Evidence for phosphatidylcholine hydrolysis by phospholipase C in rat platelets.

Production of [3H]1,2-dipalmitoylglycerol ([3H]DAG) from 1-palmitoyl-2-[9,10-3H]palmitoyl-sn-glycero-3-phosphocholine and [3H]phosphorylcholine from 1,2-dipalmitoyl-sn-glycero-3-[Me-3H]phosphocholine was studied using sonicated rat platelets. The formation of [3H]DAG and [3H]phosphorylcholine occurred at a comparable rate. [3H]Phosphorylcholine formation was dependent on the concentration of the substrate, platelet sonicates and calcium in the incubation medium. The [3H]phosphorylcholine formation increased in presence of 0.01% deoxycholate and 0.01% Triton X-100. The phosphatidylcholine-phospholipase C (PC-PLC) in the platelet sonicates was recovered in both the supernatant and particulate fractions obtained after ultracentrifugation at 105,000 x g for 1 h. The PC-PLC activity in both fractions was inhibited by 2 mM EDTA. In the presence of 0.01% deoxycholate and 0.01% Triton X-100 the activity in the particulate fraction increased compared to the activity in the supernatant, which was inhibited by 0.01% Triton X-100. The pH optima for PC-PLC in both fractions was between pH 7.2 and 7.6. PC-PLC activity was also found in rabbit and human platelet sonicates, but the activity was significantly lower than in rat platelet sonicates. There was no evidence to suggest presence of phosphatidylcholine-specific phospholipase D activity in rat sonicated platelets. This data, therefore, provides direct evidence for the presence of PC-PLC activity in rat platelets.     

Determination of proteolytic activity using L-[4,5-3H]leucine-labelled globin as a substrate

A method is described for the assay of proteolytic activity, based on the digestion of L-[4,5-3H]leucine globin. L-[4,5-3H]Leucine was incorporated into the substrate at the stage of haemoglobin biosynthesis, using rabbit erythrocytes. Assay methods for proteolytic enzymes have been based on the digestion of haemoglobin, serum albumin or casein, and the determination of the trichloroacetic acid-soluble products [1,2]. More sensitive methods have been developed by using haemoglobin labelled with a fluorescent [3-5] or radioactive marker [6,7]. These methods avoid the errors which beset the Anson procedure, such as interference by impurities (purines at 280 nm and reducing compounds at 700 nm) [8]. However, methods using labelled proteins as a substrate present a number of problems, the most troublesome of which are the high blank values and the use of non-physiological substrates when chemically modified proteins are employed. In the present communication a simple and sensitive method for the assay of proteolytic enzyme activity is described. This is based on the digestion of L-[4,5-3H]leucine globin by proteolytic enzymes and radioactivity measurement of the trichloroacetic acid soluble cleavage products.     

The measurement of mitochondrial beta-oxidation by release of 3H2O from [9,10-3H]hexadecanoate: application to skeletal muscle and the use of inhibitors as models of metabolic disease.

We describe the use of a simple assay for beta-oxidation which depends on the release of 3H2O from [9,10-3H]hexadecanoate. This was compared with the use of [1-14C]hexadecanoate which gave comparable results when all the products of beta-oxidation were measured. The prediction that 75% of the tritium is released as 3H2O and 25% as [2-3H]acetyl units was confirmed. The assay was used successfully to demonstrate impaired beta-oxidation in tissue preparations from rats treated with etomoxir and methylenecyclopropylpyruvate which are known inhibitors of beta-oxidation. Abnormalities of beta-oxidation were also detected in skeletal muscle from patients with defects of mitochondrial oxidation.     

Differential effect of valproate and its Delta2- and Delta4-unsaturated metabolites, on the beta-oxidation rate of long-chain and medium-chain fatty acids

Overall fatty acid oxidation rates were investigated in rat hepatocytes using [9,10-3H]-palmitic, [9,10-3H]-oleic, [9,10-3H]-myristic and [2,3-3H]-phenylpropionic acids. The effect of both valproate (VPA) (0-10 mM) and two of its unsaturated metabolites, Delta2(E)-VPA and Delta4-VPA (0-10 mM), on the overall 3H2O production rate was studied. The results give evidence of a general inhibitory effect of VPA on the beta-oxidation rate of all the tested substrates. Similar effects were observed with both VPA metabolites but these effects appeared to be dependent on the chain length of the substrate. When the effect on the oxidation of the medium-chain fatty acid 3-phenylpropionate (PPA) was studied, Delta2(E)-VPA at 0.5 mM caused a 94% inhibition of the overall beta-oxidation rate. However, with long-chain substrates, 0.5 mM Delta(4)-VPA was a more potent inhibitor (20-30% of control activity) than 0.5 mM Delta(2E)-VPA (60-80% of control activity). Our results suggest that VPA and/or its metabolites inhibit fatty acyl-CoA metabolism within the mitochondrion by two different mechanisms. The first mechanism involves CoASH sequestration, which affects the oxidation rate of all fatty acids with different chain length. The second mechanism is more specific in nature and involves selective inhibition of particular enzymes implicated in fatty acid beta-oxidation.     

A rapid assay for activity of phospholipase A2 using radioactive substrate

A rapid method for the assay of phospholipase A2 has been developed using a radioactive substrate, L-alpha-dipalmitoyl-(2-[9,10(N)-3H]palmitoyl)-phosphatidylcholine. The substrate diluted with cold carrier (1 mM) is dissolved in 80% ethanol containing 25 mM sodium deoxycholate. The enzymatic reaction is performed in 1.0 ml 0.1 M glycine-NaOH buffer, pH 9.0, containing 2 mumol CaCl2, 10 micrograms bovine serum albumin, 2.5 mumol sodium deoxycholate, 0.01 unit (or less) phospholipase A2, and 40-100 nmol substrate. The enzymatic reaction is terminated by adding 0.2 ml 5% Triton X-100 solution containing 40 mumol EDTA. The product of the enzymatic reaction, radioactive palmitic acid, is extracted by 10 ml hexane containing 0.1% acetic acid in the presence of anhydrous sodium sulfate (0.5 g/ml). Activity of phospholipase A2 is directly determined from the radioactivity in the hexane extract. The present method achieves a quick separation of the radioactive product, [3H]palmitic acid, from the radioactive substrate, L-alpha-dipalmitoyl-(2-[3H]palmitoyl)-phosphatidylcholine, without the need of separation by TLC.     

Morphological transforming activity and metabolism of cyclopenta-fused isomers of benz[a]anthracene in mammalian cells

4 isomeric cyclopenta-derivatives of benz[e]anthracene (benz[a]aceanthrylene, benz[j]aceanthrylene, benz[l]aceanthrylene, and benz[k]acephenanthrylene) were examined for their ability to morphologically transform C3H10T1/2CL8 mouse-embryo fibroblasts. All of these polycyclic aromatic hydrocarbons studied except benz[k]acephenanthrylene transformed C3H10T1/2CL8 cells to both type II and type III foci in a concentration-dependent fashion. Benz[j]aceanthrylene was the most active, equivalent in activity to benzo[a]pyrene on a molar basis, in producing dishes of cells with transformed foci (94% at 1.0 microgram/ml). Benz[e]aceanthrylene, and benz[l]aceanthrylene produced 58% and 85% of the dishes with foci respectively at 10 micrograms/ml. Metabolism studies with [3H]benz[j]aceanthrylene in C3H10T1/2CL8 cells in which unconjugated, glucuronic acid conjugated, and sulfate conjugated metabolites were measured indicated that the dihydrodiol precursor to the bay-region diol-epoxide, 9,10-dihydroxy-9,10-dihydrobenz[j]aceanthrylene, was the major dihydrodiol formed (55%). Smaller quantities of the cyclopenta-ring dihydrodiol, 1,2-dihydroxy-1,2-dihydrobenz[j]aceanthrylene (14%), and the k-region dihydrodiol, 11,12-dihydroxy-11,12-dihydrobenz[j]aceanthrylene (5%) were also formed. Similar studies with [14C]benz[l]aceanthrylene indicated that the k-region dihydrodiol, 7,8-dihydroxy-7,8-dihydrobenz[l]aceanthrylene was the major metabolite formed (45%). The cyclopenta-ring dihydrodiol, 1,2-dihydroxy-1,2-dihydrobenz[l]aceanthrylene and 4,5-dihydroxy-4,5-dihydrobenz[l]aceanthrylene were formed in minor amounts (less than 6%). Therefore, metabolism at the cyclopenta-ring of B(j)A and B(l)A is a minor pathway in C3H10T1/2CL8 cells in contrast to previously reported studies with cyclopenta[cd]pyrene in which the cyclopenta-ring dihydrodiol was the major metabolite. These results suggest that routes of metabolic activation other than oxidation at the cyclopenta-ring such as bay region or k-region activation may play an important role with these unique polycyclic aromatic hydrocarbons in C3H10T1/2CL8 cells.     

Dual-label high-performance liquid chromatographic assay for femtomole levels of benzo[a]pyrene metabolites

A dual-label HPLC assay to measure femtomole quantities of ethyl acetate-extractable [3H]benzo[a]pyrene metabolites was developed. 14C-labeled metabolites of benzo[a]pyrene formed by rat liver 9000g supernatant were used as both internal standards and chromatographic markers. The percentage deviation between assays was determined to be between 11 and 13% for 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, benzo[a]pyrene-3,6-quinone, benzo[a]pyrene-1,6-quinone, and 9-hydroxybenzo[a]pyrene, 22% for 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, and less than 5% for 3-hydroxybenzo[a]pyrene. The detection limit of this assay was between 3 and 10 fmol per metabolite. The application of this technique to the metabolism of [3H]benzo[a]pyrene by microsomes of hamster and human oral cavity tissue is described.     

Lipid chain length alterations during placental transfer in the guinea pig

Using an in situ perfusion of the fetal side of the guinea-pig placenta the modification of a non-esterified fatty acid during transfer across the placenta was investigated. Simultaneous constant infusions of [9,10(3)H] palmitic acid and [1-14C] palmitic acid (3 animals) or [9,10(3)H] and [6-14C] palmitic acids (3 animals) or [9,10(3)H] and universal [14C] palmitic acids (3 animals) were given to the mothers and blood samples and perfusion fluid collected over 90 min in each experiment. When expressed as a ratio of perfusion fluid/maternal plasma radioactive counts, no difference between [3H] isotopes results were found for the 3 triplets of experiments. However significant differences were found between the [14C] isotope ratios. More radioactive lipid was found in the perfusion fluid when the label was positioned away from the C1 terminal of the fatty acid chain, i.e. the ratios were [1-14C] less than [6-14C] less than [9,10(3)H] less than universal [14C] palmitic acid. It was concluded that this indicates release of partially oxidised fatty acid products from the fetal side of the placenta, and it was speculated that this partial oxidation takes place in placental peroxisomes.     

A new tritium-release assay for 25-hydroxyvitamin D-1 alpha-hydroxylase

A new, rapid assay for 1 alpha-hydroxylase has been developed using 25-hydroxy-[1 alpha-3H]vitamin D3 as the substrate. Using the solubilized and reconstituted chick 1 alpha-hydroxylase, conversion of this substrate to 1,25-dihydroxyvitamin D3 causes the release of tritium into the aqueous medium. This 3H2O can be easily separated from the labeled substrate by passing the reaction mixture through a reverse-phase silica cartridge. The release of tritium is stereospecific as evidenced by the lack of 3H2O formed when 25-hydroxy-[1 beta-3H]vitamin D3 is used as the substrate. In parallel reactions containing the 25-hydroxy-[26,27-3H]vitamin D3 substrate, production of labeled 1,25-dihydroxyvitamin D3 was assessed by extraction and high-performance liquid chromatography and found to agree very closely with the amount of 3H2O produced from 25-hydroxy-[1 alpha-3H]vitamin D3, validating the accuracy of the new assay. Finally, a major advantage of the tritium-release assay for 1 alpha-hydroxylase is that the results are not affected by further metabolism of the 1,25-dihydroxyvitamin D formed in the incubations.     

Role of carnitine and carnitine palmitoyltransferase as integral components of the pathway for membrane phospholipid fatty acid turnover in intact human erythrocytes.

The deacylation and reacylation process of phospholipids is the major pathway of turnover and repair in erythrocyte membranes. In this paper, we have investigated the role of carnitine palmitoyltransferase in erythrocyte membrane phospholipid fatty acid turnover. The role of acyl-L-carnitine as a reservoir of activated acyl groups, the buffer function of carnitine, and the importance of the acyl-CoA/free CoA ratio in the reacylation process of erythrocyte membrane phospholipids have also been addressed. In intact erythrocytes, the incorporation of [1-14C]palmitic acid into acyl-L-carnitine, phosphatidylcholine, and phosphatidylethanolamine was linear with time for at least 3 h. The greatest proportion of the radioactivity was found in acyl-L-carnitine. Competition experiments using [1-14C]palmitic and [9,10-3H]oleic acid demonstrated that [9,10-3H]oleic acid was incorporated preferentially into the phospholipids and less into acyl-L-carnitine. When an erythrocyte suspension was incubated with [1-14C]palmitoyl-L-carnitine, radiolabeled palmitate was recovered in the phospholipid fraction, and the carnitine palmitoyltransferase inhibitor, 2-tetradecylglycidic acid, completely abolished the incorporation. ATP depletion decreased incorporation of [1-14C]palmitic and/or [9,10-3H]oleic acid into acyl-L-carnitine, but the incorporation into phosphatidylcholine and phosphatidylethanolamine was unaffected. In contrast, ATP depletion enhanced the incorporation into phosphatidylcholine and phosphatidylethanolamine of the radiolabeled fatty acid from [1-14C]palmitoyl-L-carnitine. These data are suggestive of the existence of an acyl-L-carnitine pool, in equilibrium with the acyl-CoA pool, which serves as a reservoir of activated acyl groups. The carnitine palmitoyltransferase inhibition by 2-tetradecylglycidic acid or palmitoyl-D-carnitine caused a significant reduction of radiolabeled fatty acid incorporation into membrane phospholipids, only when intact erythrocytes were incubated with [9,10-3H]oleic acid. These latter data may be explained by the differences in rates and substrates specificities between acyl-CoA synthetase and the reacylating enzymes for palmitate and oleate, which support the importance of carnitine palmitoyltransferase in modulating the optimal acyl-CoA/free CoA ratio for the physiological expression of the membrane phospholipids fatty acid turnover.     

t-Butyl hydroperoxide alters fatty acid incorporation into erythrocyte membrane phospholipid

Because the ability of cells to replace oxidized fatty acids in membrane phospholipids via deacylation and reacylation in situ may be an important determinant of the ability of cells to tolerate oxidative stress, incorporation of exogenous fatty acid into phospholipid by human erythrocytes has been examined following exposure of the cells to t-butyl hydroperoxide. Exposure of human erythrocytes to t-butyl hydroperoxide (0.5-1.0 mM) results in oxidation of glutathione, formation of malonyldialdehyde, and oxidation of hemoglobin to methemoglobin. Under these conditions, incorporation of exogenous [9,10-3H]oleic acid into phosphatidylethanolamine is enhanced while incorporation of [9,10-3H]oleic acid into phosphatidylcholine is decreased. These effects of t-butyl hydroperoxide on [9,10-3H]oleic acid incorporation are not affected by dissipating transmembrane gradients for calcium and potassium. When malonyldialdehyde production is inhibited by addition of ascorbic acid, t-butyl hydroperoxide still decreases [9,10-3H]oleic acid incorporation into phosphatidylcholine but no stimulation of [9,10-3H]oleic acid incorporation into phosphatidylethanolamine occurs. In cells pre-treated with NaNO2 to convert hemoglobin to methemoglobin, t-butyl hydroperoxide reduces [9,10-3H]oleic acid incorporation into phosphatidylcholine by erythrocytes but does not stimulate [9,10-3H]oleic acid incorporation into phosphatidylethanolamine. Under these conditions oxidation of erythrocyte glutathione and formation of malonyldialdehyde still occur. These results indicate that membrane phospholipid fatty acid turnover is altered under conditions where peroxidation of membrane phospholipid fatty acids occurs and suggest that the oxidation state of hemoglobin influences this response.     

Turnover of arachidonic acid in the major diacyl and ether phospholipids of human platelets

In this work, the uptake and release of [3H]arachidonic acid by the diacyl and ether species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in human platelets were studied. Uptake of [3H]arachidonic acid into 1,2-diacyl-PC and 1,2-diacyl-PE was much greater than into the ether phospholipids of the same class. In [3H]arachidonoyl-labeled platelets stimulated by thrombin, there was a decrease in total [3H] arachidonoyl-PC. This was accounted for mostly by a decrease in 1-acyl-2-[3H]arachidonoyl-PC while the level of 1-O-alkyl-2-[3H]arachidonoyl-PC (a precursor for platelet-activating factor) increased slightly. However, in ionophore A23187-stimulated platelets, the reduction of total [3H]arachidonoyl-PC was due to a decrease in both 1-acyl-2-[3H]arachidonoyl-PC and 1-O-alkyl-2-[3H] arachidonoyl-PC, suggesting that ionophore should yield more platelet-activating factor than thrombin. In both thrombin- and ionophore-stimulated platelets, there was a net increase in total [3H]arachidonoyl-PE. This consisted of a decrease in 1,2-diacyl-PE, which was essentially complete by 1 min, followed by an increase in 1-O-alk-1'-enyl-2-[3H]arachidonoyl-PE, which was slower and not apparent until 3-5 min after thrombin. During reincubation of labeled platelets with saline, the 1-O-alkyl-2-[3H]arachidonoyl-PC increased by a factor of 2, between 0 and 4 h, with no significant change in the radioactivity of any other phospholipid. Thus, upon stimulation of human platelets, arachidonic is released from both 1,2-diacyl-PC and 1,2-diacyl-PE for metabolism by platelet cyclooxygenase and lipoxygenase, while certain ether pools of PC and PE also collect arachidonic acid.     

Metabolism of platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and 1-alkyl-2-acetyl-sn-glycerol by human endothelial cells

The metabolism of platelet activating factor (1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine) and 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol was studied in cultures of human umbilical vein endothelial cells. Human endothelial cells deacetylated 1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine to the corresponding lyso compound (1-[1,2-3H]alkyl-2-lyso-sn-glycerol-3-phosphocholine) and a portion was converted to 1-[1,2-3H]alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholine. Lyso platelet activating factor (lyso-PAF) (1-[1,2-3H]alkyl-2-lyso-sn-glycero-3-phosphocholine) was detected in the media very early during the incubation and the amount remained higher than the level of the lyso product observed in the cells. Cellular levels of 1-[1,2-3H]alkyl-2-lyso-sn-glycero-3-phosphocholine were significantly higher than the acylated product (1-[1,2-3H]alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholine) at all times during the 60-min incubation period, which suggests that the ratio of acetylhydrolase to acyltransferase activities is greater in endothelial cells than in most other cells. When endothelial cells were incubated with 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol, a known precursor of PAF, 1-[1,2-3H]alkyl-sn-glycerol was the major metabolite formed (greater than 95% of the 3H-labeled metabolites during 20- and 40-min incubations). At least a portion of the acetate was removed from 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol by a hydrolytic factor released from the endothelial cells into the medium during the incubations. Only negligible amounts of the total cellular radioactivity (0.2%) was incorporated into platelet activating factor (1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine); therefore, it is unlikely that the previously observed hypotensive activity of 1-alkyl-2-acetyl-sn-glycerols can be explained on the basis of the conversion to platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by endothelial cells. Results of this investigation indicate that endothelial cells play an important role in PAF catabolism. Undoubtedly, the endothelium is important in the regulation of PAF levels in the vascular system.     

Utilization of disaturated and unsaturated phosphatidylcholine and diacylglycerols by cholinephosphotransferase in rat lung microsomes

1. Cholinephosphosphotransferase catalyzes the conversion of diacylglycerol and CDPcholine into phosphatidylcholine and CMP. Incubation of rat lung microsomes containing phosphatidyl[Me-14C]choline with CMP resulted in an increase in water-soluble radioactivity, suggesting that also in rat lung microsomes the cholinephosphotransferase reaction is reversible. 2. Microsomes containing 14C-labeled disaturated and 3H-labeled monoenoic phosphatidylcholine were prepared by incubation of these organelles with [1-14C]palmitate and [9,10-3H2]oleate in the presence of 1-palmitoyl-sn-glycero-3-phosphocholine, ATP, coenzyme A and MgCl2. Incubation of these microsomes with CMP resulted in an equal formation of 14C- and 3H-labeled diacylglycerols, indicating that disaturated and monoenoic phosphatidylcholines were used without preference by the backward reaction of the cholinephosphotransferase. When in a similar experiment the phosphatidylcholine was labeled with [9,10-3H2]palmitate and [1-14C]linoleate, somewhat more 14C- than 3H-labeled diacylglycerol was formed. 3. The backward reaction was used to generate membrane-bound mixtures of [1-14C]palmitate- and [9,10-3H2]oleate- or of [9,10-3H2]palmitate- and [1-14C]linoleate-labeled diacylglycerols. When the microsomes containing diacylglycerols were incubated with CDPcholine, both 3H- and 14C-labeled diacylglycerols were used for the formation of phosphatidylcholine, indicating that there is no absolute discrimination against disaturated diacylglycerols. This observation is in line with our previous findings and indicates that also the CDPcholine pathway may contribute to dipalmitoylphosphatidylcholine synthesis in lung.     

Metabolism of the diacetyl derivatives of stereoisomeric monoacyl-sn-glycerols by rat adipocytes in vitro.

Diacetyl long-chain 1(3)- and 2-acyl-sn-glycerols containing either [9,10-3H]oleic acid or [1-14C]palmitic acid were synthesized by partial hydrolysis of the corresponding labelled triacylglycerols and acetylation. They were obtained in a high degree of stereochemical purity by preparative h.p.l.c. on a column containing a diol bonded phase. Each compound was rapidly metabolized by adipocyte preparations in vitro, and a high proportion of the label was recovered in the unesterified fatty acid and triacylglycerol fractions. Negligible amounts of intermediate products of hydrolysis were detected. Triacylglycerols were formed from [9,10-3H]oleic acid and from diacetyl-1(3)-[9,10-3H]oleoyl glycerol precursors at about the same rate, but the 2-isomer was metabolized rather more slowly. The results were consistent with the hypothesis that essentially complete hydrolysis occurred in the medium or at the plasma membrane, through the actions of lipoprotein lipase and monoacylglycerol lipase, and that subsequent esterification took place within the cell. To confirm that no putative intermediate monoacylglycerols were utilized for triacylglycerol biosynthesis via the monacylglycerol pathway, the positional distributions of fatty acids in triacylglycerols from each substrate were determined. No positional selectivity was observed. It was concluded that monoacylglycerols, of an origin exogenous to the tissue, e.g. those derived from plasma triacylglycerols, were not utilized to a significant degree for triacylglycerol biosynthesis in adipose tissue. The diacetyl derivatives of monoacylglycerols may serve as useful stereochemical probes in studies of triacylglycerol biosynthesis via the monoacylglycerol pathway in other tissues.     

Rapid assay for hormone-sensitive lipase activity of adipose tissue

A highly specific and rapid assay for hormone-sensitive lipase activity of rat adipose tissue is described. The method employs emulsified 2,3-di-O-oleyl-[9,10-(3)H(2)]oleoyl glycerol as a substrate; it is very sensitive and is suitable for serial sampling.     

Hydrolysis of triacylglycerol arachidonic and linoleic acid ester bonds by human pancreatic lipase and carboxyl ester lipase

The hydrolysis of polyenoic fatty acid ester bonds with pure human colipase-dependent lipase, with carboxyl ester lipase (CEL) and with these enzymes in combination was studied, using [3H]arachidonic- and [14C]linoleic acid-labelled rat chylomicrons as a model substrate. During the hydrolysis with colipase-dependent lipase, the amount of 3H appearing in 1,2-X-diacylglycerol (DG) markedly exceeded that of 14C. When CEL was added in addition this [3H]DG was efficiently hydrolyzed. CEL alone hydrolyzed the triacylglycerol (TG) at a low rate. The hydrolysis pattern with human duodenal content was similar to that seen with colipase-dependent lipase and CEL in combination. Increasing the concentration of taurodeoxycholate (TDC) and taurocholate (TC) or of TDC alone stimulated the hydrolysis of [3H]- and [14C]TG, but increased the accumulation of labelled DG that could act as substrate for CEL. It is suggested that very-long-chain polyenoic fatty acids of DG formed during the action of the colipase-dependent lipase on TG containing these fatty acids may be a physiological substrate for CEL.     

Steric course of the hydration of D-gluco-octenitol catalyzed by alpha-glucosidases and by trehalase

Crystalline Aspergillus niger alpha-glucosidase and highly purified preparations of rice alpha-glucosidase II and Trichoderma reesei trehalase were found to catalyze the hydration of [2-(2)H]-D-gluco-octenitol, i.e., (Z)-3,7-anhydro-1,2-dideoxy-[2-2H]-D-gluco-oct-2-enitol, to yield 1,2-dideoxy-[2-2H]-D-gluco-octulose. In each case, the stereochemistry of the reaction was elucidated by examining the newly formed centers of asymmetry at C-2 and C-3 of the hydration product. The C-1 to C-3 fragment of each isolated [2-2H]-D-gluco-octulose product was recovered as [2-2H]propionic acid and identified by its positive optical rotatory dispersion as the S isomer, showing that each enzyme had protonated the octenitol (at C-2) from above its re face. 1H NMR spectra of enzyme/D-gluco-octenitol digests in D2O showed that the alpha-anomer of [2-2H]-D-gluco-octulose was exclusively produced by each alpha-glucosidase, whereas the beta-anomer was formed by action of the trehalase. The trans hydration catalyzed by the alpha-glucosidases was found to be very strongly inhibited by the substrate; the cis hydration reaction catalyzed by the trehalase showed no such inhibition. Special importance is attached to the finding that in hydrating octenitol each enzyme creates a product of the same anomeric form as in hydrolyzing an alpha-D-glucosidic substrate. This result adds substantially to the growing evidence that individual glycosylases create the configuration of their reaction products by a means that is independent of donor substrate configuration, that is, by a means other than "retaining" or "inverting" substrate configuration.     

Competitive, reversible inhibition of cytosolic phospholipase A2 at the lipid-water interface by choline derivatives that partially partition into the phospholipid bilayer.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA2 using membranes isolated from [3H]arachidonate-labeled U937 cells as substrate, 2-(2'-benzyl-4-chlorophenoxy)ethyl-dimethyl-n-octadecyl-ammonium chloride (compound 1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 5 microM). It was over 70 times more selective for the cPLA2 as compared with the human nonpancreatic secreted phospholipase A2, and it did not inhibit other phospholipases. Additionally, it inhibited arachidonate production in N-formyl-methionyl-leucyl-phenylalanine-stimulated U937 cells. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl-sn -glycero-3-phosphomethanol containing 6-10 mol % of 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid-water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (KI*app) was determined to be 0.097 +/- 0.032 mol % versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.3 +/- 0.1 mol %. Thus, compound 1 represents a novel structural class of inhibitor of cPLA2 that partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site. Shorter n-alkyl-chained (C-4, C-6, C-8) derivatives of compound 1 were shown to have even smaller KI*app values. However, these short-chained analogs were less potent in terms of bulk inhibitor concentration needed for inhibition when using the [3H]arachidonate-labeled U937 membranes as substrate. This discrepancy was reconciled by showing that these shorter-chained analogs did not partition into the [3H]arachidonate-labeled U937 membranes as effectively as compound 1. The implications for in vivo efficacy that result from these findings are discussed.     

Indeno[1,2-b]indole derivatives as a novel class of potent human protein kinase CK2 inhibitors

Herein we describe the synthesis and properties of indeno[1,2-b]indole derivatives as a novel class of potent inhibitors of the human protein kinase CK2. A set of 19 compounds was obtained using a convenient and straightforward synthesis protocol. The compounds were tested for inhibition of human protein kinase CK2, which was recombinantly expressed in Escherichia coli. New inhibitors with IC(50) in the micro- and sub-micromolar range were identified. Compound 4b (5-isopropyl-7,8-dihydroindeno[1,2-b]indole-9,10(5H,6H)-dione) inhibited human CK2 with an IC(50) of 0.11 μM and did not significantly inhibit 22 other human protein kinases, suggesting selectivity towards CK2. ATP-competitive inhibition by compound 4b was shown and a K(i) of 0.06 μM was determined. Our findings indicate that indeno[1,2-b]indoles are a promising starting point for further development and optimization of human protein kinase CK2 inhibitors.