Binding of Complement Inhibitor C4b-binding Protein to a Highly Virulent Streptococcus pyogenes M1 Strain Is Mediated by Protein H and Enhances Adhesion to and Invasion of Endothelial Cells

Streptococcus pyogenes AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the complement inhibitor C4b-binding protein (C4BP). We found a strong correlation between the ability of AP1 and its isogenic mutants lacking protein H to inhibit opsonization with complement C3b and binding of C4BP. C4BP bound to immobilized protein H or AP1 bacteria retained its cofactor activity for degradation of ¹²⁵I-C4b. Furthermore, C4b deposited from serum onto AP1 bacterial surfaces was processed into C4c/C4d fragments, which did not occur on strains unable to bind C4BP. Recombinant C4BP mutants, which (i) lack certain CCP domains or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. Using recombinant protein H fragments, we narrowed down the binding site to the N-terminal domain A. With a peptide microarray, we identified one single 18-amino acid-long peptide comprising residues 92–109, which specifically bound C4BP. Biacore was used to determine K_D = 6 × 10⁻⁷ m between protein H and a single subunit of C4BP. C4BP binding also correlated with elevated levels of adhesion and invasion to endothelial cells. Taken together, we identified the molecular basis of C4BP-protein H interaction and found that it is not only important for decreased opsonization but also for invasion of endothelial cells by S. pyogenes.     

Studies on the subcellular localization of the membrane-bound fraction of intestinal calcium-binding protein

Sorbitol density gradient centrifugation applied to intestinal mucosa homogenates resulted in a complete separation of soluble calcium-binding protein from the bound fraction of calcium-binding protein, providing further documentation of the bound pool of calcium-binding protein. The peak of the bound calcium-binding protein was not associated with the major peaks of any of the markers used, but was associated with minor peaks of alkaline phosphatase, RNA, and glucose-6-phosphatase. Lack of association of bound calcium-binding protein with (Na⁺ + K⁺)-ATPase indicated that the bound calcium-binding protein is not on the basolateral membrane. Differential centrifugation fractionation indicated that the bound calcium-binding protein is not associated with nuclei or mitochondria. The bound calcium-binding protein also could not be detected in partially pyrified brush borders. Exclusion of the brush border and basolateral membranes as the location of the bound calcium-binding protein suggests an intracellular locate.     

Metabolism of benzo(a)pyrene to reactive intermediate(s) via prostaglandin biosynthesis.

We have examined the oxidation of benzo (a) pyrene (BP) to electrophilic metabolites during the formation of prostaglandins (PG) and thromboxanes (TX) from arachidonic acid (AA) by guinea pig lung microsomal protein. In the presence of NADPH or AA, electrophilic metabolites of [¹⁴C]-BP were generated which were non-extractable from microsomal protein and thus assumed to be covalently bound. The total amount of BP metabolized in the presence of NADPH was 2–2 $\text{1}\text{2}$ times the amount of BP metabolized in the presence of AA. Only 4–5% of BP metabolized by the NADPH mediated mixed-function oxidase system was covalently bound, whereas 12–15% of the BP metabolized in the presence of AA was covalently bound to tissue protein and DNA. Quinones were the major metabolites produced by the AA dependent system, while dihydrodiols were the major metabolites formed by the NADPH dependent system. 7, 12-Dimethyl-benzanthracene, and 7,8-BP-dihydrodiol, but not 3 hydroxy-BP were also oxidized by PG synthetase to reactive metabolites.     

Grb2 dominantly associates with dynamin II in human hepatocellular carcinoma HepG2 cells

The two SH3 domains and one SH2 domain containing adaptor protein Grb2 is an essential element of the Ras signaling pathway in multiple systems. The SH2 domain of Grb2 recognizes and interacts with phosphotyrosine residues on activated tyrosine kinases, whereas the SH3 domains bind to several proline‐rich domain‐containing proteins such as Sos1. To define the difference in Grb2‐associated proteins in hepatocarcinoma cells, we performed coprecipitation analysis using recombinant GST‐Grb2 fusion proteins and found that several protein components (p170, p125, p100, and p80) differently associated with GST‐Grb2 proteins in human Chang liver and hepatocarcinoma HepG2 cells. Sos1 and p80 proteins dominantly bind to Grb2 fusion proteins in Chang liver, whereas p100 remarkably associate with Grb2 in HepG2 cells. Also GST‐Grb2 SH2 proteins exclusively bound to the p46^Shc, p52^Shc, and p66^Shc are important adaptors of the Ras pathway in HepG2 cells. The p100 protein has been identified as dynamin II. We observed that the N‐SH3 and C‐SH3 domains of Grb2 fusion proteins coprecipitated with dynamin II besides Sos1. These results suggest that dynamin II may be a functional molecule involved in Grb2‐mediated signaling pathway on Ras activation for tumor progression and differentiation of hepatocarcinoma cells. J. Cell. Biochem. 84: 150–155, 2002. © 2001 Wiley‐Liss, Inc.     

Applications of biotinylated V3 loop peptides of human immunodeficiency virus type 1 to flow cytometric analyses and affinity chromatographic techniques

A principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) lies within the V3 loop of gp120, the external major envelope glycoprotein. V3 loop peptides derived from two HIV-1 strains, HTLV-III BH-10 (V3-BH10) and LAV_ELI (V3-ELI), were synthesized and biotinylated. The binding of both biotinylated V3-BH10 and V3-ELI to the surfaces of MOLT-4 clone 8 cells was demonstrated by flow cytometric analyses. Both the peptides (more than 2 μM) bound to the cells (2 · 10⁵) in a dose-dependent manner. The binding of biotinylated V3-BH10 was specifically inhibited by a neutralizing monoclonal antibody (0.5β). The binding of both of the biotinylated V3 loop peptides was enhanced by the addition of unlabeled V3-BH10. In addition, the peptides were employed as ligands on affinity columns. A major V3 loop binding protein (V3BP) was purified from the membrane soluble fraction of MOLT-4 cells by successive application to two different V3 loop columns. V3BP consisted of two major polypeptides (32 and 33 kDa). The SDS-PAGE profile of V3BP did not change under non-reducing conditions, but only a single band was observed after analysis on native PAGE. The major peak of the eluate as determined by size exclusion chromatography was abroad and the estimated relative molecular mass was much larger than 33 kDa, suggesting that V3BP comprises several subunits. Taken together, we confirmed that the V3 loop peptides are useful in the characterization of V3BP(s) of which they are conformational ligands.     

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations¹. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in²). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones^3,4, forming foci within 5-15 minutes⁵. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair⁶. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins^7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs⁶. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis¹⁰.In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1''s behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs¹¹. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis¹². Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.     

A critical role of STAT1 in streptozotocin-induced diabetic liver injury in mice: Controlled by ATF3

It is well-established that the administration of streptozotocin accelerates diabetic liver injury as well as type-I diabetes, however the underlying mechanisms are poorly understood. Here we investigated the molecular mechanisms of diabetic liver injury in a model of streptozotocin (STZ)-induced type-I diabetes. STZ administration induced type-1 diabetes and chronic liver injury was associated with increased STAT1, which is implicated in diabetic liver injury by virtue of its ability to promote hepatocyte apoptosis, in the liver and pancreas, which were all strongly inhibited in STAT1^−/– mice. Similarly, STZ-induced ATF3, a stress-inducible gene, was completely abolished in the liver of IFN-γ^−/− mice, but not in STAT1^−/− mice. Inhibition of STAT1 by siRNA or dominant-negative DNA did not affect ATF3 protein expression but blocked IFN-γ-induced ATF3 translocation from the cytosol into the nucleus. In contrast, inhibition of ATF3 by using siRNA diminished STAT1 protein expression and IFN-γ/STZ-induced hepatocyte apoptosis. Furthermore, GST pull-down and co-IP assay showed that STAT1 bound to C-terminal domain of ATF3. Such direct interaction increased the stability of STAT1 by inhibiting its ubiquitination as well as proteasome activity. Our results suggest that STAT1 is a common signaling pathway contributing to STZ-induced diabetes and diabetic liver injury. ATF3 functions as a potent regulator of STAT1 stability, accelerating STZ-induced diabetes and diabetic liver injury.     

Inhibition by Cu2+ and Cd2+ of a mu‐class glutathione S‐transferase from shrimp Litopenaeus vannamei

Glutathione S‐transferases (GSTs) are a family of detoxifying enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thereby increasing the solubility of xenobiotics and aiding its excretion from the cell. The present work presents the inhibition of a mu‐class GST of the marine shrimp Litopenaeus vannamei by copper (Cu²⁺) and cadmium (Cd²⁺). The protein was overexpressed in bacteria and its enzymatic activity measured using 1‐chloro‐2,4‐dinitrobenzene. The mean inhibitory concentration (IC₅₀) for shrimp GST against Cu²⁺ was 4.77 μM and for Cd²⁺ was 0.39 μM. A molecular model of the protein based on the crystal structure of a maize GST bound to cadmium showed that the metal binds in the GSH‐binding site by coordination with Asp and Gln residues. These results are consistent with the experimental data and suggest that sublethal concentration of metals may affect the capacity of the organism to detoxify pesticides or xenobiotics. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:218–222, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20326     

Formation of Mutagens by Heating the Aqueous Solution of Amino Acids and Some Nitrogenous Compounds with Addition of Glucose

A ³H-labeled bicyclic phosphate (BP), 4-[2,3-³H]propyl-2,6,7-trioxa-1-phosphabicyclo-[2.2.2]octane 1-oxide, bound specifically to rat brain synaptic membrane preparations. The apparent dissociation constant and the quantity of the BP bound at saturation were estimated to be about 30 μm and about 2 pmol per mg of protein, respectively. BP analogs except 4-amino BP, which is extremely low in toxicity, inhibited the binding but no obvious correlation was observed between the toxicity and the inhibitory potency of the BP analogs. The binding was also inhibited by high concentrations of GABA and picrotoxinin, whereas it was enhanced by bicuculline methiodide.     

The DNA binding of benzo[alpha]pyrene metabolites catalysed by rat lung microsomes in vitro and in isolated perfused rat lung.

When [³H]benzo[a]pyrene is incubated in vitro together with DNA, NADPH and rat lung microsomes, covalent binding of benzo[a]pyrene (BP) metabolites to DNA occurs. These metabolite-nucleoside complexes can be resolved into several distinct peaks by elution of a Sephadex LH-20 column with a water-methanol gradient. 3-Methylcholanthrene (MC) pretreatment of animals induces the total covalent binding in vitro several-fold and increases the amounts of at least five metabolite-nucleoside complexes associated with the 7,8-diol-9,10-epoxidcs, the 7,8-oxide or quinones oxygenated further, the 4,5-oxide and phenols oxygenated further. These increases correspond well with the increases in the production of both non-K-region and K-region metabolites of BP by lung microsomes, as determined by highpressure liquid chromatography (HPLC). On the other hand, when [³H]BP is metabolized in isolated perfused rat lung, only the peak representing the 7,8-diol-9,10-epoxide bound to nucleoside(s) is readily detectable and then only in lungs from MC-treated animals. The extent of binding of BP metabolites to lung DNA is very low, about 0.0004% of the total dose applied to the perfusion medium; more than 60% of this can be accounted for by the binding of the 7,8-diol-9,10-epoxides to nucleoside(s). It is suggested that the further metabolism leading to metabolites not available to covalent binding, (e.g. conjugation) of primary BP metabolites in the intact tissue is responsible for the differences in the metabolite-nucleoside patterns observed in vivo, as compared with microsomal metabolism in vitro.     

Liver glutathione S-transferase polymorphism in Japanese and its pharmacogenetic importance

Summary A total of 168 autopsy liver extracts from Japanese individuals were examined for the glutathione S-transferase (GST) isozymes by means of starch gel electrophoresis. The gene frequencies of GST1^*1, GST1^*2, and GST1^*0 in Japanese were 0.252, 0.057, and 0.691, respectively. GST1^*3 was detected as a rare variant allele. The incidence of GST1 0 in 41 liver biopsy samples from patients suffering from various liver diseases was investigated using polyacrylamide gel isoelectric focusing. The GST1 0 phenotype was found more frequently in livers with hepatitis and carcinoma than in control livers. The isozymes coded by different GST loci were partially purified and characterized to study their biochemical properties. The apparent Km values with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate for the isozymes at the GST1, GST2, GST3, and GST4 loci were 604, 1345, 776 and 591 M, respectively.     

Inhibition of human glutathione transferases by multidrug resistance chemomodulators in vitro

Reversal of the drug-resistance phenotype in cancer cells usually involves the use of a chemomodulator that inhibits the function of a resistance-related protein. The aim of this study was to investigate the effects of MDR chemomodulators on human recombinant glutathione S-transferase (GSTs) activity. IC₅₀ values for 15 MDR chemomodulators were determined using 1-chloro-dinitrobenzene (CDNB), cumene hydroproxide (CuOOH) and anticancer drugs as substrates. GSTs A1, P1 and M1 were inhibited by O⁶-benzylguanine (IC₅₀s around 30 μM), GST P1-1 by sulphinpyrazone (IC₅₀ = 66 μM), GST A1-1 by sulphasalazine, and camptothecin (34 and 74 μM respectively), and GST M1-1 by sulphasalazine, camptothecin and indomethacin (0.3, 29 and 30 μM respectively) using CDNB as a substrate. When ethacrynic acid (for GST P1-1), CuOOH (for A1-1) and 1,3-bis (2-chloroethyl)-1-nitrosourea (for GST M1-1) were used as substrates, these compounds did not significantly inhibit the GST isoforms. However, progesterone was a potent inhibitor of GST P1-1 (IC₅₀ = 1.4 μM) with ethacrynic acid as substrate. These results suggest that the target of chemomodulators in vivo could be a specific resistance-related protein.     

Contrasting effects of IGF binding protein-3 expression in mammary tumor cells and the tumor microenvironment

IGFBP-3 has both stimulatory and inhibitory effects on cancer progression. The growth of EO771 mammary carcinoma cells as syngeneic tumors in C57BL/6 mice is reduced in Igfbp3-null (BP3KO) mice, suggesting that systemic IGFBP-3 enhances tumor progression. In this study we assessed the growth of EO771 cells expressing human IGFBP-3 in BP3KO mice. Cells expressing hIGFBP-3 showed decreased proliferation in vitro and increased levels of IGF-1 receptor (IGF1R) protein but not mRNA, consistent with sequestration of endogenous IGF by IGFBP-3. The growth rate of these cells was restored by exposure to IGF-1 or analogues with reduced affinity for IGFBP-3 (long Arg³-IGF-1) or IGF1R (Leu²⁴-IGF-1). In EO771 cells implanted orthotopically into mice, hIGFBP-3 expression by the cells inhibited tumor establishment in BP3KO but not wild-type mice. For tumors that successfully established, final weight was not affected significantly by hIGFBP-3 expression. However, final tumor weight was inversely related to intratumoral T cell counts, and sera from BP3KO mice with tumors showed low-titer immunoreactivity against IGFBP-3. The contrasting effects on tumor establishment and progression of IGFBP-3 expressed by mammary carcinoma cells, compared to systemic stromal and circulating IGFBP-3, highlights the complexity of growth regulation by IGFBP-3 in mammary tumors.     

Partial Characterization of Glutathione S-Transferase Isozymes Induced by the Herbicide Safener Benoxacor in Maize

The effects of the dichloroacetamide safener benoxacor on maize (Zea mays L. var Pioneer 3906) growth and glutathione S-transferase (GST) activity were evaluated, and GST isozymes induced by benoxacor were partially separated, characterized, and identified. Protection from metolachlor injury was closely correlated with GST activity, which was assayed with metolachlor as a substrate, as benoxacor concentration increased from 0.01 to 1 [mu]M. GST activity continued to increase at higher benoxacor concentrations (10 and 100 [mu]M), but no further protection was observed. Total GST activity with metolachlor as a substrate increased 2.6- to 3.8-fold in response to 1 [mu]M benoxacor treatment. Total GST activity from maize treated with or without 1 [mu]M benoxacor was resolved by fast protein liquid chromatography anion-exchange chromatography into four major activities, designated activity peaks A, B, C, and D in their order of elution. These GST activity peaks were enhanced to varying degrees by benoxacor. Activity peak B showed the least induction, whereas activity peak A was absent constitutively and thus highly induced by benoxacor. In contrast to earlier reports, there appear to be not one, but at least two, major constitutive isozymes (activity peaks A and D) having activity with metolachlor as substrate; there were at least three such isozymes in benoxacor-treated maize (activity peaks A, C, and D). The elution volumes of activity peaks A, B, C, and D were compared with those of partially purified maize GST I and GST II; also, the reactivity of polypeptides in these activity peaks with antisera to GST I or GST I/III (mixture) was evaluated. Evidence from these experiments indicated that activity peak B contained GST I, and activity peak C contained GST II and GST III. Activity peaks A and D contained unique GSTs that may play a major role in metolachlor metabolism and in the safening activity of benoxacor in maize. Isozymes present in activity peaks A and D were not detected in earlier reports because of the very low activity with the artificial substrate 1-chloro-2,4-dinitrobenzene. Immunoblotting experiments also indicated the presence of numerous unidentified GST subunits, including multiple subunits in chromatography fractions containing single peaks of GST activity; this is indicative of the likely complexity and diversity of the maize GST enzyme family.     

A simple enzyme–substrate localized conjugation method to generate immobilized,functional glutathione S-transferase fusion protein columns for affinity enrichment

Immobilized protein receptors and enzymes are tools for isolating or enriching ligands and substrates based on affinity. For example, glutathione S-transferase (GST) is fused to proteins as a tag for binding to its substrate glutathione (GSH) linked to solid supports. One issue with this approach is that high-affinity interactions between receptors and ligands require harsh elution conditions such as low pH, which can result in leached receptor. Another issue is the inherent nonspecific chemical conjugation of reactive groups such as N-hydroxysuccinimide (NHS) that couple lysines to solid supports; the nonspecificity of NHS may result in residue modifications near the binding site(s) of the receptor that can affect ligand specificity. In this study, a simple conjugation procedure is presented that overcomes these limitations and results in immobilized GST fusion proteins that are functional and specific. Here, the affinity of GST for GSH was used to generate an enzyme–substrate site-specific cross-linking reaction; GSH–Sepharose was preactivated with 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) and then incubated Fc gamma receptor IIIa (FcγRIIIa)–GST. The immobilized FcγRIIIa–GST more specifically bound glycosylated immunoglobulin G1s (IgG1s) and was used to enrich nonfucosylated IgG1s from weaker binding species. This technique can be used when modifications of amino acids lead to changes in activity.     

From flakes to grooves: a technical shift in antlerworking during the last glacial maximum in southwest France

The evolution of antlerworking technology in Paleolithic and Mesolithic Europe, especially the production of splinters, is usually described as a cumulative process. A progressive increase in blank standardization and productivity was prompted by the application of a key technical process, the groove and splinter technique (GST). The Badegoulian, however, appears as an interruption in this continuum. According to the original definition of this post-Solutrean, pre-Magdalenian archeological culture, one of its distinctive features is the absence of the GST and the manufacture of antler blanks by knapping only. However, this conception has been recently questioned, leading to an alternative hypothesis suggesting that both GST and knapping were used during the Badegoulian. In this article, we present new evidence from several sites in southwest France, which sheds new light on the issue of Badegoulian antlerworking and the transition with the subsequent Lower Magdalenian. Our study is based on two complementary methods: the technological analysis of antler assemblages well-dated to the Badegoulian (Le Cuzoul de Vers) or to the Lower Magdalenian (La Grotte des Scilles, Saint-Germain-la-Rivière), and the direct ¹⁴C dating of specific antler artifacts from mixed or problematic contexts (Cap-Blanc, Reverdit and Lassac). The results firmly establish that, in southwest France, knapping is the only method used for the production of antler splinters during the Badegoulian, before ca. 20,500 cal BP (calibrated years before present), and that it is rapidly replaced by the GST at the beginning of the Lower Magdalenian, after ca. 20,500 cal BP. This technical shift is not linked to an influx of new human populations, environmental change or the supposed economic advantages of the GST. Instead, it must be understood as one of the expressions of a broader reconfiguration of the technical world that starts to take shape in the middle of the Last Glacial Maximum.     

Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells

Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr¹⁷⁴, Tyr¹⁸³ and Tyr⁴⁴⁶ in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr¹⁸³ and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutational analysis showed that Tyr¹⁷⁴, Tyr¹⁸³ and Tyr⁴²⁶ of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr⁴²⁶ is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr⁴²⁶ was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr⁴²⁶ following BCR stimulation.     

Vitamin K1 amplification of benzo(a)pyrene metabolism in chick embryos

Seventeen-day-old chick embryos were used as a test system to assess the effect of vitamin K1(K1) on benzo(a)pyrene (BP) metabolism as measured by the induction of arylhydrocarbon hydroxylase (AHH) and cytochrome P-450 and the levels of glutathione (GSH) and glutathione S-transferase (GST) in liver. Twenty-four hours after injection of BP into the air sac there was a sharp rise in AHH and P-450 and a drop in GSH. When K1 was injected 24 hr prior to BP there was a decrease in GST activity as compared with the control plus an augmented increase in AHH induction. This augmentation in BP metabolism (Phase I) together with a concomitant decrease in at least one mechanism of Phase II conjugation is in keeping with other evidence that K1 can play an adjuvant role in BP induced mutagenicity and carcinogenicity. Ubiquinone has a much lesser effect on BP metabolism than does K1 in equimolar concentration.     

Different binding sites for glucose and sorbose at the erythrocyte membrane,studied by gel filtration and infrared spectroscopy

Summary Human red blood cell membranes were solubilized with sodium dodecylsulfate and incubated with various concentrations of¹⁴C-glucose and¹⁴C-sorbose. After gel filtration on Sephadex G-100, which separated lipoproteins of differing lipid content, it was observed that the radioactivity of the bound glucose coincided with the protein peak. Radioactivity of bound sorbose was found mainly before and after the protein peak. This distribution of bound sugars was confirmed by double labeling experiments in which³H-glucose and¹⁴C-sorbose were applied simultaneously. Infrared spectroscopy revealed differences between the membranes loaded with sorbose and glucose. Particularly, the band in the C–O–C and P=O region at 1,225 cm^–1 was intensified in the sorbose-loaded membranes. Compared to serum albumin, the erythrocyte membranes were found to bind 4 times as much¹⁴C-glucose per mg of protein. It is concluded from the results obtained by gel filtration that glucose and sorbose preferentially bind at different sites of the erythrocyte membrane. The results obtained by infrared spectroscopy correspond with this conclusion.