Golgi Apparatus-rich Fraction Research Articles

The assembly of hepatic very low density lipoproteins: evidence of a role for the Golgi apparatus.

Previous studies from our laboratory have suggested that the assembly of lipoproteins by the liver is not completed in the rough endoplasmic reticulum but continues while the particles are en route to or within the Golgi apparatus. To investigate further the role of the Golgi apparatus in lipoprotein assembly, mice were injected with [3H]glycerol and killed 7.5 to 45 min after injection. Microsomes and Golgi apparatus-rich fractions were isolated from the livers and separated into membrane and content fractions. TG within microsomal and Golgi membranes were labeled, rapidly reaching peak specific activity within 7.5 min of isotope injection. The specific activity of TG in microsomal membranes decreased to approximately 40% of peak values by 45 min, whereas the specific activity of TG in the Golgi membranes decreased to approximately 30% of peak values by 45 min. To determine whether the turnover of the Golgi membrane TG pool was dependent on microsomal TG transfer protein (MTP), mice were gavaged with an MTP inhibitor, and the labeling experiments were repeated. Inhibition of MTP attenuated the turnover of newly synthesized Golgi membrane TG by approximately 50% and the turnover of microsomal membrane TG by approximately 40%. Based on the rapid turnover of the Golgi membrane TG pool and the attenuation of the turnover of this pool by MTP inhibitor, we propose that lipid is transferred from the Golgi membrane to luminal lipoproteins in an MTP-dependent manner. The results support our hypothesis that assembly of VLDL continues within the Golgi apparatus.

Assembly of very low density lipoproteins in mouse liver: evidence of heterogeneity of particle density in the Golgi apparatus

The assembly of very low density lipoproteins (VLDL) by hepatocytes is believed to occur via a two-step process. The first step is the formation of a dense phospholipid and protein-rich particle that is believed to be converted to VLDL by the addition of bulk triglyceride in a second step. Previous studies in our laboratory led us to hypothesize a third assembly step that occurs in route to or in the Golgi apparatus. To investigate this hypothesis, nascent lipoproteins were recovered from Golgi apparatus-rich fractions isolated from mouse liver. The Golgi fractions were enriched 125-fold in galactosyltransferase and contained lipoprotein particles averaging approximately 35 nm in diameter. These lipoproteins were separated by ultracentrifugation into two fractions: d < 1.006 g/ml and d1.006–1.210 g/ml. The d < 1.006 g/ml fraction contained apolipoprotein B-100 (apoB-100), apoB-48, and apoE, while the d1.006–1.210 g/ml fraction contained these three apoproteins as well as apoA-I and apoA-IV. Both fractions contained a 21-kDa protein that was isolated and sequenced and identified as major urinary protein. Approximately 50% of the apoB was recovered with the denser fraction. To determine if these small, dense lipoproteins were secreted without further addition of lipid, mice were injected with Triton WR1339 and [3H]leucine, and the secretion of apoB-100 and apoB-48 into serum VLDL (d < 1.006 g/ml) and d1.006–1.210 g/ml fractions was monitored over a 2-h period. More than 80% of the newly synthesized apoB-48 and nearly 100% of the apoB-100 were secreted with VLDL. These studies provide the first characterization of nascent lipoproteins recovered from the Golgi apparatus of mouse liver. We conclude that these nascent hepatic Golgi lipoproteins represent a heterogeneous population of particles including VLDL as well as a population of small, dense lipoproteins. The finding of the latter particles, coupled with the demonstration that the primary secretory product of mouse liver is VLDL, suggests that lipid may be added to nascent lipoproteins within the Golgi apparatus. —Swift, L. L., K. Valyi-Nagy, C. Rowland, and C. Harris. Assembly of very low density lipoproteins in mouse liver: evidence of heterogeneity of particle density in the Golgi apparatus.

Role of the Golgi Apparatus in the Phosphorylation of Apolipoprotein B

Rat hepatic Golgi apparatus-rich fractions were utilized in an in vitro phosphorylation system containing [gamma-32P]ATP to investigate the phosphorylation of apolipoproteins (apo) B48 and B100. Our results demonstrate that the Golgi apparatus contains a kinase(s) that phosphorylates both apoB48 and apoB100 as well as 290- and 460-kDa proteins recognized by antibody to apoB. We refer to the latter proteins as apoB57 and apoB90, respectively. Phosphorylations in the presence of Triton X-100, which increases the permeability of the membranes, or alamethicin, an ionophore that facilitates transmembrane diffusion of ATP, indicate that the active site of the kinase is on the luminal side of the membranes. However, studies with EDTA and EGTA, which are inhibitory to the kinase, suggest binding sites for Mg2+ and perhaps Ca2+ on the cytosolic membrane face. Phosphorylation of apoB was not stimulated by cAMP nor inhibited by protein kinase inhibitor peptide (5-24), indicating that cAMP dependent protein kinase was not involved in the phosphorylation process. Sodium carbonate treatment of the phosphorylated fraction, which permits separation of membrane and luminal contents, revealed that phosphorylated apoB90 and apoB57 are associated primarily with the membrane, whereas phosphorylated apoB48 is found in luminal contents as well as with the membranes. Phosphorylated apoB100 was found primarily with the membrane fraction. No evidence was found for phosphorylation of apoB in rough endoplasmic reticulum fractions. These studies demonstrate the importance of the Golgi apparatus as a subcellular site for the phosphorylation of apoB and suggest that apoB phosphorylation may be important in the assembly and secretion of apoB-containing lipoproteins.

Comparative study of the N-glycoprotein synthesis through dolichol intermediates in mitochondria, golgi apparatus-rich fraction and endoplasmic reticulum-rich fraction

1. 1. Glycosylation of endogenous dolichol acceptors was higher in mitochondria than in C 30,000 g (Golgi apparatus-rich fraction) and C 100.000 g (endoplasmic reticulum-rich fraction). 2. 2. In mitochondria, N-glycoprotein biosynthesized were composed of high mannose type and nonfucosyalted biantennary complex type while in C 30,000 g and C 100,000 g preparations, they contained biantennary complex type as tri and tetraantennary complex type oligodaccharides in both fucosylated and non-fucosylated forms.

Nascent lipoproteins from recirculating and nonrecirculating liver perfusions and from the hepatic Golgi apparatus of African green monkeys

Perfusate apoB-100-containing lipoproteins from the isolated, perfused livers of African green monkeys consist of significant amounts of d greater than 1.006 g/ml particles in addition to very low density lipoproteins (VLDL). Distinguishing characteristics of these perfusate lipoproteins are the relative abundance of surface lipids and deficiency of core lipids. The present studies were performed to determine the likelihood that the d greater than 1.006 g/ml perfusate lipoproteins are secretion products instead of products of post-secretory modification (e.g., lipolysis) of secreted VLDL. [14C]Leucine from the perfusate became incorporated into the apoB of each of the perfusate lipoprotein classes to a similar extent in both recirculating and nonrecirculating perfusions. When endogenously radiolabeled perfusate VLDL from one liver was recirculated through a second liver, only about 15% of the radiolabeled protein appeared in the d greater than 1.006 g/ml fraction. The particle morphology and the cholesterol and apoB distribution between VLDL and d greater than 1.006 g/ml fractions were similar in recirculating and nonrecirculating perfusions. A Golgi apparatus-rich fraction was isolated from the homogenates of fresh liver samples and the isolated Golgi VLDL and d greater than 1.006 g/ml lipoproteins exhibited morphologic evidence of extra surface material analogous to that seen in perfusate. Taken together, these data support the possibility that significant amounts of d greater than 1.006 g/ml lipoproteins, many with surface-rich properties, are nascent, secretory products of the primate liver. The low level of lecithin:cholesterol acyltransferase (LCAT) in this perfusion system appears to permit detection of these secretion products and it is significant to note that the perfusate lipoprotein profile, which is unlike that of normal plasma, is similar to that of LCAT-deficient patients.

Xylosylation and glucuronosylation reactions in rat liver Golgi apparatus and endoplasmic reticulum.

We have studied in rat liver the subcellular sites and topography of xylosylation and galactosylation reactions occurring in the biosynthesis of the D-glucuronic acid-galactose-galactose-D-xylose linkage region of proteoglycans and of glucuronosylation reactions involved in both glycosaminoglycan biosynthesis and bile acid and bilirubin conjugation. The specific translocation rate of UDP-xylose into sealed, "right-side-out" vesicles from the Golgi apparatus was 2-5-fold higher than into sealed right-side-out vesicles from the rough endoplasmic reticulum (RER). Using the above vesicle preparations, we only detected endogenous acceptors for xylosylation in the Golgi apparatus-rich fraction. The specific activity of xylosyltransferase (using silk fibroin as exogenous acceptor) was 50-100-fold higher in Golgi apparatus membranes than in those from the RER. Previous studies had shown that UDP-galactose is translocated solely into vesicles from the Golgi apparatus. In these studies, we found the specific activity of galactosyltransferase I to be 40-140-fold higher in membranes from the Golgi apparatus than in those from the RER. The specific translocation rate of UDP-D-glucuronic acid into vesicles from the Golgi apparatus was 10-fold higher than into those from the RER, whereas the specific activity of glucuronosyltransferase (using chondroitin nonasaccharide as exogenous acceptor) was 12-30-fold higher in Golgi apparatus membranes than in those from the RER. Together, the above results strongly suggest that, in rat liver, the biosynthesis of the above-described proteoglycan linkage region occurs in the Golgi apparatus. The specific activity of glucuronosyltransferase, using bile acids and bilirubin as exogenous acceptor, was 10-25-fold higher in RER membranes than those from the Golgi apparatus. This suggests that transport of UDP-D-glucuronic acid into the RER lumen is not required for such reactions.

Intestinal lipoprotein synthesis. Comparison of nascent Golgi lipoproteins from chow-fed and hypercholesterolemic rats.

Hypercholesterolemia, induced by a cholesterol-enriched diet, is associated with distinctive modifications in the serum lipoproteins of a variety of species. Present in the serum of these animals are several classes of lipoproteins enriched in cholesteryl esters and apolipoprotein E. To investigate the role of intestinal lipoprotein synthesis in diet-induced hypercholesterolemia, we characterized nascent lipoproteins retrieved from Golgi apparatus-rich fractions of intestinal epithelial cells from chow-fed control and hypercholesterolemic rats. To eliminate chylomicrons from the preparations, rats were fasted overnight prior to the experiments. Golgi very low density lipoproteins (d less than 1.006 g/ml) from control rats were triglyceride-rich lipoproteins that migrated slightly slower than pre-beta migrating serum very low density lipoproteins. These particles contained apoproteins B-240, A-IV, and A-I. Golgi very low density lipoproteins from hypercholesterolemic rats were likewise triglyceride-rich lipoproteins migrating electrophoretically like control Golgi very low density lipoproteins and they contained apoproteins B-240, A-IV, and A-I. However, these latter particles contained less triglyceride and more cholesterol compared to control Golgi very low density lipoproteins. In addition, by radioisotope incorporation studies, Golgi very low density lipoproteins from hypercholesterolemic rats contained relatively more apoprotein A-IV (21.6 vs. 11.0%) and less apoprotein B-240 (17.0 vs. 27.0%) than found in control Golgi very low density lipoproteins. Approximately 60% of the total apoprotein radioactivity was found in apoprotein A-I in both preparations. We conclude that intestinal lipoprotein synthesis is modified by diet-induced hypercholesterolemia. The significance of these modifications with respect to the marked hypercholesterolemia observed in these animals remains to be determined.

Citrate accumulation by a Golgi apparatus-rich fraction from lactating bovine mammary gland

1. 1. Golgi apparatus-rich fractions from lactating bovine mammary gland rapidly accumulated citrate from incubation medium. Characteristics of this process suggested that a citrate transport system may be present in Golgi apparatus membranes. 2. 2. Endoplasmic reticulum fractions accumulated citrate at nearly the same rate as Golgi apparatus; secretory vesicle fractions displayed lower ability to accumulate citrate. Intact epithelial cells (acini) from lactating mammary gland did not accumulate citrate. 3. 3. Citrate accumulation by Golgi apparatus was pH and temperature sensitive but was not altered by metabolic inhibitors. 4. 4. These observations suggest a role for Golgi apparatus in packaging intracellular citrate for secretion into milk.

Hepatic Golgi lipoproteins: precursors to plasma lipoproteins in hypercholesterolemic rats

Two classes of nascent lipoproteins can be isolated from Golgi apparatus-rich fractions of liver from hypercholesterolemic rats. Golgi very low density lipoproteins (VLDL, d less than 1.006 g/ml) are enriched in cholesteryl esters and are similar in many respects to hypercholesterolemic plasma B-VLDL. Golgi low density lipoproteins (LDL, d 1.006-1.05 g/ml) are cholesteryl ester-rich beta-migrating lipoproteins similar to hypercholesterolemic plasma LDL. To determine if this latter lipoprotein is a precursor to plasma LDL, control and hypercholesterolemic rats were injected with Triton WR 1339 (400 mg/kg) to block intravascular lipoprotein catabolism, followed in 30 min with 100 microCi [3H]leucine. At time intervals up to 3 hours after [3H]leucine injection, rats were killed, and plasma lipoproteins and, in some experiments, Golgi lipoproteins were isolated. Three hours after radioisotope injection, 52% of the total lipoprotein radioactivity was found in the plasma VLDL of hypercholesterolemic rats compared to 82% in chow-fed control rats. Twenty-four percent of the total lipoprotein radioactivity appeared in the plasma IDL fraction in hypercholesterolemic rats, while only 3% was found in the same fraction in control rats. After Triton, the time course of specific activities of the Golgi and plasma lipoproteins was consistent with Golgi VLDL and LDL being precursors to plasma VLDL and IDL, respectively. The time course of specific activities of the tetramethylurea-insoluble proteins of plasma and Golgi lipoproteins provided additional evidence in support of this relationship. Furthermore the composition of plasma VLDL and IDL after Triton injection resembled their hepatic Golgi counterparts. We conclude that the liver of the hypercholesterolemic rat synthesizes, assembles, and secretes a cholesteryl ester-enriched VLDL and a cholesteryl ester-rich, beta-migrating LDL. The former is a precursor to plasma VLDL while the latter is a precursor to plasma IDL.

Energy-dependent calcium sequestration activity in a Golgi apparatus fraction derived from lactating rat mammary glands

A fraction isolated from lactating rat mammary glands was shown by marker enzyme assays to be rich in Golgi apparatus vesicles. This Golgi apparatus-rich fraction was shown to accumulate calcium in the presence of ATP but not in its absence. Other nucleoside triphosphates were only partially effective in promoting calcium transport. Mg 2+ was required for the uptake which was also temperature and pH dependent. The uptake was sustained by the use of oxalate and phosphate as intravesicular trapping agents. In the presence of 10 mM oxalate the apparent K m for calcium uptake was 0.24 μM ionized calcium. The V was 4.45 nmol calcium/min per mg protein. Preloaded calcium could be rapidly released by the addition of the ionophore A23187 indicating an intravesicular location for the sequestered ion. Addition of ethylene glycol bis( β-aminoethyl ether)- N, N′-tetraacetic acid resulted in a slower release of preaccumulated calcium, indicating the existence of one or more efflux routes by which calcium leaves the vesicles in the presence of MgATP. Ruthenium red partially inhibited the uptake but lanthanum and particularly the sulphydryl inhibitor p-hydroxymercuribenzoate were much more effective. The properties of the calcium-sequestering system in the Golgi apparatus-rich fraction were similar to those reported for other non-muscular tissues and lend support to the hypothesis that calcium is secreted into milk via the Golgi apparatus of the mammary gland secretory cell.

The ATP-dependent concentration of calcium by a Golgi apparatus-rich fraction isolated from rat liver.

Fractions rich in Golgi apparatus were isolated from female rat liver and incubated with different concentrations of MgATP and calcium. Calcium was sequestered by the fraction in the presence but not in the absence of ATP. The kinetics of the uptake process showed an exponential type accumulation to a maximum value which could be explained by a 'pumpleak' mechanism where Vmax was estimated at 6.7 nmol calcium/mg protein/min. Apparent Km for ATP was 1 mM and for calcium was 85 micron. The uptake is not inhibited in 5 mM azide, nor is it enhanced in 5 mM oxalate, suggesting that the sequestration is not caused by contamination from mitochondria or microsomes. Parallel experiments on a fraction rich in plasma membrane show at least a 10-fold reduction of activity over the Golgi apparatus-rich fraction, which is interpreted as evidence that plasma membrane contamination in the Golgi apparatus-rich fraction is not primarily responsible for the calcium sequestration activity. Morphometric analysis showed that about 85% of the recognizable membrane profiles in the fraction was associated with elements of the Golgi apparatus. Further evidence is given which suggests that at least 90% of the fraction was uniform in its ability to sequester calcium. In preliminary experiments it was not found possible to separate galactosyltransferase activity (a secific enzyme associated with the Golgi apparatus) from the calcium-sequestration mechanism. The stoichiometry of the hydrolysis of ATP associated with the uptake of calcium is the same as that shown by other isolated membrane systems.

Composition and synthesis of three higher ganglioside homologs in bovine mammary tissue.

Three higher gangliosides were identified as constituents of bovine mammary gland. The structures of these three gangliosides were shown to be ceramide-glucose-galactose-(sialic acid)-N-acetylgalactosamine-galactose, ceramide-glucose-galactose-(sialic acid)2-N-acetylgalactosamine, and ceramide-glucose-galactose-(sialic acid)2-N-acetylgalactosamine-galactose. These gangliosides accounted for only a small fraction (less than 20%) of the lipid-bound sialic in mammary gland. White fatty acids with even carbon numbers from C14 to C26 were predominant in these gangliosides, they also contained C23 and C25 fatty acids. Mammary gland Golgi apparatus-rich fractions had all glycosyltransferases required for synthesis of these gangliosides starting with ceramide.

Similarities of the golgi apparatus membrane and the plasma membrane in rat liver cells

A Golgi apparatus-rich fraction and a plasma membrane-rich fraction were isolated from a common homogenate of rat liver. Their respective buovant densities, appearances in the electron microscope and 5'-nucleotidase and UDP-galactose ovalbumin galactosyltransferase activities were in accord with published data on separately isolated Golgi apparatus-rich and plasma membrane-rich fractions. Contamination by endoplasmic reticulum and mitochondria was low. Gel electrophoresis of the membrane proteins of the Golgi apparatus-rich and plasma membrane-rich fractions (separately and mixed) showed a close similarity. After Neville's demonstration that electrophoretic patterns of membrane protein subunits from different subcellular fractions are easily distinguishable, the present work demonstrates an unusually close relationship between the Golgi apparatus membrane and the cell membrane. It is possible that membrane similarity may be mediated by the transfer of membrane-bound vesicles from the Golgi apparatus to the cell membrane.

Membranes of mammary gland: X. Adenosine triphosphate dependent calcium accumulation by golgi apparatus rich fractions from bovine mammary gland

Golgi apparatus rich fractions from lactating bovine mammary gland had an Mg 2+-dependent, Ca 2+-stimulated adenosine triphosphatase. These Golgi apparatus fractions also accumulated Ca 2+ in vitro. Accumulation of Ca 2+ required ATP and could be abolished by treatment either with low concentrations of deoxycholate followed by ultrasound, or by heating at 100 °C for 10 min. The adenosine triphosphatase activity of Golgi apparatus was strongly stimulated by low concentrations of Ca 2+ and moderately stimulated by high concentrations of K +. This activity was unaffected by Na + and was not inhibited by ouabain. The pH optimum for the Mg 2+-dependent hydrolysis of ATP was 7.5, the K m was 5 × 10 −5 M and the activation energy was 6 000 calories/mole. This Mg 2+-dependent adenosine triphosphatase activity was also found in rough endoplasmic reticulum, smooth microsomes and milk fat globule membrane, the latter membrane being derived directly from the apical plasma membrane. All of these membrane fractions had the ability to specifically accumulate Ca 2+. Specific accumulation was highest with smooth microsomes and lowest with milk fat globule membrane with Golgi apparatus and rough endoplasmic reticulum being intermediate. These observations provide one plausible explanation for intracellular Ca 2+ accumulation and secretion into milk. Further, these results help explain the ultrastructural observations of casein micelle formation in secretory vesicles elaborated by Golgi apparatus.

Glycoprotein Biosynthesis: INCORPORATION OF GLYCOSYL GROUPS INTO ENDOGENOUS ACCEPTORS IN A GOLGI APPARATUS-RICH FRACTION OF LIVER

Abstract A Golgi apparatus-rich fraction from rat liver was examined for the ability to mediate steps involved in the biosynthesis of glycoproteins. Five enzymatic activities were studied: the transfer of glucosamine, galactose, glucuronic acid, mannose, and N-acetylneuraminic acid from their sugar nucleotide precursors to endogenous trichloracetic acid-precipitable proteins. The glucosaminyl transfer was enriched 23-fold in the Golgi membrane fraction, and represented 17% of the total activity of the homogenate. The initial rate of transfer of glucosamine exhibits optima both at pH 6.1 and 7.1. UDP-glucuronic acid stimulates the net transfer of glucosamine at pH 7.1, but not at pH 6.1. GDP-mannose and GTP are also stimulatory at pH 7.1. UDP-glucose, UDP-galactose, UTP, UDP, and UMP are inhibitory. The Golgi-associated galactosyl transfer was enriched over 24-fold, and accounted for 13% of the original activity of the homogenate. This transfer activity is stimulated by GDP-mannose. UDP-glucose, UDP-N-acetylglucosamine, and UMP inhibit the transfer of galactose. The Golgi membrane fraction also transfers glucuronic acid from UDP-glucuronic acid to trichloracetic acid-insoluble material. This transfer is stimulated by UDP-N-acetyl-glucosamine. It represents 5% of the activity of the original homogenate, and is enriched approximately 8-fold in the Golgi fraction. The Golgi-associated transfer of N-acetylneuraminic acid represents 10% of the activity of the homogenate, and is enriched 18-fold. This transfer is stimulated by UDP-galactose, and is inhibited by CMP. Although the transfer of mannose from GDP-mannose to trichloracetic acid-precipitable material is very active in the crude homogenate, little transfer, and rapid destruction of GDP-mannose is observed in the Golgi fraction. Mild alkaline treatment (0.5 n NaOH, 24°, 90 min) results in the release of 88 to 92% of the radioactivity of the 14C-glucuronic acid-labeled product to the trichloracetic acid-soluble fraction. On the other hand, only 32 to 35% of the 14C-glucosamine-labeled product is converted into a trichloracetic acid-soluble form by this treatment. When glucosamine transfer is stimulated by the addition of UDP-glucuronic acid, however, approximately 50% of the product is released by alkali. It is suggested that the alkali-stable products might represent glycoproteins, while the more alkali-labile products may be glycosaminoglycans. Bacterial heparinase degraded 70% of the glucuronic acid-labeled product, and 25% of the glucosamine-labeled product. Testicular hyaluronidase degraded 35% of the glucuronic acid-labeled, and 7% of the glucosamine-labeled material. Pronase digestion converted all of the radioactivity of glucosamine-, glucuronic acid-, or galactose-labeled trichloracetic acid-precipitable products into trichloracetic acid-soluble forms. These digestion products are still of relatively high molecular weight, as they are eluted in the void volume of a Sephadex G-50 column. Acid hydrolysis released all of the radioactivity of the glucosamine-labeled, pronase-digested product as free glucosamine, as identified by paper chromatography. The radioactive product of the galactosyl transferase reaction was shown to be 14C-galactose by the same method. The results of this study are consistent with the hypothesis that the N-acetylglucosamine, galactose, and N-acetyl-neuraminic acid residues of the terminal sialic acid → galactose → N-acetylglucosamine sequence of many glycoproteins are added to the nascent glycoprotein within the Golgi apparatus, while the sugar residues of the inner core are inserted at other sites in the endoplasmic reticulum.

Isolation of a Golgi apparatus-rich fraction from rat liver. II. Enzymatic characterization and comparison with other cell fractions.

Enzymatic activities associated with Golgi apparatus-, endoplasmic reticulum-, plasma membrane-, mitochondria-, and microbody-rich cell fractions isolated from rat liver were determined and used as a basis for estimating fraction purity. Succinic dehydrogenase and cytochrome oxidase (mitochondria) activities were low in the Golgi apparatus-rich fraction. On the basis of glucose-6-phosphatase (endoplasmic reticulum) and 5'-nucleotidase (plasma membrane) activities, the Golgi apparatus-rich fraction obtained directly from sucrose gradients was estimated to contain no more than 10% endoplasmic reticulum- and 11% plasma membrane-derived material. Total protein contribution of endoplasmic reticulum, mitochondria, plasma membrane, microbodies (uric acid oxidase), and lysosomes (acid phosphatase) to the Golgi apparatus-rich fraction was estimated to be no more than 20-30% and decreased to less than 10% with further washing. The results show that purified Golgi apparatus fractions isolated routinely may exceed 80% Golgi apparatus-derived material. Nucleoside di- and triphosphatase activities were enriched 2-3-fold in the Golgi apparatus fraction relative to the total homogenate, and of a total of more than 25 enzyme-substrate combinations reported, only thiamine pyrophosphatase showed a significantly greater enrichment.

Localization of glycosyl transferase activities in a Golgi apparatus-rich fraction isolated from rat liver

A Golgi apparatus-rich fraction isolated from rat liver catalyzed the transfer of galactose from UDP-galactose to N-acetylglucosamine with the formation of N-acetylaminolactose as well as the transfer of glucosamine from UDP-N-acetyl- glucosamine to endogenous protein acceptors. Based on enzymatic and morphological criteria, Golgi apparatus fractions were estimated to contain at least 80% Golgi apparatus-derived material. Approximately half of the total glycosyl transferase activities of the original homogenates was recovered in the Golgi apparatus fraction. The glycosyl transferase activities of purified endoplasmic reticulum fractions were much lower than those of the Golgi apparatus. Plasma membrane fractions as well as the soluble supernatant fraction contained little or no activity.

Enzymatic transfer of 14C-glucosamine from UDP-N-acetyl- 14C-glucosamine to endogenous acceptors in a golgi apparatus-rich fraction from liver

A Golgi apparatus-rich fraction isolated from rat liver catalyzes the transfer of glucosamine from UDP-N-acetylglucosamine to endogenous protein acceptors. The Golgi-associated transfer accounts for almost half of the total activity of the original homogenate. Plasma membrane and endoplasmic reticulum fractions are practically devoid of activity. GDP-mannose and GTP stimulate this transfer, possibly by sparing UDP-N-acetylglucosamine from enzymatic hydrolysis. UDP-glucose, UDP-galactose, UTP, UDP, and UMP are inhibitory. Digestion with pronase converts the radioactive product of this transfer into a trichloroacetic acid-soluble form. All of the radioactive material cochromatographs with glucosamine, following acid hydrolysis of pronase glycopeptides.