Pierre Louisot

Evidence for glycosyl-transferases in rat liver nuclei

Abstract Five glycosyl-transferases have been found present in purified hepatocyte nuclei of the rat (mannosyl-, galactosyl-, N-acetyl-glucosaminyl-, N-acetyl-galactosaminyl- and sialyl-transferases); these are capable of fixing specific carbohydrates on to endogenous or exogenous protein acceptors.

Biosynthèse des glycoprotéines dans les mitochondries: I. Etude de l'activité mannosyl-transfèrase mitochondriale

Summary Glycoprotein biosynthesis in mitochondria has been approached by studying the mannosyl — transferase activity. This enzyme incorporates mannose, from GDP — mannose, the coenzymic form, onto an endogenous acceptor. Studied on «in vitro acellular systems, the optimal pH is 7,4 and the optimal temperature is 30°C. The affinity constant for the GDP-mannose substrate is 110 nM. The enzyme is inhibited by ADP, GDP and GTP. Mn 2+ and Mg 2+ ions are activators, and maximal activity is reached at 5.10 −3 M concentration of Mn 2+ . Puromycin and cycloheximide have no effect. Introduction of energetic substrates (glutamate, succinate) do not modify the mannosyl-transferase activity. The same is true for inhibitors (rotenone, amytal, antimycin and cyanide) and an uncoupler (2,4-dinitrophenol). Mitochondrial mannosyl-transferase is different from microsomic homologous systems, and its activity is independent from the energetic potentialities of mitochondria.

Biosynthèse des glycoprotéines. XXI. Étude de la mannosyl-transférase microsomique des splénocytes

Summary An enzymatic system catalyzing mannose transfer to endogenous proteins from the coenzymatically active form of the ose, the GDP-mannose, is present in splenic cellular microsomes. This mannosyltransferase shows an optimum pH of 5.8, an optimum temperature of 30°C, and is highly sensitive to any temperature or ionic strength increase. Its Km for GDP-mannose is 0.1 μ M. The enzyme is activated by Mn++ and Mg++, by mono- and triphosphate nucleosides and inhibited by diphosphate nucleoside and X-100 Triton. Splenic microsomic mannosyl-transferase proves very sensitive to any alternation of subcellular architecture at the microsomic level.

A new pathogenic hypothesis for cystic fibrosis: Hyperactivity of glycosyl transferases at microsomic level

Abstract In cystic fibrosis, glycosyl transferase activities at lung microsome level are very enhanced. In the same conditions, no disturbance of amino acid incorporation is observed. It seems that the disturbance of glycosyl transferase regulation is perhaps the prime cause of this disease. In vitro , guanosine diphosphate is a very strong inhibitor for normal and cystic ubrosis glycosyl transferases.

Biosynthesis of glycoproteins in the aortic wall study of intima microsomic mannosyl-transferase activity

Abstract Glycoprotein biosynthesis in the aortic wall has been approached by studying the mannosyl-transferase activity in the intimal microsomes. Studied on “in vitro” acellular systems, the optimal pH is 5.8 and the optimal temperature is 27°C. This enzyme shows thermal inactivation which is compensated by an increased reaction rate, under the influence of its exposure to supra-optimal temperature. The affinity constant for the GDP-mannose substrate is 15 μ m . The enzyme is inhibited by nucleoside-diphosphates, GTP and its structural analogue, β-γ-methylene-GTP at concentration of 10−3 to 10−4 m . Mn2+ and Mg2+ ions are activators, and maximal activity is reached at a 5 × 10−3 m concentrations of Mn2+. Cemulsol, deoxycholate and Triton X-100 are inhibitors in strong concentrations. The same is true for cycloheximide and puromycin. The exposure of the enzyme to supra-optimal temperatures uncovers a varying behaviour as a function of low or high concentrations of substrate. There is an activating effect by the substrate in the presence of a high GDP-mannose concentration, after preincubation at supra-optimal temperature only.

Subcellular distribution and metabolic fate of exogenous ceramides taken up by HL-60 cells.

Ceramides (Cer) are key intermediates in the metabolism of sphingomyelin and are also important second messengers. We report that natural long-chain ceramides added to the incubation medium in microgram amounts are internalized in HL-60 cells as well as the short-chain analogue C2-Cer and targeted to various subcellular compartments. No significant difference was detected in the ability of HL-60 cells to metabolize exogenous Cer containing a short (acetyl) versus long (palmitoyl or oleoyl) acyl chain. After a 2-h incubation time with [14C]-C16 ceramides, most of the cell-bound radioactivity was found in free ceramides. Sphingomyelin was the major metabolized sphingolipid containing labeled ceramides and only a small proportion of exogenous ceramides were converted to neutral glycolipids and gangliosides. Up to 20% of the exogenous ceramides taken up by the cells were recovered in mitochondria, mostly as authentic C16 ceramides and C16 sphingomyelin, along with a trace amount of labeled GM3 ganglioside. These results are consistent with the notion that exogenous natural ceramides enter cells, can be further metabolized in situ and partly targeted to mitochondria, which are known to be involved in the control of programmed cell death.

Glycoprotien biosynthesis in liver mitochondria. II. Mitochondrial localization of mannosyltransferase

Abstract The preparation of liver mitochondria in the pure state, and submitochondrial fractionation in inner membrane, outer membrane and matrix, show that the mannosyltranferase of the mitochondria is localized in the inner membrane.

Impaired glycosylation in liver microsomes of orotic-acid-fed rats.

In rats fed orotic acid, the incorporation in liver subcellular fractions of sugars injected intraperitonealy is altered only for mannose, but not for fucose or galactose. Direct determinations of several glycosyltransferases are done in smooth and rough microsomes: fucosyl-, glactosyl-, N-acetylglucosaminyltransferase activities are at quite similar levels in normal and fatty livers. By contrast, sialyltransferase activity is increased (+50%) in smooth microsomes of fatty livers, while mannosyltransferase activity is inhibited by 30%. These alterations are not caused by interfering reactions (pyrophosphatases or proteases). For the mannosyltransferase activity, the inhibition is found in the dolichylphosphorylmannose intermediates. Kinetic studies suggest that there is deficiency of both enzyme and endogenous dolichyl phosphate.

Characterization of a rat brain fucosyl-transferase.

Rat brain fucosyl‐transferase was solubilized using Triton X–100 detergent, and then purified by electrofocusing.

Further characterization of mitochondrial contact sites: Effect of short-chain alcohols on membrane fluidity and activity

Two mitochondrial contact site-enriched fractions were further characterized by freeze-fracture electron microscopy. Examination of the replicas revealed that these two fractions are in the form of vesicles with membrane sheets attached to the vesicles. The physical state of these fractions has been investigated by means of steady-state fluorescence polarization to assess the effects of alcohols on their fluidity properties and activity. Comparison between intact membranes and their extracted lipids shows that butanol and hexanol induce a differential increase of the overall membrane fluidity in the two contact site-enriched fractions. This alteration in the membrane dynamics leads to a complex modulation of cytochrome c oxidase activity in both fractions. These results bring further evidence for the existence of morphologically and functionally distinct domains in mitochondrial membranes.