Does rat liver Golgi have the capacity to synthesize phospholipids for lipoprotein secretion?

Phosphatidylcholine and phosphatidylethanolamine in lipoproteins secreted from cultured rat hepatocytes are derived from specific biosynthetic pools (Vance, J. E., and Vance, D. E. (1986) J. Biol. Chem. 261, 4486-4491). We have tested the hypothesis that some of the phospholipids destined for secretion with lipoproteins may be made in the Golgi. Golgi fractions were prepared by three different procedures. Although each procedure yielded membranes highly enriched in galactosyltransferase, the protein profiles on polyacrylamide gels were distinct for each preparation. Similarly, the presence of phospholipid synthetic enzyme activities differed among the preparations of Golgi. Two of the preparations were judged to be contaminated by no more than 15% with endoplasmic reticulum. Although an unequivocal conclusion that Golgi contains phospholipid biosynthetic enzymes is not possible, the available evidence is consistent with this hypothesis. Golgi prepared by one method (Croze, E. M., and Morré, D. J. (1984) J. Cell. Physiol. 119, 46-57) was studied in detail. This preparation contained activities for CTP:phosphocholine cytidylyltransferase, CDP-choline:1,2-diacylglycerol cholinephosphotransferase, CDP-ethanolamine:1,2-diacylglycerol ethanolamine-phosphotransferase, phosphatidylethanolamine-N-methyltransferase, and phosphatidylserine synthase. These enzyme activities in the Golgi displayed properties similar to the enzyme activities in endoplasmic reticulum with respect to Km values for substrates, pH optima, cofactor requirements, and inhibition by metabolites. Topology experiments suggested that these enzymes on endoplasmic reticulum and Golgi are all exposed to the cytosolic surface. Phosphatidylserine decarboxylase was not detected in the Golgi preparation. The results support the hypothesis that Golgi has the capacity to make certain phospholipids for lipoprotein secretion: phosphatidylcholine via the CDP-choline and methylation pathways, phosphatidylethanolamine by the CDP-ethanolamine pathway, and phosphatidylserine. Synthesis of phosphatidylethanolamine via decarboxylation of phosphatidylserine does not appear to occur in Golgi.     

Effect of acute ethanolic intoxication on the neuraminidase activity of rat liver golgi apparatus

Neuraminidase and galactosyltransferase were investigated in total Golgi apparatus and in the three fractions of increasing densities (GF₁, GF₂ and GF₂) isolated from the microsomal fraction of rat liver homogenates by flotation in a discontinuous sucrose density gradient (Ehrenreich, J.H., Bergeron, J.J.M., Siekevitz, P. and Palade, G.E. (1973) J. Cell Biol. 59, 45–72). About 50% decreases in neuraminidase content (units/g liver) and specifixc activity (units/ mg protein) were observed in total Golgi as well as in the three fractions isolated at 45 min, 90 min, 180 min and 16 h after administration of a single oral dose of 50% aqueous ethanol (0.6 g/100 g body weight). Colchicine administration (intraperitoneal injection, 0.5 mg/100 g body weight) caused a similar loss of neuraminidase activity; however, the effect of ethanol plus colchicine was not additive. Golgi galactosyltransferase, on the other hand, experienced marked increases of activity following ethanol administration but, unlike the results reported by others (Gang, H., Lieber, C.S. and Rubin, E. (1973) Nat. New Biol. 243, 123–125), significant increases in total activity and specific activity were already quite evident at 90 min after ethanol ingestion. In contrast with the decreased values observed in Golgi, the total particle-bound neuraminidase was significantly elevated following ethanol administration. Ultrastructural studies revealed increased lysosomal content and detachment of polysomes from the rough endoplasmic reticulum. A model, which takes into account these enzymological and ultrastructural findings and their biological significance, is proposed.     

Biochemical studies on rat liver Golgi apparatus. II. Further characterization of isolated Golgi fraction.

Although the preparation of rat liver Golgi apparatus isolated by our method contains appreciable activities of NADH- and NADPH-cytochrome c reductases and glucose-6-phosphatase, these enzymes as well as thiamine pyrophosphatase of the extensively fragmented Golgi fraction are partitioned in aqueous polymer two-phase systems quite differently from those associated with microsomes. Similarly, the partition patterns of acid phosphatase and 5'-nucleotidase of the Golgi fragments differ from those of homogenized lysosomes and plasma membrane, respectively. It is concluded that most, if not all, of these marker enzymes in the Golgi fraction cannot be ascribed to contamination by the non-Golgi organelles. In sucrose density gradient centrifugation the NADH- and NADPH-cytochrome c reductase activities of the Golgi fraction behave identically with galactosyltransferase but differently from the reductase activities of microsomes, again indicating that the reductases are inherently associated with the Golgi apparatus. NADPH-cytochrome c reductase of the Golgi preparation is immunologically identical with that of microsomes. The marker enzymes mentioned above and galactosyltransferase behave differently from one another when the Golgi fragments are subjected to partitioning in aqueous polymer two-phase systems, suggesting that these enzymes are not uniformly distributed in the Golgi apparatus structure.     

The subcellular distribution of terminal N-acetylglucosamine moieties. Localization of a novel protein-saccharide linkage, O-linked GlcNAc

Previously our laboratory reported the discovery of a novel protein-saccharide linkage in which single N-acetylglucosamine (GlcNAc) residues are attached in O-linkages to protein (Torres, C. R., and Hart, G. W. (1984) J. Biol. Chem. 259, 3308-3317). This linkage was first found on plasma membrane proteins of living cells by galactosylation with bovine milk galactosyltransferase. Here we report the distribution of O-linked GlcNAc in highly enriched rat liver subcellular organelles. Nonidet P-40 solubilized organelles were labeled by galactosyltransferase with UDP-[3H]galactose, and the amount of radiolabel occurring on GlcNAc residues in O-linkages was assessed by its sensitivity to beta-elimination and by its resistance to deglycosylation with endo-beta-N-acetylglucosaminidase F. The presence of galactose-labeled O-linked GlcNAc residues was confirmed by high voltage paper electrophoresis. There is a 17-fold range per mg of protein in the amount of galactosylatable terminal GlcNAc residues found in the various organelles, as well as a wide range in the organelles' apparent content of O-linked GlcNAc residues. Nuclei and the soluble fraction of rat liver cells are particularly enriched with proteins bearing O-linked GlcNAc residues, although these residues are demonstrable in virtually all organelles tested. Furthermore, examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that many different organelle-specific proteins are glycosylated with O-linked GlcNAc residues. Because of the wide occurrence of this unique linkage, these data suggest that glycosylation with O-linked GlcNAc residues is not an exclusive marker for a particular organelle. In addition, we have surveyed the organelles for their content of glycoproteins bearing GlcNAc-terminated N-linked oligosaccharides. Our data demonstrate that there are significant amounts of these oligosaccharides in rough and stripped microsomes, nuclei, and nuclear envelopes. In light of evidence that terminal GlcNAc transferases are localized to the Golgi complex, these data suggest that there are glycoproteins which enter into the Golgi for processing and then are transported back into the rough endoplasmic reticulum, and possibly the nucleus.     

Receptor-mediated endocytosis in rat liver: purification and enzymic characterization of low density organelles involved in uptake of galactose-exposing proteins

Rat liver organelles involved in receptor-mediated endocytosis were labeled with a conjugate of galactosylated BSA to horseradish peroxidase [( 3H]galBSA-HRP), injected 10 min before sacrifice. These organelles were recovered at low density (1.11-1.13 g/ml) in sucrose gradients (Quintart, J., P. J. Courtoy, J. N. Limet, and P. Baudhuin, 1983, Eur. J. Biochem., 131:105-112). Upon incubation of such low density fractions in 3,3'-diaminobenzidine (DAB) and H2O2 and equilibration in a second sucrose gradient, galBSA-HRP-containing particles selectively shifted towards heavier densities (Courtoy, P. J., J. Quintart, and P. Baudhuin, 1984, J. Cell Biol., 98:870-876, companion paper), resulting in up to 250-to 300-fold purification with respect to the homogenate. The most purified preparations, wherein DAB- stained structures represented approximately 85% of the total volume of particles, contained only trace activities of enzymes usually regarded as markers for other subcellular entities. These minor activities could reflect either contamination or true enzyme association to the ligand- containing structures. Considering the latter hypothesis, at most 1.0% of alkaline phosphodiesterase I and 2.6% of 5'-nucleotidase (markers for plasma membrane), 3.6% of N-acetyl-beta-glucosaminidase (lysosomes), and 6.0% of galactosyltransferase (Golgi complex) from the homogenate would be associated with the whole population of ligand- containing organelles. After DAB cytochemistry on liver fixed 10 min after galBSA-HRP injection, ligand-containing structures accounted for 0.78-0.89% of the fractional volume of the hepatocytes and displayed a membrane area of 2,100 cm2/cm3, compared with 6,700 cm2/cm3 for the pericellular membrane. Altogether, our data support the hypothesis that these ligand-containing organelles are structurally distinct from plasma membrane, lysosomes, and Golgi complex.     

Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate

Chicken liver plasma membranes, minimally contaminated with Golgi apparatus-derived vesicles, were prepared from a low-speed (400 g) pellet by means of flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous sucrose gradient. Based on the analysis of suitable marker enzymes, alkaline phosphatase and alkaline phosphodiesterase, two plasma membrane fractions were isolated with enrichments, depending on the equilibrium density and marker of 28-97 and with a total yield of 4-5%. Golgi apparatus fractions were prepared by flotation of microsomes, obtained from the same homogenate as the low-speed pellet, in a discontinuous sucrose gradient. The trans-Golgi marker galactosyltransferase was 27-fold enriched in a fraction of intermediate density (d=1.077-1.116 g/ml). Approximately 12% of galactosyltransferase was recovered in the membranes equilibrating d=1.031-1.148 g/ml. Contamination with plasma membrane fragments was low in the light (d=1.031-1.077 g/ml) and intermediate density Golgi vesicles. The isolation of purified plasma membranes and Golgi vesicles from one liver homogenate will enable future studies on receptor cycling between these cell organelles.     

The topographical location and unique nature of a glucokinase associated with the Golgi apparatus of rat liver.

A particulate glucokinase was recovered in the Golgi-rich fraction of rat liver prepared by the method of Morré [Methods Enzymol. (1971) 22, 130-148], thus extending the demonstration by Berthillier et al. [Biochim. Biophys. Acta (1973), 293, 370-378] of particulate glucokinase activity in a microsomal subfraction that showed enrichment in Golgi characteristics. The purity of this fraction was examined and it was then subjected to several treatments, the action of Triton X-100, freezing and thawing, and sonication to establish the topographical location of the glucokinase activity thus solubilized. The evidence suggests that the glucokinase activity is either soluble in the lumen of the Golgi apparatus or loosely associated with the inside of the Golgi membranes.     

Isolation and Characterization of an Enriched Golgi Fraction from Neurons of Developing Rat Brains

We report a method for the isolation of enriched fractions of intact Golgi apparatus from neurons of 10- to 12-day-old rat brains. Neurons were prepared according to a modified method of Farooq and Norton [J. Neurochem. 31, 887-894 (1978)]. Golgi-enriched fractions were obtained after centrifugation of postmitochondrial supernatants in a discontinuous sucrose gradient. Golgi fractions 1 and 2, recovered at the interfaces of 28-34% and 34-36% sucrose densities, respectively, were examined with morphometric and enzymatic methods. Morphometric analyses showed that 21-34% of fraction 1 and 11-29% of fraction 2 consisted of intact Golgi apparatus. Lysosomes, mitochondria, ribosomes, and rough endoplasmic reticulum contaminated fraction 1 (6-10%) and fraction 2 (14-26%). Golgi fraction 1 showed a 25- to 65-fold enrichment over neurons of UDP Gal:GlcNAc galactosyltransferase, CMP-sialic acid:lactosylceramide sialyltransferase, and PAPS:cerebroside sulfotransferase activities. Golgi fraction 2 showed a 8- to 23-fold enrichment over neurons of the activities of the above glycolipid- and glycoprotein-synthesizing enzymes. The activities of the possible marker enzymes rotenone-insensitive NADH-cytochrome c reductase, succinate-cytochrome c reductase, and arylsulfatase were low or minimally elevated in the Golgi fractions. A sevenfold enrichment of Na+, K+-ATPase activities was found in the Golgi fractions. This is consistent either with significant plasma membrane contamination or with the presence of this enzyme in the neuronal Golgi apparatus.     

Isolation of a Golgi rich fraction from rat ventral prostate

Unmodified procedures for isolation of fractions rich in Golgi elements from other tissues have not proved applicable to the rat ventral prostate because of the tendency of membranous material to aggregate. We have devised a new procedure whereby: 1) a Golgi rich fraction from rat ventral prostate was released by a gentle two-step homogenization and isolated by centrifugation through discontinuous sucrose density gradients; 2) the specific activity of UDP-galactose: glycoprotein galactosyltransferase increased 69-fold in this fraction; 3) the isolated Golgi fraction was reasonably free from mitochondria, lysosomes, endoplasmic reticulum and plasma membranes as shown by the relatively low activities of marker enzymes; 4) the specific activities of acid phosphatase and 5'-nucleotidase in the Golgi rich fraction was 4 times greater than that in prostate homogenate. Both enzymes are secretory products and their presence in Golgi elements is probably associated with their packaging in secretory granules.     

Localization of cerebroside-sulfotransferase activity in the Golgi apparatus of rat kidney

A Golgi-rich fraction that contains both uridine diphosphogalactose: N-acetylglucosamine galactosyltransferase activity and 3′-phosphoadenosine-5′-phosphosulfate:cerebroside sulfotransferase activity has been isolated from rat kidney. Both activities are increased about 80-fold in the Golgi fraction compared to the homogenate. Little or no galactosyltransferase or sulfotransferase activity was found in purified nuclei, mitochondria, rough endoplasmic reticulum, plasma membranes and supernatant. The results indicate that both galactosyltransferase and sulfotransferase are localized in Golgi apparatus from rat kidney. This is the first evidence that Golgi apparatus functions to modify a lipid component of the cell.     

Mechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant

The two preceding papers [Powers, V. M., Koo, C. W., Kenyon, G. L., Gerlt, J. A., & Kozarich, J. W. (1991) Biochemistry (first paper of three in this issue); Neidhart, D. J., Howell, P. L., Petsko, G. A., Powers, V. M., Li, R., Kenyon, G. L., & Gerlt, J. A. (1991) Biochemistry (second paper of three in this issue)] suggest that the active site of mandelate racemase (MR) contains two distinct general acid/base catalysts: Lys 166, which abstracts the alpha-proton from (S)-mandelate, and His 297, which abstracts the alpha-proton from (R)-mandelate. In this paper we report on the properties of the mutant of MR in which His 297 has been converted to asparagine by site-directed mutagenesis (H297N). The structure of H297N, solved by molecular replacement at 2.2-A resolution, reveals that no conformational alterations accompany the substitution. As expected, H297N has no detectable MR activity. However, H297N catalyzes the stereospecific elimination of bromide ion from racemic p-(bromomethyl)mandelate to give p-(methyl)-benzoylformate in 45% yield at a rate equal to that measured for wild-type enzyme; the unreacted p-(bromomethyl)mandelate is recovered as (R)-p-(hydroxymethyl)mandelate. At pD 7.5, H297N catalyzes the stereospecific exchange of the alpha-proton of (S)- but not (R)-mandelate with D2O solvent at a rate 3.3-fold less than that observed for incorporation of solvent deuterium into (S)-mandelate catalyzed by wild-type enzyme. The pD dependence of the rate of the exchange reaction catalyzed by H297N reveals a pKa of 6.4 in D2O, which is assigned to Lys 166.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compartmentation of asparagine-linked oligosaccharide processing in the Golgi apparatus

Golgi-associated processing of complex-type oligosaccharides linked to asparagine involves the sequential action of at least six enzymes. By equilibrium sucrose density gradient centrifugation of membranes from Chinese hamster ovary cells, we have partially resolved the set of four initial enzymes in the pathway (Mannosidase I, N-acetylglucosamine (GlcNAc) Transferase I, Mannosidase II, and GlcNAc Transferase II) from two later-acting activities (galactosyltransferase and sialyltransferase). In view of the recent demonstration that galactosyltransferase is restricted to the trans face of the Golgi complex in HeLa cells (Roth, J., and E.G. Berger, 1982, J. Cell Biol., 93:223-229), our results suggest that removal of mannose and attachment of peripheral N-acetylglucosamine may occur in some or all of the remaining cisternae on the cis side of the Golgi stack.     

Biochemical, kinetic and cytochemical approaches resolve Golgi subcompartments of IgM-secreting rat myeloma cells

The rat myeloma cells chosen for study (IR202) are highly specialized toward the synthesis and secretion of immunoglobulin M (IgM). In [35S]methionine pulse-chase protocols the half-time for secretion of newly synthesized [35S]Ig at 37 degrees C is approximately 2 1/2 h. No degradation of [35S]Ig was detected in such experiments. Pulse-chase experiments with [3H]galactose show that addition of this terminal sugar occurs only approximately 2 min before discharge. The intracellular pool of Ig bearing mature oligosaccharides is therefore very small. Incubation at 20 degrees C stops secretion of the [35S]- and [3H]Ig. We describe a subcellular fractionation protocol for these cells which results in the recovery of a total microsomal fraction by gel filtration. This fraction includes approximately 1/4 of the galactosyltransferase and uridine diphosphatase (UDPase) of the homogenate. By employing two cytological Golgi markers (an "overosmicatable material" and UDPase), galactosyltransferase activity and [35S]methionine and [3H]galactose pulse-chase protocols with the chase at 15 degrees C we document the partial resolution of Golgi subcompartments in isopycnic sucrose gradients used to subfractionate the total microsomal fraction. Electron microscopic and enzymologic examination of the fractions resolved by these gradients confirm that rough microsomes are well separated from Golgi membranes and that the fractions most highly enriched in galactosyltransferase activity have a protein-based specific activity approximately 10 times that of the total microsomal fraction. These studies, therefore, form the basis for an analysis of the composition of the membranes of the Golgi Complex and document the location of proximal Golgi elements, as defined by cytological criteria, in isopycnic gradients.     

Sphingomyelin synthesis in rat liver occurs predominantly at the cis and medial cisternae of the Golgi apparatus

The intracellular site of sphingomyelin (SM) synthesis was examined in subcellular fractions from rat liver using a radioactive ceramide analog N-([1-14C]hexanoyl)-D-erythro-sphingosine. This lipid readily transferred from a complex with bovine serum albumin to liver fractions without disrupting the membranes, and was metabolized to radioactive SM. To prevent degradation of the newly synthesized SM to ceramide, all experiments were performed in the presence of EDTA to minimize neutral sphingomyelinase activity and at neutral pH to minimize acid sphingomyelinase activity. An intact Golgi apparatus fraction gave an 85-98-fold enrichment of SM synthesis and a 58-83-fold enrichment of galactosyltransferase activity. Controlled trypsin digestion demonstrated that SM synthesis was localized to the lumen of intact Golgi apparatus vesicles. Although small amounts of SM synthesis were detected in plasma membrane and rough microsome fractions, after accounting for contamination by Golgi apparatus membranes, their combined activity contributed less than 13% of the total SM synthesis in rat liver. Subfractions of the Golgi apparatus were obtained and characterized by immunoblotting and biochemical assays using cis/medial (mannosidase II) and trans (sialyltransferase and galactosyltransferase) Golgi apparatus markers. The specific activity of SM synthesis was highest in enriched cis and medial fractions but far lower in a trans fraction. We conclude that SM synthesis in rat liver occurs predominantly in the cis and medial cisternae of the Golgi apparatus and not at the plasma membrane or endoplasmic reticulum as has been previously suggested.     

Purification of an alanine racemase from Streptococcus faecalis and analysis of its inactivation by (1-aminoethyl)phosphonic acid enantiomers

An alanine racemase has been purified some 30 000-fold almost to homogeneity from Gram-positive Streptococcus faecalis NCIB 6459; the enzyme has been purified to the same extent (4000-fold) from an O-carbamyl-D-serine-resistant mutant with a 7-fold higher enzyme level in crude extract. The racemase has one pyridoxal phosphate molecule per 42-kDa subunit, has a Vmax of 3570 units/mg and a Km of 7.8 mM in the L to D direction, and has a Vmax of 1210 units/mg and a Km of 2.2 mM in the D to L direction. The Keq is 0.8 and kcat/Km values are ca. 3 X 10(5) M-1 s-1. The purified enzyme is inhibited in a time-dependent manner by both L- and D-(l-aminoethyl)phosphonates (Ala-P), confirming observations of Atherton et al. in crude extracts of this organism [Atherton, F. R., Hall, M. J., Hassal, C. H., Holmes, S. W., Lambert, R. W., Lloyd, W. J., & Ringrose, P. S. (1980) Antimicrob. Agents Chemother. 18, 897]. Studies with [1-2H]-, [1-3H]-, and [1,2-14C]Ala-P rule out enzymic activation and processing as the basis for irreversible inhibition. Thus, enzyme after exposure to [14C]Ala-P or [alpha-3H]Ala-P and gel filtration contains stoichiometric amounts of radioactive label, but denaturation quantitatively releases intact Ala-P into solution as revealed by high-performance liquid chromatography and cocrystallization with authentic material. The Ala-P isomers are slow binding inhibitors of this racemase as is the alpha,alpha'-dimethyl analogue but not the D or L isomers of the corresponding phosphinate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of sodium pump beta 1-subunit mRNA expression during hepatocellular growth transitions in vitro and in vivo.

Previous suggestions (Hubert, J. J., Schenk, D. B., Skelly, H., and Leffert, H. L. (1986) Biochemistry 25, 4156-4163) of tissue-specific isoforms or nonexistence of hepatic Na,K-ATPase beta 1-subunits were reevaluated by quantifying beta 1-subunit mRNA levels in quiescent and proliferating liver. RNA was extracted from caudate liver lobes of sham or 67% hepatectomized adult rats and from primary cultures of adult rat hepatocytes that simulate developmental and regenerating growth transitions. Northern blot analysis with a 32P-labeled full-length Na,K-ATPase beta 1-cDNA probe (Mercer, R. W., Schneider, J. W., Savitz, A., Emmanuel, J., Benz, T.J., and Levenson, R. (1986) Mol. Cell. Biol. 6, 3884-3890) revealed four (approximately 2.7, 2.4, 1.7-1.8, and 1.5 kilobases) low abundance mRNA species in quiescent tissue, freshly isolated hepatocytes, and cultured hepatocytes derived from lag or late stationary phase (1-2 days or 11-12 days postplating, respectively). In contrast, proliferating liver from 4 h post-67% hepatectomized rats or cultured hepatocytes in logarithmic growth phase contained levels of beta 1-subunit mRNA which exceeded quiescent levels by 4-35-fold. Membrane Na,K-ATPase activity also increased 2-3-fold during liver regeneration 12-24 h after partial hepatectomy. When proliferation in vitro was augmented by transforming growth factor-alpha, a hepatocyte mitogen, or reinitiated in late stationary phase by a change to fresh culture medium containing rat serum, beta 1-subunit mRNA expression was restimulated 4-20-fold. Parallel measurements of alpha-tubulin mRNA induction showed relatively nonsynchronous or invariant changes during hepatocyte proliferative transitions; similar results were obtained after Northern blots with a sodium pump alpha I-subunit cDNA probe. No detectable hybridization signals were observed when either rat kidney or hepatocyte RNAs from freshly isolated and cultured cells or regenerating tissues were probed for the sodium pump 3.4-kilobase mRNA beta 2-isoform. These observations suggest that enhanced hepatic beta 1-subunit gene expression is linked specifically to growth-associated increases in Na,K-ATPase activity, hepatocyte proliferation, and mitogen activation.     

Studies on the effect of inflammation on rat liver and serum sialyltransferase. Evidence that inflammation causes release of Gal beta 1 leads to 4GlcNAc alpha 2 leads to 6 sialyltransferase from liver

Turpentine induced inflammation has been shown to elevate liver sialyl- and galactosyltransferase activities (Turchen, B., Jamieson, J.C., Huebner, E., and van Caeseele, L. (1977) Can. J. Zool. 55, 1567-1571; Lombart, C., Sturgess, J., and Schachter, H. (1980) Biochem. Biophys. Acta 629, 1-12). We now report that serum sialyl-, but not galactosyltransferase activities are significantly elevated in turpentine inflammation. A liver slice system is used to demonstrate that liver releases large amounts of sialyltransferase activity into medium after inflammation, whereas only a low level of galactosyltransferase activity is released. Studies with rat and human asialo-alpha 1-acid glycoprotein as acceptors, coupled with the use of lactose to confirm the nature of the linkages formed, showed that Gal beta 1 leads to 4GlcNAc alpha 2 leads to 6 sialyltransferase is released from liver in turpentine inflammation and is mainly responsible for the elevated sialyltransferase activity found in serum. The alpha 2 leads to 6 sialyltransferase is exhibiting the properties of a typical acute phase reactant.     

Tetrodotoxin receptors in membrane fragments: purification from Electrophorus electricus electroplax and binding properties

A tetrodotoxin receptor-rich preparation of membrane fragments from the electric organ of Electrophorus electricus is described. The specific binding of neurotoxins and freeze-fracture electron microscopy are used as tools to identify and to characterize membrane fractions. Freeze-fracture electron micrographs of the electric organ demonstrate a high density of membrane particles in the extrasynaptic regions. Density gradient fractions show a broad distribution of [3H]tetrodotoxin, [3H]saxitoxin and 125I-labelled bungarotoxin binding in the range of 1.04--1.15 g/ml sucrose densities, with specific neurotoxin binding up to approx. 5 pmol/mg protein. Carrier-free column electrophoresis of density gradient fractions yields a subfraction with tetrodotoxin and alpha-neurotoxin binding up to 30 pmol/mg protein. The major part of the membrane fragments forms vesicles, which are separated by lectin chromatography into an outside-out and inside-out population. The latter represents at least 50% of the material of a density gradient fraction. For the association of tetrodotoxin, a bimolecular kinetic constant kf greater than or equal to 3.10(5) M-1.s-1 is determined. The dissociation constant is k'b = 2.5.10(-2)s-1. These data are in agreement with a thermodynamic dissociation constant of Kd = 20 nM as determined earlier for E. electricus membrane fragments by equilibrium methods (Grünhagen, H.H., Rack, M., St?mpfli, R., Fasold, H. and Reiter, P. (1981) Arch. Biochem. Biophys. 206, in the press). However, these association kinetics of tetrodotoxin binding in vitro are significantly different from kinetics determined electrophysiologically in Rana (Wagner, H.H. and Ulbricht, W. (1975) Pflügers Arch. 359, 297--315) or Xenopus (Schwarz, J.R., Ulbricht, W. and Wagner, H.H. (1973) J. Physiol. 233, 167--194).     

The activity of Golgi-membrane galactosyltransferase in the liver of streptozotocin-diabetic rats.

The Golgi-rich membrane fraction isolated from streptozotocin-diabetic rat liver had a lower protein content than the corresponding fraction from normal liver. Its UDPgalactose-N-acetylglucosamine galactosyltransferase activity calculated per 1 g of liver or whole liver was decreased. The electron-microscopic examination of the negatively stained fraction revealed morphological changes. The morphology of the Golgi complex in thin sections of diabetic liver was also changed.     

Formation of cation channels in planar lipid bilayers by brefeldin A.

Brefeldin A (BFA) is a novel agent with the unique property of effecting a rapid increase of Golgi cisternae volume and subsequent loss of a recognizable Golgi apparatus in treated cells. Although a receptor-mediated mechanism has been proposed, the molecular basis of BFA action remains unknown (Lippincott-Schwartz, J., Glickman, J., Donaldson, J. G., Robbins, J., Kreis, T. E., Seamon, K. B., Sheetz, M. P., and Klausner, R. D. (1991) J. Cell Biol. 112, 567-577). Since a variety of ionophores distort Golgi architecture by initially causing osmotic swelling of the cisternae (Mollenhauer, H. H., Morre, D. J., and Rowe, L. D. (1990) Biochim. Biophys. Acta 1031, 225-246), Golgi membrane permeabilization by BFA seemed possible. We examined the effects of BFA on the conductance of planar lipid bilayers bathed in several aqueous salt solutions. Addition of BFA (1 microgram/ml) quickly augmented alkali cation conductance (K+ greater than Na+ much greater than Li+) but not anion conductance of the bilayer. Lower concentrations (1 ng/ml) indicated that BFA formed discrete, cation-selective channels in these bilayers. Given that Golgi cisternae volume increases immediately upon treatment with BFA, these findings suggest that alteration of ion gradients or Golgi membrane potential followed by an influx of water may be the mechanism by which BFA initiates disruption of Golgi structural integrity. Subsequent functional perturbations may then ensue either as a consequence of these initial structural changes or by a combination of several distinct mechanisms.