Mutagenicity of styrene and styrene oxide

Incubation of S. typhimurium strain TA 1535 with styrene increased the number of his⁺ revertants/plate in presence of a fortified S9 rat-liver fraction. Styrene was also highly cytotoxic for Salmonella cells. Styrene oxide, the presumed first metabolite, had a mutagenic effect towards strains TA 1535 and TA 100 both with and without metabolic activation. Styrene is probably mutagenic because it is metabolized to styrene oxide.     

Mutagenicity of non-K-region diols and diol-epoxides of benz(a)anthracene and benzo(a)pyrene in S. typhimurium TA 100

When incubated with a 9,000 x g rat-liver supernatant, benzo(a)pyrene 7,8-diol and benz(a)anthracene 8,9-diol were more active than the parent hydrocarbons in inducing his⁺ revertant colonies of S. typhimurium TA 100. Benzo(a) pyrene 9,10-diol was less active than benzo(a)pyrene; the K-region diols, benz(a)anthracene 5,6-diol and benzo(a)pyrene 4,5-diol, were inactive. None of the diols was active when the cofactors for the microsomal mono-oxygenase were omitted. The diol-epoxides benzo(a)pyrene 7,8-diol 9,10-oxide, benz(a)anthracene 8,9-diol 10,11-oxide and 7-methylbenz(a)anthracene 8,9-diol 10,11-oxide and the K-region epoxides, benzo(a)pyrene 4,5-oxide and benz(a)anthracene 5,6-oxide, were mutagenic without further metabolism.     

Initiation of mammalian protein synthesis. I. Purification and characterization of seven initiation factors

We have purified seven protein factors from rabbit reticulocytes, all of which are presumed to be involved in the initiation of mammalian protein synthesis. They are termed eIF-1, eIF-2, eIF-3, eIF4A, eIF-4B, eIF-4C and e-IF-5. The purification from the KCl wash of crude ribosomes involves fractionation with ammonium sulphate, ion-exchange chromatography and separation by size. The operational definition of an initiation factor was its requirement for translation of natural messenger RNA (globin mRNA) in a highly purified and fractionated system using completely defined elongation components, i.e. aminoacyl-tRNA, the two elongation factors EF-1 and EF-2, and GTP. By the same criterion ATP was also shown to be required for initiation. The initiation factors were purified to homogeneity with the exception of eIF-4B, which was 60% to 70% pure. They were characterized physically by sucrose gradient centrifugation and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. With the exception of eIF-2 and eIF-3, they consist of single polypeptide chains ranging in molecular weight from 15,000 (eIF-1) to about 160,000 (eIF-5). The factor eIF-2 has three subunits of about 35,000, 50,000 and 55,000 molecular weight. The factor eIF-3 appears to be homogeneous as judged by gel electrophoresis in non-dissociating conditions and sedimentation analysis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, however, reveals at least nine subunits ranging in molecular weight from about 35,000 to 160,000. Initiation complexes (mRNA · Met-tRNA_f · 80 S ribosome), made in the presence of the seven initiation factors, ATP and GTP were isolated on a sucrose gradient and shown to be fully active in polypeptide chain elongation when supplied with aminoacyl-tRNA, the two elongation factors and GTP.     

Binding of glycoproteins of microsomal and Golgi membranes to lectins.

Four subunits of the acetylcholine receptor molecule, obtained from the electric organ of $\text{Torpedo ocellata}$, have been isolated using polyacrylamide gel electrophoresis, and assayed by titration with a fluorescent lanthanide, terbium, and by affinity-labeling with $\text{p}$-($\text{N}$-maleimido)benzyl [trimethyl-³H] ammonium iodide. The site with which the activator-analogue affinity label reacts, as well as the terbium-binding sites, are mainly associated with the smallest of the subunits of an apparent molecular weight of 40,000. Calcium competes with terbium for these binding sites. The affinity for terbium is the same in the intact molecule as in the subunit (K_Tb ? 19 ± 1 μM), but the affinity for calcium decreases by a factor of 4 (K_Ca ? 4 mM) in the subunit. Hydrolysis of the receptor, catalyzed by trypsin and chymotrypsin, to peptides with an apparent molecular weight of 8000 or less, does not affect the terbium-binding sites. These experiments indicate that the binding sites for neural activators and for calcium are associated with the same subunit, and that the terbium- and calcium-binding sites reflect structural properties of the polypeptide chain rather than the three-dimensional structure of the protein.     

Pyridine nucleotide transhydrogenases of parasitic helminths.

Mitochondria from the parasitic helminth, Hymenolepis diminuta, catalyzed both NADPH:NAD⁺ and NADH:NADP⁺ transhydrogenase reactions which were demonstrable employing the appropriate acetylpyridine nucleotide derivative as the hydride ion acceptor. Thionicotinamide NAD⁺ would not serve as the oxidant in the former reaction. Under the assay conditions employed, neither reaction was energy linked, and the NADPH:NAD⁺ system was approximately five times more active than the NADH:NADP⁺ system. The NADH:NADP⁺ reaction was inhibited by phosphate and imidazole buffers, EDTA, and adenyl nucleotides, while the NADPH:NAD⁺ reaction was inhibited only slightly by imidazole and unaffected by EDTA and adenyl nucleotides. Enzyme coupling techniques revealed that both transhydrogenase systems functioned when the appropriate physiological pyridine nucleotide was the hydride ion acceptor. An NADH:NAD⁺ transhydrogenase system, which was unaffected by EDTA, or adenyl nucleotides, also was demonstrable in the mitochondria of H. diminuta. Saturation kinetics indicated that the NADH:NAD⁺ reaction was the product of an independent enzyme system. Mitochondria derived from another parasitic helminth, Ascaris lumbricoides, catalyzed only a single transhydrogenase reaction, i.e., the NADH:NAD⁺ activity. Transhydrogenase systems from both parasites were essentially membrane bound and localized on the inner mitochondrial membrane. Physiologically, the NADPH:NAD⁺ transhydrogenase of H. diminuta may serve to couple the intramitochondrial metabolism of malate (via an NADP linked “malic” enzyme) to the anaerobic NADH-dependent ATP-generating fumarate reductase system. In A. lumbricoides, where the intramitochondrial metabolism of malate depends on an NAD-linked “malic” enzyme which is localized primarily in the intermembrane space, the NADH:NAD⁺ transhydrogenase activity may serve physiologically in the translocation of hydride ions across the inner membrane to the anaerobic energy-generating fumarate reductase system.     

Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors

The assembly of initiation complexes is studied in a protein synthesis initiation assay containing ribosomal subunits, globin [¹²⁵I]mRNA, [³H]Met-tRNA_f, seven purified initiation factors, ATP and GTP. By omitting single components from the initiation assay, specific roles of the initiation factors, ATP and GTP are demonstrated. The initiation factor eIF-2 is required for the binding of Met-tRNA_f to the 40 S ribosomal subunit. The initial Met-tRNA_f binding to the small ribosomal subunit is a stringent prerequisite for the subsequent mRNA binding. The initiation factors eIF-3, eIF-4A, eIF-4B and eIF-4C together with ATP promote the binding of mRNA to the 40 S initiation complex. The association of the 40 S initiation complex with the 60 S ribosome subunit to form an 80 S initiation complex is mediated by the initiation factor eIF-5 and requires the hydrolysis of GTP. The factor eIF-1 gives a twofold overall stimulation of initiation complex formation. A model of the sequential steps in the assembly of the 80 S initiation complex in mammalian protein synthesis is presented.     

Ceramide and inositol content of the lipopeptidophosphoglycan from Trypanosoma cruzi.

Inositol (2.5%), 17-methyl-sphinganine (4.8%) and sphinganine (1.5%) have been identified as constituents of the lipopeptidophospholycan isolated from whole cells of epimastigote forms of $\text{Trypanosoma cruzi}$. The branched chain base was characterized by combined gas chromatography — mass spectrometry.     

A reappraisal of the relationship of phosphate-acceptor protein to parvalbumins.

A phosphate-acceptor protein thought to be related to parvalbumins was described from dogfish muscle (Blum, H.E. $\text{et al.}$, 1974 Proc. Nat. Acad. Sci. USA $\text{71}$, 2198–2202). Further examination of this material indicated that the fraction obtained contained mainly classical parvalbumin, contaminated by less than 5% of a true phosphate-acceptor protein of MW ca 18 000 that accompanies parvalbumin throughout its purification. No such acceptor could be found in hake subjected to identical purification procedures. It is concluded that a phosphate-acceptor protein such as found in dogfish muscle bears no relation to parvalbumins.     

Immunogenicity of allograft components: I. Assay for immunogenicity

We describe an assay for in vivo quantitation of the “immunogenicity” of isolated cell populations. The assay is based on the observation that if an AgB-incompatible recipient rat is primed with donor strain spleen cells 72 hr prior to transplantation, heart allograft survival is reduced from 6.2 to 3.0 days. The effect is independent of the priming cell dose at levels above 3 × 10⁵ cells, whereas doses lower than 10⁵ spleen cells are unable to reduce the survival. The effect is suboptimal if the priming-transplantation interval is less than 3 days, or is prolonged to 4–10 days. The effect is immunologically specific: priming with irrelevant AgB-incompatible spleen cells fails to reduce the survival. Priming with cell populations previously reported “less immunogenic,” such as ultrasonicated spleen cells, erythrocytes, spleen T cells, or spleen cells deriving from methotrexate or cyclophosphamide-treated rats, fails to reduce the survival, or reduces it only when given in 100-fold higher numbers than the minimal dose of intact spleen cells giving maximal reduction.     

Interaction of Bacillus Calmette--Guérin-activated macrophages and neoplastic cells in vitro II. The relationship of selective binding to cytolysis

5′-Methylthioadenosine (MTA), a degradation product of S-adenosylmethionine, inhibits DNA and protein synthesis as well as cellular proliferation in human lymphocyte cultures stimulated with mitogens, antigens, or allogeneic cells. MTA (10^?3M) inhibited [³H]Tdy uptake in PHA- or Con A-induced transformation greater than 95%, and inhibited the uptake of both [³H]Tdy and [¹⁴C]Leu to the same degree in lymphocytes stimulated with PPD or allogeneic lymphocytes. MTA inhibition was dose dependent, inhibition being lost when exogenous levels reached approximately 10^?5M. The inhibitory effects of MTA were not produced by cytotoxicity since MTA-inhibited cells washed free of this compound could be stimulated at least as well as uninhibited cells. Understanding the mode of action of MTA and the mechanisms controlling its metabolism may lead to new approaches for regulating cellular proliferation.     

Apparent cooperative effects in acetylcholine receptor-mediated ion flux electroplax membrane preparations.

The kinetics of acetylcholine receptor-mediated flux of ²²sodium ions from microsacs has been measured in the presence of activators (carbamylcholine and decamethonium) and an inhibitor (d-tubocurarine) of neural transmission. The dependence of the first-order rate constant, k_obs, for ²²sodium ion efflux on either decamethonium or carbamylcholine concentration does not exhibit cooperativity. The apparent cooperativity observed by Kasai and Changeux in dose-response curves for ²²sodium flux from the same preparation is adequately accounted for by the contribution which efflux from non-excitable microsacs, the main component of the preparation, makes to the measurements. d-Tubocurarine was found to be a non-competitive inhibitor of decamethonium-activated ²²sodium efflux. The results of the kinetic measurements are in agreement with equilibrium measurements of the interaction of decamethonium with the same microsac preparation, i.e. adherence to a classic Langmuir binding isotherm and separate binding sites for activators and inhibitors of neural activity. The results indicate a direct relationship between ligand binding and receptor-mediated ion flux. How these two processes contribute to electrophysiological measurements is not apparent.     

Amino acid esterification by alpha-chymotrypsin immobilized in spiropyran membrane.

α-Chymotrypsin was immobilized in a collagen membrane modified with a spiropyran compound. The immobilized chymotrypsin was used for the esterification of N-acetyl-l-tyrosine (AT). N-Acetyl-l-tyrosine ethyl ester (ATEE) was synthesized from AT and ethanol by immobilized chymotrypsin under visible light. The optimum pH for the esterification was 7. An increase of the chymotrypsin content in the spiropyran-collagen membrane increased the rate and the yield of ATEE. The yield of ATEE reached 40% under visible light. Initially, ATEE was synthesized in the dark. However, the ATEE synthesized was gradually hydrolyzed in the dark. The amount of ATEE in the reaction mixture increased with irradiation by visible light and decreased in the dark. Therefore, the esterification of N-acetyl-l-tyrosine was controlled by light irradiation.     

Mutagenesis and anti-mutagenesis in Salmonella: influence of ethionine and caffeine on yields of mutations induced by 2-aminopurine and 9-aminoacridine

Ethionine, the ethyl analogue of methionine, slightly reduced the yield of reversions of the hisC3076 frameshift marker induced by 9-aminoacridine (9AA) in an excision-proficient strain of Salmonella typhimurium, but completely abolished mutagenesis genesis by 9AA in the excision-deficient uvrB-deletion strain TA1537. No toxic effects of ethionine were apparent in either the excision-proficient or the excision-deficient strain. Because of the differential effects of ethionine on mutagenesis in the two strains, it seemed possible that an ethionine-sensitive step in the process(es) leading to fixation of 9AA-induced mutations might be compensated for by the uvrA,B,C⁺ excision-repair system. To further test this possibility, we used caffeine (a compound known to significantly reduce the efficacy of the excision-repair process) as a co-treatment with ethionine for cells of an excision-proficient strain exposed to 9AA. Treatment with caffeine alone or ethionine alone had very little effect on reversion yield, whereas co-treatment with the two agents abolished 9AA mutagenesis. It appeared, therefore, that either the caffeine-sensitive pathway or the ethionine-sensitive pathway needed to be functioning if 9AA-induced reversions of the hisC3076 marker were to be detected. Addition of methionine to cells of the excision-deficient strain exposed to 9AA restored their ability to be mutated by 9AA, however. In a base-pair substitution back-mutation system, ethionine slightly enhanced the yields of revertants of the trpE8 marker induced by 2-aminopurine (2AP) in both an excision-proficient strain (at all 2AP dose levels tested) and an excision-deficient strain (only at the lower dose levels). In the excision-deficient strain, doses of 2AP above 300 μg/plate were highly toxic when ethionine was also present. It was for this reason that no 2AP-induced revertants were recovered at the higher 2AP concentrations. Treatment of the trpE8 strain with methionine also enhanced the yield of 2AP-induced revertants of this marker.     

Purification and properties of mitochondrial and peroxisomal 3-hydroxyacyl-CoA dehydrogenase from rat liver

There are two 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) in rat liver, one in mitochondria (type I enzyme), and another in peroxisomes (type II enzyme). In a series of the studies on the properties and the physiological roles of fatty acid oxidation systems in both organelles, the two enzymes were purified and compared for their properties. The final preparations obtained were judged to be homogeneous based on the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation velocity analysis. Type I enzyme was composed of two identical subunits of molecular weight of 32,000, whereas type II enzyme was a monomeric enzyme having a molecular weight of 70,000–77,000. These subunit structures were confirmed by the results of fluorescence studies. Both enzymes were different in amino acid compositions, especially in the contents of tryptophan and half-cystine. Antibodies against them formed single precipitin lines for the corresponding enzymes, but not for the others when subjected to an Ouchterlony double-diffusion test. The K_m values of type II enzyme for various substrates were lower than those of type I enzyme except those for acetoacetyl-CoA. As for 3-hydroxyacyl-CoA substrates, both enzymes had lower K_m's for longer-chain substrates. The V for the substrates of C₄C₁₀ were similar for each enzyme, though the value of type II enzyme for C₁₀ substrate was rather lower. The results of fluorescence studies suggested that their dissociation constants for NADH were lower and those for NAD⁺ were higher at lower pH. Both enzymes were specific to l-form of 3-hydroxyacyl-CoA substrate. The optimal pH of the forward reaction of type I and type II enzymes was 9.6 and 9.8, and of the reverse reaction, 4.5 and 6.2, respectively. From these results they were concluded to be completely different enzymes.     

The role of copper and quaternary structure on the conformational properties of Octopus vulgaris hemocyanin

Some structural properties of Octopus vulgaris hemocyanin have been investigated by fluorescence spectroscopy. The three-dimensional structure of Octopus hemocyanin is remarkably tight, resulting in a deep burial of almost all the tryptophyl residues of the protein. The hemocyanin conformation has been studied in the two main aggregation states (11 S, 50 S) of the protein, and with respect to the presence or absence of copper in the active site. Upon changing the pH of the solution, Octopus hemocyanin in the 50 S aggregation state can assume at least three different conformations. During the transition between each conformation the fluorescence quantum yield changes, but the environment of tryptophans does not change. Dissociation of the protein from 50 S to 11 S strongly enhances its susceptibility toward denaturating agents such as pH or temperature, and modifies the effects of fluorescence quenchers such as acrylamide. Moreover, these effects are more pronounced when copper is removed from the active site. A comparative analysis of the results shows that the subunit-subunit interactions exerted within the 50 S species are more important in the maintenance of the conformational stability than the copper ions present in the active sites. This behavior can be accounted for by the large amount of Ca(II) ions linked to 50 S hemocyanin.     

Interaction between calcium and ligand-binding sites of the purified acetylcholine receptor studied by use of a fluorescent lanthanide

The acetylcholine receptor isolated from $\text{Torpedo ocellata}$ binds about 10 moles of a fluorescent lanthanide, terbium, per mole α-bungarotoxin-binding site, a process which is accompanied by a fluorescence enhancement (λexcitation 295 nm, λemission 546 nm) which allows detection of receptor-Tb³⁺ complexes at μM concentrations. In presence of calcium two types of terbium-binding site are revealed, both with terbium dissociation constants of 18 ± 0.5 μM. About 60% of the sites bind calcium with an apparent dissociation constant of 1.1 ± 0.1 mM. Sites which interact with calcium also interact with activators of neural transmission, carbamylcholine and decamethonium, but not with the inhibitors, d-tubocurarine and α-bungarotoxin. Whether the displacement of calcium by chemical mediators is directly responsible for activator-induced changes in ion permeability of neural membranes is an important question raised by our experiments. The results show that fluorescent lanthanides can be an important tool in such studies.