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C B Hirschberg

Publications and source records attributed to C B Hirschberg.

85 records · Page 5Linked to original sources
PubMed1980

CMP-N-acetylneuraminic acid: isolation from and penetration into mouse liver microsomes.

We present three lines of evidence for the existence of an intramicrosomal pool of CMP-NeuNAc in liver slices incubated with 3H-NeuNAc: first, enrichment in the microsomal fraction over the cell homogenate of CMP-3H-NeuNAc relative to CMP-14C-NeuNAc, the latter added as marker for sugar nucleotide trapped between and adsorbed onto vesicles; second, removal from microsomes by isotonic sucrose washing of only half of the CMP-3H-NeuNAc synthesized in vivo under conditions which remove all the CMP-14C-NeuNAc; and third, complete loss of both sugar nucleotides upon treatment of microsomes by hypotonic shock or with low concentrations of detergents. Incubation of CMP-NeuNAc with microsomes in vitro showed that the intact sugar nucleotide was penetrating the vesicles. When the sugar nucleotide is labeled in the sugar moiety, microsomal vesicles accumulate soluble radiolabeled products. This accumulation was saturable and dependent on time, temperature and concentration of sugar nucleotide, and was also inhibited by substrate analogues and by pretreatment of microsomes with proteases. Together, these results provide strong evidence for the existence of a specific transport system for CMP-NeuNAc in microsomes.

Animals↗
PubMed1977

Biosynthesis of phytosphingosine by the rat.

Labeled C18-phytosphingosine was detected in a glyco-lipid-rich fraction from rat intestine 4 and 24 h after injection of D-erythro[4,5-3H]C18-dihydrosphingosine. The criteria for identification were: (a) co-migration on gas-liquid chromatography of the trimethylsilyl ethers of the free and N-acetylated base with authentic standard, (b) co-migration on thin layer chromatography of the dinitrophenyl derivative with authentic standard, (c) labeled pentadecanol detection by gas-liquid radiochromatography after subjecting intact sphingolipids or a mixture of free long chain bases to periodate oxidation followed by sodium borohydride reduction, and (d) obtainment of the same fragmentation pattern as with authentic standard when the trimethylsilyl ethers of the N-acetylated base were subjected to gas-liquid chromatography-mass spectrometry. Smaller amounts of labeled C18-phytosphingosine were also detected in ceramide- and sphingomyelin-rich fractions of intestine and in liver and kidney. Experiments with germ-free rats strongly suggest that the intestinal flora is not the (sole) site for the biosynthesis of C18-phytosphingosine.

Animals↗
PubMed1976

Sialic acid uptake by fibroblasts.

The existence of surface sialytransferases that use cytidine monophosphate (CMP)-sialic acid as substrate has been postulated in previous studies. This is based on the assumption that if whole, viable cells can catalyze the transfer of sialic acid from CMP-sialic acid to endogenous acceptors, then the transferases carrying out the reaction must be on the cell surface, provided that (1) CMP-sialic acid does not enter the cells, and (2) CMP-sialic acid does not break down outside the cells, yielding free sialic acid which then may enter the cells, in amounts large enough to explain the incorporation. We now report evidence showing that after incubation of intact NIL, BHK, and 3T3 fibroblasts with CMP-sialic acid, at least 78% of the sialic acid incorporated by these cells is the result of free sialic acid uptake. When cells growing in a monolayer were incubated with a mixture of CMP-[14C]sialic acid and [3H]CMP-sialic acid with a ratio of 3H/14C=0.60, this ratio was found to be markedly increased in whole cells. Chemical analyses of the radioactive species in the incubation medium showed that a considerable portion of the radiolabeled sugar nucleotide had broken down to cytidine, phosphoric acid, and sialic acid. Upon incubation of cells with doubly labeled sugar nucleotide in the presence of a large excess of both nonradiolabeled cytidine and sialic acid, the cells incorporated less than 6% of both isotopes. Incubation of cells with a mixture of CMP-[14C]sialic acid and [3H]sialic acid resulted in only 20-40% of the radioactivity within the cells being membrane bound, and 70-90% of this incorporation could be inhibited by the addition of 10 mM azide to the incubation medium. The possibility that a small fraction of the total incorporation of sialic acid by these cells is due to surface sialytransferases cannot be completely ruled out. The uptake of free sialic acid by these fibroblasts is concentration dependent and a portion of it is incorporated into glycoproteins and glycolipids. Considerable loss of cell integrity was observed when fibroblasts grown on plates were removed by (ethylenedinitrilo)-tetraacetic acid or trypsin and subsequently incubated in buffer, indicating that these preparations are not suitable for intact cell studies.

Azides↗
PubMed1975

Synthesis of glycolipids and phospholipids in hamster cells: dependence on cell density and the cell cycle.

Sakiyama et al. ('72) reported the isolation of a line of hamster cells (NIL 1c1) which contains only three glycolipids, hematoside, ceramide monohexoside and ceramide dihexoside. The incorporation of radiolabeled palmitate into hematoside during 24 hours was three fold higher in normal confluent, non growing cells than sparse, growing ones. Polyoma transformed cells did not exhibit this effect. We have continued studies with the untransformed cell line and have found that the higher incorporation of radiolabeled palmitate into hematoside by normal confluent cells is not due to a higher rate of turnover of hematoside at confluence but represents a true chemical increase. We have also found that this increase is not a gradual process during cell growth but instead occurs only when the cells become confluent and stop growing. The increase of hematoside at confluence is not due to a higher rate of synthesis of hematoside during G1, relative to the other phases of the cell cycle. We found the rate of synthesis of hematoside to be constant throughout the cell cycle. The rate of synthesis of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol and sphingomyelin was also studied as a function of the cell cycle. We found no large differences in the synthetic rate of any given phospholipid species throughout the cell cycle although the rate of synthesis of the glycerophospholipids was somewhat higher during late G1 and S. We did, however, find major differences in the rates of synthesis of the different phospholipid species.

Animals↗
PubMed1972

Mechanism of the enzymatic synthesis of cardiolipin in Escherichia coli.

In previous studies, the enzymatic conversion of phosphatidylglycerol to cardiolipin (diphosphatidylglycerol) in cell-free preparations from E. coli was shown to be stimulated by the addition of CDP-dipalmitin, suggesting the participation of the cytidine coenzyme as phosphatidyl donor. The present communication, however, presents three lines of evidence supporting the following mechanism for the synthesis of cardiolipin in E. coli.2 Phosphatidylglycerol --> cardiolipin + glycerolWhen CDP-dipalmitin labeled with (32)P in the phosphatidyl moiety was incubated with phosphatidyl[2-(3)H]-glycerol, the cardiolipin produced in the enzymatic reaction was labeled with tritium, but not with (32)P. Thus, CDP-diglyceride stimulates the reaction but does not participate as phosphatidyl donor. When [(28)P]phosphatidyl[2-(3)H]glycerol was used as substrate, the ratio of tritium to (32)P in the cardiolipin product was only half of that in the starting phosphatidylglycerol, consistent with the elimination of 1 mol of glycerol during conversion to cardiolipin. Finally, free glycerol produced during the reaction has been unambiguously identified by phosphorylation with ATP in a reaction catalyzed by glycerol kinase (EC 2.7.1.30), followed by chromatographic isolation of labeled sn-3-glycero-3-phosphate.

Cell-Free System↗