Half–life of plasma cholinesterase

The half-life of plasma cholinesterase (acylcholine acylhydrolase EC 3.1.1.8) was determined in three patients homozygous for the atypical gene for plasma cholinesterase by measuring the rate of disappearance of enzyme activity following intravenous injection of concentrated human cholinesterase. Half-life values of 10.9, 11.1, and 11.3 days were estimated. The distribution volume was estimated to be 18.0, 18.2, and 13.8% of body weight, respectively.     

Synergistic inhibition of human leukemia cell growth by deoxyguanosine and 1-beta-D-arabinofuranosylcytosine

We studied the ability of 2'-deoxyguanosine (dGuo) to influence 1-beta-D-arabinofuranosylcytosine (ara-C) inhibition of soft agar cloning of the cultured human leukemia cell line K562. Ara-C alone inhibited cloning in concentrations of greater than 10 nM, with a steep drop in colony formation observed between 10 and 100 nM. dGuo and ara-C synergistically inhibited cloning; the combination of ineffective concentrations of dGuo (10-50 microM) and ara-C (less than or equal to nM) inhibited cloning by 40-70%. In K562 cells, dGuo is metabolized by both nucleoside kinase and purine nucleoside phosphorylase (PNP), resulting in augmentation of both the GTP pool (to more than 200% of control after a 3 hr incubation with 500 microM dGuo) and the dGTP pool (to more than 2700% of control after 3 hr with 500 microM dGuo). dGuo (50-500 microM) caused a decrease in the dCTP and dTTP pools and an increase in the dATP pool. Synergistic concentrations of dGuo plus 10 nM ara-C augmented the ara-CTP pool up to 800% of control after 3 hr to levels equivalent to those observed after incubation with 500 nM ara-C alone. Incorporation of 10 nM ara-CTP into DNA also increased in the presence of dGuo (up to a maximum of 300% of control), but only to a level that approximated the value observed with nM ara-C alone. The disparity between enlargement of the ara-CTP pool and augmentation of ara-C incorporation into DNA is consistent with the observation of Steinberg et al. [Cancer Res. 39, 4330 (1979)] that high concentrations of dGTP may inhibit DNA polymerase activity. Thus, synergy between dGuo and ara-C is multifactorial, possibly involving inhibition of DNA polymerase by elevated dGTP and ara-CTP pools and augmented incorporation of ara-C into DNA.     

Differences between small and large intestine and liver in the inducibility of microsomal enzymes in response to stimulation by phenobarbitone and betanaphthoflavone in the diet

Rats were fed either sodium phenobarbitone (PB) or betanaphthoflavone (BNF) for seven days. Deethylation of 7-ethoxyresorufin ( 7ERR ) and 7-ethoxycoumarin ( 7EC ) was measured in small and large intestine and liver, and cytochrome P-450 in liver. Our semi-purified diet was shown to produce minimal levels of intestinal deethylation activity. BNF was added to the semi purified diet and fed at levels from 0.1 to 100 mg BNF/kg of diet. Significant (P less than 0.05) induction of deethylation in small intestine was seen at all dose levels, ranging from 2-fold at 0.1 mg/kg diet to greater than 100-fold at 100 mg/kg diet. A 3-fold increase was also seen in the large intestine at 50 mg/kg. A significant increase in hepatic deethylation was only seen at 100 mg/kg. PB was administered in drinking water at 50, 100 and 1000 mg PB/l. Significant (P less than 0.05) induction of hepatic deethylation was seen at all dose levels, ranging from 2-fold at 50 mg/l to 5-fold at 1000 mg/l. Hepatic cytochrome P450 was also increased. No significant increase in intestinal deethylation was seen at any of the doses used.     

Intracellular localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in rat oligodendrocytes and C6 glioma cells, and effect of cell maturation and enzyme induction on localization

We investigated whether the membrane-associated myelin enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.37), is localized primarily inside the cell or exposed on the cell surface of rat oligodendrocytes and rat C6 glioma cells. Determinations were made by enzyme assays of intact, viable cells vs cells broken by freezing and thawing. Assay of both oligodendrocytes and C6 cells showed that the great majority of the CNP activity was localized inside the cells. Oligodendrocytes were also tested by immunofluorescence staining of unfixed, living cells whose membranes had been made permeable to antibody by fixation. Fixed oligodendrocytes showed intense fluorescence when incubated with rabbit anti-CNP antiserum and fluorescein-conjugated second antibody whereas unfixed cells were not stained. We then tested the possible influence on CNP localization of 3 conditions known to increase CNP specific activity: maturation of oligodendrocytes in vitro during a period when CNP specific activity increases 8-fold or more; growth of C6 cultures to high cell density; and induction of CNP activity in oligodendrocytes and C6 cells by dibutyryl cyclic AMP. Under all conditions, most CNP activity was intracellular. These results show that both the catalytic and major antigenic sites of CNP are localized primarily inside the cell, and suggest an intracellular role for CNP in oligodendrocytes. The results with C6 cells also show that these cells resemble oligodendrocytes with respect to CNP localization.     

Diazepam inhibits cholesterol esterification by arterial ACAT and plasma LCAT, in vitro

Benzodiazepine drugs have been reported to have antiatherosclerotic effects in rabbits and roosters and to alter the pattern of circulating lipoproteins in man. The mechanism(s) of these effects has not been elucidated. The studies presented here indicate that diazepam, the most widely used benzodiazepine, is an inhibitor of cholesterol esterification by ACAT in vitro in atheromatous rabbit aortas, in microsomes isolated from atheromatous rabbit aortas, and in normal rat aortas. Diazepam also inhibited LCAT in plasma from man, monkey, rabbit, and rat, in vitro. The ability of diazepam to inhibit these enzyme systems may offer insight into possible in vivo mechanisms of action against atherosclerosis and of lipoprotein modification.     

Degradation of biogenic oligosaccharides by beta-N-acetyl-glucosaminidase secreted by Entamoeba histolytica

beta-N-Acetylglucosaminidase secreted by Entamoeba histolytica was extracted from the growth medium by affinity chromatography on CH-Sepharose 4 B coupled to p-aminophenyl-1-thio-beta-2-acetamido-2-deoxyglucopyranoside. The enzyme was further purified by isoelectric focusing, by sequential chromatography on DEAE-cellulose and Sephadex G-150, and by preparative disc gel electrophoresis. Chitobiose (betaGlcNAc1-4GlcNAc) derived from chitin as well as the oligosaccharides betaGlcNAc1-4 betaGlcUA1-3GlcNAc, betaGlcNAc1-4 betaGlcUA1-3 betaGlcAc1-4GlcUA, and betaGlcNAc1-4 betaGlc-UA1-3 betaGlcNAc1-4 betaGlcUA1-3 betaGlcNAc1-4GlcUA derived from hyaluronic acid were tested as potential physiological substrates. All these oligosaccharides are susceptible to action of beta-N-acetylglucosaminidase from E. histolytica. Under identical conditions chitobiose is cleaved 38-48 times faster than hyhyauronate oligosaccharides. No release of N-acetylglucosamine was observed when glycopeptides from ovalbumin were used as substrate. The pH optimum of hydrolase activity was 4.5 when chitobiose was used as substrate. Optimal hydrolysis of aluronate oligosaccharides was observed at pH 3.0 for trisaccharide and pH 2.0 for tetra- and hexasaccharide, respectively. Estimation of molecular weight by means of gel filtration gave values of 75 000. The isoelectric point was 5.02 beta-N-Acetylglucosaminidase from E. histolytica does not act on macromolecular chitin and hyaluronic acid.     

The binding of sulfonamides to horse liver alcohol dehydrogenase

The binding of sulfonamides to the active site of horse liver alcohol dehydrogenase has been studied by their effect on affinity labelling and steady state kinetics. Affinity labelling with iodoacetate and BIP has been used to study binding to free enzyme. The unsubstituted sulfonamide, sulfanilamide (I), shows very weak binding compared to the other sulfonamides tested. Most important for binding is the type of substituent attached to the parent sulfonamide, particularly when as in sulfathiazole this is a heterocycle which binds to the catalytic zinc atom of the enzyme. For sulfathiazole the dissociation constant from the enzyme is pH dependent showing two pK_a values. The lower at pH 7 is the pK_a of the drug itself, while that at pH 9 agrees with the ionization of water bound to the catalytic zinc ion.Steady state kinetics have been carried out at pH 7.0 and 10.0 to examine sulfonamide binding to the enzyme when coenzyme is attached. Both NAD⁺ and NADH induce substrate competitive sulfonamide binding. Likewise sulfathiazole accelerates the dissociation of NADH from the enzyme and so V_max for alcohol oxidation. The latter like stimulation of the affinity labelling reaction with iodoacetate is considered to result from binding of the thiazole ring to the catalytic zinc ion. With all the sulfonamides examined hydrophobic binding and charge are important in determining affinity to the active site and the mode of binding. Sulfonamides containing pyrazole or imidazole rings can be important in alcohol therapy.     

Conversion of 2,6-diamino-9-(2-hydroxyethoxymethyl)purine to acyclovir as catalyzed by adenosine deaminase

Adenosine deaminase (ADA) was partially purified from several sources using affinity chromatography. These enzymes have the capacity to catalyze the deamination of 2,6-diamino-9-(2-hydroxyethoxymethyl)purine (A134U) to form the antiviral agent acyclovir [9-(2-hydroxyethoxymethyl)guanine]. Their relative substrate efficiencies (Vmax/Km) with A134U (standardized to adenosine = 100) were: dog ADA, 0.092; human ADA, 0.015-0.029; rat ADA, 0.025; calf ADA, 0.016; and Escherichia coli ADA, 0.0003. In addition to having the lowest efficiency with A134U, the bacterial ADA was also distinguished by its lack of binding of the mammalian ADA inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine and by its weak binding to the 9-(p-aminobenzyl)adenine-agarose affinity column. Four minor metabolites of A134U and acyclovir have been reported to be produced in the rat. These compounds are oxidized on either the C-8 position of the ring or the terminal carbon of the side chain. Neither acyclovir nor any of these metabolites produced significant inhibition of calf intestine ADA. The oxidized metabolites containing an N-6 amino group were extremely slow substrates of this enzyme.     

Renal secretion of purine nucleosides and their analogs in mice

Previous results have indicated that 2'-deoxyadenosine (dAdo) and 2'-deoxytubercidin (dTub) are secreted by the mouse kidney. Secretion of dTub appeared to occur via the organic cation carrier [J. F. Kuttesch, Jr. et al., Biochem. Pharmac. 31, 3387 (1982)]. In the current study, the structural specificity of the secretory system for d Tub was probed by evaluating the renal clearance of several sugar-modified dTub analogs. The following sugar-modified derivatives also underwent apparent secretion: 3'-deoxy, arabinosyl, and xylosyl. These results suggest a lack of structural specificity of the secretory system for dTub. Tubercidin was apparently reabsorbed, analogous to the observation in mice that adenosine clearance is less than that of inulin. In related experiments, a transport maximum for dAdo could not be demonstrated due to the marked pharmacologic activity of dAdo. Cimetidine was found to selectively inhibit the organic cation secretory system since it blocked the renal secretion of tetraethylammonium but not that of p-amminohippurate in mice. Correspondingly, cimetidine prevented the renal secretion dTub; however, cimetidine did not inhibit the renal secretion of dAdo nor the renal reabsorption of Ado. These results suggest that renal secretion of dTub occurs via the organic cation carrier. The mechanisms for the renal secretion of dAdo and for the renal reabsorption of Ado may be unique and independent of the organic cation system.     

In vitro interactions of di-, tetra- and hexa-chlorobiphenyl with rabbit liver monooxygenase.

2,4,6,2',4',6'-Hexa,- 2,5,2',5'-tetra- and 2,2'-dichloro-biphenyl (HCB, TCB and DCB) caused a type-I-binding spectrum with liver microsomes of phenobarbital treated rabbits. They had a high affinity to cytochrome P-450, which increased with decreasing chlorine content of the compounds. In parallel, the O-demethylation of p-nitroanisole was most effectively inhibited by DCB, to a smaller extent by TCB and HCB. Inhibition was competitive. While DCB was rapidly metabolized, metabolism of TCB was slow. No metabolites have been detected of HCB. Although HCB was not metabolized it stimulated the microsomal NADPH- and oxygen-consumption and also the reduction of cytochrome P-450. The stoichiometry of the NADPH- and O₂-consumption of 2:1 in the presence of HCB is compatible with the notion that NADPH is oxidized to H₂O, i.e. HCB may act as uncoupler of electron transport from monooxygenation.