Spatial activities and induction of glutamate-cysteine ligase (GCL) in the postimplantation rat embryo and visceral yolk sac.
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 gamma-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 K(m) values for glutamate of 0.75 and 1.38 mM and for cysteine 0.03 mM in both tissues. Apparent V(max) values were higher in the VYS in each case, corresponding with a lower apparent K(m) 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 A(2) (PGA(2)) and t-butylated hydroxytoluene (BHT). Basal expression of GCL catalytic subunit (GCL(C)) and regulatory subunit (GCL(R)) 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, GCL(C) 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 GCL(R) in the embryo (8-fold). Densitometry of immunoblots revealed approximately 75% more GCL(C) in the VYS than in the embryo. Following treatments, a marked increase was induced in embryonic GCL(C) content with both DEM (85%) and diamide (19%), but in the VYS, only DEM caused an increase in GCL(C) protein (38%).
Hansen
Jason M
JM
Department of Biochemistry, Emory University, Atlanta, Georgia 30322, USA.
Lee
Eunyong
E
Harris
Craig
C
D000818:Animals; D001755:Blastocyst / Q000201:enzymology* / Q000473:pathology; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D004151:Dipeptides / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D005260:Female; D005721:Glutamate-Cysteine Ligase / Q000096:biosynthesis* / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D015151:Immunoblotting; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D011247:Pregnancy; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015017:Yolk Sac / Q000201:enzymology* / Q000473:pathology
D017931:DNA Primers; D004151:Dipeptides; D012333:RNA, Messenger; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; C017341:gamma-glutamylcysteine