-supported electron flows, complete inhibition of photophosphorylations and electron transport activities through photosystem II + I, onset of the decay N,N,N',N-tetramethyl-rho-phenylenediamine-supported activity of photosystem activity and of the stimulation of photosystem activity (from reduced dichloroindophenol to NADP+ by exogenous plastocyanin. 3. Lipolytic acyl hydrolase catalyzed a limited hydrolysis of each lipid but in a stepwise manner, the galactolipids being attacked before the ionic lipids. The extent of the hydrolysis was not more than 50% for each lipid. Most of the hydrolytic process occurred before any significant change in photochemical activities could be observed. 4. In subchloroplast particles I, a treatment by lipolytic acyl hydrolase did not greatly affect the electron transport whilst lipid hydrolysis was almost complete. 5. In subchloroplast particles II, neither the electron flow activities nor lipid content were significantly altered by lipolytic acyl hydrolase. 6. The sites of lipolytic acyl hydrolase action appeared to be localized between plastoquinones and P700. It is suggested that it is not possible to establish a quantitative and/or temporal correlation between the extent of lipid hydrolysis and the inhibition of photochemical activities. 7. The profile of the hydrolysis of lipids in thylakoid membranes suggests that ionic lipids are less accessible to lipolytic acyl hydrolase than galactolipids.

Chloroplasts↗
PubMed1988

Effect of dietary carbohydrates on bacterial cholyltaurine hydrolase in poultry intestinal homogenates.

The bile salt hydrolase activity in intestinal homogenates reflects composite activities of the gastrointestinal microbial consortia. We have proposed that specific transformations of conjugated bile acids by the intestinal microflora result in the production of metabolites which depress the growth of poultry. The influence of dietary carbohydrates on the physical and kinetic properties of cholyltaurine hydrolase activity, one such bile acid-transforming enzyme in gastrointestinal homogenates of young chickens, was characterized by using a sensitive radiochemical assay. Cholyltaurine hydrolase activity in crude extracts of ileal homogenates was increased twofold by 0.25% Triton X-100 and a freeze-thaw cycle. The pH optimum for cholyltaurine hydrolase from ileal homogenates was very broad and reflected the pH range of poultry intestinal contents (i.e., 5.8 to 6.4). The carbohydrate component of the diet did not affect the apparent temperature optimum (41 degrees C) or stability profile, nor did it affect the apparent Km for taurocholic acid hydrolysis (approximately 0.43 mM). The enzymes in intestinal homogenates were active on all taurine-conjugated bile acids tested. The carbohydrate component of the diet did, however, affect the specific activity of cholyltaurine hydrolase in ileal homogenates from chickens. The levels of cholyltaurine hydrolase activity (rye greater than sucrose greater than corn) in homogenates from birds fed the different diets were directly related to the amount of growth depression (rye greater than sucrose greater than corn) associated with feeding these dietary carbohydrates. These data suggest that intestinal levels of cholyltaurine hydrolase are correlated with the amount of carbohydrate-induced growth depression in poultry.

Amidohydrolases↗
PubMed1999

Screening of lactic acid bacteria for bile salt hydrolase activity.

Bile salt hydrolysis is an important metabolic reaction in the bile salt metabolism of mammals. This reaction has a facilitating effect for bile salt excretion but can also be involved in various illnesses. In recent years interest has increased to use bile salt hydrolysis to influence the cholesterol metabolism of humans and farm animals. To understand the distribution and range of bile salt hydrolase activity in lactic acid bacteria, we screened more than 300 strains of the genera Bifidobacterium and Lactobacillus and the species Lactococcus lactis, Leuconostoc mesenteroides, and Streptococcus thermophilus. Results obtained for 273 strains showed that bile salt hydrolase activity is common in Bifidobacterium and Lactobacillus but absent in L. lactis, Leu. mesenteroides, and S. thermophilus. Nearly all bifidobacteria species and strains have bile salt hydrolase activity, whereas this activity can only be found in selected species of lactobacilli. A strong correlation can be observed between the habitat of a genus or species and the presence of bile salt hydrolase activity. Most often bile salt hydrolase activity is found in strains that have been isolated from the intestines or from feces from mammals--an environment rich in conjugated and unconjugated bile acids. Strains and species from other habitats like milk or vegetables--environments from which bile salts are absent--do normally not have bile salt hydrolase activity. In two independent assays, we established that bile salt hydrolase activity in bifidobacteria is, in general, much higher than in lactobacilli.

Amidohydrolases↗
PubMed1984

Isolation and characterization of Escherichia coli strains defective in CDP-diglyceride hydrolase.

CDP-diglyceride, an obligatory intermediate in the biosynthesis of the glycerophospholipids in Escherichia coli, is cleaved in vitro to phosphatidic acid and CMP by a membrane-bound hydrolase. Previous work from our laboratory (Bulawa, C.E., Hermes, J.D., and Raetz, C. R. H. (1983) J. Biol. Chem. 258, 14974-14980) has demonstrated that this enzyme also catalyzes the transfer of CMP from CDP-diglyceride to phosphate and numerous phosphomonoesters. We now report the isolation of E. coli mutants which are defective in CDP-diglyceride hydrolase. These mutations, designated cdh, map at minute 88 between pfkA and tpi. This information permitted the identification of a ColE1 hybrid plasmid, pLC16-4, which causes the overproduction of hydrolase activity. The isolation of deletion and Tn10 insertion mutants at cdh suggests that the hydrolase is nonessential for cell growth. Hydrolase mutants are defective in both CDP-diglyceride hydrolysis and CDP-diglyceride-dependent cytidylylation, indicating that both activities are encoded by the cdh gene. Although previously described as a ribospecific enzyme, we have found that incubation of the partially purified hydrolase with [alpha-32P]dCDP-diglyceride and phosphate yields two products, [32P]dCMP and [alpha-32P]dCDP. That a single enzyme utilizes both CDP- and dCDP-diglyceride is demonstrated by the following. (i) The hydrolysis of [alpha-32P]CDP-diglyceride is inhibited by nonradioactive dCDP-diglyceride and vice versa. (ii) Utilization of both liponucleotides is inhibited by AMP. (iii) Mutants in the cdh gene are defective in both CDP- and dCDP-diglyceride hydrolysis, while cdh clones overproduce both activities. (iv) Hydrolase mutants accumulate both CDP- and dCDP-diglyceride.

CDPdiacylglycerol-Serine O-Phosphatidyltransferase↗
PubMed1983

Induction of hepatic long-chain acyl-CoA hydrolase by clofibric acid administration.

The induction of hepatic long-chain acyl-CoA hydrolase in the cytosolic fraction by administration of clofibric acid (p-chlorophenoxyisobutyric acid) was compared in rats, mice and guinea-pigs. In rats, two long-chain acyl-CoA hydrolases were induced by the administration of clofibric acid. In mice, only one long-chain acyl-CoA hydrolase was induced, and this hydrolase had properties similar to those of the lower-molecular-weight hydrolase induced in the hepatic cytosol of rats. In hepatic cytosol of guinea-pig, no hydrolase was induced by the administration of clofibric acid.

Animals↗