Mechanism and stereochemistry of enzymic reactions involved in porphyrin biosynthesis.

5-Aminolaevulinate synthetase cataylses the condensation of glycine and succinyl-CoA to give 5-aminolaevulinic acid. At least two broad pathways may be considered for the initial C--C bond forming step in the reaction. In pathway A the Schiff base of glycine and enzyme bound pyridoxal phosphate (a) undergoes decarboxylation to give the carbanion (b) which then condenses with succinyl-CoA with the retention of both the original C2 hydrogen atoms of glycine. In pathway B, loss of a C2 hydrogen atom gives another type of carbanion (c) that reacts with succinyl-CoA. Evidence has been presented to show that the initial C--C bond forming event occurs via pathway B which involves the removal of the pro R hydrogen atom of glycine. Subsequent mechanistic and stereochemical events occurring at the carbon atom destined to become C5 of 5-aminolaevulinate have also been delineated.(Carticle) Several mechanistic alternatices for the formation of the two vinyl groups of haem from the propionate residues of the precursor, coproporphyrinogen III, have been examined. (see article). It is shown that during the biosynthesis both the hydrogen atoms resident at the alpha positions of the propionate side chains remain undisturbed thus eliminating mechanisms which predict the involvement of acrylic acid intermediates. Biosynthetic experiments performed with precursors containing stereospecific labels have shown that the two vinyl groups of haem are formed through the loss of pro S hydrogen atoms from the beta-positions of the propionate side chains. In the light of these results, three related mechanisms for the conversion, propionate leads to vinyl, have been considered. In order to study the mechanism of porphyrinogen carboxy-lyase reaction, stereo-specifically deuterated, tritiated-succinate was incorporated into the acetate residues of uroporphyrinogen III which on decarboxylation generated asymmetric methyl groups in coproporphyrinogen III and then in haem. Degradation of the latter yielded chiral acetate deriving from C and D rings of haem. Configurational analysis of this derivate acetate shows that the carboxy-lyase reaction proceeds with a retention of configuration.     

The substrate radical of Escherichia coli oxygen-independent coproporphyrinogen III oxidase HemN

During porphyrin biosynthesis the oxygen-independent coproporphyrinogen III oxidase (HemN) catalyzes the oxidative decarboxylation of the propionate side chains of rings A and B of coproporphyrinogen III to form protoporphyrinogen IX. The enzyme utilizes a 5'-deoxyadenosyl radical to initiate the decarboxylation reaction, and it has been proposed that this occurs by stereo-specific abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chains leading to a substrate radical. Here we provide EPR-spectroscopic evidence for intermediacy of the latter radical by observation of an organic radical EPR signal in reduced HemN upon addition of S-adenosyl-L-methionine and the substrate coproporphyrinogen III. This signal (g(av) = 2.0029) shows a complex pattern of well resolved hyperfine splittings from at least five different hydrogen atoms. The radical was characterized using regiospecifically labeled (deuterium or 15N) coproporphyrinogen III molecules. They had been generated from a multienzyme mixture and served as efficient substrates. Reaction of HemN with coproporphyrinogen III, perdeuterated except for the methyl groups, led to the complete loss of resolved proton hyperfine splittings. Substrates in which the hydrogens at both alpha- and beta-positions, or only at the beta-positions of the propionate side chains, or those of the methylene bridges, were deuterated showed that there is coupling with hydrogens at the alpha-, beta-, and methylene bridge positions. Deuterium or 15N labeling of the pyrrole nitrogens without labeling the side chains only led to a slight sharpening of the radical signal. Together, these observations clearly identified the radical signal as substrate-derived and indicated that, upon abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chain by the 5'-deoxyadenosyl radical, a comparatively stable delocalized substrate radical intermediate is formed in the absence of electron acceptors. The observed hyperfine constants and g values show that this coproporphyrinogenyl radical is allylic and encompasses carbon atoms 3', 3, and 4.     

Number and activity of active ribosomes in bacterial polyribosomes.

Sirohaem is a new type of haem that has been detected as a prosthetic group of several bacterial and plant enzymes that catalyse the six-electron reductions of sulphite to sulphide or of nitrite to NH(3). When a methionine-requiring mutant of Escherichia coli K12 was grown on a minimal medium supplemented with d-glucose and l-[Me-(3)H]methionine, 2.4 methyl groups per spectrophotometrically detectable haem group were incorporated into the sirohaem prosthetic group of the NADPH-sulphite reductase isolated from the organism. When the same strain of cells was grown on minimal medium supplemented with d-[U-(14)C]glucose and l-[Me-(3)H]methionine, the sirohaem isolated was found to contain a ratio of glucose-derived carbon/methionine-derived methyl of 19.8. This ratio is in excellent agreement with the value of 20 predicted by the iron-dimethyl-urotetrahydroporphyrin structure for sirohaem proposed by Murphy, Siegel, Kamin & Rosenthal [(1973) J. Biol. Chem.248, 2801-2814]. It can be concluded that sirohaem is indeed methylated, with the methyl groups derived from methionine (rather than by modification of existing side chains, as in protohaem). The structure proposed by Murphy et al. (1973) is therefore probably correct in its essential features. A possible relationship between the pathway for biosynthesis of sirohaem and that for synthesis of vitamin B(12) is discussed.     

A note concerning acetate activation of peroxidative activity of catalases using 2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid as a substrate

Beef liver catalases showed peroxidative activity using 2,2'-azino-bis-(3-ethylbenzthiazoline)-6-sulfonic acid as the electron donor and hydrogen peroxide as the acceptor at a pH of 5. This activity was not observed at pH 7. The reaction depended on acetate concentration, although succinate and propionate could partly replace the acetate as a catalyst. Other haem proteins also catalyzed a peroxidative effect. The reaction using syringaldazine or the coupling between dimethylaminobenzoic acid and 3-methyl-2-benzothiazolinone hydrazone was less effective and less sensitive. Evidence is presented that the reaction is associated with a conformational change of the catalase.     

Gut tract microorganisms supply the precursors for methyl-branched hydrocarbon biosynthesis in the termite,Zootermopsis nevadensis

In vivo and in vitro experiments were performed to examine the role of succinate and other potential precursors of the methylmalonyl-CoA used for methyl-branched hydrocarbon biosynthesis in the termite Zootermopsis nevadensis. The in vivo incorporation of [1,4-¹⁴C]succinate and [2,3-¹⁴C]succinate into hydrocarbon confirmed that succinate is a direct precursor to the methyl branch unit. The other likely precursors, the branched chain amino acids valine and isoleucine, were not efficiently incorporated into hydrocarbon. Carbon-13 NMR showed that one of the labeled carbons of [1,4-¹³C]succinate labeled position 6 of 5-methylalkanes and positions 6 and 18 of 5,17-dimethylalkanes, indicating that succinate, as a methylmalonyl-CoA unit, was incorporated as the third unit to form 5-methylheneicosane and as both the third and ninth units to form 5,17-dimethylheneicosane. Analysis of organic acids after the in vivo metabolism of [2,3-¹⁴C]succinate showed that succinate was converted to propionate and methylmalonate. Labeled succinate injected into the hemolymph was readily taken up by the gut tract. Isolated gut tissue efficiently converted succinate to acetate and propionate, both of which were released into the incubation media. Mitochondria from termite tissue (minus gut tract) converted succinate to methylmalonate and propionate only in the presence of malonic acid, an inhibitor of succinate dehydrogenase. The results of these studies show that while termite mitochondria are able to convert succinate to propionate and methylmalonate, most of the propionate used for methyl-branched hydrocarbon biosynthesis is produced by gut tract microorganisms. The propionate is then presumably transported through the hemolymph to epidermal cells for use in methyl-branched hydrocarbon biosynthesis.     

The murein of Thiobacillus neapolitanus

Amino acid analysis of pure murein isolated from cells of T. neapolitanus revealed the typical constituents of most mureins form Gram-negative bacteria. i.e. glutamic acid, alanine and diaminopimelic acid, but the molecular ratio ot these was unusual, being approximately 1: 1: 1. The reduced amount of alanine was explained by the absence of monomers containing tetrapeptide side chains, as revealed by h. p. 1. c. analysis, [(3)H]glutamic acid, [(3)H]diaminopimelic acid and [(3)H]N-acetylglucosamine were incorporated into the murein and allowed to determine the degree of its crosslinkage (28%) and the occurrence of turnover.     

Synthesis and biological evaluation of a radiolabeled analog of methyl 2-acetamido-2,4-dideoxy-beta-D-xylo-hexopyranoside directed towards influencing cellular glycosaminoglycan biosynthesis.

Two methods are presented for the synthesis of methyl 2-acetamido-2,4-dideoxy-beta-D-xylo-hexopyranoside. The first method employs the Barton-McCombie deoxygenation methodology, and the second method utilizes an oxidation-beta-elimination methodology that allows for the incorporation of hydrogen isotopes into the title compound. Hence, methyl 2-acetamido-2,4-dideoxy-beta-D-xylo-hexopyranoside (4) and methyl 2-acetamido-2,4-dideoxy-beta-D-xylo-hexopyranoside-6-t (14) were synthesized and evaluated for their ability to inhibit hepatocyte, cell-surface glycosaminoglycan biosynthesis and to incorporate a [(3)H] radiolabel into isolated glycosaminoglycans, respectively. Compound 4, at a concentration of 1.0 mM, demonstrated a reduction of D-[(3)H]glucosamine and [(35)S]sulfate incorporation into isolated glycosaminoglycans by 69 and 59%, of the control cultures, respectively. At 10 and 20 mM, 4 demonstrated a maximum inhibition of incorporation of both radiolabels to approximately 10% of the control cultures. Compound 14 demonstrated a maximum incorporation of a [(3)H] radiolabel into isolated cell-surface glycosaminoglycans at 10 and 20 mM. The mechanism of inhibition of glycosaminoglycan biosynthesis is due, in part, to the incorporation of a 4-deoxy moiety into glycosaminoglycan chains resulting in premature chain termination.     

Production of a co-polyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from succinic acid by Rhodococcus ruber: biosynthetic considerations

The biosynthesis of the 3-hydroxyvalerate (3HV) monomer of polyhydroxyalkanoate by Rhodococcus ruber from succinic acid was investigated using nuclear magnetic resonance analysis. Polymer produced from [2,3-¹³C]- and [1,4-¹³C]succinate showed that the C-1-C-2 and C-4-C-5 fragments of 3HV were derived from carbons 2 and 3 of succinate, essentially without bond cleavage, and carbon 3 of 3HV was derived from a carboxyl carbon of succinate. Using [1,2-¹³C]succinate it was demonstrated that the C-1-C-2 bond of succinate was cleaved during polymer biosynthesis. Methylmalonyl-coenzyme A (CoA) mutase activity was detected in cell-free extracts of R. ruber by enzyme assay and HPLC analysis of reaction products. A pathway, involving the known methylmalonyl-CoA pathway for propionate formation in Propionibacteria, followed by the established pathway for PHA biosynthesis from propionyl-CoA and acetyl-CoA, is proposed for the biosynthesis of 3HV from succinate by R. ruber. Correspondence to: A. J. Anderson     

Hidden stereospecificity in the biosynthesis of divinyl ether fatty acids

Incubations of [8(R)-2H]9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid, [14(R)-2H]13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid and [14(S)-2H]13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid were performed with preparations of plant tissues containing divinyl ether synthases. In agreement with previous studies, generation of colneleic acid from the 8(R)-deuterated 9(S)-hydroperoxide was accompanied by loss of most of the deuterium label (retention, 8%), however, the opposite result (98% retention) was observed in the generation of 8(Z)-colneleic acid from the same hydroperoxide. Formation of etheroleic acid and 11(Z)-etheroleic acid from the 14(R)-deuterated 13(S)-hydroperoxide was accompanied by loss of most of the deuterium (retention, 7-8%), and, as expected, biosynthesis of these divinyl ethers from the corresponding 14(S)-deuterated hydroperoxide was accompanied by retention of deuterium (retention, 94-98%). Biosynthesis of omega5(Z)-etheroleic acid from the 14(R)- and 14(S)-deuterated 13(S)-hydroperoxides showed the opposite results, i.e. 98% retention and 4% retention, respectively. The experiments demonstrated that biosynthesis of divinyl ether fatty acids from linoleic acid 9- and 13-hydroperoxides takes place by a mechanism that involves stereospecific abstraction of one of the two hydrogen atoms alpha to the hydroperoxide carbon. Furthermore, a consistent relationship between the absolute configuration of the hydrogen atom eliminated (R or S) and the configuration of the introduced vinyl ether double bond (E or Z) emerged from these results. Thus, irrespective of which hydroperoxide regioisomer served as the substrate, divinyl ether synthases abstracting the pro-R hydrogen generated divinyl ethers having an E vinyl ether double bond, whereas enzymes abstracting the pro-S hydrogen produced divinyl ethers having a Z vinyl ether double bond.     

Measurement of bile acid synthesis by 14CO2: the metabolism of propionyl CoA.

The relationship between 14CO2 evolution from the catabolism of [26 or 2714C] cholesterol to bile acids was studied in rats with biliary fistulae. When equal quantities of [26 or 2714C] cholesterol and [414C] cholesterol were administered, there was a significant linear relationship between 14CO2 expiration in the breath and [414C] bile acid excreted in the bile. Bile acid synthesis calculated as the ratio of 14CO2: molar specific activity of biliary cholesterol correlated highly with biliary bile acid excretion in the bile acid depleted rat. Phenobarbital, a known inducer of gamma-amino levulenic acid formation from succinyl CoA did not alter the relationship between the 14CO2 estimation of bile acid synthesis and biliary bile acid excretion, indicating that the relationship between [26 or 2714C] cholesterol side chain cleavage and 14CO2 formation was not altered. Phenobarbital, however, did cause a reduction in bile acid synthesis measured by 14CO2 evolution and by biliary bile acid excretion. The 14CO2 method underestimated bile acid excretion. 8.7% in untreated and phenobarbital treated rats respectively. Since 11% of the radioactivity which was expired as 14CO2 was isolated as bile acids, radioactivity cleaved as [1 or 314C] propionyl CoA may enter cholesterol-bile acid biosynthesis resulting in the underestimation of bile acid synthesis. To test whether radioactivity from propionyl CoA enters steroid biosynthesis [114C] propionate and [214C] propionate were given to untreated biliary fistula rats and the biliary lipids excreted in 60 hours were analyzed. Incorporation of radioactivity into cholesterol and bile acids was greater after the administration of [214C] propionate than after [114C] propionate than after [114C] propionate, suggesting that radioactivity from propionyl CoA may enter steroid biosynthesis by metabolic events in which the methylene and carboxyl carbon atoms are differentiated. Although the use of 14CO2 expiration from [26 or 2714C] cholesterol catabolism underestimates the rate of bile acid synthesis, it should have many applications because of the constant relationship between 14CO2 formation and cholesterol side chain cleavage.     

Metabolism of propionate to acetate in the cockroach Periplaneta americana

Carbon-13 NMR and radiotracer studies were used to determine the precursor to methylmalonate and to study the metabolism of propionate in the cockroach Periplaneta americana. [3,4,5-13C3]Valine labeled carbons 3, 4, and 26 of 3-methylpentacosane, indicating that valine was metabolized via propionyl-CoA to methylmalonyl-CoA and served as the methyl branch unit precursor. Potassium [2-13C]propionate labeled the odd-numbered carbons of hydrocarbons and potassium [3-13C]propionate labeled the even-numbered carbons of hydrocarbons in this insect. This labeling pattern indicates that propionate is metabolized to acetate, with carbon-2 of propionate becoming the methyl carbon of acetate and carbon-3 of propionate becoming the carboxyl carbon of acetate. In vivo studies in which products were separated by HPLC showed that [2-14C]propionate was readily metabolized to acetate. The radioactivity from sodium [1-14C]propionate was not incorporated into succinate nor into any other tricarboxylic acid cycle intermediate, indicating that propionate was not metabolized via methylmalonate to succinate. Similarly, [1-14C]propionate did not label acetate. An experiment designed to determine the subcellular localization of the enzymes involved in converting propionate to acetate showed that they were located in the mitochondrial fraction. Data from both in vivo and in vitro studies as a function of time indicated that propionate was converted directly to acetate and did not first go through tricarboxylic acid cycle intermediates. These data demonstrate a novel pathway of propionate metabolism in insects.     

Propionate inhibits hepatocyte lipid synthesis

Oat bran lowers serum cholesterol in animals and humans. Propionate, a short-chain fatty acid produced by colonic bacterial fermentation of soluble fiber, is a potential mediator of this action. We tested the effect of propionate on hepatocyte lipid synthesis in rats using [1-14C]acetate, 3H2O, and [2-14C]mevalonate as precursors. Propionate produced a statistically significant inhibition of cholesterol biosynthesis from [1-14C]acetate at a concentration of 1.0 mM and from 3H2O and [2-14C]mevalonate at concentrations of 2.5 mM. Propionate also produced a significant inhibition of fatty acid biosynthesis at concentrations of 2.5 mM using [1-14C]acetate as a precursor. The demonstration of propionate-mediated inhibition of cholesterol and fatty acid biosynthesis at these concentrations suggests that propionate may inhibit cholesterol and fatty acid biosynthesis in vivo and may mediate in part the hypolipidemic effects of soluble dietary fiber. Further studies are needed to clarify this action of propionate and to establish the exact mechanisms by which the inhibition occurs.     

Stereochemistry and kinetic isotope effects in the bovine plasma amine oxidase catalyzed oxidation of dopamine.

The stereochemistry of the bovine plasma amine oxidase catalyzed oxidation of 2-(3,4-dihydroxyphenyl)-ethylamine (domapine) has been investigated by comparing 3H/14C ratios of 3,4-dibenzyloxyphenethyl alcohols, derived from 3,4-dihydroxyphenylacetaldehydes, to starting dopamines chirally labeled at C-1 and C-2. The oxidation of [2RS-3H]-, [2R-3H]-, and [2S-3H]dopamine leads to products which have retained 53, 59, and 47% of their tritium. Similarly, oxidation of [1RS-3H]-, [1R-3H]-, and [1S-3H]dopamine leads to an 80, 80, and 92% retention of tritium. The configurational purity of tritium at C-2 of dopamine and C-1 of the dopamine precursor 3-methoxy-4-hydroxyphenethylamine has been confirmed employing dopamine-beta-hydroxylase (specific for the pro-R hydrogen at C-2) and pea seedling amine oxidase (specific for the pro-S hydrogen at C-1). In addition, chromatographically resolved isozymes of bovine plasma amine oxidase have been demonstrated to lead to the same stereochemical result as pooled enzyme fractions. We have been able to rule out carbon interchange and tritium transfer in the ethylamine side chain of dopamine as the source of the apparent nonstereospecificity. Estimated primary tritium isotope effects are 1 for [2-3H]dopamines and 5--6 and 26--34 for [1R-3H]- and [1S-3H]dopamine, respectively. We propose the presence of alternate dopamine binding modes, characterized by absolute but opposing stereochemistries and differential primary tritium isotope effects at C-1.     

Intermolecular tritium transfer in the transcarboxylase reaction.

Transfer of tritium from [3-3H]pyruvate into propionyl-CoA is found during the reaction of transcarboxylase: Methylmalonyl-CoA + pyruvate leads to oxalacetate + propionyl-CoA. About 5% of the tritium counts that are labilized in the reaction are found in a position of the propionate that exchanges rapidly with water in the presence of transcarboxylase. Transfer from [2-3H]propionate of propionyl-CoA to pyruvate is real but only about one-tenth as great. The tritium transfers between reactants on two subunits are difficult to explain by a "carbanion" mechanism of --C--H bond cleavage and support the cyclic mechanism in which carboxybiotin itself is the base and the enol form of biotin is the proton-transferring agent.     

Oxysterols: 27-hydroxycholesterol and its radiolabeled analog

We describe a convenient and stereoselective route to the synthesis of 27-hydroxycholesterol. Also its radiolabeled analog, 22, 23 di [(3)H]-27-hydroxycholesterol with high specific radioactivity (55 Ci/mmol) was synthesized by this method. Julia condensation of steroidal 22-sulfone with aldehyde, led to the addition of the 23-27 carbon side chain building block to the steroid backbone. Formed in this reaction beta-hydroxysulfone moiety was reduced by sodium amalgam generate 22-23 unsaturated bond. Further reduction either by hydrogen or tritium furnished substrates for the synthesis of title compounds.     

The stereospecific removal of a C-19 hydrogen atom in oestrogen biosynthesis

1. The synthesis of a number of 19-substituted androgens is described. 2. A method for the partially stereospecific introduction of a tritium label at C-19 in 19-hydroxyandrost-5-ene-3beta,17beta-diol was developed. The 19-(3)H-labelled triol produced by reduction of 19-oxoandrost-5-ene-3beta,17beta-diol with tritiated sodium borohydride is tentatively formulated as 19-hydroxy[(19-R)-19-(3)H]androst-5-ene-3beta,17beta-diol and the 19-(3)H-labelled triol produced by reduction of 19-oxo[19-(3)H]-androst-5-ene-3beta,17beta-diol with sodium borohydride as 19-hydroxy[(19-S)-19-(3)H]-androst-5-ene-3beta,17beta-diol. 3. In the conversion of the (19-R)-19-(3)H-labelled compound into oestrogen by a microsomal preparation from human term placenta more radioactivity was liberated in formic acid (61.6%) than in water (38.4%). In a parallel experiment with the (19-S)-19-(3)H-labelled compound the order of radioactivity was reversed: formic acid (23.4%), water (76.2%). 4. These observations are interpreted in terms of the removal of the 19-S-hydrogen atom in the conversion of a 19-hydroxy androgen into a 19-oxo androgen during oestrogen biosynthesis. 5. It is suggested that the removal of C-19 in oestrogen biosynthesis occurs compulsorily at the oxidation state of a 19-aldehyde with the liberation of formic acid.     

Elucidation of the pathways of catabolic glutamate conversion in three thermophilic anaerobic bacteria

The glutamate catabolism of three thermophilic syntrophic anaerobes was compared based on the combined use of [(13)C] glutamate NMR measurements and enzyme activity determinations. In some cases the uptake of intermediates from different pathways was studied. The three organisms, Caloramator coolhaasii, Thermanaerovibrio acidaminovorans and strain TGO, had a different stoichiometry of glutamate conversion and were dependent on the presence of a hydrogen scavenger (Methanobacterium thermoautotrophicum Z245) to a different degree for their growth. C. coolhaasii formed acetate, CO(2), NH(4)(+) and H(2) from glutamate. Acetate was found to be formed through the beta-methylaspartate pathway in pure culture as well as in coculture. T. acidaminovorans converted glutamate to acetate, propionate, CO(2), NH(4)(+) and H(2). Most likely, this organism uses the beta-methylaspartate pathway for acetate formation. Propionate formation occurred through a direct oxidation of glutamate via succinyl-CoA and methylmalonyl-CoA. The metabolism of T. acidaminovorans shifted in favour of propionate formation when grown in coculture with the methanogen, but this did not lead to the use of a different glutamate degradation pathway. Strain TGO, an obligate syntrophic glutamate-degrading organism, formed propionate, traces of succinate, CO(2), NH(4)(+) and H(2). Glutamate was converted to propionate oxidatively via the intermediates succinyl-CoA and methylmalonyl-CoA. A minor part of the succinyl-CoA was converted to succinate and excreted.     

Identification of a novel UDP-Glc:GlcNAc beta1-->4-glucosyltransferase in Lymnaea stagnalis that may be involved in the synthesis of complex-type oligosaccharide chains

Several studies suggest, that the snail Lymnaea stagnalis contains glycoproteins whose oligosaccharide side chains have structural features not commonly found in mammalian glycoproteins. In this study, prostate glands of L. stagnalis were incubated in media containing either [(3)H]-mannose, [(3)H]-glucosamine, or [(3)H]-galactose, and the metabolically radiolabeled protein-bound oligosaccharides were analyzed. The newly synthesized diantennary-like complex-type asparagine-linked chains contained a considerable amount of glucose, next to mannose, GlcNAc, fucose, galactose, and traces of GalNAc. Since glucose has not been found before as a constituent of diantennary N-linked glycans as far as we know, we assayed the prostate gland of L. stagnalis for a potential glucosyltransferase activity involved in the biosynthesis of such structures. We report here, that the prostate gland of L. stagnalis contains a beta1-->4-glucosyltransferase activity that transfers glucose from UDP-glucose to acceptor substrates carrying a terminal N-acetylglucosamine. The enzyme prefers substrates carrying a terminal GlcNAc that is beta6 linked to a Gal or a GalNAc, structures occurring in O-linked glycans, or a GlcNAc that is beta2 linked to mannose, as is present in N-linked glycans. Based on combined structural and enzymatic data, we propose that the novel beta1-->4-gluco-syltransferase present in the prostate gland may be involved in the biosynthesis of Glcbeta1-->4GlcNAc units in complex-type glycans, in particular in N-linked diantennary glycans.     

Biosynthesis of lasalocid A: biochemical mechanism for assembly of the carbon framework

Labeling experiments on the biosynthesis of the polyether antibiotic lasalocid A (1) using carboxylic acid precursors bearing 13C, 2H, and 3H labels at various positions established the following: (1) 2H or 3H at C-2 of propionate or 2H at C-2 of butyrate was partially retained at C-12 and C-14 of 1, respectively. (2) 2H at C-2 of propionate or at C-2 and C-3 of succinate did not label C-10. These and earlier data [Hutchinson, C. R., Sherman, M. M., Vederas, J. C., & Nakashima, T. T. (1981) J. Am. Chem. Soc. 103, 5953; Hutchinson, C. R., Sherman, M. M., McInnes, A. G., Walter, J. A., & Vederas, J. C. (1981) J. Am. Chem. Soc. 103, 5956] are consistent with a hypothesis for the stereochemical control of lasalocid A biosynthesis, whose main tenets are that the configuration of C-12 and C-14 is determined by the stereoselectivity of the carbon chain forming condensation between acyl thio ester and 2-carboxyacyl thio ester intermediates and that the configuration of C-11 and C-15 results from the reduction of 2-keto thio ester intermediates with opposing stereospecificities.     

Different metabolic recycling of the lipid components of exogenous sulphatide in human fibroblasts.

Cultured human fibroblasts were fed with two differently labelled sulphatide molecules [one labelled on C-3 of the sphingosine (Sph) moiety [( Sph-3H]sulphatide), the second on C-1 of stearic acid [( stearoyl-14C]sulphatide)], and the intracellular metabolic fate of radioactivity was monitored. Incorporated radioactivity was almost all recovered in the total lipid extract, regardless of the labelling position of the added sulphatide; however, large differences in the level of incorporation occurred among labelled glycosphingolipids. For example, sphingomyelin was present as the major radiolabelled lipid after [Sph-3H]-sulphatide incubation, but was detectable only in trace amounts after [stearoyl-14C]sulphatide administration; in the latter case the radioactivity was located predominantly in glycerophospholipids. From this finding it can be inferred that the free long-chain base (sphingosine) that originates from lysosomal catabolism of sulphatide is mainly, and quite specifically, utilized for sphingomyelin biosynthesis, whereas the ceramide moiety is not; conversely the fatty acid released from ceramide is non-specifically re-utilized for phospholipid biosynthesis.