Articulo glyoxylato

FEBS Letters 390 (1996) 258-260

FEBS 17277

Induction of glyoxylate cycle enzymes in rat liver upon food starvation
Vasily N. Popov a, Abir U. Igamberdiev a,*, Claus Schnarrenberger b,**, Sergei V. Volvenkin ~
aDepartment of Plant Physiology and Biochemistry, Voronezh State University, Voronezh 394693, Russian Federation blnstitute for Plant Physiology and Microbiology, Freie UniversitdtBerlin, KOnigin-Luise-Str. 12-16a, D-14195 Berlin, Germany
Received 24 May 1996; revised version received 13 June 1996

Abstract The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, have been detected in liver of foodstarved rats. Activities became measurable 3 days and peaked 5 days after the beginning of starvation. Both enzymes were found in the peroxisomal cellfraction after organelle fractionation by isopycnic centrifugation. Isocitrate lyase was purified ll2-fold by ammonium sulfate precipitation, and chromotography on DEAE-cellulose and Toyopearl HW-65. The specific activity of the purified enzyme was 9.0 units per mg protein. The Km(isocitrate) was 68 Iz2VIand the pH optimum was at pH 7.4. Malate synthase was enriched 4-fold by ammonium sulfateprecipitation. The enzyme had a Km(acetyl-CoA) of 0.2 [aM, a Km(glyoxylate) of 3 mM and a pH optimum of 7.6.
Key words: Isocitrate lyase; Malate synthase; Rat liver; Glyoxylate cycle

a mechanism of secondary glycogen production, possibly by conversion of fat to carbohydrates. Although other reactions could also account for glycogen synthesis in the liver of starved rats, p r o o f of the presence orabsence of glyoxylate activities in liver of starved rats would be of prime interest because this would document the induction of the glyoxylate cycle in mammalian rat liver. Here, we report the discovery of the key enzymes of the glyoxylate cycle in liver of starved rats as support for the suggestion that prolonged food starvation leads to glycogen formation in liver. We have characterized theactivities of partially purified malate synthase and isocitrate lyase and demonstrate their compartmentalization in the peroxisomal cell fraction.

2. Materials and methods
3-month-old rats (Rattus rattus L.) were fed regularly and then transferred to food starvation and excessive water supply for up to 7 days. Animals were anaesthetized with diethyl ether before decapitation. 1 g of liver wasexcised and homogenized in 10 ml of 0.4 M sucrose, 50 mM Tris-HC1, pH 7.5, 1 mM MgC12, 1 mM dithiothreitol, and 1 mM EDTA using a mortar and pestle for preparation of crude homogenates. Centrifugation was at 4000xg for 5 min. The supernatant was used for enzyme assays. For preparation of cell organelles, 0.1% Triton X-100 was omitted from the grinding medium, 0.3 M sucrose being included instead.After the preliminary sedimentation of unruptured tissue fragments, nuclei, and cell debris, the supernatant was applied to a continuous sucrose gradient (25-57%) buffered with 50 mM Tris-HC1, pH 7.5, 1 mM MgC12, 1 mM dithiothreitol, and 0.5 mM EDTA. Particles were centrifuged to equilibrium for 2.5 h at 100000Xg. 2-ml fractions were collected, diluted to 0.25 mM sucrose and centrifuged for 15 min at20 000 x g. The pellets were resuspended, each in 50 mM Tris-HC1, pH 7.5, 1 mM MgC12, and 0.1% Triton X-100, and used for enzyme measurements. Enzyme assays were performed at 25°C and followed photometrically. Isocitrate lyase was determined according to the method of Dixon and Kornberg [9]. The assay mixture of 1 ml volume consisted of 50 mM Tris-HC1, pH 7.5, 0.5 mM isocitrate, 5 mM MgCI2, and 4mM phenylhydrazine-HCl. The reaction was followed at 324 nm. An extinction coefficient of 1.67× 10 4 M -i cm -i was used. Malate synthase was measured according to Hock and Beevers [10]. The assay mixture consisted of 50 mM Tris-HC1, pH 7.6, 0.15 mM acetyl-CoA, 2 mM glyoxylate, and 1 mM 5,5'-dithiobis(2-nitrobenzoic acid). The activity was followed at 412 nm. The extinction coefficient was...