The halobacterial H+-translocating ATP synthase relates to the eukaryotic anion-sensitive H+-ATPase

The H+-translocating ATP synthase of Halobacterium halobium (Y. Mukohata and M. Yoshida (1987) J. Biochem. 102, 797-802) includes a catalytic moiety of 320 kDa which is isolated as an azide-insensitive ATPase (T. Nanba and Y. Mukohata (1987) J. Biochem. 102, 591-598). The polyclonal antibody against this archaebacterial ATPase cross-reacts with the anion-sensitive H+-ATPase of red beet, Beta vulgaris, tonoplast as well as with another archaebacterial ATPase from Sulfolobus acidocaldarius. The affinity is much higher than to F1-ATPase from spinach chloroplasts or to Ca2+-ATPase from sarcoplasmic reticulum of rabbit skeletal muscle.     

Single site catalysis of the F1-ATPase from Saccharomyces cerevisiae and the effect of inorganic phosphate on it

The kinetical characteristics of ATP hydrolysis by mitochondrial F1-ATPase from Saccharomyces cerevisiae (yeast) have been studied under conditions where only a single catalytic site per enzyme molecule bound ATP. Four major features were observed, that is, fast ATP binding to the enzyme, slow product release from the enzyme, an equilibrium close to unity between ATP and products on the enzyme, and promotion of ATP hydrolysis on the second addition of a large excess of ATP (cold chase). These are essentially the same as the kinetical characteristics observed for beef heart mitochondrial F1-ATPase, which were called as unisite catalysis by Grubmeyer et al. (Grubmeyer, C. et al. (1982) J. Biol. Chem. 257, 12092-12100), although the release of a hydrolysis product, Pi, from the yeast enzyme appeared to occur significantly faster than that from the beef enzyme, which resulted in a decreased extent of cold chase promotion of ATP hydrolysis of the yeast enzyme. The yeast F1-ATPase showed unisite catalysis even in the absence of Pi in the reaction mixtures, while it was reported for the beef F1-ATPase that the presence of Pi in the reaction mixture was essential for unisite catalysis (Penefsky, H.S. & Grubmeyer, C. (1984) in H+-ATPase (ATP Synthase) (Papa, S. et al., eds.) pp. 195-204, The ICSU Press). Another difference in the Pi effect on the kinetics was that ATP hydrolysis was initiated without a lag time in the absence of Pi in the case of the yeast enzyme when a 1,000-fold molar excess of ATP per enzyme molecular was mixed with the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of carcinogenic N-nitrosopropylamines to mutagens by lung and pancreas S9 fractions from various animal species and man

The mutagenic potential of 7 carcinogenic N-nitrosopropylamines was examined by the Ames liquid incubation assay, using lung and pancreas 9000 × g supernatant (S9) fractions from rats, hamsters, mice, rabbits, monkeys and humans for metabolic activation. N-Nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP), N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitrosomethyl(2-oxopropyl)amine (MOP) showed positive mutagenicity in strain TA100 in the presence of lung S9 from each of the uninduced animals and humans. Besides the 3 N-nitrosopropylamines, N-nitrosomethyl(2-hydroxypropyl)amine (MHP) was also positive in the presence of lung S9 from polychlorinated biphenyl (PCB)-induced rats, hamsters and mice. On the other hand, in the presence of pancreas S9 from uninduced or PCB-induced animals, only HPOP and BOP showed positive mutagenicity. In contrast, N-nitrosobis(2-hydroxypropyl)amine (BHP), N-nitrosobis (2-acetoxypropyl)amine (BAP) and N-nitroso-2,6-dimethylmorpholine (NDMM) showed negative mutagenicity in the presence of lung and pancreas S9 from either uninduced or PCB-induced animals and humans. HPOP was a direct-acting mutagen, and lung and pancreas S9 from 5 animal species and man did not affect the activity. BOP was mutagenic even in the presence of bovine serum albumin. The mutagenic activation of MHP by lung S9 from PCB-induced rats, hamsters and mice was completely inhibited by preincubation in an atmosphere of carbon monoxide or by addition of cytochrome c or metyrapone to the S9 mixture, whereas 7,8-benzoflavone totally lacked this effect. However, that of MOP was insensitive to these inhibitors. These results of mutagenicity assay indicate that only the methyl derivatives of N-nitrosopropylamines, MHP and MOP are activated by the lung from 5 animal species and man, whereas the pancreas from all the tested animals did not activate the 7 N-nitrosopropylamines to mutagens, and that the phenobarbital-inducible major cytochrome P-450 in the lung of rodents is involved in the mutagenic activation of MHP.     

Constriction of fetal ductus arteriosus by non-steroidal anti-inflammatory drugs: Study of additional 34 drugs

Our study on transplacental effects of 24 non-steroidal anti-inflammatory drugs (NSAID) on the fetal ductus arteriosus of full-term pregnant rats was extended to 34 other NSAID using the same whole-body freezing technique (1). In total, 58 NSAID were evaluated, and their potency in usual clinical doses was classified into 4 grades. Indomethacin and 15 other NSAID caused strong fetal ductal constriction, phenylbutazone and 14 other NSAID caused moderate, and aspirin and 16 other NSAID caused mild constriction of the fetal ductus arteriosus. Salicylamides, and six out of eight basic NSAID did not constrict the fetal ductus arteriosus. Further clinical implications of these results are discussed.     

Metabolic conversion of N-methyl carbon of {gamma}-glutamylmethylamide to caffeine in tea plants

Tea seedlings were treated with ¹⁴C-methylamine to cause synthesisof ¹⁴C--glutamylmethylamide (N-methyl-¹⁴C). The metabolic conversionof -glutamylmethylamide was studied by tracing ¹⁴C. ¹⁴C--Glutamylmethylamide (N-methyl-¹⁴C) translocated from rootsand cotyledons to shoots of tea seedlings, was converted almostentirely into caffeine. Conversion was greater in light-exposedsamples. For those grown in the dark, the converted amount didnot correspond to the total caffeine produced. More ¹⁴C--glutamylmethylamidewas present in stems than in leaves, but with ¹⁴C-caffeine,the opposite was found. When ¹⁴C--glutamylmethylamide or ¹⁴C-methylamine was appliedto leaf disks, ¹⁴C-caffeine was biosynthesized from both substances. ¹ Present address: Department of Agricultural Chemistry, ShizuokaUniversity, Iwata, Shizuoka 438, Japan. (Received September 25, 1971; )     

Carotenoids and fatty alcohols as non-ionic hydrophobic inhibitors of photochemical activities of chioroplasts

Carotenoids and fatty alcohols were added to intact spinachchloroplasts in suspension, and the effects of these reagentson the absorption spectrum and photochemical activities of chloroplastswere examined. The addition of lutein, ß-caroteneand neoxanthin transformed a chlorophyll a form (C684) havingan absorption maximum at 684 nm into C666, and the activityof photosystem II decreased parallel with this transformation.The activity of photosystem I was completely unaffected by theeffect of added carotenoids, whereas both cyclic and non-cyclicphotophosphorylations were inhibited by the additions. Theseeffects were reproducibly observed only with a "petroleum ether-soluble"fraction of each carotenoid, monomeric and less polymerizedforms, which are soluble in petroleum ether at –25°C;the "insoluble" fraction of more polymerized forms was ineffective.Comparative experiments with lutein, DCMU and CCCP as inhibitorsand the dependence of inhibition on light intensity suggestedthat the site of inhibition by carotenoids is on the water sideof photosystem II between its reaction center and the CCCP-inhibitingsite. Fatty alcohols with carbon atoms between 8 and 12 producedstronger but less specific effects on the spectrum and activities.At low concentrations the uncoupling of photophosphorylationoccurred and, in a middle concentration range, the activityof photosystem II decreased with the transformation of C684into C666 and with a change in carotenoid bands. At higher concentrations,where the enhanced activity of photosystem I decreased froma maximum to zero, the red band of chlorophyll a further changedin a complex manner and the absorption around 503 nm decreased.We, thus, deduced that C684 and some endogeneous carotenoidsare associated with photosystem II on its water side and thatadded carotenoids and fatty alcohols at lower concentrationschange their states, resulting in the inhibition of this photosystem. (Received December 20, 1971; )     

Effects of cationic and anionic chain compounds on absorption spectra and photochemical activities of chloroplasts

The effects of ionic chain compounds on absorption spectra andphotochemical activities of spinach chloroplasts in suspensionwere investigated and compared with the effects of non-ionicchain compounds previously studied. Two types of spectral changestook place when chloroplasts were treated with ionic compounds.One type of change, not observable with non-ionic chain compoundssuch as carotenoids and fatty alcohols, was in the efficiencyof light absorption; absorption bands over the whole visibleregion were either intensified or flattened at low reagent concentrations.Intensification was observed with anionic fatty acids with 10–18carbon atoms and ascribed to swelling of whole chloroplasts,while flattening was observed with cationic primary and quarternaryamines and ascribed to aggregation of chloroplasts. The othertype of spectral change, transformation of bands observed athigh concentrations of ionic chain compounds, was essentiallysimilar to that found with non-ionic chain compounds. The redand Soret bands of chlorophylls were transformed and absorptionin the range of 490–520 nm decreased on treatment withionic chain compounds. Activity of photosystem I was enhancedby reagents at low concentrations and inhibited at high concentrations,while activity of photosystem II decreased in a middle concentrationrange. These activity changes were correlated to the spectralchanges, although the correlations were less marked than thosefound with non-ionic compounds. This is probably because ofoverlapping of the two types of changes which occur with ionicchain compounds. (Received March 8, 1972; )     

Hepatic collagenolytic activity in rats after carbon tetrachloride poisoning

1. Collagenolytic activity towards acid-soluble collagen labelled with [(14)C]-proline was assayed in rat liver with and without carbon tetrachloride poisoning. The products of enzymic digestion were found to be free amino acids and peptides. 2. The hepatic collagenolytic activity increased under conditions of single-dose and subacute carbon tetrachloride poisoning, and correlated with hydroxyproline content. The highest activity was found during recovery from subacute poisoning. 3. Under the same experimental conditions, hepatic acid-proteinase activity changed independently of the collagenolytic activity and also of hepatic hydroxyproline content. 4. The increased collagenolytic activity during carbon tetrachloride poisoning was found mainly in the supernatant fraction. 5. The ratio of the collagenolytic activity to hepatic hydroxyproline content increased during recovery from single-dose and subacute poisoning, and decreased during subacute poisoning.