Characteristics of the protein carrier of the peptide-transport system in the scutellum of germinating barley embryos

Through the use of the protein reagents N-ethylmaleimide, p-chloromercuribenzenesulphonic acid and phenylarsine oxide, it is shown that in the scutellum of the germinating barley embryo, the transport of peptides, but not the transport of amino acids or glucose is specifically thiol-dependent. Furthermore, these essential thiol groups are shown to exist as redox-sensitive, vicinal-dithiols that lie at the substrate-binding sites of the peptide-transport proteins. The binding of N-ethylmaleimide to these dithiols is shown to be very fast, matching the kinetics of inhibition of peptide transport by this reagent. A technique for the specific labelling of the dithiols with N-ethyl[2,3-¹⁴C]maleimide is described, which allows the carrier proteins to be visualized at the scutellar epithelium using radioautography and permits calculation of the approximate amount of peptide-transport protein present per scutellum. In related studies, the importance of arginyl and histidyl residues to both amino-acid and peptide transport is shown, although other residues, e.g. carboxyl ligands do not seem to be critically involved.Abbreviations Ala alanine - Gly glycine - Leu Leucine - NEM N-ethylmaleimide - PAO phenylarsine oxide - PCMBS p-chloromercuribenzenesulphonic acid - Phe phenylalanine     

Modification of oligosaccharide-lipid synthesis and protein glycosylation in glucose-deprived cells

Incubation of vesicular stomatitis virus-infected glucose-starved baby hamster kidney cells with [³⁵S]methionine results in the synthesis of all viral proteins. However, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping, the G protein is abnormally glycosylated. Metabolic labeling of the oligosaccharide-lipid precursors with [³H]mannose for 15 min, followed by Chromatographic and enzymatic analysis, indicates that the radiolabeled lipid-linked oligosaccharides are devoid of glucose in contrast to the glucosylated oligosaccharide-lipids synthesized by cells grown in the presence of glucose. Also, in contrast to control cells, examination of the glycopeptide fraction reveals the presence of [³H]mannose-labeled glycopeptides which are resistant to erado-β-N-acetylglucos-aminidase H and are smaller in size than glycopeptides from mature vesicular stomatitis virus. In order to observe these effects, a minimum time of 5 h of glucose deprivation is necessary and the addition of 55 μm glucose or mannose to the medium reverses these effects. These results indicate that vesicular stomatitis virus-infected BHK cells deprived of glucose are unable to glucosylate the oligosaccharide-lipid intermediates and, consequently, are unable to glycosylate the G protein normally.     

In vivo and in vitro inhibition of lipid-linked saccharide and glycoprotein synthesis in plants by amphomycin

The effect of the polypeptide antibiotic, amphomycin, on the in vitro and in vivo synthesis of polyprenyl-linked sugars and glycoproteins in plants was examined. This antibiotic blocked the transfer of mannose from GDP-[¹⁴C]mannose into mannosyl-phos-phoryl-dolichol by a particulate enzyme preparation from mung beans and also inhibited the transfer of GlcNAc from UDP-[³H]GlcNAc to GlcNAc-pyrophosphoryl-polyisoprenol. The in vitro incorporation of these sugars into trichloroacetic acid-insoluble material was also markedly inhibited by this antibiotic. Since most of the radioactivity incorporated into this insoluble material is rendered water-soluble by treatment with pronase, it seems likely that these sugars are incorporated into glycoproteins whose synthesis is sensitive to amphomycin. Amphomycin also inhibited the transfer of glucose from UDP-[¹⁴C]glucose to steryl glucosides, although this system was less sensitive to antibiotic than was synthesis of the polyprenyl-linked sugars. The antibiotic did not block the in vitro transfer of glucose from UDP-[¹⁴C]glucose to β-glucans. In carrot slice cultures, amphomycin also inhibited the incorporation of [¹⁴C]mannose into glycolipid and glycoprotein, but it did not prevent the incorporation of [¹⁴C]lysine into protein.     

Modification of yeast 3-phosphoglycerate kinase: isolation and sequence determination of a nitrated active-site peptide and isolation of a carboxyl modified active-site peptide.

Previous studies have shown that yeast 3-phosphoglycerate kinase is inhibited by nitration of a single tyrosine residue. Chymotryptic fragmentation of the nitrated protein followed by peptide mapping revealed approximately fifty peptides, one of which was shown to contain a nitrotyrosine residue. Isolation of this unique peptide was accomplished by gel filtration and high voltage paper electrophoresis. The sequence as established by Edman degradation and carboxypeptidase hydrolysis is: Lys-NO₂Tyr-Phe-Phe-Lys. Independent observations on the X-ray crystallographic model of yeast phosphoglycerate kinase provides supportive evindence of this sequence. Additionally, a peptide has been isolated containing an active-site carboxyl residue following modification of the enzyme with [¹⁴C]methoxyamine.     

The mechanism of dextransucrase action: Biosynthesis of branch linkages by acceptor reactions with dextran

Dextransucrase from Leuconostoc mesenteroides B-512 catalyzes the polymerization of dextran from sucrose. The resulting dextran has 95% α-1 → 6 linkages and 5% α-1 → 3 branch linkages. A purified dextransucrase was insolubilized on Bio-Gel P-2 beads (BGD, Bio-Gel-dextransucrase). The BGD was labeled by incubating it with a very low concentration of [¹⁴C]sucrose or it was first charged with nonlabeled sucrose and then labeled with a very low concentration of [¹⁴C]sucrose. After extensive washings with buffer, the ¹⁴C label remained attached to BGD. This labeled material was previously shown to be [¹⁴C]dextran and was postulated to be attached covalently at the reducing end to the active site of the enzyme. When the labeled BGD was incubated with a low molecular weight nonlabeled dextran (acceptor dextran) all of the BGD-bound label was released as [¹⁴C]dextran whereas essentially no [¹⁴C]dextran was released when the labeled BGD was incubated in buffer alone under comparable conditions. The released [¹⁴C]dextran was shown to be a slightly branched dextran by hydrolysis with an exodextranase. Acetolysis of the released dextran gave 7.3% of the radioactivity in nigerose. Reduction with sodium borohydride, followed by acid hydrolysis, gave all of the radioactivity in glucose, indicating that the nigerose was exclusively labeled in the nonreducing glucose unit. These results indicated that [¹⁴C]dextran was being released from BGD by virtue of the action of the low molecular weight dextran and that this action gave the formation of a new α-1 → 3 branch linkage. A mehanism for branching is proposed in which a C₃-OH on an acceptor dextran acts as a nucleophile on C₁ of the reducing end of a dextranosyl-dextransucrase complex, thereby displacing dextran from dextransucrase and forming an α-1 → 3 branch linkage. It is argued that the biosynthesis of branched linkages does not require a separate branching enzyme but can take place by reactions of an acceptor dextran with a dextranosyl-dextransucrase complex.     

Characterization of an antiserum against the glucocorticoid receptor

An immunoglobulin (IgG) fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor from rat liver cytosol contained specific antibodies to glucocorticoid receptor. This was shown following incubation of the [³H]triamcinolone acetonide-glucocorticoid receptor (TA-GR) complex with the IgG fraction by (I) adsorption of the [³H]TA-GR-antibody complex to protein A linked to Sepharose, (II) an increased sedimentation rate of the [³H]TA-GR-antibody complex compared to that of the [³H]TA-GR complex, and (III) an increased molecular size of the [³H]TA-GR-antibody complex when compared to that of the [³H]TA-GR complex as judged from gel filtration. The antibody fraction was characterized with regard to titer, cross-reactivity and specificity. The antibodies cross-reacted with the glucocorticoid receptor from various rat tissues (liver, thymus and hippocampus), as well as with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus. In the rat liver, the antibody bound to both the nuclear and the cytosolic glucocorticoid receptor (Stokes radius 6.1 nm). It did not cross-react with the proteolytic fragments of the glucocorticoid receptor, the 3.6 nm complex or the 1.9 nm complex. Binding of the antibodies was not seen to the androgen, estrogen or progestin receptors in rat to rat serum transcortin. With an indirect competitive ELISA (enzyme-linked immunosorbent assay) combined with various separation techniques, based on different physiocochemical principles, it was shown that the glucocorticoid receptor was the only detectable antibody binding protein from rat liver cytosol using this assay system. These findings also indicate an immunochemical similarity between glucocorticoid receptors in different tissues as well as in different species, but not between glucocorticoid receptors and other steroid hormone receptor proteins. The cytosolic and nuclear glucocorticoid receptors in rat liver were shown to be immunochemically similar.     

Peculiarities of interaction of monoclonal antibody B2 with polycyclic aromatic hydrocarbons and peptide-mimotope of benzo[<Emphasis Type="Italic">a</Emphasis>]pyrene

In order to determine the possible mechanisms of interactions of monoclonal antibody B2 with haptens and early synthesized peptide-mimotope of benzo[a]pyrene using the phage display method, amino acid sequences of variable fragments of heavy and light chains are determined and a model of Fab-fragment is constructed. The structure of the antibody active center is determined using molecular docking with polycyclic aromatic hydrocarbons. It is identified that the active center of monoclonal antibody B2 brings the two pockets of binding. The correlation between preliminarily obtained experimental data on the cross-reactivity of monoclonal antibody B2 with some ligands and calculated bond energy is found. It is shown that synthetic peptide-mimotope of benzo[a]pyrene is weak competing with the conjugate of benzo[a]pyrene for binding with monoclonal antibody B2. The immunization of mice with the conjugate of peptide and bovine serum albumin results in creation of antibodies to benz[a]anthracene and anthracene but not to benzo[a]pyrene. The model of peptide-mimotope of benzo[a]pyrene from pIII protein of bacteriophage is built. It is determined that tryptophan included into peptide composition can be exposed on the surface and be available for antibody. The data of modeling obtained in this study can be applicable for further optimization as both the structure of peptide-mimotope of benzo[a]pyrene and for active center of monoclonal antibody B2.     

Characterization and solubilization of the cytochalasin B binding component from human placental microsomes

Human placental microsomes exhibit uptake of d-[³H]glucose which is sensitive to inhibition by cytochalasin B (apparent K_i = 0.78 /gm M). Characterization of [³H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by d-glucose with an ED₅₀ = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a K_d = 0.15μM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60–70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [³H]cytochalasin B to the soluble protein displays a pattern of inhibition by d-glucose similar to that observed for intact membranes, and the measurement of an ED₅₀ = 37.5 mM d-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [³H]cytochalasin B incorporation into soluble protein (M_r range 42 000-68 000) is prevented by the presence of d-glucose. An identical photolabelling pattern is observed for incorporation of [³H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions.     

Optimal tracers for parallel labeling experiments and 13C metabolic flux analysis: A new precision and synergy scoring system

¹³C-Metabolic flux analysis (¹³C-MFA) is a widely used approach in metabolic engineering for quantifying intracellular metabolic fluxes. The precision of fluxes determined by ¹³C-MFA depends largely on the choice of isotopic tracers and the specific set of labeling measurements. A recent advance in the field is the use of parallel labeling experiments for improved flux precision and accuracy. However, as of today, no systemic methods exist for identifying optimal tracers for parallel labeling experiments. In this contribution, we have addressed this problem by introducing a new scoring system and evaluating thousands of different isotopic tracer schemes. Based on this extensive analysis we have identified optimal tracers for ¹³C-MFA. The best single tracers were doubly ¹³C-labeled glucose tracers, including [1,6-¹³C]glucose, [5,6-¹³C]glucose and [1,2-¹³C]glucose, which consistently produced the highest flux precision independent of the metabolic flux map (here, 100 random flux maps were evaluated). Moreover, we demonstrate that pure glucose tracers perform better overall than mixtures of glucose tracers. For parallel labeling experiments the optimal isotopic tracers were [1,6-¹³C]glucose and [1,2-¹³C]glucose. Combined analysis of [1,6-¹³C]glucose and [1,2-¹³C]glucose labeling data improved the flux precision score by nearly 20-fold compared to widely use tracer mixture 80% [1-¹³C]glucose +20% [U-¹³C]glucose.     

In vitro complex formation between bacteriophage φ29 terminal protein and deoxynucleotide

It has previously been shown that the 5′-terminal deoxyadenosine residue of each φ29 DNA strand is linked covalently to the 30,000 dalton terminal protein. When extracts prepared from φ29-infected$\text{Bacillus}\text{subtilis}$ cells are incubated with [α-³²p]dATP, complexes consisting of the nucleotide covalently linked to a 30,000 dalton protein can be detected. The formation of this complex requires the presence of φ29 DNA containing the bound 30,000 dalton terminal protein and Mg⁺⁺. When uninfected cell extracts were used, there was no complex formation. When [α-³²p]dCTP was used in place of [α-³²p]dATP, no complex was formed. DNA-protein templates prepared from φ29 related phages, φ15, Nf, M2Y and GA-1, also supported the complex formation in various degrees. These results support the hypothesis that the terminal protein serves as a primer for the initiation of φ29 DNA replication.     

LABELLING OF BRAIN PROTEINS AT EARLY PERIODS AFTER SUBCUTANEOUS INJECTION OF A MIXTURE OF [U-14C]GLUCOSE AND [3H]GLUTAMATE

To obtain evidence of the site of conversion of [U-¹⁴C]glucose into glutamate and related amino acids of the brain, a mixture of [U-¹⁴C]glucose and [³H]glutamate was injected subcutaneously into rats. [³H]Glutamate gave rise to several ³H-labelled amino acids in rat liver and blood; only ³H-labelled glutamate, glutamine or γ-aminobutyrate were found in the brain. The specific radioactivity of [³H]glutamine in the brain was higher than that of [³H]glutamate indicating the entry of [³H]glutamate mainly in the ‘small glutamate compartment’. The ¹⁴C-labelling pattern of amino acids in the brain and liver after injection of [U-¹⁴C]glucose was similar to that previously reported (Gaitonde et al., 1965). The specific radioactivity of [¹⁴C]glutamine in the blood and liver after injection of both precursors was greater than that of glutamate between 10 and 60 min after the injection of the precursors. The extent of labelling of alanine and aspartate was greater than that of other amino acids in the blood after injection of [U-¹⁴C]glucose. There was no labelling of brain protein with [³H]glutamate during the 10 min period, but significant label was found at 30 and 60 min. The highest relative incorporation of [¹⁴C]glutamate and [¹⁴C]aspartate in rat brain protein was observed at 5 min after the injection of [U-¹⁴C]glucose. The results have been discussed in the context of transport of glutamine synthesized in the brain and the site of metabolism of [U-¹⁴C]glucose in the brain.     

Isolation ofn-ethylmaleimide-labelled phlorizin-sensitived-glucose binding protein of brush border membrane from rat kidney cortex

A glucose receptor with high affinity for phlorizin from isolated brush border of rat kidney was labelled specifically withN-[¹⁴C]ethylmaleimide and then extracted from the membranes.After the solubilization of the brush borders with sodium dodecyl sulphate theN-[¹⁴C]ethylmaleimide-labelled receptor protein was isolated and was found to have a molecular weight of approximately 30 000 as determined by sodium dodecyl sulphate-polyacrylamide gel disc electrophoresis. The receptor protein eluted from the sodium dodecyl sulphate-containing gels migrates as a single band on sodium dodecyl sulphate-free polyacrylamide gels.The receptor protein can also be released from the brush borders with low concentrations of sodium deoxycholate. Under these conditions the molecular weight of theN-[¹⁴C]ethylmaleimide-labelled receptor protein is approximately 60 000 in contrast to the protein component solubilized with sodium dodecyl sulphate. Since this detergent is known to dissociate the brush border membrane into its protein components, our results suggest that the phlorizin- sensitive glucose receptor protein has a molecular weight of about 30 000.     

MonoADP-Ribosylation of the NAD+-Dependent Alcohol Dehydrogenase from Entamoeba histolytica

The human parasite Entamoeba histolytica is an amitochondrial protozoan whose metabolism depends on glucose fermentation. Among the metabolic enzymes absolutely required for amoeba growth is the NAD⁺-dependent alcohol dehydrogenase (EhADH2). The polymeric form of EhADH2 was sedimented at 160,000g, and in this fraction we observed [³²P]-labeling of a 96-kDa protein under mono-ADP-ribosylation conditions with [³²P]NAD⁺. The [³²P]-labeled protein had the same molecular weight as the EhADH2 monomer. Because of the importance of monoADP-ribosylation in the regulation of many physiological processes, the aim of this study was to determine whether EhADH2 is ADP-ribosylated, and what would be the consequence of this modification on its alcohol and aldehyde dehydrogenase enzymatic activities. This study describes the ADP-ribosylation of EhADH2. This modification did not have an effect on the enzymatic activities, but it may regulate other functions of EhADH2.     

Hyperglycaemic activity and metabolic effects of 3-aminopicolinic acid

Administering 3-aminopicolinate to rats starved for 24h immediately initiated a progressive increase in blood glucose concentration. Hyperglycaemia was not the result of glycogenolysis, nor was it due to an inhibition of insulin release, since it caused marked hyperinsulinaemia. The rate of [6-³H]glucose disappearance from the blood of the intact rat was not altered by 3-aminopicolinate, indicating that it does not cause hyperglycaemia by inhibiting glucose utilization or by causing a redistribution of total body glucose. 3-Aminopicolinate increased the rate of fall in the specific radioactivity of blood [6-³H]-glucose, indicating dilution of the glucose pool by newly synthesized glucose. The rate of ¹⁴C incorporation into blood glucose from [¹⁴C]alanine and [¹⁴C]lactate was increased 90 and 35% respectively, whereas that from [¹⁴C]glycerol and [¹⁴C]xylitol was either unaffected or slightly decreased by 3-aminopicolinate administration. Liver phosphoenolpyruvate of rats was increased to four to seven times the normal concentration 10min to 1h after injections of 50–300mg of 3-aminopicolinate/kg body wt. and the amounts of 2-phosphoglycerate and 3-phosphoglycerate were increased to three to four times normal. The high concentrations of liver phosphoenolpyruvate, 2-phosphoglycerate and 3-phosphoglycerate, as well as the enhancement of gluconeogenesis from lactate and alanine, but not from glycerol or xylitol, is compatible with an enhancement of gluconeogenesis at a step between pyruvate and the triose phosphates. After injections of 3-aminopicolinate, liver malate, citrate, aspartate, alanine, lactate and pyruvate were also increased, but to lesser extents than was phosphoenolpyruvate. The increases in some of these metabolites were approximated after an intravenous infusion of glucose, so their elevated concentration after 3-aminopicolinate administration could have been, in part, a consequence of the hyperglycaemia. The possibility is considered that 3-aminopicolinate stimulates gluconeogenesis in vivo by facilitating Fe²⁺ activation of phosphoenolpyruvate carboxykinase as it does with the purified enzyme in vitro [MacDonald & Lardy (1978) J. Biol. Chem. 253, 2300–2307]. In this effect 3-aminopicolinate may simulate the physiological role of the naturally occurring ferroactivator protein [Bentle & Lardy (1977) J. Biol. Chem. 252, 1431–1440].     

Structural Plasticity of NFU1 Upon Interaction with Binding Partners: Insights into the Mitochondrial [4Fe-4S] Cluster Pathway

In humans, the biosynthesis and trafficking of mitochondrial [4Fe-4S]²⁺ clusters is a highly coordinated process that requires a complex protein machinery. In a mitochondrial pathway among various proposed to biosynthesize nascent [4Fe-4S]²⁺ clusters, two [2Fe-2S]²⁺ clusters are converted into a [4Fe-4S]²⁺ cluster on a ISCA1-ISCA2 complex. Along this pathway, this cluster is then mobilized from this complex to mitochondrial apo recipient proteins with the assistance of accessory proteins. NFU1 is the accessory protein that first receives the [4Fe-4S]²⁺ cluster from ISCA1-ISCA2 complex. A structural view of the protein–protein recognition events occurring along the [4Fe-4S]²⁺ cluster trafficking as well as how the globular N-terminal and C-terminal domains of NFU1 act in such process is, however, still elusive. Here, we applied small-angle X-ray scattering coupled with on-line size-exclusion chromatography and paramagnetic NMR to disclose structural snapshots of ISCA1-, ISCA2- and NFU1-containing apo complexes as well as the coordination of [4Fe-4S]²⁺ cluster bound to the ISCA1-NFU1 complex, which is the terminal stable species of the [4Fe-4S]²⁺ cluster transfer pathway involving ISCA1-, ISCA2- and NFU1 proteins. The structural modelling of ISCA1-ISCA2, ISCA1-ISCA2-NFU1 and ISCA1-NFU1 apo complexes, here reported, reveals that the structural plasticity of NFU1 domains is crucial to drive protein partner recognition and modulate [4Fe-4S]²⁺ cluster transfer from the cluster-assembly site in the ISCA1-ISCA2 complex to a cluster-binding site in the ISCA1-NFU1 complex. These structures allowed us to provide a first rational for the molecular function of the N-domain of NFU1, which can act as a modulator in the [4Fe-4S]²⁺ cluster transfer.     

Use of hydrophilic interaction chromatography for the study of tyrosine protein kinase specificity

A new HPLC method has been developed to assay tyrosine protein kinase activity. Using hydrophilic interaction chromatography, it is possible to resolve the four components of the incubation medium: substrate peptide, [³²P]phosphorylated peptide, unreacted [γ-³²P]ATP, and ³²P-labelled inorganic phosphate. ATP interacts so strongly with the stationary phase material that it can be removed selectively from the incubation medium with solid-phase extraction cartridges packed with the same type of material. The three remaining components of interest can then be resolved by reversed-phase or hydrophilic interaction HPLC. This procedure permits the evaluation of almost every type of peptide as a substrate of tyrosine protein kinase.     

Effects of intestinal insulin-like peptide on glucose catabolism in male adult Locusta migratoria

An insulin-like peptide (ILP) extracted from midgut of 25-day-old male adult Locusta migratoria can modify the relative activity of the two main pathways of glucose catabolism. The effect of ILP on the activity of the pentose cycle and the glycolytic-citric acid cycle in Locusta migratoria was evaluated by a radiorespirometric method by means of [1-¹⁴C] glucose and [6-¹⁴C]glucose as substrates. The time course of the ILP effect was determined. The insulin-like peptide increases the relative activity of the pentose cycle. This effect is rapid and of short duration. Injected mammalian insulin has a similar effect.     

Ontogenetic Study of the Effects of Energetic Nutrients on Amino Acid Metabolism of Rat Cerebral Cortex

We studied the effect of various energetic nutrients on metabolism of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in cerebral cortex of rats at different ages. At gestational age, glucose and lactate stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine and from l-[U-¹⁴C]leucine, respectively; glucose, -OH-butyrate and lactate stimulated lipid synthesis from l-[U-¹⁴C]leucine. At 10 days of age, glucose, mannose, and fructose stimulated protein synthesis, and glucose and mannose stimulated oxidation to CO₂ as well as lipid synthesis from l-[U-¹⁴C]leucine. In adult rats, glucose, mannose, and fructose stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine; glutamine also markedly decreased the oxidation of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in 10–day-old and adult rats.     

Studies of the residual glycogen branching enzyme activity present in human skin fibroblasts from patients with Type IV glycogen storage disease

Human skin fibroblasts from patients with Type IV glycogen storage disease, in which there is a demonstrable deficiency of glycogen branching enzyme, were shown to be able to synthesize [¹⁴C]glycogen containing [¹⁴C]glucose at branch points when sonicates containing endogenous glycogen synthase $\text{a}$ were incubated with UDP[¹⁴C]glucose. The branch point content of the glycogen synthesized by the Type IV cells was essentially the same as that formed by normal cells, but the total synthetic capacity of the Type IV cells was lower. A new assay for the branching enzyme using glycogen synthase as the indicator enzyme has been developed. Using this assay it has been shown that the residual branching enzyme of affected children and of their heterozygote parents is less easily inhibited by an IgG antibody raised in rabbits against the normal human liver enzyme than is the branching enzyme of normal fibroblasts.     

Multi-step metabolic activation of benzene. Effect of superoxide dismutase on covalent binding to microsomal macromolecules,and identification of glutathione conjugates using high pressure liquid chromatography and field desorption mass spectrometry

Incubation of [¹⁴C]benzene or [¹⁴C]phenol with liver microsomes from untreated rats, in the presence of a NADPH-generating system, gave rise to irreversible binding of metabolites to microsomal macromolecules. For both substrates this binding was inhibited by more than 50% by addition of superoxide dismutase to the incubation mixtures. The decrease in binding was compensated for by accumulation of [¹⁴C]hydroquinone, indicating superoxide-mediated oxidation of hydroquinone as one step in the activation of benzene to metabolites binding to microsomal macromolecules. Since our previous work had shown that binding occurred mainly with protein rather than ribonucleic acid and was virtually completely prevented by glutathione, suggesting identity of metabolite(s) responsible for binding to protein and glutathione, a conjugate was chemically prepared from p-benzoquinone and reduced glutathione (GSH) and identified by field desorption mass spectrometry (FDMS) as 2-(S-glutathionyl) hydroquinone. Microsomal incubations, containing an NADPH-generating system, with benzene, phenol, hydroquinone or p-benzoquinone in the presence of [³H]glutathione or, alternatively, with [¹⁴C]benzene or [¹⁴C]phenol in the presence of unlabeled glutathione, were performed. All of these incubations gave rise to a peak of radioactivity eluting from the high pressure liquid chromatograph (HPLC) at a retention time identical to that of the chemically prepared 2-(S-glutathionyl) hydroquinone, whilst microsomal incubation of catechol in the presence of [³H]glutathione led to a conjugate with a very different retention time which was not observed after incubation of benzene or phenol. The microsomal metabolites of p-benzoquinone, hydroquinone and phenol thus eluting from the HPLC were further identified as the 2-(S-glutathionyl) hydroquinone by field desorption mass spectrometry. The glutathione adduct formed from benzene during microsomal activation eluted from HPLC with the same retention time and its mass spectrum also contained the molecular ion (MH⁺) (m/e 416) of this conjugate as an intense peak, but the fragmentation patterns did not allow definite assignments probably due to the considerably smaller amounts of ultimate reactive metabolites formed from this pre-precursor and thus relatively larger amounts of impurities.The results indicate that rat liver microsomes activate benzene via phenol and hydroquinone to p-benzosemiquinone and/or p-benzoquinone as quantitatively important reactive metabolites.