Monoclonal antibody-directed immunopurification and identification of cytochromes P-450

A 28 amino acid peptide with diuretic and natriuretic activity has been purified from rat atrial muscle. The primary structure of this atrial peptide is H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly- (sequence in text) Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-(Arg)-Tyr-OH. The biological activity of this peptide is identical to that of atrial natriuretic factor and cardionatrin I isolated from rat atria.     

Induction of 7-ethoxycoumarin o-deethylase activity in cultured human epithelial cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): evidence for TCDD receptor

The responsiveness of 5 human squamous cell carcinoma (SCC) lines derived from tumors of the epidermis and tongue to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) was assessed by measuring the induction of the cytochrome P1-450-mediated monooxygenase activity, 7-ethoxycoumarin O-deethylase (ECOD). In 4 of the SCC lines the EC50 for this response was approximately 10(-9)M, whereas in one line the EC50 was 10(-10)M. In each of the less sensitive lines a concentration of 10(-10)M TCDD elicited less than 5% of the maximal enzyme activity. Specific binding of radiolabeled TCDD was detected in the cytosol fraction from all the SCC lines. The relative amount of receptor measured in each line correlated with maximally-induced ECOD activity. The data indicate that human cell lines derived from a target tissue for TCDD toxicity contain the TCDD receptor and show differential sensitivity to TCDD analogous to the murine strain differences in sensitivity regulated by the Ah locus.     

The orientation of the magnetic axes of membrane-bound iron-sulfur clusters and a cytochrome b-559 in the green halophilic alga Dunaliella parva

Photosynthetic membrane fragments separated from whole cells of the green alga Dunaliella parva, were oriented by incorporation into multilayers on thin Mylar films. These partially dehydrated films were then examined by EPR spectroscopy for evidence of orientation of paramagnetic components. Five previously identified paramagnetic components, the reduced states of iron-sulfur clusters A and B, the intermediate acceptor X^?, the reduced Rieske iron-sulfur cluster, and oxidized cytochrome b-559, displayed EPR signals showing orientation. In addition, several previously unknown paramagnetic components were also observed to be oriented. Four components, previously characterized in spinach chloroplast preparations, the iron-sulfur clusters A and B, the intermediate acceptor X^?, and cytochrome b-559, were shown to be similar in the green alga, D. parva. The orientations of iron-sulfur clusters A and B, however, were determined unambiguously in this preparation; this was not possible in previous work with spinach. The heme plane orientation of cytochrome b-559 was found to be perpendicular to the membrane plane in agreement with the results in spinach preparations. A new photoinduced EPR signal with g values of 1.88, 1.97 and 2.12 was seen only in the oriented preparations and was indicative of a reduced iron-sulfur cluster with an orientation different from that of iron-sulfur cluster A or B. This suggests the existence of a previously unidentified acceptor in Photosystem I of green plants. These studies clearly show that the orientation of these components in bioenergetic membranes are conserved over a large span of evolutionary development and are, therefore, an important aspect of the mechanism of electron transfer.     

Isolation and characterization of light- and dithiothreitol-modulatable glucose-6-phosphate dehydrogenase from pea chloroplasts

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, EC 1.1.1.49) has been purified to electrophoretic homogeneity from pea chloroplasts. The enzyme, which has a Stokes radius of 52 Å, is a tetramer made up of four 56000 Da monomers. The pH optimum is around 8.2. The enzyme is absolutely specific for NADP. The apparent K_m(NADP) is 2.4 ± 0.1 μM. NADPH inhibition of the enzyme is competitive with respect to NADP (mean K_i, 18 ± 5 μM) and is mixed (K_p >K_m, V_max >V_p) with respect to glucose 6-phosphate (mean crossover point, 0.5 ± 0.1 mM). The apparent K_m(glucose 6-phosphate) is 0.37 ± 0.01 mM. The purified enzyme is inactivated in the light in the presence of dilute stroma and washed thylakoids, and by dithiothreitol. Enzyme which has been partially inactivated by treatment with dithiothreitol can be further inactivated in the light in the presence of dilute stroma and washed thylakoids and reactivated in the dark, but only to the extent of the reverse of light inactivation. Dithiothreitol-inactivated enzyme is not reactivated further by addition of crude stroma or oxidized thioredoxin. Dithiothreitol-dependent inactivation of the enzyme follows pseudo-first-order kinetics and shows rate saturation. The enzyme which has been partially inactivated by treatment with dithiothreitol does not differ from the untreated control with respect to thermal and tryptic inactivation. However, enzyme which has been partially light inactivated shows different thermal and tryptic inactivation patterns as compared to the dark control. These observations suggest that the changes in the enzyme brought about by light modulation are not necessarily identical with those brought about by dithiothreitol inactivation.     

Effects of N,N′-dicyclohexylcarbodiimide on isolated and reconstituted cytochrome b-c1 complex from bovine heart mitochondria

N,N′-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstitued mitochondrial cytochrome b-c₁ complex. DCCD inhibits equally electron flow and proton translocation (i.e., the $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratio is not affected) catalysed by the enzyme reconstituted into phospholipid vesicles. The inhibitory effects are accompanied by structural alterations in the polypeptide pattern of both isolated and reconstituted enzyme. Cross-linking was observed between subunits V (iron-sulfur protein) and VII, indicating that these polypeptides are in close proximity. A clear correlation was found between the kinetics of inhibition of enzymic activity and the cross-linking, suggesting that the two phenomena may be coupled. Binding of [¹⁴C]DCCD was also observed, to all subunits with the isolated enzyme and preferentially to cytochrome b with the reconstituted vesicles; in both cases, however, it was not correlated kinetically with the inhibition of the enzymic activity.     

Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: A novel assay

Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (I) and glycyl-glycyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (Δ? = 860 m^?1 cm^?1) and by ninhydrin colorimetry (substrate I, ?₅₇₀ = 2.31 × 10⁴m^?1 cm^?1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5–6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.     

Conversion of a primary amine to a labeled secondary amine by the addition of phenolic group and radioiodination

To preserve the nucleophilicity of amino compounds during conjugative radioiodination, a new method for converting primary amines to phenolic secondary amines was developed. Amino acids were used as model compounds for establishing optimal conditions for the reductive amination. In the first step of the reaction, the aldehyde group of 4-hydroxybenzaldehyde (formylphenol) was reacted reversibly with an amino group to form an imine. The irreversible attachment of formylphenol to the amino group was accomplished by reduction of the imine with sodium cyanoborohydride. The pH optimum for the reaction was 5.0. Higher temperature has favorable effects on the rate and extent of the conjugation. Phenolic derivatives of amino compounds suitable for radioiodination are produced by the reactions described.     

Characterization and Location of Met5-Enkephalin-Arg6-Phe7 Stored in Various Rat Brain Regions

A specific antiserum against met⁵-enkepha-lin-arg⁶-phe⁷ was raised and used to study the distribution and characterization of met⁵-enkephalin-arg⁶-phe⁷-like immunoreactive material in rat brains by radioimmunoassay and immunohistochemical procedures. The antiserum appears to be directed to the COOH-terminus of the peptide, as it fails to cross-react with met⁵-enkeph-alin, met³-enkephalin-arg⁶, met⁵-enkephalin-arg⁶-arg⁷, met⁶-enkephalin-lys⁶, and leu-enkephalin. However, it cross-reacts with phe-met-arg-phe by about 10% and with phe-met-arg-phe-NH₂ to an insignificant degree. The highest content of met⁵-enkephalin-arg⁶-phe⁷ was found in the striatum, which contains a dense network of immunoreactive varicose fibers and terminals, as well as immunoreac tive cell bodies. The met⁵-enkephalin-arg⁶-phe⁷ in striatum can be released in a Ca²⁺-dependent manner by a depolarizing concentration of KC1, raising the possibility of a neu-roregulatory role for met⁵-enkephalin-arg⁶-phe⁷. Characterization of the immunoreactive material by gel filtration and high pressure liquid chromatography revealed the presence of multiple forms of immunoreactive material in some brain regions.     

Neutral and Acid Sphingomyelinases of Rat Brain: Somatotopographical Distribution and Activity Following Experimental Manipulation of the Dopaminergic System In Vivo

The activities of neutral, magnesium-stimulated, and acid sphingomyelinases were measured in five regions of rat brain. Neutral enzyme activity was 2-3-fold higher in striatum than in parietal cortex and 13-fold higher than in cerebral white matter. Acid sphingomyelinase activity was more evenly distributed throughout these regions. Striatal neutral sphingomyelinase activity was not affected by treatment of rats with reserpine or haloperidol and was reduced (16%) by 6-hydroxydopamine. Striatal acid sphingomyelinase was unaffected by reserpine and 6-hydroxydopamine, and was increased (17%) by haloperidol. We conclude that neutral, magnesium-stimulated sphingomyelinase activity differs in various regions of rat brain and is particularly enriched in the corpus striatum. However, it appears to be a constitutive component of tissue rather than a readily modulated regulatory element of the catecholaminergic system.     

Mutations resistant to bromodeoxyuridine in mouse lymphoma cells selected by repeated exposure to EMS characteristics of phenotypic instability and reversion to HAT resistance by 5-azacytidine

Mutations induced by repeated EMS treatments were investigated by using mouse L5178Y cells. The frequency of TG^r mutations increased linearly with the number of EMS treatments whereas the yield of BrdUrd^r mutations showed a curvilinear dose-response curve. The BrdUrd^r frequency was roughly proportional to the square of the TG^r frequency and the results were compatible with the hypothesis that BrdUrd^r cells were induced by two mutational events within a cell. Most of the BrdUrd^r colonies isolated after 6 EMS treatments, however, were unstable. When BrdUrd^r colonies that had arisen in BrdUrd medium after 2 weeks' incubation were isolated in normal medium, the descendant cells showed a nearly normal level of thymidine incorporation and low plating efficiencies of about 1% in BrdUrd medium. In contrast, after isolation of the same colonies in BrdUrd medium, a low level of thymidine incorporation and high plating efficiencies in BrdUrd medium were observed in the descendant cells.

Reverse selection from BrdUrd^r to HAT^r was accomplished with frequencies of 10⁻⁶−10⁻³ for the descendants grown in BrdUrd medium, and AzaCyd treatment drastically increased the reversion frequency to nearly 10⁻¹. Further re-revertants from HAT^r to BrdUrd^r were also found with frequencies of 10⁻³−10⁻² without treatment. These results indicate that the initial BrdUrd^r cells did not result from inactivation of the thymidine-kinase gene but that the mode of gene expression was altered in some way.