Expression of a mutant prohibitin from the aP2 gene promoter leads to obesity-linked tumor development in insulin resistance-dependent manner.

A critical unmet need for the study of obesity-linked cancer is the lack of preclinical models that spontaneously develop obesity and cancer sequentially. Prohibitin (PHB) is a pleiotropic protein that has a role in adipose and immune functions. We capitalized on this attribute of PHB to develop a mouse model for obesity-linked tumor. We achieved this by expressing Y114F-PHB (m-PHB) from the aP2 gene promoter for simultaneous manipulation of adipogenic and immune signaling functions. The m-PHB mice develop obesity in a sex-neutral manner, but only male mice develop impaired glucose homeostasis and hyperinsulinemia similar to transgenic mice expressing PHB. Interestingly, only male m-PHB mice develop histiocytosis with lymphadenopathy, suggesting that metabolic dysregulation or m-PHB alone is not sufficient for the tumor development and that both are required for tumorigenesis. Moreover, ovariectomy in female m-PHB mice resulted in impaired glucose homeostasis, hyperinsulinemia and consequently tumor development similar to male m-PHB mice. These changes were not observed in sham-operated control m-Mito-Ob mice, further confirming the role of obesity-related metabolic dysregulation in tumor development in m-PHB mice. Our data provide a proof-of-concept that obesity-associated hyperinsulinemia promotes tumor development by facilitating dormant mutant to manifest and reveals a sex-dimorphic role of PHB in adipose-immune interaction or immunometabolism. Targeting PHB may provide a unique opportunity for the modulation of immunometabolism in obesity, cancer and in immune diseases.

Chlorophenol production by anaerobic microorganisms: transformation of a biogenic chlorinated hydroquinone metabolite.

Chlorinated hydroquinones of biological origin are fully dechlorinated to 1,4-dihydroquinone by anaerobic bacteria such as Desulfitobacterium spp. (C. E. Milliken, G. P. Meier, J. E. M. Watts, K. R. Sowers, and H. D. May, Appl. Environ. Microbiol. 70:385-392, 2004). In the present study, mixed microbial communities from Baltimore Harbor sediment and a pure culture of Desulfitobacterium sp. strain PCE1 were discovered to demethylate, reductively dehydroxylate, and dechlorinate chlorinated hydroquinones into chlorophenols. Mixed microbial cultures from a freshwater source and several other desulfitobacteria in pure culture did not perform these reactions. Desulfitobacterium sp. strain PCE1 degraded 2,3,5,6-tetrachloro-4-methoxyphenol, a metabolite of basidiomycete fungi, to 2,3,5,6-tetrachlorophenol and 2,3,5-trichlorophenol, recalcitrant compounds that are primarily synthesized anthropogenically.

Microbial anaerobic demethylation and dechlorination of chlorinated hydroquinone metabolites synthesized by basidiomycete fungi.

The synthesis and degradation of anthropogenic and natural organohalides are the basis of a global halogen cycle. Chlorinated hydroquinone metabolites (CHMs) synthesized by basidiomycete fungi and present in wetland and forest soil are constituents of that cycle. Anaerobic dehalogenating bacteria coexist with basidiomycete fungi in soils and sediments, but little is known about the fate of these halogenated fungal compounds. In sediment microcosms, the CHMs 2,3,5,6-tetrachloro-1,4-dimethoxybenzene and 2,3,5,6-tetrachloro-4-methoxyphenol (TCMP) were anaerobically demethylated to tetrachlorohydroquinone (TCHQ). Subsequently, TCHQ was converted to trichlorohydroquinone and 2,5-dichlorohydroquinone (2,5-DCHQ) in freshwater and estuarine enrichment cultures. Screening of several dehalogenating bacteria revealed that Desulfitobacterium hafniense strains DCB2 and PCP1, Desulfitobacterium chlororespirans strain Co23, and Desulfitobacterium dehalogenans JW/DU1 sequentially dechlorinate TCMP to 2,3,5-trichloro-4-methoxyphenol and 3,5-dichloro-4-methoxyphenol (3,5-DCMP). After a lag, these strains demethylate 3,5-DCMP to 2,6-DCHQ, which is then completely dechlorinated to 1,4-dihydroquinone (HQ). 2,5-DCHQ accumulated as an intermediate during the dechlorination of TCHQ to HQ by the TCMP-degrading desulfitobacteria. HQ accumulation following TCMP or TCHQ dechlorination was transient and became undetectable after 14 days, which suggests mineralization of the fungal compounds. This is the first report on the anaerobic degradation of fungal CHMs, and it establishes a fundamental role for microbial reductive degradation of natural organochlorides in the global halogen cycle.

Radioiodination of the stable metabolic fragment of bradykinin, RPPGF.

Arg-Pro-Pro-Gly-Phe (RPPGF, BK[1-5]), is a stable metabolite of the peptide hormone bradykinin. Considering the short half-life of bradykinin (BK, approximately 15 secs), RPPGF has been used as a marker for BK's endogenous generation. A lack of a radioiodinated RPPGF has precluded the development of a radioimmunoassay for this peptide. The present study describes a two-step reaction that allows for the incorporation of 125I into the aromatic ring of the phenylalanine of RPPGF. This radioiodinated analog is recognized by an antibody to RPPGF, demonstrating its utility for the development of a radioimmunoassay for measurements of RPPGF, a stable metabolic product of bradykinin.

Expression, characterization, and purification of C-terminally hexahistidine-tagged thromboxane A2 receptors.

Thromboxane A2 (TxA2) receptors belong to the class of G-protein-coupled receptors. Knowledge of the relationship of structure to function for TxA2 receptors is limited because of their low levels of expression, lengthy purification procedures and poor recoveries. A C-terminal hexahistidine-tag (C-His) was ligated to the alpha-isoform of TxA2 receptors and expressed in COS-7 and Chinese hamster ovary cells. The C-His-TxA2 receptors bound the radioligands 125I-7-[(1R,2S,3S,5R)-6, 6-dimethyl-3-(4-benzenesulfonylamino)bicyclo[3.1. 1]hept-2-yl]-5(Z)-heptenoic acid, an antagonist, and 125I-[1S-1alpha, 2beta(5Z),3alpha(1E,3S*), 4alpha]-7-[3[(3-hydroxy-4-(4'-phenoxy)-1butenyl)-7-oxabicycl o-[2.2. 1]heptan-2-yl]-5-heptanoic acid, an agonist, with affinities not significantly different from those of the wild type (wt)-TxA2 receptors. LipofectAMINE transfection of the cDNAs resulted in high levels of expression (Bmax = 95 +/- 6 pmol/mg) of the C-His-TxA2 receptors. In competition binding studies the IC50 values of five different ligands were not significantly different between C-His-TxA2 and wt-TxA2 receptors. Agonist-induced stimulation of cAMP and total inositol phosphate formation were not significantly different between the two receptors. Purification on a Ni2+-NTA column resulted in a rapid (within 4 h) purification with a 36 +/- 2% recovery and a 30 +/- 6-fold purification (n = 5). The partially purified receptors were resolved on SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, dissolved in acetone/trifluoroacetic acid/hexafluoroisopropanol/sinapinic acid, and successfully subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The results suggest that the combination of a high level of expression of C-His-TxA2 receptors and a rapid purification procedure followed by SDS- polyacrylamide gel electrophoresis may provide a useful approach for mass-spectrometry based structure-function and other studies of TxA2 receptors.

Utilization of alcohols by plant and mammalian phospholipase D.

The transphosphatidylation reaction is a unique property of phospholipase D (PLD). In this study, the abilities of plant and mammalian PLDs to utilize straight chain and branched alcohols for transphosphatidylation were analyzed and compared. PLD from peanut utilizes C1 to C8 primary alcohols and gives maximal reaction with butanol. In contrast, PLD from A7r5 vascular smooth muscle cells gives maximal reaction with pentanol and does not utilize octanol. Secondary and tertiary alcohols are not substrates for either enzyme. For branched alcohols, activity increases with distance from the alcohol to the branch point. Competition studies indicated that secondary alcohols cannot access the binding pocket. Thus, PLDs have a water/alcohol binding site with defined steric and hydrophobic parameters.

Characterization of the cloned HEL cell thromboxane A2 receptor: evidence that the affinity state can be altered by G alpha 13 and G alpha q.

Thromboxane A2 (TXA2) induces activation of platelets and vascular smooth muscle contraction via cell surface receptors. A platelet type TXA2 receptor from the megakaryocyte-like HEL cell was cloned with a deduced amino acid sequenced identical to that previously reported for the human placental TXA2 receptor. Transient expression of the HEL cell TXA2 receptor cDNA and radioligand binding studies with the agonist 125I-BOP showed a single class of binding sites with an affinity comparable to a low affinity platelet TXA2 receptor. Using a series of 13-azapinane TXA2 analogs, which discriminate between TXA2 receptor subtypes in platelets and vascular smooth muscle, we found that the cloned HEL cell TXA2 receptor is characteristic of a platelet type TXA2 receptor and that its binding characteristics are different from those of vascular smooth muscle cells. The affinity of the HEL cell TXA2 receptor for 125I-BOP was significantly (P < .05) increased upon co-transfection with G alpha 13 alone, or with G alpha q alone and with G alpha 13 and G alpha 12 together (n = 4-6). GTP gamma S significantly (P < .05) decreased the affinity of the receptor for 125I-BOP in COS-7 cell membranes coexpressing HEL-TXR and G alpha 13 to a value comparable to HEL-TXA2 receptor alone. We conclude that 1) the cloned HEL cell TXA2 receptor has pharmacological characteristics of a low affinity platelet type receptor and 2) that the affinity state of this receptor may be influenced by interaction with G alpha 13 and G alpha q.

Prochiral sulfides as in vitro probes for multiple forms of the flavin-containing monooxygenase.

A homologous series of alkyl-substituted p-tolyl sulfides have been synthesized and evaluated as in vitro, isozyme-selective substrate probes for the microsomal flavin containing monooxygenases. Straight-chain and branched-chain alkyl homologs were metabolized to the corresponding (R)- and (S)-sulfoxides which were analyzed by chiral phase high-performance liquid chromatography. Initial studies demonstrated that the stereochemical composition of alkyl p-tolyl sulfoxides generated by FMO2, purified from rabbit lung, was a function of the degree of steric crowding about the prochiral center. In contrast, purified rabbit liver FMO1 formed the (R)-sulfoxide from the n-alkyl series of substrates in a highly stereoselective manner (> 90%). Similar results were obtained with these two rabbit cDNAs expressed in E. coli. In contrast to rabbit FMO1 and FMO2, a characteristic feature of catalysis by cDNA-expressed rabbit FMO3 was the lack of stereoselectivity observed for formation of methyl p-tolyl sulfoxide. Collectively, these data demonstrate that the stereochemical composition of sulfoxides generated from the n-alkyl series of sulfides is isozyme-dependent. Metabolism of methyl p-tolyl sulfide by detergent-solubilized hepatic microsomes from a wide variety of experimental animals yielded predominantly (R)- methyl p-tolyl sulfoxide, which, at least in rabbit liver, is indicative of catalysis dominated by FMO1. However, solubilized human and macaque liver preparations catalyzed this reaction in a relatively non-stereoselective manner. Macaque liver FMO was purified and the metabolite profile generated from the n-alkyl p-tolyl sulfides was found to be most similar to rabbit FMO3. Moreover, antibodies directed against macaque liver FMO selectively reacted with rabbit FMO3 and a microsomal protein expressed in adult human, but not fetal human liver, adult human kidney or adult human lung. Therefore, an FMO isoform expressed selectively in adult primate liver has catalytic and immunochemical properties consistent with its classification in the FMO3 family.

Prochiral sulfoxidation as a probe for multiple forms of the microsomal flavin-containing monooxygenase: studies with rabbit FMO1, FMO2, FMO3, and FMO5 expressed in Escherichia coli.

Multiple forms of the microsomal flavin-containing monooxygenase (FMO) exist in rabbit tissues. In order to better understand the catalytic properties of these isoforms, we have expressed rabbit FMO1, FMO2, FMO3, and FMO5 in Escherichia coli and examined their kinetic parameters and prochiral selectivities for the sulfoxidation of methyl-, ethyl-, n-propyl-, and n-butyl-substituted p-tolyl sulfides. FMO1 and FMO2 exhibited high affinities for these substrates (Km < 10 microM), in contrast to the low-affinity FMO3 form for which Km values ranged between 100 and 280 microM. FMO5 did not form quantifiable levels of sulfoxide metabolites at the concentrations used. The individual stereochemical metabolite profiles generated by FMO1, FMO2, and FMO3 were unique and served to distinguish among these three cDNA-expressed isoforms. To investigate the relationship between the kinetic parameters for the cDNA-expressed enzymes and the native microsomal enzymes, we examined the kinetics and stereoselectivity of metabolism of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes. We analyzed these data with respect to FMO1 and FMO3, the two predominant hepatic isoforms. Sulfoxidation of methyl p-tolyl sulfide by FMO1 and FMO3 solubilized from E. coli microsomes proceeded with apparent Kms of 18 and 270 microM, respectively. FMO1 was essentially stereospecific for formation of (R)-methyl p-tolyl sulfoxide, whereas FMO3 generated this metabolite with little prochiral selectivity. Sulfoxidation of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes was best described by a two-enzyme model, with apparent Km values of 11 and 340 microM. The enantiomeric purity of the (R)-methyl p-tolyl sulfoxide metabolite, generated by detergent-solubilized rabbit liver microsomes, decreased progressively with increasing substrate concentration, from a high of 96% enantiomeric excess at a substrate concentration of 5 microM to a low of 63% enantiomeric excess at a substrate concentration of 2 mM. The kinetic and stereochemical properties of the high-affinity and low-affinity components of detergent-solubilized rabbit liver microsomes were similar to those exhibited by cDNA-expressed FMO1 and FMO3, respectively. Therefore, methyl p-tolyl sulfide, used at the appropriate substrate concentrations, is useful for discriminating between FMO1- and FMO3-mediated catalysis in rabbit liver microsomal preparations.

A fluorescent assay for agonist-activated phospholipase D in mammalian cell extracts.

Phospholipase D (PLD) is activated in mammalian cells in response to a wide variety of stimuli. A rapid assay for agonist-activated PLD activity in cell extracts was developed, utilizing a fluorescent derivative of phosphatidylcholine as substrate. Utilization of the substrate was assessed following thin-layer chromatography of the reaction mixture. Hydrolysis products generated by phospholipases D, C, and A2 could be visualized in the same reaction. Phorbol ester and vasopressin increased PLD activity in intact A7r5 vascular smooth muscle cells, as measured by an isotopic labeling method. Using the in vitro fluorescent assay, enhanced PLD activity was detected in membranes prepared from A7r5 cells that had been treated with phorbol ester or vasopressin. The agonist-activated activity was independent of phosphorylation occurring during the course of the assay. PLD activity was detected, in varying amounts, in membranes prepared from a variety of different mouse tissues. These results show that a fluorescent assay can be used to rapidly assess the activity of PLD and other phosphatidylcholine-utilizing phospholipases in cell and tissue extracts. The effects of agonists on PLD activity can be retained and quantitated in a broken cell preparation, permitting characterization of the agonist-activated form of the enzyme.

Chemical analysis and hemolytic activity of the fava bean aglycon divicine.

Divicine is an unstable aglycon metabolite of the fava bean pyrimidine beta-glucoside vicine. Divicine has long been thought to be a mediator of an acute hemolytic crisis, known as favism, in susceptible individuals who ingest fava beans (Vicia faba). However, a recent report has questioned the chemical identity of the divicine that was used in most of the studies on divicine hemotoxicity. The present study was undertaken to examine the hemolytic potential of synthetic divicine. Divicine was synthesized and its identity and purity were confirmed by HPLC, mass spectrometry, and NMR spectroscopy. The stability and redox behavior of divicine, under physiological conditions, were examined by HPLC and cyclic voltammetry. The data indicate that divicine is readily oxidized under aerobic conditions; however, it was sufficiently stable at pH 7.4 to permit its experimental manipulation. When 51Cr-labeled rat erythrocytes were exposed in vitro to the parent glucoside, vicine (5 mM), and then readministered to rats, no decrease in erythrocyte survival was observed. In contrast, erythrocyte survival was dramatically reduced by in vitro exposure to divicine (1.5 mM). These data demonstrate that divicine is a direct-acting hemolytic agent and thus may be a mediator of the hemolytic crisis induced by fava bean ingestion.

Stereoselective sulfoxidation by human flavin-containing monooxygenase. Evidence for catalytic diversity between hepatic, renal, and fetal forms.

The stereoselective formation of p-tolyl methyl sulfoxide from the corresponding sulfide has been examined in detergent-solubilized human adult liver, adult kidney, and fetal liver microsomes, in order to compare the functional activities of human flavin-containing monooxygenase(s). Solubilization with detergent was performed to eradicate the contribution that cytochrome P-450 would make to the net stereochemistry. Consistent with studies in experimental animal livers, solubilized human fetal liver and adult kidney microsomes formed (R)-p-tolyl methyl sulfoxide in greater than 86% enantiomeric excess. These enzyme activities were sensitive to methimazole inhibition and were markedly thermolabile in the absence of NADPH, attributes that are consistent with a flavin-containing monooxygenase-mediated process. However, solubilized adult human liver microsomes displayed little stereoselectivity (0-40% enantiomeric excess) for the formation of (R)-p-tolyl methyl sulfoxide, although this reaction also displayed several of the characteristics of a flavin-containing monooxygenase-dependent process, including sensitivity to methimazole inhibition and NADPH protection against heat inactivation. Furthermore, this lack of stereoselectivity was not attenuated by the inclusion of activated oxygen scavengers in reaction mixtures. Human tissue metabolite profiling was further studied by using the ethyl, propyl, and isopropyl p-tolyl sulfides. Parallel changes in product stereochemistry as a function of increasing steric bulk were observed with the fetal liver and adult kidney tissue, whereas an anomalous profile was again observed with adult human liver. These data are consistent with the presence of functionally discrete complements of the flavin-containing monooxygenase in detergent-solubilized adult and fetal human liver microsomes.

Stereoselective sulfoxidation of a series of alkyl p-tolyl sulfides by microsomal and purified flavin-containing monooxygenases.

The enantioselective sulfoxidation of a series of alkyl p-tolyl sulfides was compared using purified rabbit lung and mini-pig liver flavin-containing monooxygenase (FMO). Analysis was performed by chiral-phase high pressure liquid chromatography, which afforded baseline resolution of each pair of enantiomers. The extent of enantioselective sulfoxidation was found to be a function of (a) the isozyme employed, (b) the steric bulk of the alkyl substituent, and (c) pH. At pH 8.5, rabbit lung FMO catalyzed the oxidation of methyl, ethyl, propyl, and isopropyl sulfides to products with greater than 99, 91, 85, and 63% (R)-(+)-stereochemistry, respectively. Corresponding values for the mini-pig liver form were 91, 82, 72, and 41% (R)-(+)-sulfoxide. The stereochemical profile obtained with the isolated rabbit lung form could be duplicated exactly in microsomal preparations if precautions were taken to abolish the contribution that P-450 makes to net stereochemistry. It was noted that increasing the reaction mixture pH from 8.5 to 10 led to a decrease in the stereochemical purity of products obtained from the lung form. In contrast, the stereochemical profile obtained with the isolated mini-pig liver form could not be exactly duplicated in suitably treated microsomal preparations. No evidence for multiple forms of mini-pig liver FMO was obtained, and it was concluded that discrepancies between microsomal and purified FMO metabolic profiles were most consistent with a minor modification to active site geometry occurring during purification of the mini-pig form. These data show that the active site chirality of rabbit lung and mini-pig liver FMO is largely retained following removal from microsomal membranes. Qualitative similarities in the structure-activity relationships exhibited by microsomal or purified FMO from rabbit lung and mini-pig liver suggest some conservation of active site geometry between these two otherwise distinct FMOs. Quantitative differences in the structure-activity relationships exhibited by the two FMO forms indicate that analysis of product stereochemistry may be a useful method for the discrimination of catalytically distinct FMO isozymes.

Isolation and identification of a novel human metabolite of cyclosporin A: dihydro-CsA M17.

A novel metabolite of cyclosporin A was observed in human blood and urine. An analytical sample of this metabolite was isolated from human urine and the structure was determined to be (8-hydroxy-6,7-dihydro-MeBMT1) cyclosporin based on the 1H-NMR, 13C-NMR, FAB-MS, and HPLC characteristics of the biological sample as well as by comparison with a synthetically derived authentic sample. The significance of this metabolite in terms of the pathway by which cyclosporin A is metabolized is discussed.

Formation of cyclic 1,N2-propanodeoxyguanosine and thymidine adducts in the reaction of the mutagen 2-bromoacrolein with calf thymus DNA.

The interaction of the mutagen 2-bromoacrolein (2BA) with DNA and thymidine was studied in vitro by reaction of [3-3H]2BA with thymidine, RNA, single-stranded DNA, and double-stranded DNA in phosphate buffer (pH 7.4). After purification of the nucleic acids, they were incubated at alkaline pH to convert any (hydroxybromo)propano(deoxy)-guanosine adducts to their dihydroxy analogues. After acid or enzymatic hydrolysis, the hydrolysates were analyzed by reversed-phase high-performance liquid chromatography. At a concentration of 1.6 mM, the fraction of 2BA that became covalently bound to DNA was 2.3% of the amount added. Only 3% of the radioactivity bound to DNA after extensive purification could be accounted for as cyclic 1,N2-(6,7-dihydroxy)-propanoguanine adducts. More 2BA became covalently bound to single-stranded DNA and RNA as compared with double-stranded DNA. However, high-performance liquid chromatographic analyses showed that formation of cyclic 1,N2-(6,7-dihydroxy)propanoguanine adducts was also a minor reaction with these macromolecules. Because these data showed that other type(s) of reaction(s) are more important in the reaction of 2BA with nucleic acids, we have investigated the reaction of 2BA with other nucleosides. It was found that 2BA reacted well with thymidine in vitro, and the major product was identified by 500 MHz 1H and 75.43 MHz 13C nuclear magnetic resonance and thermospray mass spectrometry as 3-(2"-bromo-3"-oxopropyl)thymidine. This adduct was unstable and decomposed upon storage. After enzymatic hydrolysis of [3H]2BA-modified double-stranded DNA and subsequent analysis of the hydrolysate by high-performance liquid chromatography, 22% of the covalently bound radioactivity to DNA coeluted with decomposition products of the 3-(bromooxypropyl)thymidine adduct. This indicates that reaction of 2BA with this nucleotide in DNA is a major reaction.

Enantiotopic differentiation during the biotransformation of valproic acid to the hepatotoxic olefin 2-n-propyl-4-pentenoic acid.

The enantiomers of 2-[( 3-13C]-n-propyl)pentanoic acid [(R)- and (S)-[13C]VPA] were employed as metabolic probes to investigate stereochemical aspects of the biotransformation of valproic acid (VPA) to 2-n-propyl-4-pentenoic acid (delta 4-VPA), a hepatotoxic metabolite of VPA. When incubated with hepatocytes freshly isolated from untreated male rats, each labeled substrate (initial concentration 1.0 mM) underwent metabolism to [13C]-delta 4-VPA, the formation of which was time-dependent and occurred at a rate of ca. 20 ng/(10(6) cells.4-h incubation). Analysis of this unsaturated metabolite by GC-MS techniques revealed that, following incubation of (R)-[13C]VPA, desaturation had taken place preferentially (by a factor of approximately 4) on the labeled propyl group (i.e., on the R side chain). Parallel incubations with (S)-[13C]VPA supported this conclusion, in that metabolism of this isotopic variant of VPA led to a terminal olefin that also was predominantly (83 +/- 2%) of R configuration (in this case oxidized selectively on the unlabeled side chain). Hence, biotransformation of VPA to delta 4-VPA in rat hepatocytes occurs with marked enantiotopic differentiation, favoring production of the R enantiomer of this chiral metabolite. When rats were pretreated with phenobarbital (80 mg kg-1 day-1 ip for 3 days) prior to isolation of hepatocytes, the overall rate of metabolism of VPA to delta 4-VPA over the 4-h incubation period increased approximately 3-fold, while the degree of product enantioselectivity was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of hydration state and thiol-disulfide status in the control of thermal stability and protein synthesis in wheat embryo.

Reduced (GSH), oxidized (GSSG), and protein-bound (PSSG) glutathione were determined in dry and hydrated wheat embryos. Dry embryos contained about 0.6 mumoles per gram dry weight each of GSSG and PSSG, and these levels declined 5- to 10-fold within minutes after the onset of imbibition. GSH declined from about 8 to 2 mumoles per gram over a period of 90 minutes. Similar changes occurred when embryos were hydrated by storage at 100% relative humidity. The decline in glutathione levels was not reversed upon redrying hydrated embryos. About 40% of the cysteine residues of embryo protein was found to be in the disulfide form in both dry and imbibed embryos. The ability of wheat embryos to withstand heat shock was shown to correlate with water content but not GSSG content. Incorporation of [(35)S]methionine into protein was studied using a system based upon wheat embryo extract (S23). Incorporation rate was found to be sensitive to the nature of thiol added to the system and to be decreased by GSSG. S23 exhibited a substantial capacity to reduce GSSG and preparation of S23 having a GSSG content comparable to dry embryos required addition of large amounts of GSSG to the extraction buffer S23 prepared in this fashion exhibited a marked decrease in ability to support protein synthesis. These results suggest that the early decrease in GSSG during germination is necessary for optimal protein synthesis in wheat embryo.

The glutathione thiol-disulfide status in the sea urchin egg during fertilization and the first cell division cycle.

The intracellular levels of GSH, GSSG, and protein-glutathione disulfide (protein-SSG) have been measured in the eggs and developing embryos of the sea urchins Lytechinus pictus and Stronglyocentrotus purpuratus. Total cellular glutathione is maintained in a very highly reduced state during these initial stages of development. Thus for unfertilized eggs of L. pictus the results (mumol/g dry weight) were 11 +/- 1 for GSH, 0.02 +/- 0.01 for GSSG, and 0.07 +/- 0.02 for protein-SSG. No significant change in these values was observed upon fertilization of the eggs or during the first cell division cycle. The values obtained with S. purpuratus were somewhat greater, but were also found to exhibit no significant variations upon fertilization or cell division. These observations indicate that changes in the total cellular glutathione thiol-disulfide status are not involved in the control mechanisms which operate during fertilization of the first cell division cycle in the sea urchin egg.