Diethyl P-nitrophenyl Phosphate Research Articles

Inhibition of pancreatic lipase by mixed micelles of diethyl p-nitrophenyl phosphate and bile salts

Solubility and Sephadex filtration assays have shown that dissolved diethyl p-nitrophenyl phosphate can be included into bile salt micelles with a partition coefficient of 32:1. This inclusion is probably a prerequisite for the organophosphate to inhibit lipase. The essential role played by colipase confirms that the primary step of the inhibition is an interaction of lipase with bile salt containing micelles. Therefore, it appears that the requirements of lipase towards specific substrates and inhibitors are very similar. The inhibition rate strongly depends on the total bile salt concentration and on the micellar concentration of the organophosphate. This effect may be explained, at least qualitatively, by a competition between simple and mixed micelles for the binding of colipase and lipase.

Mechanism of pancreatic lipase action. 2. Catalytic properties of modified lipases.

Reaction of lipase with diethyl pyrocarbonate results in the modification of three histidine residues. One is highly reactive, although without affecting the activity, while the two others react more slowly with a concomitant loss of activity on both dissolved and emulsified substrates. As previously shown, lipase can also be modified either by reaction of five carboxyl groups with carbodiimide (5N-lipase) or by esterification of one serine residue with diethyl p-nitrophenyl phosphate (DP-lipase). In the three cases, the activity on emulsified substrates is abolished. The modification of histidine residues results also in a loss of activity on dissolved substrates, suggesting that the essential histidine is at (or close to) the active site. The ability of lipase to be adsorbed on siliconized glass beads is not impaired in this reaction. By contrast, 5N-lipase is still able to hydrolyze dissolved monomeric substrates and to adsorb on siliconized glass beands. Therefore, the essential carboxyl group is assumed to play an important role in the interfacial activation. Finally, since DP-lipase is still fully active on dissolved p-nitrophenyl acetate, the serine residue, which has been previously suggested to be the acylable one, is more likely implicated in the recognition and the binding to interfaces, as confirmed by the inability of DP-lipase to be adsorbed on siliconized glass beads.

Catalytic effects of polar molecules and their ability to form hydrogen bonds

A study was made of the reaction rate between sodium ethylate and p-nitrophenyl diethyl phosphate in the alcohol-benzene system, in the presence of electron-donor additives, the effect of which on the reaction rate at low alcohol concentrations is determined by their ability to form hydrogen bonds.

Malathion a and b esterases of mouse liver—I: Separation and properties

The hydrolysis of malathion [S-(1,2-dicarbethoxyethyl) O, O-dimethyl phosphorodithioate] is catalysed by two types of enzymes in the mouse liver. The first is malathion B-esterase which hydrolyses one of the carboxyethyl ester groups giving malathion mono-acid. This is a B type esterase, being sensitive to low concentrations of DFP (diisopropyl phosphorofluoridate), paraoxon (diethyl p-nitrophenyl phosphate) and EPNO the oxygen analogue of EPN ( O-ethyl O- p-nitrophenyl phenylphosphorothionate). It is predominently in the microsomes, acts at pH 7.4–7.6 and is probably the same as the non-specific carboxylesterases (EC 3.1.1.1). The second enzyme is malathion A-esterase which is insensitive to DFP and other organophosphates even at high concentrations. It is predominently in the cell-sap, has an optimum pH of 8.8, requires 2-mercaptoethanol, reduced glutathione or similar SH compounds for activation, and is inhibited by p-chloromercuribenzoate and heavy metals. The A esterase degrades malathion at the P-S linkage giving O, O-dimethyl phosphorothioate.

Structure and Function of the Holdfast Organ and Lappets of Alaria mustelae Bosma, 1931, with Further Studies on Esterases of A. marcianae (La Rue, 1917) (Trematoda: Diplostomatidae)

In the intestine of experimentally infected mink, the scoop-shaped forebody of Alaria mustelae grasps a single villus, holding it tightly against the holdfast organ. Some breakdown of villous tissue occurs there but not near the invaginated lappets. Adult worms apparently feed on this tissue but also probably ingest intact tissue directly. Histochemical staining revealed nonspecific esterases in the holdfast organ gland cells, forebody gland cells and ducts, forebody tegumentary cells, and ceca. From effects of the organophosphate inhibitor diethyl p-nitrophenyl phosphate (E600), nonspecific esterase activity in the ceca is due to A-esterases and in other areas to B-esterases. Using E600 with A. marcianae adults (experimentally infected dog), A and B types occur in the holdfast organ, A, B, and C types in the forebody gland cells and ducts, and only B in other areas. A comparison of the structure and function of the holdfast organ and lappets of A. mustelae and A. marcianae is presented. Three recent studies (Johnson et al., 1971; Bhatti and Johnson, 1971, 1972) have been made on the structure and function of the holdfast organ and lappets of the strigeoid trematode Alaria marcianae (La Rue, 1917). These studies have indicated the importance of the lappets in extracorporeal digestion, with a secondary role in attachment. The holdfast organ was implicated in extracorporeal digestion and also possibly in nutrient absorption. Attachment of adults to the host mucosa was accomplished primarily by the forebody rather than by the holdfast organ and lappets. The subject of the present study is the closely related species, A. mustelae Bosma, 1931. In this preliminary investigation, histological studies were made as well as histochemical staining for esterases. Additionally, further histochemical staining of esterases in A. marcianae adults was carried out. Histochemical staining has demonstrated nonspecific esterases in the holdfast organ gland cells and in gland cells associated with the lappets, if present, of all adult strigeoids studied (Lee, 1962; Erasmus and Ohman, 1963; Ohman, 1965, 1966a, b; Bogitsh, 1966; Reid and Harkema, 1970; Johnson et al., 1971; Bhatti and Johnson, 1972). In studies attempting to identify these enzymes further, Erasmus and Ohman (1963) in Cyathocotyle bushiensis and Received for publication 9 July 1973. * Supported in part by Graduate School Research Grant, University of South Dakota. Ohman (1965) in Diplostomum spathaceum reported a B esterase and an unclassified resistant type. Ohman (1966a) in Apatemon gracilis minor and Ohman (1966b) in Holostephanus luehei found a type resembling B esterases, and Bhatti and Johnson (1972) reported a C esterase in A. marcianae. MATERIALS AND METHODS Mesocercariae of A. mustelae and A. marcianae were from naturally infected leopard frogs, Rana pipiens (Steinhilber and Co., Oshkosh, Wisc.). Two 7-month-old female mink (ranch-reared) were each injected intraperitoneally with about 400 A. mustelae larvae and killed 23 and 28 days after infection. One dog was injected intraperitoneally with 2,400 A. marcianae larvae and killed 34 days after infection. Portions of the small intestine with attached adult worms were immediately removed and fixed in cold (4 C) fixatives. Fixation for histological studies was for 24 to 48 hr in Bouin's fixative, neutral phosphatebuffered 5% glutaraldehyde, or neutral phosphatebuffered formalin. Free and attached adults were dehydrated in ethanol, cleared in benzene, embedded in Tissuemat, sectioned at 10 /t, and stained with hematoxylin and eosin. Fixation for staining of esterases was for 16 hr in formol-calcium (Barka and Anderson, 1962), followed by rinsing for 24 hr in cold 0.1 M cacodylate buffer (pH 7.4) containing 0.2 M sucrose and storage in this solution at 4 C until used. Before sectioning, material was infiltrated with 10% gelatin (12 hr at 37 C) to prevent loss of sections from slides. Free and attached adults were sectioned at 8 A in an International Harris cryostat (-22 C) and mounted on slides.

The biochemistry of virus-induced cell fusion. Changes in membrane integrity

1. Phospholipids prelabelled with [(14)C]acetate, [(32)P]phosphate, [(3)H]- or [(14)C]-choline or [(3)H]inositol are not significantly degraded during fusion of Lettrée cells mediated by Sendai virus, nor are carbohydrates prelabelled with [(3)H]fucose, [(14)C]galactose or [(3)H]glucosamine. Less than 1nmol of lysophosphatidylcholine/10(7) cells is formed during fusion. Diethyl p-nitrophenyl phosphate, which inhibits phospholipase A by more than 95% has no effect on fusion. It is concluded that none of the events leading to cell fusion is accompanied by significant turnover of phospholipids or other membrane components. 2. Intracellular K(+) leaks out during virally mediated cell fusion; the loss is not as extensive as that of intracellularly accumulated choline or deoxyglucose. Movement of Ca(2+) into or out of cells could not be detected. 3. At concentrations of Lettrée cells insufficient to be agglutinated by virus, intracellularly accumulated choline and deoxyglucose leak out. Agglutination caused by concanavalin A does not result in leakage of intracellular metabolites. 4. P815Y cells, which agglutinate but do not fuse in the presence of virus, show leakage of intracellularly accumulated metabolites. The extent of leakage does not alter during the G(1) and S periods of the cell cycle. 5. Leakage is inhibited by Ca(2+), but is unaffected by EDTA. 6. It is concluded that the interaction of Sendai virus with mammalian cells causes a weakening of membrane integrity so that intracellular metabolites leak out. Such destabilization may facilitate viral entry and is therefore an interesting system for further biochemical studies.

Studies of the binding of sulfur released in the mixed-function oxidase-catalyzed metabolism of diethyl p-nitrophenyl phosphorothionate(parathion) to diethyl p-nitrophenyl phosphate (paraoxon)

Incubation of [ 35S]labeled parathion with rat liver microsomes in the presence of an NADPH-generating system leads to the covalent binding of sulfur to the macromolecules of the microsomal membrane. The maximal binding of sulfur to microsomes requires the presence of NADPH, is increased using microsomes from phenobarbital and 3-methyl-cholanthrene-treated animals, and is inhibited by carbon monoxide. The majority, if not all, of the sulfur bound is in a form free of the remainder of the parathion molecule. These findings, coupled with the fact that the apparent K m and V max for sulfur binding are not statistically different from the apparent K m and V max for metabolism of parathion to paraoxon, indicate the sulfur bound is that released in the mixed-function oxidase-catalyzed metabolism of parathion to paraoxon. Under the conditions of the experiments described in this report, the binding of sulfur to microsomes decreases the concentration of cytochrome P-450 in the microsomes and inhibits slightly the rate of the mixed-function oxidase-catalyzed metabolism of benzphetamine.

Further characterisation of lipoprotein lipase and hepatic post-heparin lipase from rat plasma

1. 1. The pre-heparin plasma of the rat, and the perfusate obtained from isolated rat liver following perfusion with heparin, showed lipolytic activities principally against monoglyceride and tributyrin substrates, and minor triglyceride and diglyceride hydrolase activities. Rates of activity with 1- and 2-monoglycerides were similar. 2. 2. Each activity in these preparations was characterised by resistance to inhibition by NaCl and protamine, and by sensitivity to inhibition by diethyl p-nitrophenyl phosphate. The pH optimum was 7.25–7.4 for the different lipid substrates. 3. 3. Purified lipoprotein lipase, the post-heparin perfusate of extrahepatic tissues and whole post-heparin plasma were characterised by major lipolytic activities against di- and triglycerides, and by monoglyceride hydrolase activity directed mainly against the I-isomer. Each activity was strongly inhibited by NaCI and by protamine, and was comparatively resistant to inhibition by diethyl p- nitrophenyl phosphate. pH optima were 8.0–8.3 for the different lipid substrates, in the presence of whole serum as activator. 4. 4. A principal function of the monoglyceride hydrolase activity of pre-heparin plasma, and released from the liver by heparin, may be to hydrolyse the 2-monoglyceride residue, which is the major product of lipoprotein lipase activity against triglyceride substrates.

Action of organophosphates and sulfonyl halides on porcine pancreatic lipase

Pancreatic lipase (glycerol-ester hydrolase, EC 3.1.1.3) is inhibited by emulsions of diethyl p-nitrophenyl phosphate (E 600) or by solutions of this organophosphate in the presence of bile salts. The inhibited enzyme contained a single phosphoryl radical per mole. This radical was shown to be bound to a serine residue in the sequence Leu-Ser-Gly-His. Therefore, lipase is a serine-histidine enzyme like ordinary carboxylesterases and a number of proteases. However, the specific properties of lipase were confirmed when it was observed that the enzyme was not inhibited by E 600 solutions in the absence of bile salts. Inhibition in the presence of bile salts is assumed to be caused by inclusion of E 600 into micelles of these compounds. Moreover, in contrast with other serine enzymes, lipase is not inhibited by diisopropylfluorophosphate (DFP) even in the presence of bile salts. It is also not inhibited by sulfonyl halides unless the nucleophilicity of the sulfur atom is enhanced by an electrophilic substituant. These results are in perfect agreement with the observations already made with the substrates of lipase. In addition, concentrated DFP solutions were found to react with a single, non-essential tyrosine residue in native lipase. This residue is located in the sequence: Thr-Asn-Gln-Asn-Glx-Asx(Asx, Glx)-Tyr-Glx-Leu.

Stability, inhibition and reactivation of acetylcholinesterase covalently coupled to glass

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was covalently coupled to a silanized fritted glass disc by a diazonium salt reaction and by a diimide condensation. The diimide-coupled adduct survived numerous assays and chemical studies for a period of 55 days at room temperature in contrast to the diazonium-coupled adduct which rapidly lost activity. Both adducts exhibited a lesser pronounced dependence of activity on pH than the free enzyme, reflecting the weak buffering action of the silanol-silane surface. The diimide adduct is inhibited by diethyl p-nitrophenyl phosphate and can be reactivated by either Toxigonin or alkaline phosphatase (orthophosphoric monoester phosphohydralase, EC 3.1.3.1).

Deformylation of 4,4'-diformamidodiphenyl sulfone (DFD) by plasma of certain mammals

Enzymatic defonnylation of 4,4'-diformamidodiphenyl sulfone (DFD) to 4,4'-diaminodiphenyl sulfone (DDS) occurs rapidly and completely in the mouse and in vitro in mouse plasma. Within 1 hr all DPD is present as DDS. The intermediate product in the stepwise defonnylation is 4-arnino-4'-formamidodiphenyl sulfone (MFD). DFD is also deformylated in plasma from other animals with species variation in the rates of defonnylation; mouse > rat > guinea pig > rabbit. DFD is not deformylated in plasma from dog or man. Defonnylation is inhibited by diethyl p-nitrophenylphosphate (paraoxon), but not by p-chloromercuribenzoate or neostigmine. The reaction also shows marked substrate specificity: of other formamido compounds tested only those linked to the diphenylsulfone structure were substrates. The rapid defonnylation of DFD to DDS indicates that the antimalarial efficacy of DPD in Plasmodium berghei in the mouse is due to its conversion to DDS. Reported superiority of DFD to DDS in treatment of P. berghei is considered to be related to solubility differences in the two compounds and repository effects of certain routes of administration.

Effect of irreversible inhibitors on transport ATPase

Preincubation of brain Na + K ATPase with diisopropylfluorophosphate, methane sulfonyl chloride or diethyl p-nitrophenyl phosphate inhibited irreversibly Na + K ATPase, Na +-dependent phosphorylation and ATP-ADP exchange. It is concluded that inhibition is direct, and that the active center of the enzyme contains a “reactive” serine residue.

Microsomal Esterase of Rat Liver

Abstract Rat liver microsomal esterase has been purified 254-fold by a combination of solubilization in a mechanical cell homogenizer, acetone and ammonium sulfate precipitations, and hydroxylapatite column chromatography. The enzyme hydrolyzed glyceryl 1-monodecanoate at a rapid rate; the Km is 1.61 x 10-3 m. The hydrolysis rates of corresponding di- and triglycerides were one-third and one-hundredth of that of glycerol 1-monodecanoate, respectively. The enzyme showed little hydrolytic activity on long chain mono-, and di- and triglycerides. p-Nitrophenyl esters of short chain fatty acids were hydrolyzed at appreciable rates. Diethyl p-nitrophenyl phosphate, at a concentration of 1 x 10-4 m, inhibited the enzyme activity completely; thus the enzyme was tentatively classified as the B-type described by Aldridge.

Characterization of cathepsins in cartilage.

The presence of a cathepsin B-like enzyme in rabbit ear cartilage was established by the use of the synthetic substrates benzoyl-l-arginine amide and benzoyl-dl-arginine 2-naphthylamide. This was facilitated by using a technique that permits the incubation of a fixed weight of thin (18mu) cartilage sections with an appropriate exogenous substrate. The enzymic properties of cathepsin B in cartilage have been compared with an endogenous enzyme that liberates chondromucopeptide by degrading the cartilage matrix autocatalytically at pH5. Besides being maximally active at pH4.7, these cartilage enzymes are enhanced in activity by cysteine and inhibited by arginine analogues, iodoacetamide, chloroquine and mercuric chloride. They are not inhibited by EDTA, di-isopropyl phosphorofluoridate and diethyl p-nitrophenyl phosphate. When inhibiting the release of chondromucopeptide from cartilage at pH5, the arginine-containing synthetic substrates are hydrolysed simultaneously. These enzymes also share the same heat-inactivation characteristics at various pH values, being stable at acid pH and unstable at neutral and alkaline pH. The experimental evidence indicates that a cathepsin B-like enzyme may be partly responsible for the autolytic degradation of cartilage matrix at pH5.

Postnatal changes of activity and electrophoretic pattern of jejunal and ileal nonspecific esterase and alkaline phosphatase of the rat. Effect of adrenalectomy.

During postnatal development of the rat, the activity of nonspecific esterase (substrate, β-naphthyl acetate) in the jejunum and ileum increased. Electrophoresis showed that distribution of this enzyme was different in these two parts of the intestine in the suckling rat, whereas the zymogram was the same for both parts in the adult rat. In intact animals, the degree of inhibition of nonspecific esterase activity by 10−4 M eserin, 5 × 10−8 M di-ethyl p-nitrophenyl phosphate (E.600), and 0.4 M NaCl was different, but there were no differences with any one inhibitor between the jejunum and the ileum of suckling and weanling rats. Adrenalectomy at 7 days of age did not influence the activity of the enzyme, but at 14 days of age it inhibited the rise of activity in jejunum and ileum. Adrenalectomy influenced the sensitivity of nonspecific esterase activity to inhibition by eserin and E.600. When cortisone was administered to adrenalectomized rats, certain characteristics of the enzyme (specific activity, sensitivity to various inhibitors, zymogram) were, in most case, comparable with those of preparations from normal controls.During postnatal development, the activity of alkaline phosphatase (substrate, β-naphthyl phosphate) in the jejunum showed practically no change, but in the ileum it decreased. Change in the zymogram was of the same type in both jejunum and ileum. Adrenalectomy at 7 days of age was followed by a fall in activity in the ileum that was apparent at 14 days of age, but the zymogram was not affected. Adrenalectomy on day 14 did not produce an apparent change by day 21, but there was retention of the earlier electrophoretic pattern. When cortisone was administered to adrenalectomized rats, certain characteristics of the enzyme (specific activity, zymogram) were comparable with those of preparations from normal controls.The results are discussed from the point of view of difference between the jejunum and ileum of suckling animals, and the influence of the adrenal glands.

Dissociatiion of esterase from proteinase activity of Bacillus subtilis spores.

Preparations of ballistically disintegrated Bacillus subtilis spores have been shown to possess proteinase in addition to esterase activities. Both proteinase and esterase activities have been suggested as trigger for the initiation of germination, but information on these two activities in bacterial endospores is scant. Since some proteinases catalyze the hydrolysis of esters, there exists a possible identity of some esterases with proteinases. To get information on the esterase and proteinase activities of B. subtilis spores, the sensitivity to heat and to inhibitors was explored. The thermal effect and the effect of certain inhibitors on esterase activities towards several esters suggested that an unspecific esterase was responsible for these activities.Differential effects of heat, pH, and inhibitors on esterase and proteinase activities indicated the dual nature of these two activities. In absence of convincing experimental evidence to the contrary, the present indication seems to be that the esterase activities observed in B. subtilis spores were dissociated from the activity which hydrolyzes casein. The esterase activity was readily inhibited by diethyl p-nitrophenyl phosphate, diisopropylphosphofluoridate, eserine, sodium fluoride, and Zn ions, and possessed properties of a B-esterase. The proteinase activity was inhibited only by diisopropylphosphofluoridate showing properties, so far studied, similar to subtilopeptidase.

Oxime Reactivation of Diethylphosphoryl Human Serum Cholinesterase

Abstract Reactivation of diethyl p-nitrophenyl phosphate inhibited human serum cholinesterase by pyridine-2-aldoxime methiodide and isonitrosoacetophenone has been investigated as a function of reactivator concentration, pH, and temperature in boric acid-borax buffer and in salt solution. Constants shown were the dissociation constants of the diethylphosphoryl cholinesterase reactivator complex for the reactivation by pyridine-2-aldoxime methiodide and the bimolecular rate constants for that by isonitrosoacetophenone. The protonation constants of the diethylphosphoryl cholinesterase were evaluated from the relationship between pH and the dissociation constants of the complex; they indicated a value of 8.4 in buffer medium and 7.8 in salt solution. Changes in reactivation rate and in apparent activation energy of reactivation due to the medium changes may be explained by the nature of the activated complex formed in the respective reactivation processes.

A chemical and histochemical study on the significance of the nonspecific esterase activity in the adult rat testis.

In frozen sections of fresh and formol-calcium chloride fixed testes 2 functionally different types of nonspecific esterase activity can be demonstrated histochemically using various combinations of the substrates anaphthyl acetate and indoxyl acetate and the inhibitor diethylp-nitrophenyl phosphate (E600). The activity of the majority of interstitial cells (Leydig cells) can be visualized on fresh-frozen sections with α-naphthyl acetate as substrate. This activity decreases markedly after hypophysectomy and is sensitive to 1O-6M concentration of the inhibitor. The Sertoli cells and a few of the interstitial cells (possibly macrophages), on the other hand, prefer indoxyl acetate as substrate and activity is demonstrable only in formalinfixed frozen sections. It resists E600, and the number of cells exhibiting this E600-resistant activity increases greatly after hypophysectomy both in the seminiferous tubules and in the interstitium. In chemical quantitative determinations of testicular nonspecific esteras...

Introduction of mercury into the active site of chymotrypsin p-chloromercuribenzene sulfonyl-chymotrypsin

Pancreatic lipase (glycerol-ester hydrolase, EC 3.1.1.3) is inhibited by emulsions of diethyl p-nitrophenyl phosphate (E600) or by solutions of this organophosphate in the presence of bile salts. The inhibited enzyme contained a single phosphoryl radical per mole. This radical was shown to be bound to a serine residue in the sequence Leu-Ser-Gly-His. Therefore, lipase is a serine-histidine enzyme like ordinary carboxylesterases and a number of proteases.However, the specific properties of lipase were confirmed when it was observed that the enzyme was not inhibited by E600 solutions in the absence of bile salts. Inhibition in the presence of bile salts is assumed to be caused by inclusion of E600 into micelles of these compounds.Moreover, in contrast with other serine enzymes, lipase is not inhibited by diisopropylfluorophosphate (DFP) even in the presence of bile salts. It is also not inhibited by sulfonyl halides unless the nucleophilicity of the sulfur atom is enhanced by an electrophilic substituant. These results are in perfect agreement with the observations already made with the substrates of lipase.In addition, concentrated DFP solutions were found to react with a single, non-essential tyrosine residue in native lipase. This residue is located in the sequence: Thr-Asn-Gln-Asn-Glx-Asx(Asx, Glx)-Tyr-Glx-Leu.

Role and oxidation pathway of poly-beta-hydroxybutyric acid in Micrococcus halodenitrificans.

Cells of Micrococcus halodenitrificans have a high poly-β-hydroxybutyric acid (PHBA) content, the oxidation of which accounts for the high endogenous respiration of this organism. The amount of polymer in resting cells decreased with time, and viability could be correlated with the polymer content of the cells. PHBA-rich cells maintained their viability for longer periods than PHBA-poor cells. Cells oxidized PHBA in the presence of sodium chloride but not in water; optimal oxidation occurred in 0.33 to 0.75 M NaCl.The results suggest the following oxidation pathway. A diethyl p-nitrophenyl phosphate sensitive depolymerase catalyzes the hydrolysis of the polyester to β-hydroxybutyrate. This soluble substrate is oxidized by a DPN-linked D(−)-β-hydroxybutyric acid dehydrogenase to acetoacetate. Acetoacetate is further oxidized in the presence of oxalacetate, ATP, and coenzyme A. Citrate is formed, indicating the presence of the condensing enzyme, and PHBA oxidation is strongly inhibited by malonate. These findings suggest that oxidation proceeds via the tricarboxylic acid cycle.