Endogenous phosphorylation of retinal photoreceptor outer segment proteins by calcium phospholipid-dependent protein kinase

A calcium phospholipid-dependent protein kinase (C-kinase) activity was detected in the soluble fraction of rod outer segments (ROS) of the bovine retina. The enzyme required calcium, phosphatidylserine (PS) and diacylglycerol for maximal activity. In the presence of calcium and PS, C-kinase endogenously phosphorylated proteins with molecular weights of 95,000, 91,000, 31,000, 21,000, 19,000, 18,000, 16,000, 14,000 and 11,000. Addition of diolein in the reaction mixture further enhanced the endogenous phosphorylation of these proteins. Retinal was found to inhibit the phosphorylation of endogenous proteins by C-kinase in a concentration dependent manner. Half-maximal inhibition of enzyme activity was obtained at a retinal concentration of about 12μM. These results suggest that calcium, phospholipids and the C-kinase enzyme may play an important role in the functional regulation of rod photoreceptors and, with retinal, perhaps in the visual process as well.     

Electrolysis-mediated irreversible inactivation of lipoxygenase directed toward electroaffinity labelling

Irreversible inhibition of soybean lipoxygenase-1 (SL-1) was accomplished via a controlled potential oxidative electrolysis of 1,5-dihydroxynaphthalene (1,5-DHN) at +0.8 V vs SCE. The inactivation of SL-1 with this known inhibitor was greatly enhanced under these electrolytic conditions to which the enzyme itself was stable. Electrolyses were run at 0 degree C in a 0.05 M phosphate buffer, pH 7.0, using graphite cloth electrodes. The rate of inactivation was observed to be limited by and dependent on the anodic oxidation of 1,5-DHN. The non-oxidizable (at this potential) inhibitor indomethacin was shown to protect the enzyme from irreversible inactivation, however, an external nucleophile (2-mercaptoethanol) had little effect. These initial studies support the capability of such electrochemical methods for the site-specific covalent modification (affinity labelling) of lipoxygenase, and perhaps other enzymes.     

Highly potent and specific inhibitors of human renin

We designed aldehyde derivatives of small peptides representing the C-terminal portion of angiotensin I sequence as an inhibitor of human renin. Among compounds that we synthesized, benzyloxycarbonyl (Z)-Phe-His-Leucinal (compound V), Z-Pro-Phe-His-Leucinal (Compound IV) and Z-[3-(1'-naphthyl)Ala]-His-Leucinal (compound VII) markedly inhibited human renin (IC50, 7.5 X 10(-7), 3.2 X 10(-7) and 8.0 X 10(-8) mol/l, respectively). Compound VII was shown to be noncompetitive (Ki = 2.4 X 10(-7) mol/l). It did not inhibit either cathepsin D or pepsin. Compound V had slight or no inhibitory effect at the concentration of 10(-5) mol/l on six animal renins except for monkey and rabbit renins. Results obtained show that these aldehyde compounds are highly selective and species specific inhibitors for human and monkey renins.     

Zinc is a constituent of bovine heart cytochrome c oxidase preparations

Cytochrome c oxidase preparations from bovine heart muscle contain 1 zinc per 2 irons. Metal contents of nine preparations determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) show that Cu, Fe and Zn are the only metals present in significant amounts with average Cu/Fe, Fe/Zn, and Cu/Zn atom ratios of 1.3, 2.1 and 2.8, respectively. Removal of adventitious copper results in a Cu:Fe:Zn stoichiometry of 2:2:1. The zinc is tightly bound. Dialysis against a solution of 1,10-phenanthroline at pH 7.4 or an acidic buffer (pH 4.4) does not remove Zn. Dialysis against 0.8 M KCN at pH 10 causes partial loss of both Cu and Zn. This is the first evidence for the presence of Zn in a cytochrome c oxidase.     

Receptor-mediated regulation of calcium mobilization and cyclic GMP synthesis in neuroblastoma cells

In neuroblastoma N1E 115 cells, carbachol, histamine and PGE1 elevated cyclic GMP content and, induced the efflux of preloaded 45Ca2+, the release of membrane-bound Ca2+ measured by fluorescent CTC, and the increase in [Ca2+]i as measured by Quin 2 fluorescence. The time course of the responses, the absolute requirement of extracellular Ca2+, the inhibition by receptor blockers, and the concentration dependency on histamine were all similar between these responses. The observation indicates that the mobilization of Ca2+, especially the increase of [Ca2+]i, may be intimately linked to the synthesis of cyclic GMP in the cells.     

Chronic administration of an organophosphorus insecticide to rats alters cholinergic muscarinic receptors in the pancreas

Male rats were treated for 10 days with the organophosphorus insecticide, acetylcholinesterase inhibitor, O,O-diethyl S-[2-(ethylthio)ethyl]phosphorodithioate (disulfoton, 2 mg/kg/day by gavage). At the end of the treatment, binding of [³H]quinuclidinyl benzilate ([³H]QNB) to cholinergic muscarinic receptors and cholinesterase (ChE) activity were assayed in the pancreas. Functional activity of pancreatic muscarinic receptor was investigated by determining carbachol-stimulated secretion of α-amylase in vitro. ChE activity and [³H]QNB binding were significantly decreased in the pancreas from disulfoton-treated rats. The alteration of [³H]QNB binding was due to a decrease in muscarinic receptor density with no change in the affinity. Basal secretion of amylase from pancreas in vitro was not altered, but carbachol-stimulated secretion was decreased. The effect appeared to be specific since pancreozymin was able to induce the same amylase release from pancreases of control and treated rats. The results suggest that repeated exposures to sublethal doses of an organophosphorus insecticide lead to a biochemical and functional alteration of cholinergic muscarinic receptors in the pancreas.     

In vitro alkylation of calf thymus DNA by acrylonitrile. Isolation of cyanoethyl-adducts of guanine and thymine and carboxyethyl-adducts of adenine and cytosine

Reaction of the rodent carcinogen acrylonitrile (AN) at pH 5.0 and/or pH 7.0 for 10 and/or 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), 2'-deoxyinosine (dIno), N6-methyl-2'-deoxyadenosine (N6-Me-dAdo) and thymidine (dThd) resulted in the formation of cyanoethyl and carboxyethyl adducts. Adducts were not detected after 4 h. The adducts isolated were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo), N6-CE-dAdo, 3-CE-dCyd, 7-(2-cyanoethyl)-Gua (7-CNE-Gua), 7,9-bis-CNE-Gua, imidazole ring-opened 7,9-bis-CNE-Gua, 1-CNE-dIno, 1-CE-N6-Me-dAdo and 3-CNE-dThd. Structures were assigned on the basis of UV spectra and electron impact (EI), chemical ionization (CI), desorption chemical ionization (DCI) and Californium-252 fission fragment ionization mass spectra. Evidence is presented which strongly suggests that N6-CE-dAdo was formed by Dimroth rearrangement of 1-CE-dAdo during the reaction between AN and dAdo. The carboxyethyl adducts resulted from initial cyanoethylation (by Michael addition) at a ring nitrogen adjacent to an exocyclic nitrogen atom followed by rapid hydrolysis of the nitrile moiety to a carboxylic acid. It was postulated that the facile hydrolysis is an autocatalyzed reaction resulting from the formation of a cyclic intermediate between nitrile carbon and exocyclic nitrogen. AN was reacted with calf thymus DNA (pH 7.0, 37 degrees C, 40 days) and the relative amounts of adducts isolated were 1-CE-Ade (26%), N6-CE-Ade (8%), 3-CE-Cyt (1%), 7-CNE-Gua (26%), 7,9-bis-CNE-Gua (4%), imidazole ring-opened 7,9-bis-CNE-Gua (19%) and 3-CNE-Thy (16%). Thus a carcinogen once adducted to a base in DNA was shown to be subsequently modified resulting in a mixed pattern of cyanoethylated and carboxyethylated AN-DNA adducts. Three of the adducts (1-CE-Ade, N6-CE-Ade and 3-CE-Cyt) were identical to adducts previously reported by us to be formed following in vitro reaction of the carcinogen beta-propiolactone (BPL) and calf thymus DNA. The results demonstrate that AN can directly alkylate DNA in vitro at a physiological pH and temperature.     

Heterogeneity of hepatic microsomal UDP-glucuronosyltransferase(s) activities: comparison between human and mammalian species activities

The first comparative profiles of UDP-glucuronosyltransferase(s) (UDPGT) activities obtained under standard conditions in vitro in mammals (man, rat [Wistar and Gunn], mouse, monkey [Papio papio and Cynomolgus], pig, guinea pig, rabbit, dog) are presented for 16 aglycones. A decreasing scale of these activities was obtained from planar to bulky molecules. The scale was identical for each of the mammals studied, including man. Statistical analysis of the results revealed a division of the aglycones into three groups, one being correlated with the molecular form called GT1 the two others with the GT2 form. The profile of activities in the Gunn rat revealed very weak activity towards planar molecules (GT1). These results provide evidence that under standard conditions, human UDPGT activities are comparable to those from other animals.     

Mutagenicity of 7,12-dimethyl[a]anthracene and some other aromatic mutagens in Drosophila melanogaster

The optimal conditions for mutagenesis studies with DMBA and some other aromatic carcinogens in Drosophila were investigated in detail. The results presented in this paper indicate the following.The mutagenic effectiveness of DMBA is dependent on the route of administration, injection being far more effective when compared with feeding.The choice of the solvent is a crucial experimental condition. DMBA, when dissolved in oil/DMF, is ineffective whereas a special fat emulsion of DMBA gives high mutation frequencies.There appears to be an extreme strain dependence in the mutagenicity of DMBA. Mutagenic effectiveness in strain Berlin-K was rather low, whereas Oregon-K and Karsnäs-60 proved to be very susceptible to DMBA.Under the conditions of test, DMBA did not induce loss of a ring-X chromosome and did not produce recessive lethal mutations in such a chromosome.DMBA did not produce 2–3 translocations to any significant extent.An increase in DMBA-induced recessive lethal mutations was found upon storage of treated sperm with an optimal storage time of 4–10 days.DMBA is efficient in the production of delayed recessive lethal mutations in strain Berlin-K. Twice as many lethals were recovered with the F3 generation as compared with those in F2. In strain Oregon-K, where the frequency of F2 lethals was much higher than in strain Berlin-K, the ratio of F3/F2 lethals was clearly lower.Enzyme induction with phenobarbital reduces the mutagenic effectiveness of DMBAWith TMBA, similar strain differences in sensitivity were observed as those found for DMBA. Whereas TMBA was not mutagenic in Berlin-K, considerable mutagenicity was observed in Oregon-K and Karsnäs-60.Injection of carcinogenic polycyclic aromatic hydrocarbons and aromatic amines, when dissolved in special fat emulsions, enhances the mutagenic effectiveness of some compounds (DMBA, TMBA, DA and AcO-AAF), but this procedure does not always solve the problems-pertinent to these classes of promutagens in Drosophila.     

Forward mutation assay of V79 cells to 6-thioguanine resistance in a soft agar technique that eliminates effects of metabolic co-operation

A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1?2 × 10⁶ cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 10⁵ cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 10⁶ cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture.