Ganglioside biosynthesis. Characterization of uridine diphosphate galactose: GM2 galactosyltransferase in golgi apparatus from rat liver.

An enzyme that transfers galactose from UDP-Gal to ganglioside GM2 (Tay-Sachs ganglioside) was concentrated 50 times in Golgi apparatus from rat liver relative to total homogenates. This enzyme required detergents or phospholipids as dispersing agents. Of the numerous detergents tested, sodium taurocholate and Triton CF-54 were most effective in stimulating the reaction. Cardiolipin alone was more effective than any of the detergents tested in stimulating enzyme activity. The pH optimum for the reaction varied with the nature of the dispersing agent. With sodium taurocholate, Triton CF-54 and cardiolipin, the pH optima were 6.2, 5.9, and 5.6, respectively. The enzyme had a nearly absolute requirement for Mn2+, with maximum activity being attained at a concentration of 15 mM Mn2+. Other divalent or trivalent cations were either less effective than Mn2+ or inhibited the transferase reaction. The Km values calculated for UDP-Gal and GM2 were 1.1 X 10(-4) M and 9.9 X 10(-5) M, respectively. The enzyme could not be dissociated from Golgi apparatus fractions by treatment with ultrasound, indicating that it is tightly associated with the membrane and not part of the luminal contents. The newly synthesized GM2, the product of the reaction, was incorporated into or became tightly associated with the membranes of the Golgi apparatus.     

Ganglioside biosynthesis in rat liver. Characterization of three sialyltransferases

Three sialyltransferase activities involved in ganglioside biosynthesis were studied in Golgi-enriched preparations of rat liver: the formation of GM3, GD3 and GD1a. The conditions for the quantitative assays of these enzymatic reactions were standardized and optimized, with Triton X-100 being used as detergent. The apparent Km values of each sialyltransferase for N-acetyl-2-(5'-cytidylyl)neuraminic acid (1.5 mM with GM3 synthase, 0.2 mM with GD3 synthase, and 0.5 mM with GD1a synthase) and the respective glycolipid substrates (0.08 mM for lactosylceramide, 0.1 mM for GM3, and 0.5 mM for GM1) were determined. Competition experiments showed that the three sialyltransferase activities are three individual catalytic entities. Moreover, evidence was found that product inhibition may play a role in the regulation of the activity of sialyltransferases.     

Ganglioside biosynthesis in rat liver. Characterization of UDP-N-acetylgalactosamine -- GM3 acetylgalactosaminyltransferase

UDP-N-acetylgalactosamine--GM3 acetylgalactosaminyltransferase (GM2-synthase) was studied in a Golgi-rich fraction from rat liver. Activity in a cell-free system required the presence of detergents; octyl glucoside was found to be the most effective in stimulating the enzyme. Optimal activity of GM2-synthase was obtained at pH 7.2, in the presence of 0.8% octyl glucoside, 10 mM Mn2+ and 5 mM CDP-choline. The latter was used to counteract the rapid sugar nucleotide hydrolysis caused by a nucleotide pyrophosphatase activity in the Golgi fraction. The apparent Km values for UDP-N-acetylgalactosamine and added GM3 were 0.035 mM and 0.1 mM, respectively. Different results were obtained if endogenous GM3 only was used as the glycolipid acceptor. In this case, the apparent Km value for UDP-N-acetylgalactosamine was 0.18 mM and Co2+ and Fe2+ exceeded Mn2+ in activating GM2-synthase. Under optimal assay conditions and in the presence of added GM3 and 5 mM CDP-choline, the specific activity of the enriched Golgi fraction was measured to be 25-30 nmol X mg protein-1 X h-1; with endogenous GM3 as the sole glycolipid acceptor, V was calculated to be 9 nmol X mg protein-1 X h-1.     

Ganglioside biosynthesis. Characterization of CMP-N-acetylneuraminic acid : lactosylceramide sialyltransferase in Golgi apparatus from rat liver.

An enzyme that transfers sialic acid from GMP-sialic acid to lactosylceramide was concentrated 40-50 times in Golgi apparatus from rat liver relative to total homogenates. This enzyme required detergents as dispersing agents. Of the numerous detergents tested, the combination Tween 80-Triton CF-54 (1 : 2, w/w) was the most effective in stimulating the reaction. Two apparent pH optima, at 6.35 and 5.5, were observed. The enzyme showed no requirement for a divalent cation. The Km values calculated for CMP-N-acetylneuraminic acid and lactosylceramide were 2.7 - 10(-3) and 1.3 - 10(-4) M, respectively. The enzyme could not be dissociated from Golgi apparatus fractions by treatment with ultrasound, indicating that it is tightly associated with the membrane. The newly synthesized GM3, the product of the reaction, was incorporated into or became tightly associated with the membranes of the Golgi apparatus.     

Characterization of beta 1----4 galactosyltransferase purified from rat liver microsomes.

beta 1----4 Galactosyltransferase was purified from rat liver microsomes. Catalytic properties of the enzyme resembled those of previously purified soluble and membrane-bound beta 1----4 galactosyltransferases. The enzyme purified in the present study showed a major band around a molecular weight of 53,000 on SDS-PAGE. The NH2-terminal sequence of the enzyme was determined up to the 20th residue. The sequence was identical to the amino acid sequence from Ala-13 to Lys-32 deduced from mouse beta 1----4 galactosyltransferase cDNA. These results suggest that most of the mature enzyme in rat liver microsomes is produced by removal of the NH2-terminal 12 amino acids from a precursor polypeptide.     

Ganglioside biosynthesis in Golgi apparatus of rat liver. Stimulation by phosphatidylglycerol and inhibition by tunicamycin

Golgi vesicles were isolated and purified from rat liver, in which the specific activities of glycosyltransferases (e.g. GM3:CMP-NeuAc sialyltransferase, GD3 synthase; GM3:UDP-GalNAc galactosaminyltransferase, GM2 synthase) were 50-60-times enriched relative to microsomes or total homogenate. Synthesis of gangliosides GM2 and GM1 in such Golgi vesicles is, in the absence of any detergents, stimulated 6-fold and 20-fold respectively by phosphatidylglycerol. Other phospholipids like phosphatidylethanolamine and phosphatidylserine are also significantly stimulatory. With 50 micrograms Golgi protein and 1 nmol UDP-GalNAc, optimal stimulation of GM2 synthase was obtained with 20 micrograms of phosphatidylglycerol and 7.5 nmol of the lipid acceptor GM3. Under the same experimental conditions this stimulation exceeds (by about 40%) that obtained with optimal amount (200 micrograms) of the detergent octylglucoside. Phosphatidylglycerol, on the other hand, has virtually no stimulatory activity on the synthesis of ganglioside GD3 either in the presence of Mg2+ or Mn2+, indicating that facilitation by phospholipid of GM3 transport into Golgi vesicles was not the basis of stimulation of GM2 synthesis. Tunicamycin inhibits the synthesis of gangliosides GM2 and GM1 in isolated Golgi vesicles, but only in the absence of detergents. In the presence of phosphatidylglycerol, GM2 synthesis, for example, was inhibited by 60% by 2 micrograms tunicamycin and more than 85% by 10 micrograms tunicamycin, per 50 micrograms Golgi membrane protein. The inhibition was stronger on GM1 synthesis: 85% with 2.5 micrograms of the antibiotic. The dependence on phosphatidylglycerol and the degree of inhibition by tunicamycin of the synthetic activities are strictly dependent on the intactness of the Golgi vesicles: both phenomena become increasingly less evident when the vesicles are pelleted, and frozen and thawed several times, and completely disappear when the vesicles are solubilized by detergents or disrupted by ultrasonication. Furthermore, tunicamycin inhibition is reversible by increased concentration of phosphatidylglycerol. All these results indicate that phosphatidylglycerol does not stimulate, and tunicamycin does not inhibit, the transferases themselves; rather, the two opposing effects might relate to carrier-mediated transport, e.g. of nucleotide sugars, across Golgi vesicles.     

Ganglioside biosynthesis in rat liver: effect of UDP-amino sugars on individual transfer reactions

Several glycosyltransferases participating in ganglioside biosynthesis were measured in Golgi-rich fractions from rat liver. Addition of those UDP-amino sugars to the enzyme assays which accumulate in liver after treatment of rats with D-galactosamine inhibited the transferases to different degrees. The simultaneous presence of UDP-GalN, UDP-GalNAc, UDP-GlcN, and UDP-GlcNAc in concentrations resembling their overall content in livers 6 h after D-galactosamine administration led to an inhibition of the glycolipid galactosyltransferases, GL2 and GM1 synthases of 44 and 64%, respectively. GM2 synthase was moderately inhibited whereas the sialyltransferases (GM3, GD3, and GD1a synthases) were almost unimpaired. Induction of liver cell damage by D-galactosamine did not cause any change of glycosyltransferase activities as determined in rat liver homogenates and Golgi-rich fractions. These results indicate a possible role for UDP-amino sugars in the depression of ganglioside biosynthesis observed in vivo after GalN administration.     

Isoprenoid biosynthesis in rat liver peroxisomes. Characterization of cis-prenyltransferase and squalene synthetase.

Isolated peroxisomes were able to utilize [3H]isopentenyl diphosphate to synthesize farnesyl diphosphate, which then was utilized as substrate by both the peroxisomal squalene synthetase and cis-prenyltransferase. The specific activity of squalene synthetase in peroxisomes was as high as in microsomes, i.e. 160 pmol/mg of protein/min. If NADPH was omitted from the assay medium, presqualene diphosphate accumulated, which indicates that the reaction occurs in two steps, as in microsomes. In the presence of NADPH, incorporation from [3H]farnesyl diphosphate was stimulated 3-fold, and the major products were squalene and cholesterol. The specific activity of cis-prenyl-transferase in peroxisomes was 4-fold higher than in microsomes, i.e. 456 pmol of isopentenyl diphosphate incorporated/mg of protein/h. There were two major products formed from farnesyl diphosphate and [3H] isopentenyl diphosphate, i.e. trans,trans,cis-geranylgeranyl diphosphate and long chain polyprenyl diphosphates. The polyprenyl diphosphates had the same chain length distribution as that of dolichol derivatives in rat liver, with the dominating polyisoprenes being C90 and C95. In contrast to the microsomal enzyme, peroxisomal cis-prenyltransferase did not require detergents for optimal activity. The enzyme was associated primarily with the peroxisomal membrane after sonication of the peroxisomes.     

Effect of cyclic nucleotides in vitro assays of rat liver galactosyltransferase.

Experiments with rat liver microsomal galactosyltransferase has been developed to test the effect of cyclic nucleotides on the transfer activity. An overall stimulation is observed when cAMP or cGMP (concentration higher that 10(-6) M) are added to the incubation medium. However, more detailed experiments show that the cyclic nucleotides do not act as direct effectors of the enzyme but present the precursors degradation by the glycosylnucleotide pyrophosphatases.     

Cell-free biosynthesis of lipophosphoglycan from Leishmania donovani. Characterization of microsomal galactosyltransferase and mannosyltransferase activities

Incubation of microsomal preparations from Leishmania donovani parasites with UDP-[3H]galactose or GDP-[14C]mannose resulted in incorporation of radiolabel into an endogenous product that exhibited the chemical and chromatographic characteristics of the parasite's major surface glycoconjugate, lipophosphoglycan. The [3H]galactose- or [14C]mannose-labeled product was (i) cleaved by phosphatidylinositol-specific phospholipase C; (ii) deaminated by nitrous acid; and (iii) degraded into radioactive, low molecular weight fragments upon hydrolysis with mild acid. Analysis of the products of mild acid hydrolysis revealed the presence of phosphorylated Gal-beta-Man as the major fragment with lesser amounts of mono-, tri-, and tetrasaccharides. The incorporation of the two isotopic precursors was neither stimulated by the addition of dolichylphosphate nor inhibited by amphomycin, indicating that dolichol-saccharide intermediates are not involved in assembly of the repeating units of lipophosphoglycan. Development of this cell-free glycosylating system will facilitate further studies on the pathway and enzymes involved in lipophosphoglycan biosynthesis.     

Mucin biosynthesis. Characterization of UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase from human tracheal epithelium

We have characterized the UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase in human tracheal epithelium using asialo ovine submaxillary mucin as the acceptor. Maximal enzyme activity was obtained at pH 6.0-7.5 and at 20-25 mM MnCl2 and at 2% Triton X-100. Cd2+ could substitute for Mn2+ as the divalent ion cofactor. Spermine, spermidine, putrecine, cadaverine, and poly-L-lysine stimulated the enzyme activity at low (2.5 mM) MnCl2 concentration. The apparent Michaelis constants for N-acetylgalactosamine, asialo ovine submaxillary mucin, and UDP-galactose were 15.5, 1.14, and 1.36 mM, respectively. The enzyme activity was not affected by alpha-lactalbumin. The alpha-N-acetygalactosaminide beta 3 galactosyltransferase was shown to be different from the N-acetylglucosamine galactosyltransferase by acceptor competition studies. The product of galactosyltransferase was identified as Gal beta 1 leads to 3GalNAc alpha Ser (Thr) by (a) isolation of [14C]Gal-GalNAc-H2 after alkaline borohydride treatment of the 14C-labeled product, (b) establishment of the beta-configuration of the newly synthesized glycosidic bond by its complete cleavage by bovine testicular beta-galactosidase, and (c) assignment of the 1 leads to 3 linkage by identification of threosaminitol obtained from the oxidation of the disaccharide with periodic acid followed by reduction with sodium borohydride, hydrolysis in 4 N HCl, and analysis on an amino acid analyzer. The 1 leads to 3 linkage was confirmed by its resistance to jack bean beta-galactosidase and by the presence of a m/e 307 ion fragment and the absence of a m/e 276 ion by gas-liquid chromatography-mass spectrometry analysis. When acid and beta-galactosidase-treated human tracheobronchial mucin was used as the acceptor, 3.3% of the product was found as [14C]Gal-GalNAc-H2. The remainder of the [14C]Gal was found in longer oligosaccharides formed by a different beta-galactosyltransferase. This galactosyltransferase is slightly inhibited by alpha-lactalbumin and stimulated by spermine.     

Characterization and biosynthesis of cytochrome b(5) in rat liver microsomes

1. Cytochrome b₅ is released from rat liver microsomes by both proteolytic enzymes and by treatments that disrupt phospholipids. Cytochrome P-420 is only released to a marked extent by treatments that disrupt phospholipids. 2. Cytochrome b₅ was isolated in a pure state from both the rough and smooth fractions of rat liver microsomes after treatment with trypsin, and was shown to contain two cytochrome components with identical spectral properties. 3. Amino acid analyses of the two components are presented, together with peptide `fingerprint' patterns of tryptic digests of the two components. 4. Studies based on the direct isolation of cytochrome b₅ after administration of a single dose of radioactive amino acid to rats demonstrate that the cytochrome is synthesized initially in the rough fraction of microsomes and only subsequently appears in the smooth fraction. 5. Isolated rat liver microsomes are capable of incorporating radioactive amino acids into cytochrome b₅ under standard conditions. 6. Under these conditions the amino acid is incorporated into peptide linkage in the cytochrome.     

The effect of cycloheximide on galactosyltransferase of rat liver Golgi membranes.

An inhibitory effect of cycloheximide on the initial rate of galactosyltransferase of rat liver Golgi membranes has been demonstrated. Cycloheximide was effective in inhibiting the activity of the enzyme when added directly to the assay medium or after pre-incubation of the membranes with the drug. The inhibition observed with different concentrations of nucleotide sugar was shown to be competitive at higher concentrations of the nucleotide sugar (0.10-1.0 mumol). The inhibition observed with different concentrations of acceptor, N-acetylglucosamine was complex and could not be analysed further with the present data. Washing the Golgi membranes previously incubated with cycloheximide with water failed to reverse the inhibition. Washing with UDPgalactose partially reversed the inhibition only. These results, together with the observation that serum galactosyltransferase was not inhibited by cycloheximide supported the view that the cycloheximide effect may be primarily on the membrane system.     

Purification of membrane-bound galactosyltransferase from rat liver microsomal fractions

1. Rat liver microsomal preparations incubated in 1% Triton X-100 at 37°C for 1h released about 60% of the membrane-bound UDP-galactose–glycoprotein galactosyltransferase (EC 2.4.1.22) into a high-speed supernatant. The supernatant galactosyltransferase which was solubilized but not purified by this treatment had a higher molecular weight than the serum enzyme as shown by Sephadex G-100 column chromatography. 2. The galactosyltransferase present in the high-speed supernatant was purified 680-fold by an affinity-column-chromatographic technique by using a column of activated Sepharose 4B coupled with α-lactalbumin. The galactosyltransferase ran as a single band on polyacrylamide gels and contained no sialyltransferase, N-acetylglucosaminyltransferase or UDP-galactose pyrophosphatase activities. 3. The purified membrane enzyme had properties similar to serum galactosyltransferase. It had an absolute requirement for Mn²⁺ that could not be replaced by Ca²⁺, Mg²⁺, Zn²⁺ or Co²⁺, and was active over a wide pH range (6–8) with a pH optimum of 6.5. The apparent K_m for UDP-galactose was 10.8μm. The protein α-lactalbumin modified the enzyme to a lactose synthetase by increasing substrate specificity for glucose in preference to N-acetylglucosamine and fetuin depleted of sialic acid and galactose. 4. The molecular weight of the membrane enzyme was 65000–70000, similar to that of the purified serum enzyme. Amino acid analyses of the two proteins were similar but not identical. 5. Sephadex G-100 column chromatography of the purified membrane enzyme showed a small peak (2–5%) of higher molecular weight than the purified serum enzyme. Inclusion of 1mm-ε-aminohexanoic acid in the isolation procedures increased this peak to as much as 30% of the total enzyme recovered. Increasing the ε-aminohexanoic acid concentration to 100mm resulted in no further increase in this high-molecular-weight fraction.     

Ganglioside biosynthesis in rat liver: different distribution of ganglioside synthases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells

The activities of five glycolipid-glycosyltransferases, GL2, GM3, GM2, GM1, and GD1a synthase, were determined in a cell-free system with homogenate protein of total rat liver, isolated hepatocytes, Kupffer cells, and sinusoidal endothelial cells. In rat liver parenchymal and nonparenchymal cells ganglioside synthases were distributed differently. Compared to hepatocytes, Kupffer cells expressed a nearly sevenfold greater activity of GM3 synthase, but only 14% of GM2, 19% of GM1, and 67% of GD1a synthase activity. Sinusoidal endothelial cells expressed a pattern of enzyme activities quite similar to that of Kupffer cells with the exception of higher GM2 synthase activity. Activity of GL2 synthase was distributed unifromly in parenchymal and nonparenchymal cells of rat liver, but differed by sex. It was 1 to 2 orders of magnitude below that of all the other ganglioside synthases investigated. The results indicate GL2 synthase regulates the total hepatic ganglioside content, and hepatocytes but not nonparenchymal liver cells have high enzymatic capacities to form a-series gangliosides more complex than GM3.     

Ubiquinone biosynthesis in rat liver peroxisomes

The possibility that ubiquinone biosynthesis is present in rat liver peroxisomes was investigated. The specific activity of trans-prenyltransferase was 30% that of microsomes, with a pH optimum of around 8. trans-Geranyl pyrophosphate was required as a substrate and maximum activity was achieved with Mn(2+). Several detergents specifically inactivated the peroxisomal enzyme. The peroxisomal transferase is present in the luminal soluble contents, in contrast to the microsomal enzyme which is a membrane component. The treatment of rats with a number of drugs has demonstrated that the activities in the two organelles are subjected to separate regulation. Nonaprenyl-4-hydroxybenzoate transferase has about the same specific activity in peroxisomes as in microsomes and like the transferase activity, its regulation differs from the microsomal enzyme. The results demonstrate that peroxisomes are involved in ubiquinone biosynthesis, and at least two enzymes of the biosynthetic sequence are present in this organelle.