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.     

Effects of human pancreatic lipase-colipase and carboxyl ester lipase on eicosapentaenoic and arachidonic acid ester bonds of triacylglycerols rich in fish oil fatty acids

Fish oil chylomicrons, obtained from mesenteric duct chyle of rats fed [3H]20:5 and [14C]20:4 or [3H]20:5 and [14C]18:2 in a fish oil emulsion, were incubated with human pancreatic lipase-colipase, human carboxyl ester lipase (CEL) and human duodenal contents. With duodenal contents, the triacylglycerols labelled with [3H]20:5 and [14C]20:4 were rapidly converted to free fatty acids (FFA) and monoacylglycerols. Also during incubation with lipase-colipase the [3H]- and [14C]triacylglycerols disappeared completely and at equal rates, but in this case much [3H]20:5 and [14C]20:4 accumulated in diacylglycerols. When CEL was also added, the rate of disappearance of [3H]- and [14C]triacylglycerols increased and the radioactivity of diacylglycerols decreased markedly. During incubation of chylomicrons labelled with [3H]20:5 and [14C]18:2 with lipase-colipase, the rates of hydrolysis of [3H]- and [14C]triacylglycerols were similar, but more [3H]20:5 than [14C]18:2 accumulated in diacylglycerols. The accumulation of [3H]diacylglycerol was reduced by adding CEL. Also when fatty acids were analyzed by gas chromatography, 20:5 was enriched in remaining triacylglycerol and in diacylglycerol after incubation with lipase-colipase alone. The data thus indicate that both lipase-colipase and CEL participate in the hydrolysis of 20:5 and 20:4 ester bonds of dietary triacylglycerol.     

Reduction of the beta-Cys-gamma-Glu thiol ester bond of human C3 with sodium borohydride

Further chemical evidence has been obtained using NaB3H4 to support our previous assignment of a thiol ester bond in human C3 (Tack, B. F., Harrison, R. A., Janatova, J., Thomas, M. L., and Prahl, J. W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5764-5768). Following trypsin activation of human C3 in the presence of NaB3H4, 3H was shown to have incorporated specifically into the alpha'-chain of C3b. Subsequent fragmentation of [3H]C3b with porcine elastase further localized the label to the C3d subdomain. Under identical conditions, native C3 or C3 pretreated with trypsin (C3b) showed low reactivity with NaB3H4. A tryptic peptide containing the 3H label was isolated following digestion of [3H]C3b on activated thiol-Sepharose. After hydrolysis and saponification of the peptide hydrolysate, amino acid analysis indicated that the 3H had been incorporated into alpha-amino-delta-hydroxyvaleric acid, the product expected from reduction of an ester bond involving a glutamyl residue. On sequence analysis of the labeled peptide, the 3H was shown to reside at the position of the glutamyl residue previously proposed to be involved in the thiol ester bond. The residue at this position was confirmed as alpha-amino-delta-[3H] hydroxyvaleric acid by high performance liquid chromatography analysis and, after back hydrolysis, by amino acid analysis. These data significantly strengthen earlier studies which indicated the presence of a beta-Cys-gamma-Glu thiol ester bond in human C3.     

Lecithin-cholesterol acyltransferase (LCAT) catalyzes transacylation of intact cholesteryl esters. Evidence for the partial reversal of the forward LCAT reaction

Lecithin-cholesterol acyltransferase (LCAT) catalyzes the intravascular synthesis of lipoprotein cholesteryl esters by converting cholesterol and lecithin to cholesteryl ester and lysolecithin. LCAT is unique in that it catalyzes sequential reactions within a single polypeptide sequence, a phospholipase A2 reaction followed by a transacylation reaction. In this report we find that LCAT mediates a partial reverse reaction, the transacylation of lipoprotein cholesteryl oleate, in whole plasma and in a purified, reconstituted system. As a result of the reverse transacylation reaction, a linear accumulation of [3H]cholesterol occurred during incubations of plasma containing high density lipoprotein labeled with [3H]cholesteryl oleate. When high density lipoprotein labeled with cholesteryl [14C]oleate was also included in the incubation the labeled fatty acyl moiety remained in the cholesteryl [14C]oleate pool showing that the formation of labeled cholesterol did not result from hydrolysis of the doubly labeled cholesteryl esters. The rate of release of [3H]cholesterol was only about 10% of the forward rate of esterification of cholesterol using partially purified human LCAT and was approximately 7% in whole monkey plasma. Therefore, net production of cholesterol via the reverse LCAT reaction would not occur. [3H]Cholesterol production from [3H]cholesteryl oleate was almost completely inhibited by a final concentration of 1.4 mM 5,5'-dithiobis(nitrobenzoic acid) during incubation with either purified LCAT or whole plasma. Addition of excess lysolecithin to the incubation system did not result in the formation of [14C]oleate-labeled lecithin, showing that the reverse reaction found here for LCAT was limited to the last step of the reaction. To explain these results we hypothesize that LCAT forms a [14C]oleate enzyme thioester intermediate after its attack on the cholesteryl oleate molecule. Formation of this intermediate allows [3H]cholesterol to be liberated from the enzyme by exchange with unlabeled cholesterol of plasma lipoproteins. The liberated [3H]cholesterol thereby becomes available for reesterification by LCAT as indicated by its appearance as newly synthesized cholesteryl linoleate.     

Chylomicron-remnant uptake by freshly isolated hepatocytes. Effect of heparin and of hepatic triacylglycerol lipase.

Chylomicron remnants labelled biologically with [3H]cholesterol were efficiently taken up by freshly isolated hepatocytes during a 3 h incubation in Krebs bicarbonate medium. Their [3H]cholesteryl ester was hydrolysed (74% net hydrolysis), and 0.1 mM-chloroquine could partially inhibit this hydrolysis, provided that hepatocytes were first preincubated for 2 h 30 min at 37 degrees C. This hydrolysis was also measured in preincubated cells with remnants double-labelled (3H and 14C) on their free cholesterol moiety; [3H]cholesterol arising from [3H]cholesteryl ester hydrolysis was recovered in the free [3H]cholesterol pool. A dose-response study showed saturation of remnant uptake at 180 micrograms of remnant protein/10(7) cells. Heparin (10 units/ml) increased remnant uptake by 63% (P less than 0.01), [3H]cholesteryl ester accumulation in the cell pellet by 110% (P less than 0.025) and hepatic lipase activity secreted in the medium by 2.4-fold (P less than 0.01) and by 3.3-fold (P less than 0.01) at the end of the preincubation and incubation periods respectively. Addition of 100 munits of semi-purified hepatic lipase preparation/flask stimulated remnant uptake by 44-69%, and [3H]cholesteryl ester accumulation in the presence of chloroquine by 2.1-fold (P less than 0.025). When hepatic lipase was incubated solely with the remnants, it decreased their triacylglycerol and phospholipid contents by 24% and 26% respectively. Thus freshly isolated hepatocytes may be used to study chylomicron-remnant uptake. Hepatic lipase, which seems to underly the stimulating effect of heparin, facilitates remnant uptake in vitro, and this could be mediated by at least one (or both) of its hydrolytic properties.     

Metabolism of vitamin D. A new cholecalciferol metabolite, involving loss of hydrogen at C-1, in chick intestinal nuclei

1. A comparison was made of the nature and intestinal intracellular distribution of the metabolites formed in vitamin D-deficient chicks from [4-(14)C]cholecalciferol and [1-(3)H]cholecalciferol. 2. The simultaneous administration of the two radioactive substances showed the presence in blood, liver, intestine, kidney and bone of cholecalciferol, its ester, 25-hydroxycholecalciferol and a further metabolite of cholecalciferol more polar than 25-hydroxycholecalciferol. The (3)H/(14)C ratios in these four radioactive components were the same as that of the dosed material (4.7:1) with the exception of the most polar material. The (3)H/(14)C ratio was lower in the fourth, most polar, metabolite (0.4:1-1.8:1) in all tissues examined, with the exception of blood. 3. In the chick intestine the polar metabolite accounted for almost 70% of the radioactivity in this tissue after a dose of 0.5mug. of [4-(14)C,1-(3)H]cholecalciferol. This polar metabolite from the intestine also had the lowest (3)H/(14)C ratio of all the tissues. It appears that in the chick intestine the polar metabolite reaches a maximum concentration of 1ng./g. of tissue, above which it cannot be increased irrespective of the dose of the vitamin. 4. The intestinal intracellular organelle with the highest concentration of (14)C radioactivity is the nucleus, and this radioactivity is almost entirely due to the polar metabolite with the lowered (3)H/(14)C ratio, in this case <0.2:1. It appears to be further localized in the chromatin of the nuclei. However, about half of the polar metabolite in the intestine is extranuclear. 5. Double-labelled 25-hydroxycholecalciferol was prepared and after its administration to vitamin D-deficient chicks the polar metabolite with the lowered (3)H/(14)C ratio was detected in liver, kidney, intestine, bone, muscle and heart. 6. None of the polar metabolite with the lowered (3)H/(14)C ratio was detected 16hr. after dosing with either the double-labelled vitamin or the double-labelled 25-hydroxycholecalciferol in blood and adipose tissue of vitamin D-deficient chicks, nor in the intestine, liver and kidney of supplemented birds. 7. The reasons for this loss of (3)H relative to (14)C are discussed in relation to possible chemical structures of this new polar metabolite.     

Digestion and absorption of polyunsaturated fatty acids.

Polyunsaturated fatty acids play an important part in the structure and function of cellular membranes and are precursors of lipid mediators which play a key role in cardiovascular and inflammatory diseases. Dietary sources of essential fatty acids are vegetable oils for either linoleic or alpha-linolenic acids, and sea fish oils for eicosapentaenoic and docosahexaenoic acids. Because of the specificity of the pancreatic lipid hydrolases, triglyceride fatty acid distribution is an essential parameter in the digestibility of fats. The efficiency of the intestinal uptake depends on the hydrolysis and especially on their micellarization. n-3 polyunsaturated fatty acid ethyl ester digestion is recognized to be impaired, but n-3 polyunsaturated fatty acid triglyceride hydrolysis remains a controversial point, and to some authors explains differences observed between vegetable and fish oil absorption. So additional studies are required to investigate this intestinal step. In enterocytes, morphological and biochemical absorption processes involve reesterification of long-chain fatty acids and lipoprotein formation. At this level, specific affinity of I- and L-FABPc (cytosolic fatty acid binding proteins) to polyunsaturated fatty acids requires further investigation. A better understanding of the role of these FABPc might bring to light the esterification step, particularly the integration of polyunsaturated fatty acids into phospholipids. With reference to differences published between fish and vegetable oil absorption, longer-term absorption studies appear essential to some authors. Polyunsaturated fatty acid absorption is thought to be not very dissimilar to that of long-chain mono-unsaturated fatty acid absorption. However, several digestion and absorption specific steps are worth studying with reference to the crucial role of polyunsaturated fatty acids in the organism, and for example adaptation of possible dietary supplements.     

Putative role of cholesteryl ester transfer protein in removal of cholesteryl ester from vascular interstitium, studied in a model system in cell culture

A model system to study the putative role of cholesteryl ester transfer protein in the egress of interstitial cholesteryl ester is described. Confluent cultures of bovine aortic smooth muscle cells were labeled for 24 h with [3H]cholesteryl linoleyl ether and [14C]cholesteryl linoleate by incubation with bovine milk lipoprotein lipase. This method of labeling results in the transfer of cholesteryl linoleyl ether and cholesteryl ester to three compartments: a trypsin-releasable, trypsin-resistant and catabolic compartment (Stein, O., Halperin, G., Leitersdorf, E., Olivecrona, T. and Stein, Y. (1984) Biochim. Biophys. Acta 795, 47-59). The efflux of labeled cholesteryl linoleyl ether and cholesteryl ester from the extracellular and cell-surface related compartments into a serum-free culture medium containing 1% bovine serum albumin was studied during 24 h of postincubation. The efflux was expressed as a percentage of pulse value, i.e., radioactivity retained by the cell culture at the end of the labeling period. The efflux of [3H]cholesteryl linoleyl ether, [14C]cholesteryl ester and 14C-labeled free cholesterol (formed by cellular hydrolysis of cholesterol ester) into the culture medium with 1% bovine serum albumin was about 5% of the pulse value. Addition of human lipoprotein-deficient serum resulted in a 3-10-fold increase in the efflux of [3H]cholesteryl linoleyl ether and [14C]cholesteryl ester, but did not change markedly the efflux of 14C-labeled free cholesterol. Rat lipoprotein-deficient serum which does not contain cholesteryl ester transfer protein did not increase the efflux of [3H]cholesteryl linoleyl ether or [14C]cholesteryl ester. The rate of cholesteryl ester efflux in the presence of human lipoprotein-deficient serum was linear for about 6 h and increased further up to 24 h. Addition of Intralipid to medium containing human lipoprotein-deficient serum further enhanced the efflux of [3H]cholesteryl linoleyl ether and, to a lesser extent, that of cholesteryl ester. A similar effect was observed also by addition of rat VLDL to medium containing human lipoprotein-deficient serum. Inhibition of cholesteryl linoleyl ether and cholesteryl ester efflux and marked enhancement of free cholesterol efflux occurred when rat HDL was added to medium containing human lipoprotein-deficient serum, while human HDL was only slightly inhibitory. The results obtained with human lipoprotein-deficient serum were reproduced with partially purified cholesteryl ester transfer protein. Using the partially purified cholesteryl ester transfer protein, the efflux of cholesteryl linoleate was compared to that of cholesteryl oleate and was found to be the same.     

BSSL and PLRP2: key enzymes for lipid digestion in the newborn examined using the Caco-2 cell line

In rodents, bile salt-stimulated lipase (BSSL) and pancreatic lipase-related protein 2 (PLRP2) are the dominant lipases expressed in the exocrine pancreas in early life when milk is the main food. The aim of the present study was to evaluate whether BSSL and PLRP2 are also key enzymes in neonatal intestinal fat digestion. Using Caco-2 cells as a model for the small intestinal epithelium, purified human enzymes were incubated in the apical compartment with substrates, bile salt composition and concentrations physiologic to newborn infants. Both BSSL and PLRP2 hydrolyzed triglycerides (TG) to free FA and glycerol. Released FA were absorbed by the cells and reesterfied to TG. Together, BSSL and PLRP2 had a synergistic effect, increasing cellular uptake and reesterification 4-fold compared with the sum of each lipase alone. A synergistic effect was also observed with retinyl ester as a substrate. PLRP2 hydrolyzed cholesteryl ester but not as efficiently as BSSL, and the two had an additive rather than synergistic effect. We conclude the key enzymes in intestinal fat digestion are different in newborns than later in life. Further studies are needed to fully understand this difference and its implication for designing optimal neonatal nutrition.     

Synthesis of [1,2-3H2]cholecalciferol and metabolism of [4-14C,1,2-3H2]- and [4-14C,1-3H]-cholecalciferol in rachitic rats and chicks

[1,2-(3)H(2)]Cholecalciferol has been synthesized with a specific radioactivity of 508mCi/mmol by using tristriphenylphosphinerhodium chloride, the homogeneous hydrogen catalyst. With doses of 125ng (5i.u.) of [4-(14)C,1-(3)H(2)]cholecalciferol the tissue distribution in rachitic rats of cholecalciferol and its metabolites (25-hydroxycholecalciferol and peak P material) was similar to that found in chicken with 500ng doses of the double-labelled vitamin. The only exceptions were rat kidney, with a very high concentration of vitamin D, and rat blood, with a higher proportion of peak P material, containing a substance formed from vitamin D with the loss of hydrogen from C-1. Substance P formed from [4-(14)C,1,2-(3)H(2)]cholecalciferol retained 36% of (3)H, the amount expected from its distribution between C-1 and C-2, the (3)H at C-1 being lost. 25-Hydroxycholecalciferol does not seem to have any specific intracellular localization within the intestine of rachitic chicks. The (3)H-deficient substance P was present in the intestine and bone 1h after a dose of vitamin D and 30min after 25-hydroxycholecalciferol. There was very little 25-hydroxycholecalciferol in intestine at any time-interval, but bone and blood continued to take it up over the 8h experimental period. It is suggested that the intestinal (3)H-deficient substance P originates from outside this tissue. The polar metabolite found in blood and which has retained its (3)H at C-1 is not a precursor of the intestinal (3)H-deficient substance P.     

Hydrolysis of novel diacylglycerol analogs and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [3H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, over crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-14C]palmitoyl-2-acetyl-sn-glycerol and [1-14C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-times the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[3H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.     

Identification and characterization of mannosyl retinyl phosphate occurring in rat liver and intestine invivo

A study was conducted to determine whether mannosyl retinyl phosphate occurred in rat liver and intestine in vivo, and, if so, to partially purify it and investigate its properties. After injection of [(3)H]retinol and [(14)C]mannose, a chloroform-methanol 2:1 extract of rat liver and small intestinal mucosa yielded two (3)H/(14)C-labeled peaks on DEAE-cellulose column chromatography: peak I eluted with 10 mM and peak II eluted with 29 mM ammonium acetate. Peak II, subjected to silicic acid column chromatography, gave principally two (3)H/(14)C-labeled fractions, one eluted with chloroform-methanol 2:1 and the other with chloroform-methanol 1:1. The latter showed, on thin-layer chromatography in a chloroform-methanol-water 60:25:4 system, an R(f) of 0.25 (with coincidence of the (3)H and (14)C radioactivity), which is identical to the R(f) of authentic mannosyl retinyl phosphate. The chloroform-methanol 1:1 peak, on mild acid hydrolysis, yielded [(3)H]retinol (identified by two thin-layer chromatography systems), [(14)C]mannose, and [(14)C]-mannose phosphate (identified by paper chromatography). On mild alkali hydrolysis, the peak yielded [(3)H]retinol and [(14)C]mannose phosphate. The substance eluted in the chloroform-methanol 1:1 peak from silicic acid was therefore concluded to be mannosyl retinyl phosphate. When chromatographed on silicic acid, peak I from the DEAE-cellulose column primarily showed a fraction eluted with chloroform-methanol 2:1. When chromatographed on thin-layer plates in the above solvent, this fraction showed an R(f) of 0.3, with coincidence of (3)H and (14)C radioactivity; it was resistant to mild acid hydrolysis, mild and strong alkali hydrolysis, and glucuronidase action. Mannosyl retinyl phosphate occurs, therefore, in vivo in liver and intestinal mucosa, and it is accompanied by a closely similar, though slightly less polar, compound that remains unidentified.     

Purification of a new, calcium-independent, high molecular weight phospholipase A2/lysophospholipase (phospholipase B) from guinea pig intestinal brush-border membrane

A phospholipase A2 activity directed against phosphatidylcholine was previously described in brush-border membrane from guinea pig intestine (Diagne, A., Mitjavila, S., Fauvel, J., Chap, H., and Douste-Blazy, L. (1987) Lipids 22, 33-40). In the present study, this enzyme was solubilized either with Triton X-100 or upon papain treatment, suggesting a structural similarity with other intestinal hydrolases such as leucine aminopeptidase, sucrase, or trehalase. The papain-solubilized form, which is thought to lack the short hydrophobic tail responsible for membrane anchoring, was purified 1800-fold to about 90% purity by ion exchange chromatography on DEAE-Sephacel, gel filtration on Ultrogel AcA44, and hydrophobic chromatography on phenyl-Sepharose. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a main band with an apparent molecular mass of 97 kDa was detected under reducing and nonreducing conditions. In the latter case, phospholipase A2 activity could be recovered from the gel and was shown to coincide with the 97-kDa protein detected by silver staining. The enzyme activity was unaffected by EGTA and slightly inhibited by CaCl2. The purified enzyme displayed a similar activity against phosphatidylcholine and phosphatidylethanolamine, whereas 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine hydrolysis was reduced by 50% compared to diacylglycerophospholipids. Using phosphatidylcholine labeled with either [3H]palmitic acid or [14C]linoleic acid in the 1- or 2-positions, respectively, the purified enzyme catalyzed the removal of [3H]palmitic acid, although at a lower rate compared to [14C]linoleic acid. This resulted in the formation of sn-glycero-3-phosphocholine, but only 1-[3H]palmitoyl-sn-glycero-3-phosphocholine was detected as an intermediary product. In agreement with this, 1-acyl-2-lyso-sn-[14C]glycero-3-phosphocholine was deacylated at almost the same rate as the sn-2-position of phosphatidylcholine. Since upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the two hydrolytic activities were detected at the same position as 97-kDa protein, the enzyme is thus considered as a phospholipase A2 with lysophospholipase activity (phospholipase B), which might be involved in phospholipid digestion.     

Inhibitors of sterol synthesis. A major role of chylomicrons in the metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one in the rat

The metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one (I), a potent regulator of cholesterol (Chol) metabolism which has significant hypocholesterolemic activity upon oral administration to animals, has been investigated in male rats. After intragastric administration of [2,4-3H] I and [4-14C]Chol in triolein to intestinal lymph duct-cannulated rats, most of the 3H of the lymph was associated with chylomicrons. Most of the 3H in the chylomicrons was associated with fatty acid esters of I and the oleate ester represented the major species of the esters of I. After intravenous injection of the isolated doubly-labeled chylomicrons to intact rats, rapid clearance of 3H and 14C from blood was observed which was associated with a rapid and selective uptake of 3H and 14C by liver. The rate of disappearance of 3H from blood and the rate of uptake of 3H by liver were similar, if not identical, to those for 14C. In contrast, the disappearance of 3H from the liver was much more rapid than that of 14C. Studies of the distribution of 3H in liver demonstrated rapid formation of free I and the formation of [3H]Chol. In addition, significant amounts of the 3H in liver were associated with polar materials, a finding which was not observed in the case of 14C. After intravenous administration of the doubly-labeled chylomicrons to bile duct-cannulated rats, very rapid and substantial metabolism of the administered 3H to polar biliary metabolites was observed. The bulk of the 3H not recovered in bile at 49 h after the injection of the labeled chylomicrons was recovered in blood and tissues and almost all (integral of 94%) of this material was associated with Chol and Chol esters. The combined results indicate an important role for chylomicrons in the overall metabolism of I. The selective delivery of I to liver as its oleate ester in chylomicrons (or, more probably, as chylomicron remnants) and the subsequent metabolism of the oleate ester of I in liver has important consequences with respect to the actions of I which are discussed herein.     

Reactions of 1-bromo-2-[14C]pinacolone with acetylcholinesterase from Torpedo nobiliana. Effects of 5-trimethylammonio-2-pentanone and diisopropyl fluorophosphate

1-Bromo-2-[14C]pinacolone, (CH3)3C14COCH2Br [( 14C]BrPin), was prepared from [1-14C]acetyl chloride and tert-butylmagnesium chloride with cuprous chloride catalyst, followed by bromination. It was examined as an active-site directed label for acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) (AcChE). AcChE, isolated from Torpedo nobiliana, has k(cat) = (4.00 +/- 0.04).10(3) s-1, Km = 0.055 +/- 0.008 mM in hydrolysis of acetylthiocholine, and k(cat) = (5.6 +/- 0.2).10(3) s-1, Km = 0.051 +/- 0.003 mM in hydrolysis of acetylcholine. BrPin, binding in the trimethyl cavity, acts initially as a reversible competitive inhibitor, Ki = 0.20 +/- 0.09 mM, and, with time, as an irreversible covalently bound inactivator. Introduction of 14C from [14C]BrPin into Torpedo AcChE at pH 7.0 was followed by SDS-PAGE, autoradiography and scintillation counting, in the absence and presence of 5-trimethylammonio-2-pentanone (TAP), a competitive inhibitor (Ki = 0.075 +/- 0.001 mM) isosteric with acetylcholine; 1.8-1.9 14C was incorporated per inactivated enzyme unit at 50% inactivation. TAP retarded inactivation by [14C]BrPin, and prevented introduction of 0.9-1.1 14C per unit of enzyme protected. Prior inactivation of AcChE by BrPin prevents reaction with [3H]diisopropyl fluorophosphate [( 3H]DFP). Prior inactivation by DFP or [3H]DFP does not prevent reaction with [14C]BrPin, and this subsequent reaction with BrPin does not displace the [3H] moiety. [14C]BrPin alkylates a nucleophile in the active site, and this reaction does not alkylate or utilize the serine-hydroxyl.     

Lipid metabolism during fasting

These studies were conducted to understand the relationship between measures of systemic free fatty acid (FFA) reesterification and regional FFA, glycerol, and triglyceride metabolism during fasting. Indirect calorimetry was used to measure fatty acid oxidation in six men after a 60-h fast. Systemic and regional (splanchnic, renal, and leg) FFA ([(3)H]palmitate) and glycerol ([(3)H]glycerol) kinetics, as well as splanchnic triglyceride release, were measured. The rate of systemic FFA reesterification was 366 +/- 93 micromol/min, which was greater (P < 0.05) than splanchnic triglyceride fatty acid output (64 +/- 6 micromol/min), a measure of VLDL triglyceride fatty acid export. The majority of glycerol uptake occurred in the splanchnic and renal beds, although some leg glycerol uptake was detected. Systemic FFA release was approximately double that usually present in overnight postabsorptive men, yet the regional FFA release rates were of the same proportions previously observed in overnight postabsorptive men. In conclusion, FFA reesterification at rest during fasting far exceeds splanchnic triglyceride fatty acid output. This indicates that nonhepatic sites of FFA reesterification are important, and that peripheral reesterification of FFA exceeds the rate of simultaneous intracellular triglyceride fatty acid oxidation.     

The isolation and characterization of 60 nm vesicles (''nanovesicles'') produced during ionophore A23187-induced budding of human erythrocytes.

1. Penicillin N was synthesized by coupling alpha-amino-alpha-p-nitrobenzyl-N-p-nitro-benzyloxycarbonyl-D-adipate with 6-aminopenicillanic acid benzyl ester, followed by removal of the protecting groups through hydrogenolysis. 2. alpha-Amino-alpha-p-nitrobenzyl-N-p-nitrobenzyloxycarbonyl-D-[5-14C]adipate was prepared by treating alpha-p-nitrobenzyl-N-p-nitrobenzyloxycarbonyl-D-glutamic acid with [14C]diazomethane followed by rearrangement with silver trifluoromethanesulphonate. 3. Coupling of alpha-amino-alpha-p-nitrobenzyl-N-p-nitrobenzyloxycarbonyl-D-[5-14C]adipate with 6-aminopenicillanic acid benzyl ester gave triprotected [10-14C]penicillin N. 4. 3H was introduced at C-6 of the Schiff's base derivative (10) by oxidation followed by reduction with NaB3H4. 5. The so-derived (6 alpha-3H)-labelled Schiff's base was hydrolysed to give 6-amino [6 alpha-3H]penicillanic acid benzyl ester p-toluenesulphonic acid salt, which after coupling as the free amine with alpha-amino-alpha-p-nitrobenzyl-N-pnitrobenzyloxycarbonyl-D-adipate and then hydrogenolysis, yielded [6alpha-3H]penicillin N. 6. Triprotected [10-14C]penicillin N and triprotected [6alpha-3H]penicillin N in admixture were hydrogenolysed to give [10-14C,6alpha-3H]penicillin N.     

Steric and charge factors in the resistance of uridylyl(3' - 5')N6-(N-threonylcarbonyl)adenosine to venom phosphodiesterase hydrolysis.

The contribution of steric and negative charge factors to the resistance of uridylyl(3' - 5')N6-(N-threonylcarbonyl)adenosine to venom phosphodiesterase was investigated. The hydrolysis rates of uridylyl(3'-5')N6-(N-threonylcarbonyl)-adenosine, its model derivatives, methyl ester and O-benzyl ester, together with unmodified uridyly (3'-5')adenosine, were studied. It was found that the contribution of both factors is of the same order. The steric inhibition of digestion is distinctly higher than that confirmed by N6-(delta2-isopentenyl)adenosine [1], which is ascribed to the rigid conformation of the threonylcarbonyladenosine side chain.     

Biosynthesis in vitro of Ii core glycosphingolipids from neolactotetraosylceramide by beta 1-3- and beta 1-6-N-acetylglucosaminyltransferases from mouse T-lymphoma

The N-acetylglucosaminyltransferases probably involved in the biosynthesis in vitro of Ii core glycosphingolipids have been solubilized from a membrane preparation of mouse lymphoma P-1798 and partially characterized. The detergent-extracted membrane supernatant contains both beta 1-3- and beta 1-6-N-acetylglucosaminyltransferase activities that transfer [3H]GlcNAc from UDP-[3H]GlcNAc to the terminal galactose of neolactotetraosylceramide (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-ceramide; nLcOse4ceramide), to form the Ii core structures. The linkage of [3H]N-acetylglucosamine incorporated into the terminal galactose of nLcOse4Cer was determined from identification of 2,4,6-tri-O-methyl[3H]galactose and 2,3,4-tri-O-methyl[3H]galactose after hydrolysis of the permethylated enzymatic products, GlcNAc beta-[3H]Gal-GlcNAc-Gal-Glc-ceramide. In addition to the presence of beta-N-acetylglucosaminyltransferases, we have detected a galactosyltransferase activity in this soluble supernatant fraction that catalyzes the transfer of [14C]galactose from UDP-[14C]galactose to lactotriaosylceramide (GlcNAc beta 1-3Gal beta 1-4Glc-ceramide; LcOse3ceramide) to form nLcOse4ceramide, the acceptor in the N-acetylglucosaminyltransferase-catalyzed reaction.