ToxSci Advance Access originally published online on April 28, 2004
Toxicological Sciences 2004 81(2):371-378; doi:10.1093/toxsci/kfh154
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Toxicological Sciences vol. 81 no. 2 © Society of Toxicology 2004; all rights reserved.
HIGHLIGHTED ARTICLE |
Spatial Activities and Induction of Glutamate-Cysteine Ligase (GCL) in the Postimplantation Rat Embryo and Visceral Yolk Sac
,1
* Department of Biochemistry, Emory University, Atlanta, Georgia 30322, and Toxicology Program, Department of Environmental Health Sciences, 1420 Washington Heights, University of Michigan, Ann Arbor, Michigan 48109-2029
Received March 2, 2004; accepted April 13, 2004
Glutathione (GSH) synthesis is differentially regulated in the embryo and visceral yolk sac (VYS) of the developing rat conceptus. The innate capacity to respond to environmental insult and chemical exposure by inducing de novo GSH synthesis may help to determine overall cell sensitivity and/or resistance to chemically induced malformation. Specific activities of glutamate-cysteine ligase (GCL), the rate limiting enzyme in GSH synthesis, were determined by measuring the formation of 
-glutamylcysteine (GC) in homogenates prepared from rat embryos and VYSs. GC formation increased linearly with time and with relative protein concentration. Specific activities were found to be 60.5 ± 3.2 and 118.9 ± 4.2 pmol GC/mg protein/min in the gestational day (GD) 10 embryo and VYS, respectively, and 22.7 ± 0.4 and 71.3 ± 0.6 pmoles GC/mg protein/min in the respective GD 11 embryo and VYS. Apparent kinetic constants determined from embryo and VYS homogenates gave respective apparent Km values for glutamate of 0.75 and 1.38 mM and for cysteine 0.03 mM in both tissues. Apparent Vmax values were higher in the VYS in each case, corresponding with a lower apparent Km and higher GCL activity. GCL specific activities increased significantly following a 24 h in vitro exposure to diethyl maleate (DEM) and diamide, but remained unchanged following exposure to prostaglandin A2 (PGA2) and t-butylated hydroxytoluene (BHT). Basal expression of GCL catalytic subunit (GCLC) and regulatory subunit (GCLR) was 59- and 25-fold higher in VYS, respectively, compared to the embryo. Quantitative real-time fluorescence reverse transcriptase polymerase chain reaction (RT-PCR) showed that following DEM and diamide treatment, GCLC expression increased up to 19-fold in embryonic tissues but was not induced in the VYS. Only DEM increased the expression of the light/regulatory subunit GCLR in the embryo (8-fold). Densitometry of immunoblots revealed approximately 75% more GCLC in the VYS than in the embryo. Following treatments, a marked increase was induced in embryonic GCLC content with both DEM (85%) and diamide (19%), but in the VYS, only DEM caused an increase in GCLC protein (38%).
Key Words: glutamate-cysteine ligase; glutathione; visceral yolk sac; rat conceptus; -glutamylcysteine; rat embryo.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Tsai-Turton, B. N Nakamura, and U. Luderer Induction of Apoptosis by 9,10-Dimethyl-1,2-Benzanthracene in Cultured Preovulatory Rat Follicles Is Preceded by a Rise in Reactive Oxygen Species and Is Prevented by Glutathione Biol Reprod, September 1, 2007; 77(3): 442 - 451. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Luo and D. B. DeFranco Opposing Roles for ERK1/2 in Neuronal Oxidative Toxicity: DISTINCT MECHANISMS OF ERK1/2 ACTION AT EARLY VERSUS LATE PHASES OF OXIDATIVE STRESS J. Biol. Chem., June 16, 2006; 281(24): 16436 - 16442. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Yan and B. F. Hales Activator Protein-1 (AP-1) DNA Binding Activity Is Induced by Hydroxyurea in Organogenesis Stage Mouse Embryos Toxicol. Sci., June 1, 2005; 85(2): 1013 - 1023. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. S. Henshel Control of Glutathione Synthesis in Early Embryo Development Toxicol. Sci., October 1, 2004; 81(2): 257 - 259. [Full Text] [PDF]
|
|