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C B Hirschberg

Publications and source records attributed to C B Hirschberg.

At least 55 records · Page 3Linked to original sources
PubMed1990

Topography of initiation of N-glycosylation reactions.

Previous studies on the topography of the reactions leading to the formation of dolichol-P-P-Glc-NAc2Man9Glc3 have shown that these occur on both sides of the endoplasmic reticulum membrane (Hirschberg, C. B., and Snider, M. D. (1987) Annu. Rev. Biochem. 56, 63-87). Dolichol-P-P-GlcNAc2Man5 has been detected on the cytoplasmic side of the endoplasmic reticulum membrane while the subsequent dolichol-oligosaccharide intermediates face the lumen. Less clear is the side of the membrane where dolichol-P-P-GlcNAc2 is assembled. We now present evidence strongly suggesting that the active sites of the enzymes catalyzing the synthesis of this latter intermediate are on the cytoplasmic side of the endoplasmic reticulum membrane. In addition, dolichol-P-P-GlcNAc2 has also been detected on this side. Incubations of sealed, "right side out" rat liver endoplasmic reticulum-derived vesicles with [beta-32P] UDP-GlcNAc in the presence of 5-Br-UMP resulted in the formation of radiolabeled dolichol-P-P-GlcNAc and dolichol-P-P-GlcNAc2 under conditions where there was complete inhibition of transport of the nucleotide sugar. In other experiments with the above radiolabeled nucleotide sugar and sealed vesicles, it was demonstrated that EDTA (a membrane-impermeable reagent) inhibited the N-acetylglucosamine-1-phosphate transferase under conditions where transport of the nucleotide sugar into the lumen was unaffected. Finally, sealed vesicles were first incubated with [32P]UDP-GlcNAc and subsequently with UDP-Gal and soluble galactosyltransferase. This resulted in galactosylation of dolichol-P-P-GlcNAc2. The above results, together with the previous observations, strongly suggest that all reactions leading to this latter dolichol intermediate occur on the cytosolic side of the endoplasmic reticulum membrane.

Animals↗
PubMed1990

Differential association and distribution of acetyl- and butyrylcholinesterases within rat liver subcellular organelles.

Rat liver cholinesterases were found to share properties and characteristics with those expressed in cholinergic tissues. The distribution and presence of different molecular forms of cholinesterases in different subcellular organelles of rat liver were studied. The rough and smooth endoplasmic reticulum and Golgi apparatus were enriched in the G4 molecular form of acetylcholinesterase (AChE) (relative to the G2 molecular form), while the inverse was found in the plasma membrane. The interaction of these molecular forms of AChE with the Golgi membrane was studied in detail. Approximately one-half of the G4 form was free within the lumen while the remainder was an intrinsic membrane protein; all the G2 molecular form was anchored to the membrane via phosphatidylinositol. Only the G1 and G2 molecular forms of butyrylcholinesterase (BuChE) were found in the above subcellular organelles; both molecular forms were soluble within the lumen of Golgi vesicles. These results indicate that rat liver expresses several molecular forms of AChE which have multiple interactions with membranes and that liver is unlikely to be the source of the G4 form of BuChE present in high concentration in the plasma.

Acetylcholinesterase↗
PubMed1989

Differential association of rat liver heparan sulfate proteoglycans in membranes of the Golgi apparatus and the plasma membrane.

Heparan sulfate proteoglycans (HSPG) of rat liver are associated with the plasma membrane in a hydrophobic intrinsic and a hydrophilic extrinsic form. We were interested in determining whether or not these two forms could be detected in the Golgi apparatus, the subcellular site of addition of oligosaccharides and sulfate to HSPG. In vivo and in vitro radiolabeled HSPG from rat liver Golgi apparatus membranes could only be solubilized with detergents that disrupt the membrane lipid bilayer, suggesting that they are solely associated via hydrophobic interactions. Both forms of HSPG were detected in plasma membranes of rat liver and isolated rat hepatocytes. The detergent-solubilized HSPG bound to octyl-Sepharose columns, whereas the hydrophilic form did not; this latter form, however, was released from the membrane by heparin. The hydrophobic anchor of HSPG in the Golgi and plasma membranes was insensitive to treatment with phosphatidylinositol-specific phospholipase C under conditions in which alkaline phosphatase was sensitive; this suggests that the hydrophobic anchor of HSPG is the core protein itself. Preliminary experiments suggest that the subcellular site of processing of the hydrophobic to the hydrophilic form of HSPG is the plasma membrane. A specific processing activity, probably a protease of the plasma membrane not present in serum or the endoplasmic reticulum membrane, converted hydrophobic HSPG of the Golgi membrane to the hydrophilic form. In addition, pulse-chase experiments with [35S]Na2SO4 in rats demonstrated that at short times, the bulk of the radiolabeled cellular HSPG was in the Golgi apparatus; later on, the bulk of the radioactivity was found in the plasma membrane, the only subcellular site where the hydrophilic form of HSPG was detected.

Animals↗
PubMed1989

Mechanism of phosphorylation in the lumen of the Golgi apparatus. Translocation of adenosine 5'-triphosphate into Golgi vesicles from rat liver and mammary gland.

The occurrence of phosphorylated secretory proteins such as caseins and vitellogenin and the recent characterization of phosphorylated proteoglycans, in the xylose and protein core, has raised the question of where in the cell and how this phosphorylation occurs. Previous studies have described a casein kinase activity in the lumen of the Golgi apparatus and this organelle as the site of xylose addition to the protein core of proteoglycans. We now report the translocation in vitro of ATP into the lumen of rat liver and mammary gland Golgi vesicles which are sealed and have the same membrane topographical orientation as in vivo. The entire ATP molecule was translocated into the lumen of the Golgi vesicles; this was established by using ATP radiolabeled with tritium in the adenine and gamma-32P. Translocation was temperature dependent and saturable, with an apparent Km of 0.9 microM and Vmax of 58 pmol/mg protein/min. Preliminary evidence suggests that translocation of ATP into the vesicles' lumen is coupled to exit of AMP from the lumen. Following translocation of ATP into the lumen of the vesicles, proteins were phosphorylated.

Adenosine Triphosphate↗
PubMed1989

Topography of glycosylation in yeast: characterization of GDPmannose transport and lumenal guanosine diphosphatase activities in Golgi-like vesicles.

"Outer-chain" addition of mannose residues to yeast glycoproteins occurs in the Golgi compartment of the cell. Essential steps in this process are thought to include transport of GDPmannose from the cytoplasm into the lumen of Golgi vesicles, transfer of mannose to glycoprotein acceptors, hydrolysis of the resulting GDP to GMP, and return of GMP and inorganic phosphate to the cytoplasm. We report detection and characterization of a GDPmannose transport activity and a GDPase by yeast vesicles. The active transport of GDPmannose as well as the GDPase and another presumed Golgi enzyme, alpha 1,2-mannosyltransferase, are concentrated in a subcellular fraction that can be partially separated, by velocity sucrose gradient centrifugation, from a fraction enriched in an endoplasmic reticulum marker enzyme.

Biological Transport↗
PubMed1989

Reconstitution of Golgi vesicle CMP-sialic acid and adenosine 3'-phosphate 5'-phosphosulfate transport into proteoliposomes.

We have previously shown that Golgi apparatus vesicles transport nucleotide sugars and nucleotide sulfate into their lumen. These transport activities are organelle and substrate specific and are characterized by apparent Km for nucleotide derivatives in the low micromolar range. As part of our goal of purifying and characterizing the above transport proteins, we have reconstituted a protein extract from rat liver Golgi membranes into phosphatidylcholine liposomes. The resulting proteoliposomes transport CMP-N-acetylneuraminic acid (CMP-AcNeu) and adenosine 3'-phosphate 5'-phosphosulfate with very similar affinity and inhibition characteristics as intact Golgi vesicles. Sialic acid and sodium sulfate, which are transported only very slowly into the lumen of Golgi vesicles, are transported at low rates by the reconstituted proteoliposomes. Neither rough endoplasmic reticulum-derived vesicles nor proteoliposomes made from proteins of the rough endoplasmic reticulum transport CMP-AcNeu. The above results demonstrate that this reconstituted system can be used for further purification and characterization of nucleotide sugar and nucleotide sulfate translocator proteins. This approach should also be useful to study membrane transport proteins of lysosomes and endosomes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

An intrinsic membrane glycoprotein of the golgi apparatus with O-linked N-acetylglucosamine facing the cytosol.

We have recently described the occurrence of integral membrane glycoproteins in rat liver smooth and rough endoplasmic reticulum with O-N-acetylglucosamine facing the cytosolic and luminal sides of the membrane (Abeijon, C., and Hirschberg, C. B. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1010-1014). We now report that integral membrane glycoproteins with cytosolic facing O-N-acetylglucosamine also occur in membranes of rat liver Golgi apparatus. This was determined following incubation of vesicles from the Golgi apparatus, which were sealed and of the same membrane topographical orientation as in vivo, with UDP-[14C]galactose and saturating amounts of bovine milk galactosyltransferase. This enzyme does not enter the lumen of the vesicles and specifically catalyzes the addition of galactose, in a beta 1-4 linkage, to terminal N-acetylglucosamine. Under these conditions, galactose was transferred to a glycoprotein of molecular mass of 92 kDa. This protein was insoluble in sodium carbonate, pH 11.5, conditions under which integral membrane proteins remain membrane bound and was insensitive to treatment with peptide:N-glycosidase F. beta Elimination and chromatography showed that radiolabeled galactose was part of a disaccharide which was characterized as Gal beta 1-4GlcNAcitol. This glycoprotein is specific of the Golgi apparatus membrane. Intrinsic membrane glycoproteins with this unusual carbohydrate membrane orientation thus occur in the endoplasmic reticulum and Golgi apparatus of rat liver.

Acetylglucosamine↗
PubMed1988

Purification of rat liver N-heparan-sulfate sulfotransferase.

N-Heparan-sulfate sulfotransferase catalyzes the transfer of sulfate from 3'-phosphoadenilyl sulfate to the nitrogen of glucosamine in heparan sulfate. This reaction is an obligatory step for subsequent epimerization of D-glucuronic to L-iduronic acid and of O-sulfation of the sugar chains. We have purified this sulfotransferase from rat liver membranes to apparent homogeneity using a combination of conventional and affinity chromatography on DEAE-Sephacel, heparin-agarose, 3',5'-ADP-agarose, wheat germ-Sepharose, and finally a glycerol gradient. The pure enzyme is a glycoprotein with an apparent molecular weight of 97,000. It was enriched in specific activity 65,000-fold over the homogenate. The recovery of activity was 4% of that of the homogenate. Preliminary enzymatic characterization of the purified sulfotransferase indicates a high degree of substrate specificity. Transfer of sulfate occurs to heparan sulfate, N-heparan sulfate, and N-desulfated heparin, but not to N-acetylated heparan sulfate, N-acetylated heparin, chondroitin, chondroitin sulfate, and tyrosine-containing tripeptides.

Animals↗
PubMed1988

Intrinsic membrane glycoproteins with cytosol-oriented sugars in the endoplasmic reticulum.

We have examined the topography of N-acetylglucosamine-terminating glycoproteins in membranes from rat liver smooth and rough endoplasmic reticulum (SER and RER). It was found that some of these glycoproteins are intrinsic membrane proteins with their sugars facing the cytosolic rather than the luminal side. This conclusion was reached by using vesicles from the SER and RER that were sealed and of the same topographical orientation as in vivo. These vesicles were incubated with UDP-[14C]galactose (which does not enter the vesicles) and saturating amounts of soluble galactosyltransferase from milk, an enzyme that does not penetrate the lumen of the vesicles and that specifically adds galactose to terminal N-acetylglucosamine in a beta 1-4 linkage. Radioactive galactose was mainly transferred to SER proteins of apparent molecular mass 56 and 110 kDa and to a lesser extent to RER and SER proteins of apparent molecular mass 46 and 72 kDa. These proteins are intrinsic membrane proteins, based on the inability of sodium carbonate at pH 11.5 to remove them from the membranes. Studies with peptide N-glycosidase F and chemical beta-elimination showed that the 56-kDa protein of the SER vesicles contained terminal N-acetylglucosamine in an O-linkage to the protein. The above results suggest that some sugars of glycoproteins in the endoplasmic reticulum may attain their final orientation in the membrane by mechanisms yet to be determined.

Acetylglucosamine↗
PubMed1987

Subcellular site of synthesis of the N-acetylgalactosamine (alpha 1-0) serine (or threonine) linkage in rat liver.

We have studied the subcellular site of synthesis of the GalNAc(alpha-1-0) Ser/Thr linkage in rat liver. The specific and total activities of polypeptide:N-acetylgalactosaminyltransferase (using apomucin as exogenous acceptor) were highly enriched in membrane fractions derived from the Golgi apparatus; virtually no activity was detected in membranes from the rough and smooth endoplasmic reticulum. Vesicles of the above organelles (which were sealed and of the same membrane topographical orientation as in vivo) were able to translocate UDP-GalNAc into their lumen in an assay in vitro; the initial translocation rate into Golgi vesicles was 4-6-fold higher than that into vesicles from the rough and smooth endoplasmic reticulum. Translocation of UDP-GalNAc into Golgi vesicles was temperature dependent and saturable with an apparent Km of 8-10 microM. UDP-GalNAc labeled with different radioisotopes in the uridine and sugar was used to determine that the intact sugar nucleotide was being translocated in a reaction coupled to the exit of luminal UMP. Following translocation of UDP-GalNAc, transfer of GalNAc into endogenous macromolecular acceptors was detected in Golgi vesicles and not in those from the rough and smooth endoplasmic reticulum. The above results together with previous studies on the O-xylosylation of the linkage region of proteoglycans (Nuwayhid, N., Glaser, J.H., Johnson, J.C., Conrad, H.E., Hauser, S.C., and Hirschberg, C.B. (1986) J. Biol. Chem. 261, 12936-12941) strongly suggest that, in rat liver, the bulk of O-glycosylation reactions occur in the Golgi apparatus.

Acetylgalactosamine↗
PubMed1986

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.

Animals↗
PubMed1986

3'-O-(4-benzoyl)benzoylcytidine 5'-triphosphate. A substrate and photoaffinity label for CMP-N-acetylneuraminic acid synthetase.

A photoreactive, radiolabeled pyrimidine nucleotide, 3'-O-(4-benzoyl)benzoylcytidine 5'-triphosphate was synthesized from benzoylbenzoic acid and radiolabeled CTP. Benzoylbenzoyl-[5-3H]CTP could substitute for CTP, in an enzymatic reaction with N-acetylneuraminic acid catalyzed by Escherichia coli or rat liver CMP-NeuAc synthetase, to yield radiolabeled benzoyl-benzoyl-CMP-NeuAc. E. coli CMP-NeuAc synthetase could be specifically radiolabeled using benzoylbenzoyl-[alpha-32P]CTP as a photoaffinity label. This specific covalent binding occurred using enzyme preparations of different degrees of purity. These results suggest that benzoylbenzoic acid derivatives of pyrimidines should be of general use in the identification and active site mapping of pyrimidine-requiring proteins and enzymes. These include glycosyltransferases, sugar nucleotide synthetases, and transporters, and enzymes participating in the conjugation of bile acids and biosynthesis of nucleic acids and choline nucleotides.

Affinity Labels↗
PubMed1986

Topography of glycosylation reactions in the rough endoplasmic reticulum membrane.

The translocation of UDP-glucose and GDP-mannose from an external to a luminal compartment has been examined in rat liver vesicles derived from the rough endoplasmic reticulum (RER). RER vesicles with the same topographical orientation as in vivo were incubated with a mixture of [3H]UDP-glucose and UDP-[14C]glucose to demonstrate that the intact sugar nucleotide was translocated into the lumen of the vesicles. The translocation of UDP-glucose was dependent on temperature and was saturable at high concentrations of the sugar nucleotide. The transfer of glucose to endogenous acceptors was dependent on the translocation of UDP-glucose into the lumen of the RER since leaky vesicles resulted in both a decrease in transport and transfer of glucose to endogenous acceptors. Preliminary results suggest that the mechanism of UDP-glucose transport into RER-derived vesicles is via a coupled exchange with luminal UMP. Using the same experimental approach to detect translocation of UDP-glucose into the lumen of RER vesicles, we were unable to detect transport of GDP-mannose. Incubation of leaky vesicles with GDP-mannose resulted in stimulation of the amount of mannose transferred to endogenous acceptors, in marked contrast to that observed for UDP-glucose and UDP-N-acetylglucosamine. These results suggest that whereas UDP-glucose is translocated across the RER membrane in vitro, GDP-mannose is not transported. In addition, these results tentatively suggest that there is asymmetric synthesis of the lipid-linked oligosaccharides within the membrane of the RER.

Animals↗