O-acetylation and de-O-acetylation of sialic acids. 7- and 9-o-acetylation of alpha 2,6-linked sialic acids on endogenous N-linked glycans in rat liver Golgi vesicles

We have previously shown that radioactivity from [acetyl-3H]AcCoA is concentrated into isolated intact rat liver Golgi vesicles. The incorporated radioactivity occurred in acid-soluble and acid-insoluble components, and the acid-insoluble fraction included O-acetylated sialic acids (Varki, A., and Diaz, S. (1985) J. Biol. Chem. 260, 6600-6608). Nearly all of the protein-associated radioactivity was found to be in sialic acids alpha 2-6-linked to N-linked oligosaccharides on endogenous glycoproteins. Incubation of the vesicles with CMP-[3H]sialic acid resulted in labeling of a very similar group of glycoproteins. The 3H-O-acetyl groups were found at both the 7- and the 9-positions of N-acetylneuraminic acid residues at the end of the labeling reaction. Although 7-O-acetyl groups can undergo migration to the 9-position under physiological conditions, kinetic studies using O-acetyl-14C-labeled internal and O-acetyl-3H-labeled external standards indicate that during the labeling, release, and purification, negligible migration occurred. Studies with mild periodate oxidation provided further confirmation that O-acetyl esters are added directly to both the 7- and the 9-positions of the sialic acids in this system. The acid-soluble, low molecular weight component is released from the vesicles by increasing concentrations of saponin, and its exit parallels that of CMP-[14C]sialic acid taken up during the incubation. The vesicles themselves are impermeant to free acetate. However, even after short incubations, this saponin-releasable radioactivity was almost exclusively in [3H] acetate and not in [3H]acetyl-CoA. The apparent Km for accumulation of the [3H]acetate is almost identical with that for the generation of the acid-insoluble O-acetylated sialic acids. Most of this accumulation of free acetate is also blocked by coenzyme A-SH. Only a small portion arises from the action of an endogenous esterase on the 3H-O-acetylated sialic acids. Taken together, the results indicate that accumulation of free [3H]acetate occurs within the lumen of the vesicles in parallel with O-acetylation of sialic acids and is probably a product of abortive acetylation. It is not known if this reaction occurs in vivo. Permeabilization of Golgi vesicles to low molecular weight molecules with saponin does not alter the rate of acetylation substantially. Furthermore, double label studies suggest that the intact acetyl-CoA molecule does not gain access to the lumen of the vesicles. These results indicate that the acetylation reaction may have a different mechanism from previously described Golgi glycosylation reactions, wherein specific transporters concentrate sugar nucleotides for use by luminally oriented transferases.     

O-acetylation and de-O-acetylation of sialic acids. O-acetylation of sialic acids in the rat liver Golgi apparatus involves an acetyl intermediate and essential histidine and lysine residues--a transmembrane reaction?

Isolated intact rat liver Golgi vesicles utilize [acetyl-3H]coenzyme A to add 3H-O-acetyl esters to sialic acids of internally facing endogenous glycoproteins. During this reaction, [3H]acetate also accumulates in the vesicles, even though the vesicles are impermeant to free acetate. On the other hand, entry of intact AcCoA into the lumen of the vesicles could not be demonstrated, and permeabilization of the vesicles did not alter the reaction substantially (Diaz, S., Higa, H. H., Hayes, B. K., and Varki, A. (1989) J. Biol. Chem. 264, 19416-19426). When vesicles prelabeled with [acetyl-3H] coenzyme A are permeabilized with saponin, we can demonstrate a [3H]acetyl intermediate in the membrane that can transfer label to the 7- and 9-positions of exogenously added free N-acetylneuraminic acid but not to glucuronic acid or CMP-N-acetylneuraminic acid. This labeled acetyl intermediate represents a significant portion of the radioactivity incorporated into the membranes during the initial incubation and cannot be accounted for by nonspecifically "trapped" acetyl-CoA in the permeabilized vesicles. There was no evidence for involvement of acetylcarnitine or acetyl phosphate as an intermediate. The overall acetylation reaction appears to involve two steps. The first step (utilization of exogenous acetyl-CoA to form the acetyl intermediate) is inhibited by coenzyme A-SH (apparent Ki = 24-29 microM), whereas the second (transfer from the acetyl intermediate to sialic acid) is not affected by millimolar concentrations of the nucleotide. Studies with amino acid-modifying reagents indicate that 1 or more histidine residues are involved in the first step of the acetylation reaction. Diethylpyrocarbonate (which can react with both nonsubstituted and singly acetylated histidine residues) also blocks the second reaction, indicating that the acetyl intermediate on both sides of the membrane involves histidine residue(s). Taken together with data presented in the preceding paper, these results indicate that the acetylation of sialic acids in Golgi vesicles may occur by a transmembrane reaction, similar to that described for the acetylation of glucosamine in lysosomes (Bame, K. J., and Rome, L. H. (1985) J. Biol. Chem. 260, 11293-11299). However, several features of this Golgi reaction distinguish it from the lysosomal one, including the nature and kinetics of the reaction and the additional involvement of an essential lysine residue. The accumulation of free acetate in the lumen of the vesicles during the reaction may occur by abortive acetylation (viz. transfer of label from the acetyl intermediate to water). It is not clear if this is an artifact that occurs only in the in vitro reaction.     

Translocation of adenosine 3'-phosphate 5'-phosphosulfate into rat liver Golgi vesicles

The translocation of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) across rat liver Golgi-derived vesicles has been studied. Vesicles of the same topographical orientation as in vivo were incubated with a mixture of [adenine-8-3H]PAPS and [35S]PAPS. The tritium to radiolabeled sulfur ratio of the incubation medium was 1.73 +/- 0.03 while that in the vesicles was 1.82 +/- 0.13. This strongly suggests that the entire PAPS molecule was being translocated across the Golgi vesicle membrane even though intact PAPS could not be detected within the vesicles. Translocation of PAPS resulted in accumulation of solutes within vesicles. This accumulation was temperature dependent, saturable (apparent Km = 0.7 microM; Vmax = 25 pmol/mg of protein/10 min), and inhibited by the substrate analogue 3',5'-ADP but not by 2',5'-ADP. Translocation of PAPS was inhibited following treatment of Golgi vesicles with Pronase under conditions in which the activity of a lumenal Golgi membrane marker such as sialyltransferase was not. This result is consistent with the existence of a PAPS carrier protein, portions of which face the cytoplasmic side of the Golgi membrane.     

Succinate metabolism related to lipid synthesis in the housefly Musca domestica L

The metabolism of succinate was examined in the housefly Musca domestica L. The labeled carbons from [2,3-¹⁴C]succinate were readily incorporated into cuticular hydrocarbon and internal lipid, whereas radioactivity from [1,4-¹⁴C]succinate was not incorporated into either fraction. Examination of the incorporation of [2,3-¹⁴C]succinate, [1-¹⁴C]acetate, and [U-¹⁴C]proline into hydrocarbon by radio-gas-liquid chromatography showed that each substrate gave a similar labeling pattern, which suggested that succinate and proline were converted to acetyl-CoA prior to incorporation into hydrocarbons. Carbon-13 nuclear magnetic resonance showed that the labeled carbons from [2,3-¹³C]succinate enriched carbons 1, 2, and 3 of hydrocarbons with carbon-carbon coupling showing that carbons 2 and 3 of succinate were incorporated as an intact unit. Radio-high-performance liquid chromatographic analysis of [2,3-¹⁴C]succinate metabolism by mitochondrial preparations showed that in addition to labeling fumarate, malate, and citrate, considerable radioactivity was also present in the acetate fraction. The data show that succinate was not converted to methylmalonate and did not label hydrocarbon via a methylmalonyl derivative. Malic enzyme was assayed in sonicated mitochondria prepared from the abdomens and thoraces of 1- and 4-day-old insects; higher activity was obtained with NAD⁺ in mitochondria prepared from thoraces, whereas NADP⁺ gave higher activity with abdomen preparations. These data document the metabolism of succinate to acetyl-CoA and not to a methylmalonyl unit prior to incorporation into lipid in the housefly and establish the role of the malic enzyme in this process.     

RADIOAUTOGRAPHIC STUDY OF IN VIVO AND IN VITRO INCORPORATION OF FUCOSE-3H INTO THYROGLOBULIN BY RAT THYROID FOLLICULAR CELLS

The incorporation of fucose-³H in rat thyroid follicles was studied by radioautography in the light and electron microscopes to determine the site of fucose incorporation into the carbohydrate side chains of thyroglobulin, and to follow the migration of thyroglobulin once it had been labeled with fucose-³H. Radioautographs were examined quantitatively in vivo at several times after injection of fucose-³H into rats, and in vitro following pulse-labeling of thyroid lobes in medium containing fucose-³H. At 3–5 min following fucose-³H administration in vivo, 85% of the silver grains were localized over the Golgi apparatus of thyroid follicular cells. By 20 min, silver grains appeared over apical vesicles, and by 1 hr over the colloid. At 4 hr, nearly all of the silver grains had migrated out of the cells into the colloid. Analysis of the changes in concentration of label with time showed that radioactivity over the Golgi apparatus increased for about 20 min and then decreased, while that over apical vesicles increased to reach a maximum at 35 min. Later, the concentration of label over the apical vesicles decreased, while that over the colloid increased. Similar results were obtained in vitro. It is concluded that fucose, which is located at the end of some of the carbohydrate side chains, is incorporated into thyroglobulin within the Golgi apparatus of thyroid follicular cells, thereby indicating that some of these side chains are completed there. Furthermore, the kinetic analysis demonstrates that apical vesicles are the secretion granules which transport thyroglobulin from the Golgi apparatus to the apex of the cell and release it into the colloid.     

UDP-GlcNAc transport across the Golgi membrane: electroneutral exchange for dianionic UMP

We have examined the coupling and charge stoichiometry for UDP-GlcNAc transport into Golgi-enriched vesicles from rat liver. In the absence of added energy sources, these Golgi vesicles concentrate UDP-GlcNAc at least 20-fold, presumably by exchange with endogenous nucleotides. Under the conditions used, extravesicular degradation of UDP-GlcNAc has been eliminated, and less than 15% of the internalized radioactivity becomes associated with endogenous macromolecules. Of the remaining intravesicular label, 85% remains unmetabolized UDP-[3H]GlcNAc, and approximately 15% is hydrolyzed to [3H]GlcNAc-1-phosphate. Efflux of accumulated UDP-[3H]GlcNAc is induced by addition of UMP, UDP, or UDP-galactose to the external medium. Permeabilization of Golgi vesicles causes a rapid and nearly complete loss of internal UDP-[3H]GlcNAc, indicating that the results reflect transport and not binding. Moreover, transport of UDP-[3H]GlcNAc into these Golgi vesicles was stimulated up to 5-fold by mechanically preloading vesicles with either UDP-GlcNAc or UMP. The response of UMP/UMP exchange and UMP/UDP-GlcNAc exchange to alterations in intravesicular and extravesicular pH suggests that UDP-GlcNAc enters the Golgi apparatus in electroneutral exchange with the dianionic form of UMP.     

Intracellular processes associated with glycoprotein transport and processing.

The intracellular transport of mucus glycoprotein precursor (apomucin) from endoplasmic reticulum (ER) to Golgi was quantitated by the immunoprecipitation with 3G12 antimucin monoclonal antibody and by estimation of the apomucin glycosylation using UDP-[3H]galactose. The assembly of the entities carrying apomucin to Golgi was assessed by electron microscopy and by quantitation of the incorporation of [14C]choline, [14C]ethanolamine, and [14C]oleic acid into their lipids. The microscopic image of the isolated transport components revealed a population of 80- to 100-nm vesicles with occasional membranes of the ER used for their synthesis. On the average, the vesicles contained 82 ng apomucin/microgram of protein and 80-90% of the total incorporated lipid precursors. From that, 91% of [14C]choline was detected in phosphatidylcholine, and 9% in phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin. With [14C]oleate, 54% of the label was incorporated into ceramide, diglyceride, and phosphatidic acid, 35% to phosphatidylcholine, 7% in phosphatidylethanolamine, and 2% in sphingomyelin. After incubation of the vesicles with Golgi, the apomucin was found glycosylated and the lipids of the transport vesicles incorporated into Golgi membranes. The fusion of the vesicular membranes was accompanied by the synthesis of sphingomyelin. In the Golgi, 39-55% of the radiolabeled phosphatidylcholine of transport vesicles was converted to sphingomyelin. The results indicate that the newly synthesized membranes of apomucin transporting vesicles are enriched in phosphoglycerides and ceramides. Upon fusion with the Golgi, the membranes of the vesicles are replenished with sphingomyelin by exchange reaction between phosphatidylcholine and ceramide.     

Reconstitution of the Golgi apparatus after microinjection of rat liver Golgi fragments into Xenopus oocytes

We have studied the reconstitution of the Golgi apparatus in vivo using an heterologous membrane transplant system. Endogenous glycopeptides of rat hepatic Golgi fragments were radiolabeled in vitro with [3H]sialic acid using detergent-free conditions. The Golgi fragments consisting of dispersed vesicles and tubules with intraluminal lipoprotein-like particles were then microinjected into Xenopus oocytes and their fate studied by light (LM) and electron microscope (EM) radioautography. 3 h after microinjection, radiolabel was observed by LM radioautography over yolk platelet-free cytoplasmic regions near the injection site. EM radioautography revealed label over Golgi stacked saccules containing the hepatic marker of intraluminal lipoprotein-like particles. At 14 h after injection, LM radioautographs revealed label in the superficial cortex of the oocytes between the yolk platelets and at the oocyte surface. EM radioautography identified the labeled structures as the stacked saccules of the Golgi apparatus, the oocyte cortical granules, and the plasmalemma, indicating that a proportion of microinjected material was transferred to the surface via the secretion pathway of the oocyte. The efficiency of transport was low, however, as biochemical studies failed to show extensive secretion of radiolabel into the extracellular medium by 14 h with approximately half the microinjected radiolabeled constituents degraded. Vinblastine (50 microM) administered to oocytes led to the formation of tubulin paracrystals. Although microinjected Golgi fragments were able to effect the formation of stacked saccules in vinblastine-treated oocytes, negligible transfer of heterologous material to the oocyte surface could be detected by radioautography. The data demonstrate that dispersed fragments of the rat liver Golgi complex (i.e., unstacked vesicles and tubules) reconstitute into stacked saccules when microinjected into Xenopus cytoplasm. After the formation of stacked saccules, reconstituted Golgi fragments transport constituents into a portion of the exocytic pathway of the host cell by a microtubule-regulated process.     

Biosynthesis and turnover of O-acetyl and N-acetyl groups in the gangliosides of human melanoma cells

We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with [3H]acetate and [14C]glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with [acetyl-3H]acetyl-coenzyme A, the major labeled products were disialogangliosides. [Acetyl-3H]O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in [3H]N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from [3H]acetate was exclusively in the form of [3H]N-acetyl groups, whereas the 14C-label was at the 4-position. Pulse-chase analysis of the 3H/14C ratio showed that the N-acetyl groups of both GD3 and of the monosialoganglioside GM3 were turning over faster than the parent molecules. Selective periodate oxidation showed that both the inner and outer sialic acid residues of GD3 incorporated 3H-label in the in vitro reaction, and showed similar turnover of N-acetylation in the pulse-chase study. Taken together, these results indicate that both the O- and N-acetyl groups of the sialic acid residues of gangliosides turn over faster than the parent molecules. They also demonstrate a novel re-N-acetylation reaction that predicts the existence of de-N-acetyl gangliosides in melanoma cells.     

Incorporation of galactose from UDP-galactose into microsomal and golgi membranes of rat liver

Rough and smooth microsomes and Golgi membranes were incubated with UDP[¹⁴C]galactose and the incorporation of radioactivity into the lipid extract and into endogenous protein acceptors were measured. Antagonistic pyrophosphatases were inhibited with ATP and interference from β-galactosidase activity was greatly decreased by carrying out the incubation at pH 7.8. After incubation the particles were centrifuged to remove free oligosaccharide residues. Radioactivity was found in the lipid extract from Golgi membranes but not from rough and smooth microsomes. This radioactivity, however, was not associated with dolichol or retinyl phosphates. The incorporation of radioactivity into proteins of the Golgi fraction was more than double than that of the microsomal fractions. In addition, the transferases in these two types of particles exhibited different properties. Trypsin treatment of intact rough microsomal vesicles, smooth vesicles and Golgi membranes removed about 5, 15 and 50%, respectively, of newly incorporated protein-bound galactose, indicating that the proportion of the newly galactosylated proteins, which are localized at the cytoplasmic surface of the membrane, is lowest in rough microsomes, intermediate in smooth, and highest in Golgi membranes.     

Evidence for an in vivo modification of mitochondrial proteins by coenzyme A.

Following denaturation of mitochondrial proteins by sodium dodecyl sulfate, a [1-14C]pantothenic acid-derived radioactivity proved to be acid precipitable in the outer membrane, the intermembrane space, the inner membrane and in the matrix of rat liver mitochondria, where it had the highest specific radioactivity of 541 +/- 29 cpm/100 micrograms protein. This tightly and/or covalently bound protein radioactivity could be released by incubation in the presence of dithioerythreitol; it was identified as [14C]coenzyme A by its HPLC retention time, its absorption spectrum and its radioactivity. This acid-stable and thiol-labile coenzyme A-binding apparently refers to specific protein binding sites. With the purified, homogeneous mitochondrial matrix enzymes acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase) (EC 2.3.1.9, acetyl-CoA:acetyl-CoA C-acetyltransferase) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16) coenzyme A was found exclusively, e.g., in the modified, partially-active forms A1 und A2 of acetyl-CoA acetyltransferase and not in the unmodified fully-active enzyme. Thus it is evident that this coenzyme A modification is transient. We suggest that coenzyme A-modification is a signal involved in the assembly or the degradation process of distinct mitochondrial matrix proteins.     

Topology of UDP-galactose cleavage in relation to N-acetyl-lactosamine formation in Golgi vesicles. Translocation of activated galactose.

UDP-galactose appears to be produced on one side of a membrane barrier, opposite the galactosyltransferases that use it as a sugar donor. The translocation of activated galactose across membranes was studied in rat submaxillary-gland microsomal vesicles and in rat liver Golgi vesicles. When these intact vesicles containing the acceptor, N-acetylglucosamine, were incubated in the presence of UDP-galactose and two inhibitors of galactosyltransferase activity, the product, N-acetyl-lactosamine, formed within the vesicles. Thus at least the galactose moiety of UDP-galactose crossed the membranes. When intact Golgi vesicles were incubated with UDP-galactose labelled in both the uridine and the galactose moieties, labelled N-acetyllactosamine was again produced in the vesicles, but less than stoichiometric amounts of the uridine label was found there. Calculation of internal and external concentrations of UMP, a major product released from the cleaved uridine moiety, showed that the vesicles were actually enriched in UMP. When free UMP was incubated with the vesicles, this enrichment did not occur. This result was direct evidence for facilitated transport of UDP-galactose into the Golgi for use by galactosyltransferase.     

Ultrastructural immunolocalization of apolipoprotein B within human jejunal absorptive cells

Apolipoprotein B (apoB) was localized by electron microscopy within absorptive cells of human jejunal biopsy specimens taken fasting and after micellar fat infusion. Nakane's double antibody immunoperoxidase technique was used to label apoB near open cut surfaces of 60-Micrometers fixed tissue slices sectioned by a Ralph knife in a Vibratome. In fasting tissue, apoB label was found within structurally intact peri-mitochondrial rough endoplasmic reticulum (RER) and within Golgi cisternae of absorptive cells covering the tips of jejunal villi. After fat infusion, apoB label was found adjacent to very low density lipoproteins (VLDL) and chylomicrons within apical smooth endoplasmic reticulum (SER). Less label was seen within RER than in fasting absorptive cells, and RER-SER connections containing apoB label were occasionally seen. Expanded Golgi vesicles and cisternae contained VLDL, chylomicrons, and apoB label. Vesicles containing chylomicrons and apoB label were occasionally visualized bordering the lateral plasma membrane in a configuration suggesting exocytosis. Specific apoB label was regularly seen within intercellular spaces and capillaries, but the in vivo significance of this Localization was problematical. These observations suggest that apoB is synthesized in RER, transfers to SER where it is incorporated into new VLDL and chylomicrons, and moves to Golgi cisternae and vesicles to be prepared for exocytosis through the plasma membrane.     

Identification of [1-14C]pantothenic-acid-mediated modified mitochondrial proteins

The in vivo administration of [1-14C]pantothenic acid, which is the precursor of coenzyme A, resulted in the radioactive labelling of several mitochondrial proteins in rat liver. The incorporated radioactivity could be released by glutathione or 2-mercaptoethanol. Two mitochondrial matrix proteins acetyl-CoA acetyltransferase (liver and heart), an enzyme involved in the biosynthesis or degradation of ketone bodies, and 3-oxoacyl-CoA thiolase (liver), a protein participating in fatty acid oxidation were identified as modified proteins. The radioactivity was localized exclusively in forms A1 and A2 indicating that these forms represent the modified states of the acetyl-CoA acetyltransferase protein. Kinetics of incorporation of radioactivity revealed an accumulation of the modified forms. The ratio of specific radioactivities of A2 compared to A1 was 2.41 +/- 0.15 (n = 10). After in vivo labelling with [14C]leucine, the specific radioactivity of acetyl-CoA acetyltransferase depended on the state of the enzyme protein. The unmodified enzyme exhibited a lower specific radioactivity than its modified forms suggesting different turnover rates of these proteins.     

Ion transport and respiratory control in vesicles formed from reduced nicotinamide adenine dinucleotide coenzyme Q reductase and phospholipids.

NADH-coenzyme Q reductase from bovine heart mitochondria (complex I) was incorporated into phospholipid vesicles by the cholate dialysis procedure. Mixtures of purified phosphatidylcholine and phosphatidylethanolamine were required. Oxidation of NADH by coenzyme Q1 catalyzed by the reconstituted vesicles was coupled to proton translocation, directed inward, with an H+/2e ratio greater than 1.4. Similar experiments measuring proton translocation in submitochondrial particles gave an H+/2e ratio of 1.8. The proton translocation in both systems was not seen in the presence of uncoupling agents and was in addition to the net proton uptake from the reduction of coenzyme Q1 by NADH. Electron transfer in the reconstituted vesicles also caused the uptake of the permeant anion tetraphenylboron. The rate of electron transfer by the reconstituted vesicles was stimulated about 3-fold by uncouplers or by valinomycin plus nigericin and K+ ions. The results indicate that energy coupling can be observed with isolated NADH-coenzyme Q reductase if the enzyme complex is properly incorporated into a phospholipid vesicle.     

A comparison of acetylation in vitro of microsomal, homogenate, and Golgi fractions of rat liver

The activity of acetyltransferase was detected in the microsomal fraction of rat liver by incubation with [3H]acetyl-CoA and by analyses using sodium dodecyl sulfate - polyacrylamide gel electrophoresis. Endogenous membrane proteins of relatively high molecular weight were found to serve as substrates. Optimal conditions for assay of the enzyme were defined. A deacetylase activity was also detected, which was inhibited by 2 mM ethylenediaminetetraacetic acid. Further subfractionation disclosed that the acetyltransferase activity was most enriched in the Golgi fraction, in which its specific activity was some ninefold greater than in the total homogenate. The radioactive labelling of Golgi-associated proteins observed was relatively intense, exceeding that of histone and ribosomal proteins in the homogenate. Analysis of the acetylated Golgi fraction by two-dimensional electrophoresis revealed approximately 90 radioactive polypeptides. Various treatments demonstrated that a minimum of 80% of the incorporated radioactivity was present as derivatives of N-acetylneuraminic acid, principally N-acetyl-9-mono-O-acetylneuraminic acid (Neu5,9Ac2). The sialic acid O-acetyltransferase activity detected is thus probably identical to that reported by Varki and Diaz; the intense labelling of proteins reflects the ability of Golgi apparatus fractions to take up and concentrate acetyl-CoA. Protein-bound radioactive Neu5,9Ac2 was also detected in the medium of hepatocytes incubated with N-[3H]acetylmannosamine, demonstrating that these cells synthesize certain proteins containing acetylated sialic acids, some of which may be secreted. The data confirm that the Golgi apparatus is a major site of acetylation of protein-bound sialic acids in rat liver in vitro and provide new information showing that many glycoproteins undergo this particular type of modification.     

Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila.

The carbon monoxide dehydrogenase (CODH) complex from Methanosarcina thermophila catalyzed the synthesis of acetyl coenzyme A (acetyl-CoA) from CH3I, CO, and coenzyme A (CoA) at a rate of 65 nmol/min/mg at 55 degrees C. The reaction ended after 5 min with the synthesis of 52 nmol of acetyl-CoA per nmol of CODH complex. The optimum temperature for acetyl-CoA synthesis in the assay was between 55 and 60 degrees C; the rate of synthesis at 55 degrees C was not significantly different between pHs 5.5 and 8.0. The rate of acetyl-CoA synthesis was independent of CoA concentrations between 20 microM and 1 mM; however, activity was inhibited 50% with 5 mM CoA. Methylcobalamin did not substitute for CH3I in acetyl-CoA synthesis; no acetyl-CoA or propionyl coenzyme A was detected when sodium acetate or CH3CH2I replaced CH3I in the assay mixture. CO could be replaced with CO2 and titanium(III) citrate. When CO2 and 14CO were present in the assay, the specific activity of the acetyl-CoA synthesized was 87% of the specific activity of 14CO, indicating that CO was preferentially incorporated into acetyl-CoA without prior oxidation to free CO2. Greater than 100 microM potassium cyanide was required to significantly inhibit acetyl-CoA synthesis, and 500 microM was required for 50% inhibition; in contrast, oxidation of CO by the CODH complex was inhibited 50% by approximately 10 microM potassium cyanide.     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: II. Inhibition of secretion of thyroglobulin in rat thyroid follicular cells as visualized by radioautography after 3H-fucose injection

Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for radioautography. In thyroid follicular cells of control animals, at this time interval, 57% of the total label was associated with colloid and secretory vesicles in the apical cytoplasm while 27% was localized in the Golgi apparatus and neighboring vesicles. In experimental animals, the proportion of label in colloid and apical vesicles was reduced by more than 69% after colchicine and more than 83% after vinblastine treatment. The proportion of label in the Golgi region, on the other hand, increased by more than 125% after colchicine and more than 179% after vinblastine treatment. Within the Golgi region, the great majority of the label was associated with secretory vesicles which accumulated adjacent to the trans face of the Golgi stacks. It is concluded that the drugs do not interfere with passage of newly synthesized thyroglobulin from the Golgi saccules to nearby secretory vesicles, but do inhibit intracellular migration of these vesicles to the cell apex. In most cells the number of vesicles in the apical cytoplasm diminished, but this was not always the case, suggesting that exocytosis may also be partially inhibited. The loss of microtubules in drug-treated cells suggests that the microtubules may be necessary for intracellular transport of thyroglobulin.     

Pseudoauxotrophy of Methanococcus voltae for acetate, leucine, and isoleucine.

Methanococcus voltae is a methanogenic bacterium which requires leucine, isoleucine, and acetate for growth. However, it also can synthesize these amino acids, and it is capable of low levels of autotrophic acetyl coenzyme A (acetyl-CoA) biosynthesis. When cells were grown in the presence of 14CO2, as well as in the presence of compounds required for growth, the alanine found in the cellular protein was radiolabeled. The percentages of radiolabel in the C-1, C-2, and C-3 positions of alanine were 64, 24, and 16%, respectively. The incorporation of radiolabel into the C-2 and C-3 positions of alanine demonstrated the autotrophic acetyl-CoA biosynthetic pathway in this bacterium. Additional evidence was obtained in cell extracts in which autotrophically synthesized acetyl-CoA was trapped into lactate. In these extracts, both CO and CH2O stimulated acetyl-CoA synthesis. 14CH2O was specifically incorporated into the C-3 of lactate. Cell extracts of M. voltae also contained low levels of CO dehydrogenase, 13 nmol min-1 mg of protein-1. These results further confirmed the presence of the autotrophic acetyl-CoA biosynthetic pathway in M. voltae. Likewise, 14CO2 and [U-14C]acetate were also incorporated into leucine and isoleucine during growth. During growth with [U-14C]leucine or [U-14C]isoleucine, the specific radioactivity of these amino acids in the culture medium declined, and the specific radioactivities of these amino acids recovered from the cellular protein were 32 to 40% lower than the initial specific radioactivities in the medium.Cell extracts of M. voltae also contained levels of isopropyl malate synthase, an enzyme that is specific to the leucine biosynthetic pathway, of 0.8 nmol min-1 mg of protein-1. Thus, M. voltae is capable of autotrophic CO2 fixation and leucine and isoleucine biosynthesis.     

Sialic acid incorporation into gangliosides and glycoproteins of the fish brain

—The concentration of lipid- and non-lipid-bound sialic acid in the optic nerve tract and tectum and in whole brain of fish was estimated. The incorporation of sialic acid into gangliosides and non-lipid components was studied in fish by intracranial or intraocular application of N-[³H]acetylmannosamine or N-[³H]acetylglucosamine. After intracranial injection of N-[³H]acetylmannosamine autoradiography showed lipid- and non-lipid-bound radioactivity in the tectum opticum evenly distributed over regions of nerve fibres or perikarya indicating an ubiquitous incorporation of label. Sialic acid incorporation into glycoproteins after intracranial injection of N-acetylmannosamine always exceeded that into gangliosides. TCA-precipitable non-lipid material is labelled from intracranially applied N-acetylmannosamine in the sialic acid portion and also in nonsialic acid components, whereby the percentage of label in sialic acid increases reaching 90 per cent of the total radioactivity after 90 min. After intraocular application of N-[³H]acetylmannosamine, sialic acid in gangliosides was generally found to be more highly labelled than in glycoproteins. The ratio of radioactivity in gangliosides and glycoproteins increased with time of incubation and the distance from the eye. TCA-soluble radioactivity was translocated by fast axonal transport. Cycloheximide inhibited incorporation of N-acetylmannosamine-derived radioactivity into gangliosides and proteins but not the transport of TCA-soluble material, which accumulates in the tectum. After intraocular application of N-[³H]acetylglucosamine, TCA-soluble label arrives later in the optic tectum than radioactivity of high molecular weight components. The ratio of lipid to non-lipid-bound radioactivity does not change considerably with the time after injection or the distance from the eye. There was no accumulation of TCA-soluble radioactivity after the inhibition of incorporation into high molecular weight components.