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C B Hirschberg

Publications and source records attributed to C B Hirschberg.

At least 73 records · Page 4Linked to original sources
PubMed1986

Mechanism of galactosylation in the Golgi apparatus. A Chinese hamster ovary cell mutant deficient in translocation of UDP-galactose across Golgi vesicle membranes.

The biochemical defect in the mutant Chinese hamster ovary cell lines Clone 13 (Briles, E. B., Li, E., and Kornfeld, S. (1977). J. Biol. Chem. 252, 1107-1116) and Lec8 (Stanley, P. (1980) ACS Symp. Ser. 128, 214-221) was examined. These two mutants, which belong to the same genetic complementation group, were shown in previous studies to exhibit an 80-90% reduction in galactosylation and sialylation of proteins and lipids when compared to wild-type cells. The same studies, however, demonstrated that the mutants were not deficient in the corresponding sugar nucleotides, glycosyltransferases, and endogenous acceptors for these transferases. We now provide evidence strongly suggesting that the primary defect in Lec8 and Clone 13 cells is their inability to translocate UDP-galactose into the lumen of the Golgi apparatus. Golgi vesicles from Lec8 and Clone 13 CHO glycosylation mutants translocate in vitro UDP-galactose at only 3-5% the rate of vesicles from wild-type CHO cells. The deficiency is specific because vesicles from the mutant cells can translocate adenosine 3'-phosphate 5'-phosphosulfate, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine at rates comparable to those of vesicles from wild-type cells. These studies also suggest that sugar nucleotides sharing a common uridine nucleotide utilize different translocators present in the Golgi membrane in vivo. The consequence of the above-described mutations and the resulting block in galactosylation of macromolecules in vivo on the translocation of CMP-sialic acid into the Golgi lumen was also examined. As expected, Golgi apparatus vesicles from Lec8 cells were unable to incorporate sialic acid into (endogenous) macromolecules. However, the vesicles were able to transport CMP-sialic acid into their lumen, although the rate of translocation was only 17% of that of wild-type-derived Golgi vesicles.

Animals↗
PubMed1985

Phosphoproteins and protein kinases of the Golgi apparatus membrane.

Incubation of a highly purified fraction derived from rat liver Golgi apparatus with [gamma-32P]ATP results in phosphorylation of several endogenous phosphoproteins. One phosphoprotein with an apparent Mr of 48,300 is radiolabeled to an apparent extent at least 5-fold higher than any other phosphoprotein as part of either the Golgi apparatus or highly purified rat liver fractions derived from the rough endoplasmic reticulum, mitochondria, plasma membrane, coated vesicles, cytosol, and total homogenate. Approximately 70% of the 48.3-kDa phosphoprotein appears to be a specific extrinsic Golgi membrane protein with the phosphorylated amino acid being threonine. The protein kinase which phosphorylates the 48.3-kDa protein is an intrinsic Golgi membrane protein and is dependent on Mg2+, independent of Ca2+, calmodulin, and cAMP, and is inhibited by N-ethylmaleimide. Preliminary evidence suggests that there are also intrinsic membrane protein kinases in the Golgi apparatus which are dependent on Ca2+ and cAMP. The physiological role of the above phosphoproteins and protein kinases is not known.

Adenosine Triphosphate↗
PubMed1985

Translocation of UDP-N-acetylglucosamine into vesicles derived from rat liver rough endoplasmic reticulum and Golgi apparatus.

A mixture of UDP-N-acetylglucosamine labeled with different radioisotopes in the uridine and glucosamine was used to show that the intact sugar nucleotide was translocated across the membrane of vesicles derived from rat liver rough endoplasmic reticulum (RER) and Golgi apparatus. Translocation was dependent on temperature, saturable at high concentrations of sugar nucleotide, and inhibited by treatment of vesicles with proteases, suggesting protein carrier mediated transport. Translocation of UDP-GlcNAc by RER-derived vesicles appeared to be specific since these vesicles were unable to translocate UDP-galactose, in contrast to those derived from the Golgi apparatus. Preliminary results suggest that the mechanism of UDP-GlcNAc translocation into RER-derived vesicles is via a coupled exchange with lumenal nucleoside monophosphate. This is similar to the recently postulated mechanism for translocation of sugar nucleotides into vesicles derived from the Golgi apparatus.

Animals↗
PubMed1984

Effect of nucleotides on translocation of sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate into Golgi apparatus vesicles.

Recent studies from this laboratory have suggested that rat-liver Golgi apparatus derived membranes contain different proteins which can translocate in vitro CMP-N-acetylneuraminic acid, GDP-fucose and adenosine 3'-phosphate 5'-phosphosulfate from an external compartment into a lumenal one. The aim of this study was to define the role of the nucleotide, sugar and sulfate moieties of sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate in translocation of these latter compounds across Golgi vesicle membranes. Indirect evidence was obtained suggesting that the nucleotide (but not sugar or sulfate) is a necessary recognition feature for binding to the Golgi membrane (measured as inhibition of translocation) but is not sufficient for overall translocation; this latter event also depends on the type of sugar. Important recognition features for inhibition of translocation of the above sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate were found to be the type of nucleotide base (purine or pyrimidine) and the position of the phosphate group in the ribose. Thus, UMP and CMP were found to be competitive inhibitors of translocation of CMP-N-acetylneuraminic acid, while AMP did not inhibit. Structural features of the nucleotides which were less important in inhibition of translocation (and thus presumably in binding) of the above sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate were the number of phosphate groups in the nucleotide (CDP and CMP inhibited to a similar extent), the presence of ribose or deoxyribose in the nucleotide, a replacement of hydrogen in positions 5 of pyrimidines or 8 in purines by halogens or an azido group. The sugar or sulfate did not inhibit translocation of the above sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate into Golgi vesicles and therefore appear not to be involved in their binding to the Golgi membrane.

Adenine Nucleotides↗
PubMed1984

Biosynthesis of 4D-hydroxysphinganine by the rat. En bloc incorporation of the sphinganine carbon backbone.

Rats were injected intravenously with erythro-DL-[1-3H]sphinganine. Radiolabeled 4D-hydroxysphinganine (phytosphingosine) was detected (in addition to sphinganine and 4-sphingenine) in glycolipid- and sphingomyelin-rich fractions from intestine and kidney. Identification of these bases was achieved following hydrolysis of the these fractions and conversion of the long chain bases to their N-dinitrophenyl (Dnp) derivatives. Dnp-4D-hydroxy[3H]sphinganine was characterized by: comigration on thin-layer chromatography with authentic standard and detection of pentadecanol and Dnp-[3H]serinol. These results strongly suggest that in mammalian tissues the C18 carbon backbone of 4D-hydroxysphinganine can be derived (en bloc) from sphinganine.

Animals↗
PubMed1984

Effect of atractylosides, palmitoyl coenzyme A, and anion transport inhibitors on translocation of nucleotide sugars and nucleotide sulfate into Golgi vesicles.

The effect of palmitoyl coenzyme A, atractylosides, and anion transport inhibitors on translocation into rat liver Golgi vesicles of adenosine 3'-phosphate 5'-phosphosulfate (PAPS), CMP-N-acetylneuraminic acid, and GDP-fucose was studied. Translocation of the above three nucleotide derivatives was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 50% inhibition required 10-20 microM DIDS). The inhibition of translocation of PAPS by DIDS was used to demonstrate that sulfation of macromolecules within Golgi vesicles is preceded by translocation of PAPS into the vesicles. Palmitoyl coenzyme A, at concentrations below its critical micellar concentration, specifically inhibited translocation into Golgi vesicles of PAPS but not CMP-NeuAc and GDP-fucose. Inhibition of PAPS translocation by 50% required 9 microM palmitoyl coenzyme A. Translocation of PAPS but not of CMP-NeuAc or GDP-fucose was also inhibited by atractyloside or carboxyatractyloside with 50% inhibition requiring 15 microM either glycoside. This pattern of inhibition suggests structural similarities between the putative translocator of PAPS in Golgi membranes and the ATP/ADP translocator of mitochondria.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

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

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

Adenine Nucleotides↗
PubMed1984

Translocation across Golgi vesicle membranes: a CHO glycosylation mutant deficient in CMP-sialic acid transport.

Golgi vesicle membranes from the Lec2 CHO glycosylation mutant translocate CMP-sialic acid at only 2% the rate of vesicles from wild-type CHO cells. The deficiency is specific, because vesicles from Lec2 cells can translocate UDP-N-acetylglucosamine, adenosine 3'-phosphate 5'-phosphosulfate, and UDP-galactose at rates comparable to those of vesicles from wild-type cells. Complementation analyses show that Lec2 mutants belong to the same genetic complementation group as clone 1021, a CHO mutant of similar phenotype. Both mutants have previously been shown to have a 90% reduction in the sialylation of glycoproteins and gangliosides compared with wild-type cells. However, 1021 cells appear to have normal levels of CMP-sialic acid, sialyltransferase activity, and endogenous acceptors for sialylation. It seems likely that the primary defect in Lec2 and 1021 cells is their inability to translocate CMP-sialic acid across Golgi vesicle membranes.

Animals↗
PubMed1984

Mechanisms of glycosylation and sulfation in the Golgi apparatus: evidence for nucleotide sugar/nucleoside monophosphate and nucleotide sulfate/nucleoside monophosphate antiports in the Golgi apparatus membrane.

The mechanism of translocation in vitro of sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) into the lumen of rat liver Golgi apparatus vesicles has been studied. It has been previously shown that the Golgi apparatus membrane has specific carrier proteins for PAPS and sugar nucleotides. We now report that translocation of the above nucleotide derivatives across Golgi membranes occurs via a coupled equimolar exchange with the corresponding nucleoside monophosphates. An initial incubation of Golgi vesicles with GDP-fucose radiolabeled in the guanidine ring resulted in accumulation within the lumen of radiolabeled GMP. Exit of GMP from these vesicles was specifically dependent on the entry of (additional) GDP-fucose into the vesicles (GDP-mannose and other sugar nucleotides had no effect). GDP-fucose-stimulated exit of GMP was temperature dependent, was blocked by inhibitors of GDP-fucose transport, such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and appeared to be equimolar with GDP-fucose entry. Preliminary evidence for specific, equimolar exchange of CMP-N-acetylneuraminic acid with CMP, PAPS with 3'-AMP, and UDP-galactose and UDP-N-acetylglucosamine with UMP was also obtained. These results strongly suggest the existence of different antiport proteins within the Golgi membrane that mediate the 1:1 exchange of sugar nucleotides or PAPS with the corresponding nucleoside monophosphate. Such proteins may have a regulatory role in glycosylation and sulfation reactions within the Golgi apparatus.

Adenine Nucleotides↗
PubMed1984

Conversion of erythro-D-sphinganine to its [1-2H1] and [1-3H1] derivatives.

A convenient chemical synthesis of erythro-D-[1-2H1] sphinganine and erythro-D-[1-3H1]sphinganine is described. The approach utilizes a stereospecific starting material (natural sphinganine prepared from bovine brain sphingomyelin) and applies a sequence of selective protection of functional groups yielding 2-acetamido-3-O-benzoyloctadecan-1-ol. Oxidation of the primary alcohol to an aldehyde followed by NaB2H4 or NaB3H4 reduction and hydrolysis of the protective groups yields erythro-D-[1-2H1]sphinganine or erythro-D-[1-3H1]sphinganine. The synthetic intermediates and isotopically labeled sphinganines are characterized by infrared analysis, 1H-nuclear magnetic resonance, optical rotation, and gas-liquid radiochromatographic and mass spectral fragmentation analyses. The [1-2H1] and [1-3H1] derivatives were obtained with overall yields (and isotope enrichments) of 11% (min. 84 mol% 2H1) and 8% (60 mCi/mmol), respectively.

Animals↗
PubMed1983

Subfractionation of rat liver Golgi apparatus: separation of enzyme activities involved in the biosynthesis of the phosphomannosyl recognition marker in lysosomal enzymes.

A highly purified Golgi apparatus preparation from rat liver was subfractionated on a Percoll gradient into two major protein peaks of similar size that migrated at densities of 1.028 and 1.051 g/ml. The lighter protein peak contained 70--80% of the total activities of the oligosaccharide-processing enzymes alpha-1,2-mannosidase and mannosidase II and of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyl-1-phosphotransferase (alpha-N-acetylglucosaminylphosphotransferase), an enzyme involved in the biosynthesis of the mannose 6-phosphate recognition marker of lysosomal enzymes. These enzyme activities were enriched 2-fold in specific activity over that of the heavy protein peak. In contrast, 80% of the alpha-N-acetylglucosaminylphosphodiesterase, an enzyme that exposes 6-phosphomonoesters of mannose on the oligosaccharide chains of lysosomal enzymes, migrated in a region of slightly higher density than did the protein peak of density 1.051 g/ml. Sialyltransferase (SiaTase) and galactosyltransferase (Gal-Tase) activities distributed almost equally among the two protein peaks. Controls rule out that the two protein peaks were the result of aggregation/deaggregation and that enzyme activities were altered by Percoll per se. Lysosomal enzyme activities migrated in a region essentially devoid of Golgi apparatus-associated enzyme activities. These results suggest a physical separation within the Golgi apparatus of some of the enzymes involved in the biosynthesis and processing of the oligosaccharides on glycoproteins, including those responsible for the formation of the mannose 6-phosphate recognition marker on lysosomal enzymes.

Animals↗
PubMed1982

Transport of sugar nucleotides into rat liver Golgi. A new Golgi marker activity.

Following incubation of sealed, "right side out," rat liver Golgi-derived vesicles with a mixture of [3H]GDP-fucose and GDP-[14C]fucose, the difference in the 3H to 14C ratio between the incubation medium and the lumen of the vesicles was less than 11%, suggesting that the sugar nucleotide was transported intact into the vesicles. Transport of GDP-fucose weas temperature-dependent, saturable, with an apparent Km of 7.5 microM and a Vmax of 14 pmol/mg of protein/10 min and inhibited by substrate analogues. Pretreatment of intact Golgi vesicles with pronase inhibited transport by 80% under conditions in which sialyltransferases (a lumenal marker) were not inhibited. This result is consistent with a sugar nucleotide carrier protein, portions of which face the cytoplasmic side of Golgi vesicles. Previous studies from this laboratory had shown that CMP-N-acetylneuraminic acid (CMP-NeuAc) could penetrate mouse liver microsomes in a manner suggesting carrier-mediated transport (Carey, D. J., Sommers, L. W., and Hirschberg, C. B. (1980) Cell 19, 597-605). Subsequent to transport, a portion of the N-acetylneuraminic acid moiety became covalently linked to proteins facing the lumen of Golgi vesicles (Carey, D. J., and Hirschberg, C. B. (1981) J. Biol. Chem. 256, 989-993). Upon subfractionation of rat liver into vesicles derived from the smooth and rough endoplasmic reticulum and Golgi, both the highest CMP-NeuAc specific transport activity and total transport activity were localized in the Golgi. Any transport activity in the other vesicles could be accounted for by contamination with Golgi, as determined by established marker enzyme activities. Transport of CMP-NeuAc was temperature-dependent, inhibited by pronase, substrate analogues, and saturable, with an apparent Km of 2.4 microM and a Vmax of 150 pmol/mg of protein/10 min.

Animals↗
PubMed1982

Autoradiographic detection and characterization of a Chinese hamster ovary cell mutant deficient in fucoproteins.

Autoradiography of colony replicas immobilized on filter paper was used to isolate a Chinese hamster ovary cell line deficient in incorporation of radiolabeled fucose into a trichloroacetic acid-insoluble fraction. This cell line, called 62.1, has the same growth rate at 37 degrees C as wild-type cells, but incorporates five times less fucose into acid-insoluble radioactivity. Chemical analysis of fucose bound to macromolecules also showed a fivefold reduction in the mutant. The fucoproteins of the mutant cell line differ qualitatively from those of wild-type cells as visualized by SDS gel electrophoresis fluorography; no differences were detected between total proteins as visualized by coomassie blue staining. The macromolecular sialic acid content of the mutant was somewhat higher than the wild type (20%). Studies of the synthesis of the glycoprotein of vesicular stomatitis virus in mutant and wild-type cells showed that the mutant is unable to synthesize complex-type N-linked oligosaccharides. Enzyme assays show that ths defect in the mutant is due to reduction in UDP-N-acetylglucosamine-glycoprotein N-acetyl-glucosaminyltransferase, a key enzyme in the assembly of complex glycopeptides. Hybridization studies have shown that mutant 62.1 has common mutations belonging to the same complementation group as mutant PhaR1-1. This latter mutant was previously isolated using lectin resistance by Stanley et al. (1975) and was also deficient in the above N-acetyl-glucosaminyltransferase.

Animals↗
PubMed1981

Topography of sialoglycoproteins and sialyltransferases in mouse and rat liver Golgi.

We have studied the membrane topography of N-acetylneuraminic acid (NeuAc) in sialoglycoproteins of mouse and rat liver Golgi apparatus vesicles. Purified Golgi vesicles or Golgi vesicles present in microsomes were able to incorporate radiolabeled NeuAc from CMP-NeuAc into endogenous glycoproteins in vitro. The labeled glycoproteins showed a similar profile on sodium dodecyl sulfate gel electrophoresis to those isolated from Golgi fractions of animals injected with radiolabeled NeuAc. Using this labeling procedure in vitro, we found that only 5 to 20% of the incorporated NeuAc could be removed from intact vesicles upon treatment with neuraminidase or proteases. However, disruption of vesicles by detergents or mechanical means prior to the enzymatic treatments resulted in removal of labeled NeuAc; the extent of NeuAc removal could be correlated with that of vesicle disruption as measured by loss of latency of thiamine pyrophosphatase activity. This strongly suggests that NeuAc residues of glycoproteins are on the lumenal side of the Golgi apparatus membrane and that Golgi-derived vesicles have the same membrane orientation as in vivo. A similar approach was used to infer a lumenal orientation of sialyltransferases. Not all Golgi apparatus preparations routinely used may be suitable for membrane topography experiments of the kind described here.

Animals↗
PubMed1981

Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells were screened by replica plating-fluorography for clones showing decreased incorporation of fucose into trichloroacetic acid-insoluble macromolecules. Considerable enrichment for cells deficient in fucose uptake or incorporation into proteins (or both) was found in populations surviving the later selection cycles. Two mutant clones isolated after the fourth selection cycle had the same doubling time as the wild type, but contained only 30 to 40% as much fucose bound to proteins as the wild type. Sialic acid contents of the mutants and the wild type were similar. The mutants differed quantitatively and qualitatively from the wild type and from each other with respect to total glycoprotein profiles as visualized by sodium dodecyl sulfate gel electrophoresis. Differences were also found in resistances to cytotoxicity of lectins such as concanavalin A and wheat germ agglutinin.

Animals↗
PubMed1980

Kinetics of glycosylation and intracellular transport of sialoglycoproteins in mouse liver.

We have obtained evidence in vivo for the intracellular site of sialylation of glycoproteins and have determined the kinetics of intramembranous transport of newly synthesized sialoglycoproteins. Radioactivity of sialoglycoproteins was determined at different times in homogenate, smooth and rough endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions of liver either from slices which had been incubated with radioactive N-acetylneuraminic acid or from mice which had been injected with radioactive sugar. At short times after labeling, the Golgi apparatus contained the highest specific activity of radioactive sialoglycoproteins and over half of the total cellular radioactive sialoglycoproteins. By 15 to 25 min, most of the radioactivity in the Golgi apparatus had decreased with a concomitant increase of radioactivity in the plasma membrane. Radioactivity in the smooth and rough endoplasmic reticulum accounted for less than 20% of the total cellular radioactivity throughout. Control experiments indicated that leakage of soluble, labeled sialoglycoproteins from membrane vesicles and adsorption of them onto vesicles was minimal. In contrast to the above studies, labeling for short times with radioactive mannose, showed, as expected from studies in vitro, most of the radioactivity in the rough endoplasmic reticulum. At longer times, the radioactivity in the rough endoplasmic reticulum decreased, with an increase in the plasma membrane. Throughout, the radioactivity in the smooth endoplasmic reticulum was low.

Animals↗