Comparison of the properties of detergent-solubilized NADPH-cytochrome P-450 reductases from pig liver and kidney immunochemical,kinetic, and reconstitutive properties

NADPH-cytochrome P-450 reductases from pig liver and kidney and rabbit liver microsomes were purified to a specific activity of 50–62 μmol cytochrome c reduced/min/mg. All reductase preparations were separated into one major and one minor fraction on Sephadex G-200 columns. The molecular weights of the major fractions of the reductases were estimated to be 74,000, 75,000, and 75,500 for rabbit liver, pig kidney, and liver reductases, respectively, whereas the molecular weight of the minor fractions of these reductases, 67,000, was the same as that of the steapsin-solubilized pig liver reductase on SDS-polyacrylamide gel electrophoresis. K_m values for NADPH and cytochrome c were: 20 and 29 μm or 14 and 28 μm for the pig kidney or liver reductase, respectively. Immunochemical studies, including Ouchterlony double diffusion experiments and inhibition of benzphetamine N-demethylation activity in microsomes by antibody against pig liver NADPH-cytochrome P-450 reductase, indicated the similarity of the purified liver and kidney reductases. There were no differences in the ability to reconstitute NADPH-mediated benzphetamine N-demethylation and laurate hydroxylation in reconstituted systems between the pig liver and kidney reductases, indicating that the reductase did not determine substrate specificity in these systems.     

Enantiomer selection in the reaction of N-methyl-α-amino acid N-carboxyanhydride and 3-methyl-5-substituted hydantoin: A model reaction for the stereoselective polymerization of α-amino acid N-carboxyanhydride

The addition reaction to N-methyl-(S)-alanine or N-methyl-(S)-phenylalanine N-car-boxyanhydride (NCA) of 3-methyl-5-substituted hydantoin (HDT) catalyzed by a tertiary amine was investigated as a model reaction for the propagation reaction of NCA according to the activated-NCA mechanism. Several activated HDTs having the (S)-configuration of the asymmetric carbon atom were found to react more rapidly than their activated enantiomers. This experimental result indicates that the enantiomer selection by terminal-unit control takes place in the propagation reaction according to the activated-NCA mechanism in which an activated NCA is added to a terminal acylated NCA ring of the growing chain. The enantiomer excess of the HDT recovered from the reaction mixture of N-methyl-(S)-phenylalanine NCA and racemic HDTs activated by a tertiary amine was determined. The extent of the enantiomer selection in the polymerization was found to be 3–10 times as large as that in the model reaction. From these results, it was concluded that the chirality of the penultimate unit, as well as that of the terminal NCA ring, plays an important role in determining the enantiomer selection in the NCA polymerization.     

Radical mechanism of aminopyrine oxidation by cumene hydroperoxide catalyzed by purified liver microsomal cytochrome P-450

Under identical experimental conditions, purified preparations of rabbit liver microsomal cytochrome P-450 and beef heart metmyoglobin were equally effective at stimulating the oxidation of aminopyrine to a free radical species by cumene hydroperoxide. Mannitol had no effect on radical levels produced with either hemeprotein-hydroperoxide system; however, specific ligands of the two hemeproteins, substrates of cytochrome P-450, and phospholipid affected the two systems quite differently. Only the metmyo-globindependent oxidation of aminopyrine was significantly inhibited by fluoride and cyanide. Metyrapone, a specific ligand of cytochrome P-450, and benzphetamine, which was N-demethylated by cumene hydroperoxide only in the presence of cytochrome P-450, inhibited only the cytochrome P-450-stimulated oxidation of aminopyrine. Moreover, only with the solubilized liver hemeprotein was aminopyrine radical generation markedly stimulated by phospholipid. Similar properties of aminopyrine N-demethylation and radical formation by the cytochrome P-450-cumene hydroperoxide system have strongly implicated the radical as a requisite intermediate in product formation. Micromolar concentrations of metyrapone caused parallel inhibition, by at least 50%, of both radical generation and formaldehyde production. These results support a radical pathway of N-demethylation proposed for other hemeprotein-hydroperoxide systems (B. W. Griffin and P. L. Ting, 1978, Biochemistry, 17, 2206–2211), in which the substrate undergoes two successive one-electron abstractions, followed by hydrolysis of the iminium cation intermediate. Thus, for this class of substrates, the experimental data are consistent with the oxygen atom of the product arising from H₂O and not directly from the hydroperoxide, which has been previously proposed as a general mechanism for cytochrome P-450 peroxidatic activities.     

Radioimmunoassay for β-endorphin: Presence of immunoreactive “big-big” β-endorphin (“big” β-lipotropin) in human and rat pituitaries

A sensitive and specific radioimmunoassay for β-endorphin has been developed with an antiserum obtained in a rabbit immunized with β-endorphin contained in crude porcineACTH preparations. The minimal detectable quantity was 5 pg. The antiserum used reacted slightly with ovine β-lipotropin (5.5 %), but showed negligible cross-reactivity with other fragments of β-lipotropin, α-MSH and ACTH. Using this radioimmunoassay we have observed the presence of “big-big” β-endorphin (“big” β-lipotropin) with apparent molecular weights of 37,000 and 31,000 in human and rat pituitaries respectively, in addition to β-lipotropin and β-endorphin, by Sephadex gel-chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

The differential effects of TCA-cycle acids on the growth of plant cells cultured in liquid media containing various nitrogen sources

Alfalfa (Medicago sativa L. cv. Canadian No. 1), tobacco (Nicotiana tabacum L. var. humilis) and wheat (triticum monococcum L.) cells were grown in a defined, liquid medium containing either ammonium sulfate, L-glutamine or potassium nitrate as the sole nitrogen source, and the effects of the tricarboxylic-acid (TCA) intermediates, citrate and -ketoglutarate (5, 10, 15 mM), on the growth (dry-weight increase) of these cells was observed. The three cell suspension cultures exhibited a different growth response to the TCA-cycle intermediate supplied, depending upon the concentration of the additive and the nitrogen source. Citrate (5 mM) greatly enhanced growth of alfalfa and wheat cells in an ammonium-based medium but was less effective at higher concentrations, and in the case of alfalfa cells markedly inhibited growth. Tobacco cell growth was inhibited by all citrate concentrations tested. In contrast, all concentrations of -ketoglutarate used stimulated the growth of all three cell cultures in an ammonium-based medium. Alfalfa and wheat cells grown in an L-glutamine-based medium were influenced by citrate in a manner similar to that in ammonium-based medium. The growth of tobacco cells was slightly enhanced by 5 mM citrate but inhibited by higher concentrations. -Ketoglutarate, at all concentrations tested, was stimulatory to the growth of the cells of all three species in a glutamine-based medium, except for alfalfa cells which were inhibited at 15 mM. Both TCA-cycle acids inhibited the growth of alfalfa and tobacco cells grown on a nitrate-based medium whereas the growth of wheat cells was almost unaffected.     

Histidine-containing cyclic dipeptides as catalysts in the hydrolysis of carbonic acid p-nitrophenyl esters

A histidine-containing cyclic dipeptide, cyclo(D -Leu-L -His), was almost 20 times as efficient a catalyst as imidazole in the hydrolysis of p-nitrophenyl laurate. The effect of dioxane on the hydrolysis showed that hydrophobic interaction between the cyclic dipeptide and the ester is very important. This reaction obeyed the Michaelis-Menten kinetics, and the Michaelis constant K_m was as low as 9.98 × 10^?5M. Since the linear dipeptide having D -Leu-L -His sequence was nearly inactive in the hydrolysis, the functional groups of cyclo(D -Leu-L -His) in a specific arrangement held by the rigid backbone must have cooperated in the fast hydrolysis. Very weak catalysis by the diasteremeric cyclic dipeptide, cyclo(L -Leu-L -His), in the hydrolysis supported the above view.     

Proton conduction in phosphatidylethanolamine

The dc conductivity of polycrystalline phosphatidylethanolamine (PE) was measured in the temperature range 60–120°C. Since no conclusive evidence had so far been obtained for the presence of proton conduction in this phospholipid, hydrogen gas was shown in the present experiment to evolve during the electrolysis in its premelted state between 91 and 124°C. In this temperature range molecules assume rotation around the molecular axes and proton conduction of the Grotthus type takes place possibly along two chains of intermolecular hydrogen bonds running in parallel. Zwitter-ions behave cooperatively as proton donors and acceptors in transferring proton from molecule to molecule via the hydrogen bond networks. This efficient push-pull way of proton transferring seems to account for the fact that no polarization was observed in the dc conduction experiments. The amount of evolved gas appears to be not exactly in accordance with Faraday's law and discussions are made on possible causes for this slight deviation.     

In vitro culture of a root parasite,Aeginetia indica L.

Seed germination and further growth of seedlings of an obligate root parasiteAeginetia indica L. are described. (1) Germination: dormancy of fresh seeds can be broken by sodium hypochlorite treatment, but seeds stored for 1–5 years at 5 C did not necessarily require halogen treatment for good germination. (2) Formation of the tendril, which facilitated the organic connection with the host, was stimulated greatly byMiscanthus root extract. (3) Septation (cell division) of tendril was promoted by addition of sucrose to the basal medium. (4) Rhizogenesis of the seedlingin vitro was stimulated by deproteinized coconut milk.     

Inhibitory Modulation by Sodium Ions of the N-Methyl-D-Aspartate Recognition Site in Brain Synaptic Membranes

Specific binding of radiolabeled L-glutamic acid (Glu) was examined using rat brain synaptic membranes treated with a low concentration of Triton X-100. The binding drastically increased in proportion to increasing concentrations of the detergent used up to 0.1%. Addition of 100 mM sodium acetate significantly potentiated the binding in membranes not treated with Triton X-100, whereas it markedly inhibited the binding in Triton-treated membranes. The binding in Triton-treated membranes was inversely dependent on incubation temperature and reached a plateau within 10 min after the initiation of incubation at 2 degrees C, whereas the time required to attain equilibrium at 30 degrees C was less than 1 min. Sodium acetate invariably inhibited the binding detected at both temperatures independently of the incubation time via decreasing the affinity for the ligand. The binding was significantly displaced by agonists and antagonists for an N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors, but not by those for the other subclasses. Inclusion of sodium acetate reduced the potencies of NMDA agonists to displace the binding without virtually affecting those of NMDA antagonists. Moreover, sodium ions inhibited the ability of Glu to potentiate the binding of N-[3H] [1-(2-thienyl)cyclohexyl]piperidine to open NMDA channels in Triton-treated membranes. These results suggest that sodium ions may play an additional modulatory role in the termination process of neurotransmission mediated by excitatory amino acids via facilitating a transformation of the NMDA recognition site from a state with high affinity for agonists to a state with low affinity.     

Growth-related changes in color and sex inHalichoeres poecilopterus

Coloration and sex change were studied in a temperate wrasseHalichoeres poecilopterus in the central part of the Seto Inland Sea, Japan. 1,270 examples, 45–179 mm SL, were collected from May to December both in 1983 and 1984. The species is a diandric, protogynous hermaphrodite, and has three color patterns: pale color type (A), brilliant color type (B) and intermediate color type (AB). A-fish were less than 142 mm SL and consisted of primary males (42.6%), females (55.4%), secondary males (0.3%) and fish with transitional gonads (1.7%). A-females changed their color to B, through AB, in the size range 101–131 mm SL. A-primary males changed their color to B, through AB, in the size range 103–134 mm SL. B-fish consisted of primary males (38.6%), secondary males (54.6%) and fish with transitional gonads (6.8%). The majority of females changed their sex to male in the size range 98–131 mm SL.