Stereochemistry of the acyl dihydroxyacetone phosphate acyl exchange reaction

The fatty acid of acyl dihydroxyacetone phosphate can be exchanged enzymatically for another fatty acid. It has been shown that this reaction proceeds by cleavage of the oxygen bound to C-1 of the dihydroxyacetone phosphate (DHAP) moiety rather than by the more common cleavage at the acyl to oxygen bond. In the present study, the stereochemistry of this reaction was defined further; using deuterated substrates and fast atom bombardment-mass spectrometry, it was shown that the fatty acid exchange involves the stereospecific labilization of the pro-R hydrogen at C-1 of the DHAP moiety of acyl DHAP. The mechanism of ether bond formation, in which acyl DHAP is converted to O-alkyl DHAP, also proceeds via labilization of the pro-R hydrogen and cleavage of the fatty acid at the C-1 to oxygen bond. In addition, other workers have provided evidence that the enzyme responsible for the exchange reaction is O-alkyl DHAP synthetase. Therefore, the present results support the hypothesis that the acyl exchange is the reverse reaction of the first step in O-alkyl DHAP synthesis; in both of these reactions the pro-R hydrogen of C-1 of the DHAP moiety of acyl DHAP and the fatty acid moiety are labilized with cleavage of the fatty acid at the DHAP C-1 to oxygen bond.     

Induction of the peroxisomal glycerolipid-synthesizing enzymes during differentiation of 3T3-L1 adipocytes. Role in triacylglycerol synthesis

The glycerophosphate backbone for triglyceride synthesis is commonly believed to be created through the conversion of dihydroxyacetone phosphate (DHAP) by glycerophosphate dehydrogenase (GPD) to sn-glycerol 3-phosphate (GP), which is then converted by glycerophosphate acyltransferase (GPAT) to 1-acyl-GP. Consistent with this, GPD and GPAT are highly induced during differentiation of mouse 3T3-L1 preadipocytes. While the acyl dihydroxyacetone phosphate (acyl-DHAP) pathway for glycerolipid synthesis is commonly believed to be involved only in glycerol ether lipid synthesis, we report here that during conversion of 3T3-L1 preadipocytes to adipocytes, the specific activity of peroxisomal DHAP acyltransferase (DHAPAT) is increased by 9-fold in 6 days, while acyl-DHAP:NADPH reductase is induced by 5-fold. A parallel increase in the catalase (the peroxisomal marker enzyme) activity is also seen. In contrast, the specific activity of alkyl-DHAP synthase, the enzyme catalyzing the synthesis of the ether bond, is decreased by 60% during the same period. Unlike microsomal GPAT, the induced DHAPAT is found to have high activity at pH 5.5 and is resistant to inhibition by sulfhydryl agents, heat, and proteolysis. On subcellular fractionation, DHAPAT is found to be associated with microperoxisomes whereas GPAT activity is mainly present in microsomes. Northern blot analyses reveal that induction of DHAPAT can be largely explained through increases in DHAPAT mRNA. A comparison of microsomal and peroxisomal glycerolipid synthetic pathways, using D-[3-(3)H, U-(14)C]glucose as the precursor of the lipid glycerol backbone shows that about 40-50% of triglyceride is synthesized via the acyl-DHAP pathway. These results indicate that the acyl-DHAP pathway is important not only for the synthesis of ether lipids, but also for the synthesis of triacylglycerol and other non-ether glycerolipids.     

Stereochemical specificity of the biosynthesis of the alkyl ether bond in alkyl ether lipids.

The stereochemical course of the formation of the alkyl ether bond in alkyl ether lipids was investigated through the synthesis of stereospecifically labeled acyl R- or S-[1-3H]dihydroxyacetone 3-phosphate (DHAP) starting from L-glyceraldehyde. It was demonstrated directly that the formation of the alkyl ether bond results in the stereospecific exchange of the pro-R C-1 hydrogen of DHAP with a proton of water. The configuration of the hydrogen that is retained on C-1 after formation of the alkyl ether bond was also investigated. The alkyl ether lipid was degraded, and the DHAP backbone isolated as glycerol, converted to DHAP via glycerol 3-phosphate and treated with either aldolase or triose phosphate isomerase. The results demonstrated that the retained hydrogen on C-1, which was pro-S in the starting substrate, was pro-S in the product alkyl ether.     

Acetoacetate is a cholesterogenic precursor for myelinating rat brain and spinal cord. Incorporation of label from [3-14C]acetoacetate, [14C]glucose and 3H2O

Rat pups, 3 weeks old, were injected i.p. with combinations of 3H2O and either [3-14C]acetoacetate or [14C]glucose. 3H/14C incorporation ratios were measured in lipid fractions of homogenates and myelin prepared from whole brain and spinal cord. Spinal cord synthesized at least twice as much fatty acids and 3-fold more sterols than whole brain. Both tissues used acetoacetate preferentially for sterol synthesis, whereas label from [14C]glucose was distributed between fatty acids and sterols in the same way as 3H from 3H2O. The relative contributions of acetoacetate to sterol synthesis in whole tissue and in the purified myelin fraction were about the same, both for the cerebrum and for the spinal cord.     

The mechanism of ether bond formation in O-alkyl lipid synthesis in Ehrlich ascites tumor. Unusual cleavage of the fatty acid moiety of acyl dihydroxyacetone phosphate

We have previously presented evidence for the formation of 1-O-alkyl dihydroxyacetone-P from acyl dihydroxyacetone-P via the initial formation of an intermediate 1-O-acyl endiol of acyl dihydroxyacetone-P. This reaction involves a stereospecific exchange of the pro-R hydrogen of the acyl dihydroxyacetone-P moiety without change in configuration. The fatty acid is replaced by a long chain fatty alcohol which retains the oxygen of the primary carbinol. In the absence of fatty alcohol, water substitutes and the product is dihydroxyacetone-P which has also exchanged the pro-R hydrogen with a hydrogen from the medium. An absolute requirement of the proposed mechanism is that the loss of the fatty acid must proceed via an unusual cleavage of the dihydroxyacetone-P C-1 to oxygen bond instead of the usual cleavage at the fatty acid acyl to oxygen bond. In the present investigation, we have synthesized hexadecanoyl dihydroxyacetone-P containing oxygen-18 exclusively at the dihydroxyacetone-P C-1 oxygen. Using this substrate, we have shown that cleavage of hexadecanoyl dihydroxyacetone-P at the C-1 to oxygen bond is linked to O-alkyl dihydroxyacetone-P synthesis. Inhibition of O-alkyl lipid synthesis by means of magnesium or NADPH inhibited the unusual cleavage. At the same time, we have shown that there was hydrolysis of acyl dihydroxyacetone-P which proceeded by the usual mechanism and which was not related to synthesis of O-alkyl dihydroxyacetone-P.     

Incorporation of D-[3-3H, U-14C] glucose into glycerolipid via acyl dihydroxyacetone phosphate untransformed and viral-transformed BHK-21-c13 fibroblasts.

Untransformed BHK-21-c13 fibroblasts as well as 4 polyoma-transformed strains were incubated with D-[U-14C,3-3H]glucose. This substrate generates intracellular labeled glycerol, and also [4-3H]NADPH via the phosphogluconate oxidative pathway. The latter selectively transfers hydrogen to C-2 of glycerol in glycerolipid via the acyl dihydroxyacetone phosphate pathway. After incubation, the distribution of radioactivity and the ratios of 3H/14C at the three positions of recovered glycerol were determined in sn-glycerol 3-phosphate, saponifiable glycerolipids, alkyl ether glycerolipids, and plasmalogens. In each of the cell types examined, 3H in the sn-1 position of glycerol in the recovered ether-containing glycerolipids was negligible, yet this position contained most of the recovered 3H in sn-glycerol 3-phosphate and saponifiable glycerolipids. The 3H/14C ratio in position 2 of glycerol, measured at various incubation times, was from 5- to 200-fold greater in the saponifiable glycerolipids than in free sn-glycerol 3-phosphate. The ratio in position 2 of ether-containing glycerolipids was the same or greater than that in the saponifiable glycerolipids in all of the cell types employed. A similar pattern in the 3H/14C ratio was observed when BHK-21-c13 cells were incubated with D-[U-14C,1-3H]glucose. These observations demonstrate significant participation of the acyl dihydroxyacetone phosphate pathway in glycerolipid synthesis in BHK cells.     

O-alkyl lipid synthesis: the mechanism of the acyl dihydroxyacetone phosphate fatty acid exchange reaction

We have previously provided evidence for a mechanism by which acyl DHAP is converted enzymatically to O-alkyl DHAP. This mechanism involves, in part, the formation of an endiol of acyl DHAP, loss of the fatty acid by splitting of the DHAP carbon-1 to oxygen bond and the gain of a long chain fatty alcohol. It has been shown that acyl DHAP can exchange its fatty acid for one in the medium, presumably by the mediation of O-alkyl DHAP synthase. In the present investigation we have shown that the fatty acid which is gained by acyl DHAP in the exchange process retains both carboxyl oxygens, as predicted by our postulated mechanism. This reaction is exceptional because the usual action of acyl hydrolases is to cleave at the oxygen to acyl bond.     

In vivo incorporation of [U-14C]glycerol and [2-3H]glycerol into phospholipids of brain subcellular fractions in normal and malnourished animals

A double label design was used to study the in vivo incorporation of [U-¹⁴C] and [2-³H]glycerol into total and individual phospholipids of various brain subcellular fractions isolated from 20-day old normal and undernourished rats. In control animals, synthesis of glycerophospholipids of microsomes, mitochondria and nerve endings seems to occur through the glycerol-3-phosphate (G-3-P) pathway while a large part of the synthesis of myelin glycerophospholipids appears to proceed through the dihydroxyacetone phosphate (DHAP) pathway. In starved animals, on the other hand the incorporation of phospholipid precursors through the DHAP pathway was found to be lower than in controls while synthesis of phospholipids in the other subcellular fractions was unaffected.The possible relationship between the synthesis of glycerophospholipids and especially plasmalogens of the myelin membrane and microperoxisomes of oligodendroglial cells, where the enzymes of the DHAP pathway are located, is discussed.     

Isotope effects in 3-dehydroquinate synthase and dehydratase. Mechanistic implications.

The mechanism of 3-dehydroquinate synthase was explored by incubating partially purified enzyme with mixtures of [1-14C]3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) and one of the specifically tritiated substrates [4-3H]DAHP, [5-3H]DAHP, [6-3H]DAHP, (7RS)-[7-3H]DAHP, (7R)-[7-3H]DAHP, or (7S)-[7-3H]DAHP. Kinetic and secondary 3H isotope effects were calculated from 3H:14C ratios obtained in unreacted DAHP, 3-dehydroquinate, and 3-dehydroshikimate. 3H was not incorporated from the medium into 3-dehydroquinate, indicating that a carbanion (or methyl group) at C-7 is not formed. A kinetic isotope effect kH/k3H of 1.7 was observed at C-5, and afforded support for a mechanism involving oxidation of C-5 with NAD. A similar kinetic isotope effect was found at C-6 owing to removal of a proton in elimination of phosphate, which is reasonably assumed to be the next step in 3-dehydroquinate synthase. Hydrogen at C-7 of DAHP was not lost in the cyclization step of the reaction, indicating that the enol formed in phosphate elimination participated directly in an aldolase-type reaction with the carbonyl at C-2. In the dehydration of 3-dehydroquinate to 3-dehydroshikimate the (7R) proton from (7RS)- or (7R)-[7-3H]DAHP is lost, indicating that the 7R proton occupies the 2R position in dehydroquinate. Hence the cyclization step occurs with inversion of configuration at C-7. A kinetic isotope effect kH/k3H = 2.3 was observed in the conversion of (2R)-[2-3H]dehydroquinate to dehydroshikimate. Hence loss of a proton from the enzyme-dehydroquinate imine contributed to rate limitation in the reaction.     

The disposition of [24-3H]cerebrosterol in developing rat brain.

—The uptake into subcellular fractions of developing rat brain in vivo of intracerebrally injected [4-¹⁴C]cholesterol, [24-³H]cerebrosterol, and [24-³H]24-epicerebrosterol was measured for periods up to 30 days following administration. [4-¹⁴C]cholesterol was accumulated rapidly in nuclei, nerve endings, and microsomes, more slowly in myelin and mitochondria. [24-³H]cerebrosterol was accumulated rapidly in myelin, nerve endings, and microsomes, more slowly in nuclei and mitochondria. The uptake of [24-³H]24-epicerebrosterol was essentially the same as that of [24-³H]cerebrosterol. Ratios of radioactivities of [24-³H]cerebrosterol and [4-¹⁴C]cholesterol accentuated the early accumulation of [24-³H]cerebrosterol in myelin, nerve endings, and microsomes, and declining ³H:¹⁴C ratios disclosed the rapid elimination of [24-³H]cerebrosterol and [24-³H]24-epicerebrosterol relative to [4-¹⁴C]cholesterol in nerve endings and microsomes. The data suggest that the removal of [24-³H]cerebrosterol from brain results from an enzymic metabolism of the sterol, therefore that cerebrosterol exists in brain in a dynamic state of biosynthesis and catabolism.     

Structure of the mitochondrial beta-ketoacyl-[acyl carrier protein] synthase from Arabidopsis and its role in fatty acid synthesis

Mitochondrial fatty acid synthesis is catalyzed by a dissociated fatty acid synthase similar to those of plant plastids and bacteria. The crystal structure of a mitochondrial beta-ketoacyl-[acyl carrier protein] synthase (mtKAS), namely that from Arabidopsis thaliana, has been determined for the first time. This enzyme accomplishes the vital condensation steps in constructing fatty acid carbon skeletons. The product profile of mtKAS is unusual in that C8 and C(14-16) fatty acyl chains predominate. An enzyme architecture that likely is the basis for the observed bimodal profile of mtKAS products can be derived from the shape of the acyl binding pocket.     

In vivo labeling of myelin lipids and proteolipid protein with [3H]myristate, [14C]linoleate,and [14C]linolenate

To investigate the incorporation of essential fatty acids into myelin components, 24-day-old rabbits were injected intracerebrally with [¹⁴C]linoleate, [¹⁴C]linolenate, or [³H]Myristate for comparison. Animals were killed 22 hr later and myelin was isolated. [³H]myristate labeled all myelin lipids including monogalactosyl diglyceride, with the exception of sulfatides. With¹⁴C-essential fatty acids, only glycerophospholipids were efficiently labeled and their specific activities were in the following decreasing orders: PC>PI>PE>PS with [¹⁴C]linoleate, and PE>PC>PI=PS with [¹⁴C]linolenate. Among myelin proteins, PLP and DM-20 were labeled with all 3 precursors. PLP was purified from myelin labeled with¹⁴C-essential fatty acids. The label was then cleaved from the protein by alkaline methanolysis and was identified as a dienoic ([¹⁴C]linoleate) or a tetraenoic ([¹⁴C]linolenate) fatty acid. MBP was not labeled with [³H]myristate, but was slightly labeled with both¹⁴C-essential fatty acids. The signification of the latter result is discussed.Abbreviations FA fatty acid(s) - HPTLC high-performance thin-layer chromatography - MBP myelin basic protein - PLP proteolipid protein - PC phosphatidylcholine - PE phosphatidylethanolamine and ethanolamine plasmalogens - PI phosphatidylinositol - PS phosphatidylserine - SDS sodium dodecylsulfate     

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.     

Stereochemistry of propionyl-coenzyme A and pyruvate carboxylations catalyzed by transcarboxylase.

The stereochemistry of the two half-reactions catalyzed by the biotin-containing enzyme, transcarboxy-lase from Propionobacteria shermanii, has been determined. The pro-R hydrogen at C-2 of propionyl-coenzyme A is replaced by CO2 in formation of the S isomer of methylmalonyl-CoA, defining the process as retention of configuration. This C-2 hydrogen is abstracted at a rate identical with product formation. For the other half-reaction, pyruvate to oxalacetate, the chiral methyl group methodology of Rose (I. A. Rose (1970), J. Biol. Chem. 245, 6052) was employed. First, it was determined with [3-2-He]pyruvate that a kinetic deuterium isotope effect of 2.1 occurs at Vmax in this carboxyl transfer, indicating that the necessary requirement for discrimination against heavy isotopes of hydrogen existed. Then, 3(S)-[3-2-H,3-H]pyruvate, generated from 3(S)-]E-2-H,3-H]phosphoglycerate, was carboxylated and the oxalacetate trapped as [3030H]malate using malate dehydrogenase. Exhaustive incubation of the tritiated malate (3-H/14-C = 1.95) with fumarase to labilize the pro-R hydrogen at C-3 resulted in release of 65% of the tritium into water. Reisolation of the malate after fumarase action yielded a 30H/14-C ration of 0.67, indicating 34% retention as expected. The theoretical enantiotopic distribution for the observed k1H/k2H of 2.1 is 68:32. Selective enrichment of tritium in the pro-R position at C-3 of malate indicates enzymatic carboxylation of pyruvate with retention of configuration in this half-reaction also.     

Identification and molecular characterization of the beta-ketoacyl-[acyl carrier protein] synthase component of the Arabidopsis mitochondrial fatty acid synthase

Substrate specificity of condensing enzymes is a predominant factor determining the nature of fatty acyl chains synthesized by type II fatty acid synthase (FAS) enzyme complexes composed of discrete enzymes. The gene (mtKAS) encoding the condensing enzyme, beta-ketoacyl-[acyl carrier protein] (ACP) synthase (KAS), constituent of the mitochondrial FAS was cloned from Arabidopsis thaliana, and its product was purified and characterized. The mtKAS cDNA complemented the KAS II defect in the E. coli CY244 strain mutated in both fabB and fabF encoding KAS I and KAS II, respectively, demonstrating its ability to catalyze the condensation reaction in fatty acid synthesis. In vitro assays using extracts of CY244 containing all E. coli FAS components, except that KAS I and II were replaced by mtKAS, gave C(4)-C(18) fatty acids exhibiting a bimodal distribution with peaks at C(8) and C(14)-C(16). Previously observed bimodal distributions obtained using mitochondrial extracts appear attributable to the mtKAS enzyme in the extracts. Although the mtKAS sequence is most similar to that of bacterial KAS IIs, sensitivity of mtKAS to the antibiotic cerulenin resembles that of E. coli KAS I. In the first or priming condensation reaction of de novo fatty acid synthesis, purified His-tagged mtKAS efficiently utilized malonyl-ACP, but not acetyl-CoA as primer substrate. Intracellular targeting using green fluorescent protein, Western blot, and deletion analyses identified an N-terminal signal conveying mtKAS into mitochondria. Thus, mtKAS with its broad chain length specificity accomplishes all condensation steps in mitochondrial fatty acid synthesis, whereas in plastids three KAS enzymes are required.     

Specific modification of the condensation domain of fatty acid synthase and the determination of the primary structure of the modified active site peptides

Fatty acid synthase from the uropygial gland of goose was inactivated by iodoacetamide with a second-order rate constant of 1.3 M-1 S-1 at pH 6.0 and 25 degrees C. Of the seven component activities of the synthase, only the condensation activity was significantly inhibited by iodoacetamide modification. Since preincubation of the enzyme with acetyl-CoA, but not with malonyl-CoA, protected the enzyme from inactivation by iodoacetamide, it is suggested that iodoacetamide probably modified the primer-binding thiol group at the condensation active site. Determination of the stoichiometry of modification was done using [1-14C]iodoacetamide that was purified by high-performance liquid chromatography. Graphical analysis of the data showed that binding of 1.2 carboxamidomethyl groups per subunit of fatty acid synthase would result in complete inhibition of the enzyme activity, suggesting that there is one condensation domain per subunit of fatty acid synthase. Analysis of the tryptic peptide map of the enzyme that was modified with [1-14C]iodoacetamide in the presence and absence of acetyl-CoA revealed that acetyl-CoA prevented the labeling of a major radioactive peptide and a minor radioactive peptide. These two peptides were purified by high-performance liquid chromatography. Amino acid analysis of these two peptides revealed that the major radioactive peptide contained S-carboxymethylcysteine while the minor radioactive peptide did not. However, the latter peptide contained beta-alanine, suggesting that this peptide was from the acyl carrier protein segment of fatty acid synthase and that the iodoacetamide treatment resulted in modification of the pantetheine thiol, although to a lower extent than the primer-binding thiol. The sequence of the primer-binding active site peptide from the condensation domain was H2N-Gly-Pro-Ser-Leu-Ser-Ile-Asp- Thr-Ala-Cys(carboxamidomethyl)-X-Ser-Ser-Leu-Met-Ala-Leu-Glu-Asn-A la-Tyr-Lys- COOH, the first reported sequence of the condensation active site from a vertebrate fatty acid synthase. The acyl carrier protein segment showed extensive sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the phosphopantetheine attachment, and the sequence was H2N-Asp-Val-Ser-Ser-Leu- Asn-Ala-Asp-Ser-Thr-Leu-Ala-Asp-Leu-Gly-Leu-Asp-Ser(4'-phosphopanteth ein e) -Leu-Met-Gly-Val-Glu-Val-Arg-COOH.     

Synthesis of alkyl-ether glycerophospholipids in rat glomerular mesangial cells: evidence for alkyldihydroxyacetone phosphate synthase activity

We studied the ability of rat glomerular mesangial cells and their microsomal fractions to incorporate 1-[14C]hexadecanol to glycerophospholipids via an O-alkyl ether linkage and assessed the presence and activity of the required enzyme: alkyl-dihydroxy acetone phosphate synthase. Suspensions of cultured mesangial cells incorporated 1-[14C]hexadecanol to the phosphatidyl ethanolamine and phosphatidyl choline lipid pools, via a bond resistant to acid and base hydrolysis. When cell homogenates or microsomal fractions were incubated with palmitoyl-DHAP and 1-[14C]hexadecanol, alkyl-DHAP and 1-O-alkyl glycerol were formed (alkyl:hexadecyl). The activity of the enzyme responsible for the O-alkyl product formation was calculated to be 2.5 +/- 0.3 and 544 +/- 50 pmoles/min/mg protein for mesangial cell homogenates and mesangial cell microsomes, respectively. These observations provide evidence that mesangial cells may elaborate either linked lipid precursors de novo for the biosynthesis of O-alkyl glycerophospholipids.     

The release of [3H]GABA formed from [3H]glutamate in rat hippocampal slices

to compare the storage and release of endogenous GABA, of [3H]GABA formed endogenously from glutamate, and of exogenous [14C]GABA, hippocampal slices were incubated with 5 microCi/ml [3,4-3H]1-glutamate and 0.5 microCi/ml [U-14C]GABA and then were superfused in the presence or absence of Ca+ with either 50 mM K+ or 50 microM veratridine. Endogenous GABA was determined by high performance liquid chromatography which separated labeled GABA from its precursors and metabolites. Exogenous [14C]GABA content of the slices declined spontaneously while endogenous GABA and endogenously formed [3H]GABA stayed constant over a 48 min period. In the presence of Ca+ 50 mM K+ and in the presence or absence of Ca2+ veratridine released exogenous [14C]GABA more rapidly than endogenous or endogenously formed [3H]GABA, the release of the latter two occurring always in parallel. The initial specific activity of released exogenous [14C]GABA was three times, while that of endogenously formed [3H]GABA was only 50% higher than that in the slices. There was an excess of endogenous GABA content following superfusion with 50 mM K+ and Ca2+, which did not occur in the absence of Ca2+ or after veratridine. The observation that endogenous GABA and [3H]GABA formed endogenously from glutamate are stored and released in parallel but differently from exogenous labelled GABA, suggests that exogenous [3H] glutamate can enter a glutamate pool that normally serves as precursor of GABA.     

The synthesis of high-specific-activity UDP-[6-3H]galactose, UDP-N-[6-3H]acetylgalactosamine, and their corresponding monosaccharides.

Tritiated uridine-5'-diphosphogalactose (UDP-[3H]Gal) has been widely used to study oligosaccharide biosynthesis and structure. It can be synthesized either chemically or enzymatically using galactose oxidase to oxidize the hydroxyl moiety at C-6 to an aldehyde (6-aldo-UDP-Gal), which is then reduced back to the alcohol with tritiated sodium borohydride. Although the enzymatic approach is simple and efficient, there are several problems associated with it. First, incomplete oxidation to the aldehyde reduces the final specific activity. Second, if the galactose oxidase is not removed from the 6-aldo-UDP-Gal prior to reduction, the resulting UDP-[6-3H]Gal can be reoxidized to 6-aldo-UDP-[6-3H]Gal. We present evidence for the occurrence of this compound in one commercially obtained preparation of UDP-[6-3H]Gal. Finally, if an excess of 6-aldo-UDP-Gal is used for good yield, it is necessary to quench the reduction with nonradioactive borohydride, again reducing the final specific activity. We have devised a rapid, inexpensive, and efficient synthesis of UDP-[6-3H]Gal that circumvents all of these problems. Galactose oxidase is used to produce 6-aldo-UDP-Gal and the completeness of this reaction is confirmed on polyethyleneimine (PEI) cellulose TLC plates. The 6-aldo-UDP-Gal is purified on silica gel 60 TLC plates. This purified compound is then reduced with tritiated sodium borohydride, with the aldehyde present in excess. Unreacted 6-aldo-UDP-Gal is then purified away from the product UDP-[6-3H]Gal by chromatography on PEI cellulose. Radiochemically pure UDP-[6-3H]Gal with a specific activity of 10 Ci/mmol was obtained using the above scheme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereochemistry of phytoene biosynthesis by isolated chloroplasts

The incorporation of [2-(14)C,(5R)-5-(3)H(1)]MVA* and [2-(14)C,5-(3)H(2)]MVA into geranylgeraniol and phytoene by a preparation of ;non-aqueous' bean leaf chloroplasts has been studied. In the formation of phytoene from two molecules of geranylgeranyl pyrophosphate, the loss of hydrogen is stereospecific, the hydrogen atom lost from C-1 of each molecule of geranylgeranyl pyrophosphate being that which was originally the pro-S hydrogen atom from C-5 of mevalonate. All the pro-R hydrogen atoms from C-5 of mevalonate are retained. These results with a cell-free system confirm and extend the observations made in previous work with tomato slices.