Glutathione Turnover in Cultured Astrocytes: Studies with [15N]Glutamate

The incorporation of [15N]glutamic acid into glutathione was studied in primary cultures of astrocytes. Turnover of the intracellular glutathione pool was rapid, attaining a steady state value of 30.0 atom% excess in 180 min. The intracellular glutathione concentration was high (20-40 nmol/mg protein) and the tripeptide was released rapidly into the incubation medium. Although labeling of glutathione (atom% excess) with [15N]glutamate occurred rapidly, little accumulation of 15N in glutathione was noted during the incubation compared with 15N in aspartate, glutamine, and alanine. Glutathione turnover was stimulated by incubating the astrocytes with diethylmaleate, an electrophile that caused a partial depletion of the glutathione pool(s). Diethylmaleate treatment also was associated with significant reductions of intraastrocytic glutamate, glycine, and cysteine, i.e., the constituents of glutathione. Glutathione synthesis could be stimulated by supplementing the steady-state incubation medium with 0.05 mM L-cysteine, such treatment again partially depleting intraastrocytic glutamate and causing significant reductions of 15N labeling of both alanine and glutamine, suggesting that glutamate had been diverted from the synthesis of these amino acids and toward the formation of glutathione. The current study underscores both the intensity of glutathione turnover in astrocytes and the relationship of this turnover to the metabolism of glutamate and other amino acids.     

Structure-function studies on bacteriorhodopsin. VIII. Substitutions of the membrane-embedded prolines 50, 91, and 186: the effects are determined by the substituting amino acids

To study their role in the structure and function of bacteriorhodopsin, three prolines, presumed to be in the membrane-embedded alpha-helices, have been individually replaced as follows: Pro-50 and Pro-91 each by Gly and Ala and Pro-186 by Ala, Gly, and Val. The mutants of Pro-50 and Pro-91 all showed normal chromophore and proton pumping. However, the rates of regeneration of the chromophore in Pro-50----Ala, Pro-91----Ala and ----Gly with all-trans-retinal were about 30-fold slower than that in the wild-type, whereas the chromophore regeneration rate in Pro-50----Gly was 10-fold faster than in the wild-type. While, Pro-186----Ala regenerated the wild-type chromophore, the mutants Pro-186----Val and Pro-186----Gly showed large blue shifts (about 80 nm) in the chromophore regenerated with all-trans-retinal and showed no apparent dark-light adaptation. Pro-186----Gly first regenerated the wild-type chromophore with 13-cis-retinal which was thermally unstable and rapidly converted to the blue-shifted chromophore obtained with all-trans-retinal. High salt concentration restored the wild-type purple chromophore in the Pro-186----Gly mutant. Thus, in this mutant, the protein interconverts between two conformational states. Pro-186----Ala and Pro-186----Gly showed about 65%, whereas Pro-186----Val showed 10-20% of the normal proton pumping.     

Function of plastid mRNA 3' inverted repeats. RNA stabilization and gene-specific protein binding

Plastid protein coding regions in plants are generally flanked by 3' inverted repeat (IR) sequences. In a previous work (Stern, D. B., and Gruissem, W. (1987) Cell 51, 1145-1157), we have shown that their role may be in RNA stabilization and as a processing signal that establishes the mature mRNA 3' end. In this report we have investigated the stability and protein interaction of chloroplast mRNA 3' IR-RNA sequences in more detail. Progressive deletions into the 3' IR-RNA sequences for the chloroplast cytochrome b6/f subunit IV (petD) mRNA reduce the stability of the RNA, indicating that the potential to form a stem/loop is a minimum requirement for petD 3' IR-RNA stability in vitro. Specific point mutants also destabilize the processed 3' IR-RNA, suggesting an important role for the primary sequence. Gel mobility shift and UV-cross-linking analysis has shown that 3' IR-RNAs of petD and two other chloroplast mRNAs (rbcL and psbA) interact with proteins in vitro. Comparison of the bound petD 3' IR-RNA proteins with proteins that bind to rbcL and psbA reveals that binding of certain proteins is gene-specific. Also, precursor and processed petD 3' IR-RNAs bind different sets of proteins. A single nucleotide transversion (T----A) near the base of the stem eliminates the binding of a 29-kDa protein to the petD 3' IR-RNA precursor. We discuss the possible role of 3' IR-RNA-protein interactions in plastid mRNA 3' end maturation and differential mRNA stability.     

The effects of 2-deoxy-D-glucose and sympathetic denervation of brown fat GDP binding in Sprague-Dawley rats

Central administration of 2-deoxy-D-glucose (2-DG) decreases brown fat thermogenesis. This effect is suggested to be mediated via a central control mechanism. Our study was designed to determine the importance of the sympathetic nervous system in the response of brown fat to intraperitoneal (i.p.) injection of 2-DG. Unilateral denervation of interscapular brown adipose tissue (IBAT) was performed on male Sprague-Dawley rats (300 g body weight). Nine days after surgery, rats were injected i.p. with either saline vehicle (0.9% sodium chloride) or 2-DG (360 mg/kg wt) and then killed one hour later. Sympathetic denervation resulted in 50% decreases in total IBAT protein and in mitochondrial protein recovered. In the denervated lobes, mitochondrial GDP binding (expressed as nmol/mg mitochondrial protein and as total activity recovered) was decreased to 36% and 18%, respectively. Injection of 2-DG did not change mitochondrial protein content in either the innervated or denervated IBAT. In the innervated lobes, 2-DG significantly lowered GDP binding to 55% of that in saline-treated animals, whether expressed per mg mitochondrial protein or as total recovered activity. In contrast, 2-DG did not further decrease GDP binding in the denervated lobes. In conclusion, the effects of i.p. injection of 2-DG on brown fat thermogenesis (as evidenced by GDP binding) appear to be primarily mediated via the sympathetic nervous system.