Electron transport-linked nitrous oxide synthesis and reduction by Paracoccusdenitrificans monitored with an electrode

The nitrous oxide reductase activity of $\text{Paracoccus}\text{denitrificans}$ can be conveniently measured using an electrochemical method for determining N₂O. Introduction of this procedure has shown that (i) N₂O reductase activity is reversibly inhibited by oxygen; (ii) antimycin strongly inhibits electron flow to N₂O and that the inhibition is bypassed by tetramethyl-p-phenylenediamine; (iii) ascorbate plus tetramethyl-p-phenylenediamine, presumably by donating electrons to cytochrome c, is an effective reductant for nitrous oxide reductase; (iv) in the presence of the nitrous oxide reductase inhibitor, acetylene, N₂O is promptly produced from nitrite, consistent with the product of nitrite reductase being N₂O.     

Estrogen metabolism in nonhuman primates I. In vitro biosynthesis of estrogen glucosiduronates in rhesus monkey liver

Estrone glucosiduronate, 17β-estradiol-3-glucoslduronate, 17β-estradiol-17-glucosiduronate and estriol-16α-glucoslduronate have been biosynthesized in substantial yield by incubation of radioactive estrone, 17β-estradiol, estriol and uridlne diphosphoglucosiduronic acid with rhesus monkey liver homogenates. The metabolites were characterized by chromatography on Celite and DEAE-Sephadex, enzyme hydrolysis, derivative formation and crystallization to constant specific activity. The percent conversion to 17β-estradiol-17-glucosiduronate from 17β-estradlol ranged between 56–71%; from estrone, the conversion was 49–54%. Other metabolites formed from estradiol were estrone glucosiduronate(12–21%) and 17β-estradiol-3-glucosiduronate(5–12%). The same metabolites derived from estrone accounted for 18–28% and 10–14% respectively. After estriol incubation, more than 90% of the steroid was converted to estriol-16α-glucosiduronate with no detectable estriol-3-glucosiduronate. This report represents the first time that 17β-estradiol-17-glucosiduronate has been reported as a metabolite in the rhesus monkey and this is the only known species which forms 17β-estradiol-17-glucosiduronate as the predominant metabolite of either estrone or estradiol $\text{in vitro}$.Rhesus monkey liver is similar to the human and baboon in that it metabolizes estriol exclusively to estriol-16-glucosiduronate.     

The metabolism of formycin B in Leishmaniadonovani

Formycin B, a pyrazolo(4,3-d)pyrimidine C-nucleoside, inhibited the growth of $\text{Leishmania}\text{donovani}$ promastigotes in culture with an ED₉₀ of 0.2 μg/ml. Promastigotes incubated for 24 hrs with Formycin B at 10 μg/ml were found to convert it to the ribonucleotide, formycin B 5′-monophosphate. The parasites were also capable of aminating formycin B 5′-monophosphate as evidenced by the appearance of formycin A di- and triphosphate. The RNA contained the formycin A moiety in 3′,5′-polynucleotide linkage. Succino-AMP synthetase from these parasites was able to use formycin B 5′-monophosphate as an alternate-substrate with a K'm of 26 μM and a V'm of about 1% the V'm IMP. Formycin B 5′-monophosphate was also a substrate for mammalian succino-AMP synthetase with a V_m' of 40% the V_m' of IMP.     

Naphthylmercaptobenzoquinone,a new inhibitor of photophosphorylation in Rhodospirillumrubrum chromatophores at the level of ubiquinone

Naphthylmercaptobenzoquinone (NMBQ) inhibits photophosphorylation in $\text{Rhodospirillum}$$\text{rubrum}$ chromatophores. The endogenous unsupplemented system is the most sensitive one being 50% inhibited by 0.5 μM NMBQ, whereas 20 μM are required for 50% inhibition of photophosphorylation in the presence of N-methylphenazonium methosulfate or diaminodurene. The inhibition is less effective under argon, especially in the presence of ascorbate, and is reversed on addition of various naphthoquinones, which can also reverse the inhibition of photophosphorylation by dibromothymoquinone (DBMIB). Another quinone analog, dodecylmercaptonaphthoquinone (DMNQ), inhibits endogenous photophosphorylation even more effectively than NMBQ, but its inhibition is not reversed by the added naphthoquinones. It is concluded that NMBQ and DBMIB, but not DMNQ, inhibit photophosphorylation in chromatophoresby acting as antagonists of ubiquinone.     

Biosynthesis of canine fibrinogen: In vitro synthesis of Aα, Bβ and γ precursor chains

An mRNA fraction from dog liver translated with a rabbit reticulocyte protein synthesizing system in the presence of [³⁵S]-methionine produces fibrinogen-related proteins which are immunoprecipitated with rabbit antiserum to dog fibrinogen. Analyses of these radioactive proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography indicate that the three fibrinogen chains (Aα, Bβ and γ) are synthesized separately as larger precursors. The putative pre Aα and pre Bβ chains were characterized by their susceptibility to treatment with thrombin and batroxobin. Thrombin degraded the pre Aα and pre Bβ chains, while batroxobin only acted on the pre Aα chain. The pre γ chain was not degraded by these enzymes.     

The dual effects of aluminum as activator and inhibitor of adenylate cyclase in the liver fluke Fasciola hepatica

The liver fluke, $\text{Fasciola hepatica}$, has a very active adenylate cyclase which can be stimulated by NaF or by serotonin and guanine nucleotides. Micromolar amounts of AlCl₃ augment the activation by F-. In contrast, when the enzyme is activated with serotonin and guanine nucleotides, AlCl₃ inhibits the activation. Aluminum also inhibits the activation by forskolin. Gallium mimics the effects of aluminum.     

A respiration-dependent primary sodium extrusion system functioning at alkaline pH in the marine bacterium Vibrioalginolyticus

The membrane potential generated at pH 8.5 by K⁺-depleted and Na⁺-loaded $\text{Vibrio}\text{alginolyticus}$ is not collapsed by proton conductors which, instead, induce the accumulation of protons in equilibrium with the membrane potential. The generation of such a membrane potential and the accumulation of protons are specific to Na⁺-loaded cells at alkaline pH and are dependent on respiration. Extrusion of Na⁺ at pH 8.5 occurs in the presence of proton conductors unless respiration is inhibited while it is abolished by proton conductors at acidic pH. The uptake of α-aminoisobutyric acid, which is driven by the Na⁺-electrochemical gradient, is observed even in the presence of proton conductors at pH 8.5 but not at acidic pH. We conclude that a respiration-dependent primary electrogenic Na⁺ extrusion system is functioning at alkaline pH to generate the proton conductor-insensitive membrane potential and Na⁺ chemical gradient.     

Conjugation of androgens and estrogens by human breast tumors in vitro

The metabolism of ³H-androstenedione (Δ⁴ -A) and ³H-estriol (E³) was studied in 12 human breast tumors. Part of each tumor was analyzed for estrogen receptor content. Aliquots of tumor homogenates were incubated for 2 hr separately with ³H-δ⁴-A and ³H-E₃ in the presence of appropriate cofactors. No distinct differences emerged in the profiles of the unconjugated metabolites of ³H-δ⁴-A, the major compounds in the approximate order of descendence being androsterone, androstanedione, testosterone, 5α-androstane-3α,17β-diol, epiandrosterone, and dihydrotestosterone. One tumor homogenate from an infiltrating lobular carcinoma converted ³H-Δ⁴-A to glucosiduronate metabolites (11%), of which androsterone, 6.4%; testosterone, 1.6%; and androstanediol, 0.6% predominated. The homogenate of this tumor and two other tumors converted ³H-E₃ to ³H-E₃-3S. Conversions of E₃ to E₃-3S In the other tumor homogenates were less than 0.6%. No correlation between receptor content and the capability of the tumor to conjugate Δ⁴-A or E₃ evolved. However, correlations between steroid hormone metabolism and tumor histopathology may exist.     

The DCCD-binding polypeptide alone is insufficient for proton translocation through F0 in membranes of Escherichia, coli

In contrast to membrane vesicles of wild-type strains which become leaky to protons on removal of the F₁ ATPase, those of the mutant $\text{Escherichia}\text{,}\text{coli}\text{,}\text{N}{}_{\mathrm{I44}}$, which lacks the F₁ ATPase, can maintain a proton gradient. A normal N,N′-dicyclohexylcarbodiimide (DCCD)-binding polypeptide is present in the F₀ portion of the ATPase complex of the mutant. However, the 19000 molecular weight component of F₀ is absent. We conclude that the latter polypeptide, in addition to the DCCD-binding polypeptide, is required for a functional proton channel in F₀.     

Initial membrane reaction in peptidoglycan synthesis. Interaction of lipid with phospho-N-acetylmuramylpentapeptide translocase

The initial membrane reaction in the biosynthesis of peptidoglycan is catalyzed by $\text{phospho}\text{-N-}\text{acetylmuramyl}$ (MurNAc)-pentapeptide translocase (UDP-MurNAc-Ala-γ dGlu-Lys-dAla-dAla undecaprenyl phosphate phospho-MurN Acpentapeptide transferase). In addition to the transfer reaction, the enzyme catalyzes the exchange of [³H]uridine monophosphate with the uridine monophosphate moiety of UDP-MurN Ac-pentapeptide. Two distinct discontinuities are observed in the slopes of the Arrhenius plots of the exchange and transfer activities at 22 and 30°C for the enzyme from Staphylococcus aureus Copenhagen. Anisotropy measurements of perylene fluorescence and electron spin resonance measurements of $\text{N-}\text{oxyl}\text{-4′,4′-}\text{dimethyloxazolidine}$ derivatives of 12-and 16-ketostearic acid intercalated into membranes from this organism define the lower ($\text{T}{}_1\text{= 16–22°}\text{C}$) and upper ($\text{T}{}_{\mathrm{<mtext>h</mtext>}}\text{= 30°}\text{C}$) boundaries of a phase transition. These values correlate with the discontinuities observed for the activity measurements. Thus, it is proposed that the physical state of the lipid micro-environment of phospho-MurN Ac-pentapeptide translocase has a significant effect on the catalytic activity of this enzyme.     

Evidence against proton gradient formation being the cause of chlorophyll fluorescence quenching by N-methylphenazonium methosulfate

In strong illumination, 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU)-poisoned chloroplasts exhibit a high yield of chlorophyll fluorescence while $\text{P-700}$ turnover, proton uptake, and phosphorylation are inhibited and a pH gradient is undetectable. When 10 μM $\text{N-}\text{methylphenazonium}$ methosulfate (PMS) is included, the fluorescence yield in light is substantially reduced, and when 100 μM ascorbate is also included, the yield is diminished approximately to the level in darkness. Only very slight increases in $\text{P-700}$ turnover and proton uptake (but no detectable pH gradient) accompany the fluorescence yield decline.When 10 μM PMS and 15 mM ascorbate are added to poisoned chloroplasts (the oxygen concentration being greatly reduced), $\text{P-700}$ turnover, proton uptake, the pH gradient and phosphorylation all reach high levels. In this case, the yield of chlorophyll fluorescence is low and is the same in both light and dark. Further addition of an uncoupler eliminates proton uptake, the pH gradient and phosphorylation but does not significantly elevate the fluorescence yield. From these observations we suggest that, in DCMU-poisoned chloroplasts, the fluorescence quenching with PMS occurs by a mechanism unrelated to the generation of a phosphorylation potential.With chloroplasts unpoisoned by DCMU, PMS quenches fluorescence and considerably stimulates proton uptake, the pH gradient and phosphorylation. However, in this case, PMS serves to restore net electron transport.     

In vitro synthesis of progesterone and prostaglandin F by luteal tissue and prostaglandin F by endometrial tissue from the pig

The relationship between progesterone (P₄) synthesis $\text{in vitro}$ by luteal tissue and prostaglandin F (PGF) synthesis $\text{in vitro}$ by endometrium and luteal tissue from two stages of the cycle, Days 7 to 8 and 15 to 16, was determined. Luteal and endometrial tissues were collected from pigs in three experimental groups at two stages of the cycle: (A) 6 pigs on Days 7 to 8 with spontaneous, 5 to 6 day old corpora lutea (CL); (B) 5 pigs on Days 15 to 16 with spontaneous, 13 to 14 day old CL; and (C) 6 pigs on Days 15 to 16 with spontaneous, 13 to 14 day old CL and 5 to 6 day old CL induced by pregnant mares serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) injections. Pigs with spontaneous, 13 to 14 day old CL of the cycle and PMSG-HCG induced accessory, 5 to 6 day old CL were used so that P₄ and PGF synthesis in tissue from old and new CL could be compared in the same pig on Day 15 to 16 of the cycle. Tissues (100 mg minces) were incubated in 5 ml of Krebs Ringer solution in an atmosphere of 95% 0₂:5% CO₂ for 2 hours at 0° C, 37° C, or 37° C with 1.3 x 10^?4M indomethacin (IND). An aliquot of the incubation medium and an aliquot of the supernatant after homogenization of the tissue in the remaining medium of each flask was quantified for P₄ and PGF by radioimmunoassay. P₄ and PGF release into the medium and total accumulation of P₄ and PGF in the flasks indicated that $\text{de novo}$ synthesis had occured at 37° C. Compared to tissue from 13 to 14 day old CL, tissue from 5 to 6 day old CL synthesized more P₄ per flask (53.9 $\text{vs}$ 25.0 ng/mg tissue, P<.001) and released more P₄ into the medium (20.8 $\text{vs}$ 8.8 ng/mg, P<.001). P₄ synthesis by luteal tissue from 5 to 6 day old and 13 to 14 day old CL from pigs in group C was similar to P₄ synthesis by luteal tissue from pigs in group A and group B, respectively. Luteul PGF synthesis was not affected significantly by either the age of the CL or by PMSG-HCG treatment. For endometrial samples, the synthesis of PGF was not significantly different among pigs in groups A, B and C. If uterine PGF is involved in luteal regression in the pig, the sensitivity of the CL to PGF may be more important than an increase in PGF secretion during the late luteal phase of the estrous cycle.     

A flash photolysis ESR study of Photosystem II Signal IIvf, the physiological donor to P-680+

In flash-illuminated, oxygen-evolving spinach chloroplasts and green algae, a free radical transient has been observed with spectral parameters similar to those of Signal II ($\text{g ≈ 2.0045}$, $\text{ΔH}{}_{\mathrm{<mtext>pp</mtext>}}\text{≈ 19}\text{G}$). However, in contrast with ESR Signal II, the transient radical does not readily saturate even at microwave power levels of 200 mW. This species is formed most efficiently with “red” illumination ($\text{λ < 680}\text{nm}$ and occurs stoichiometrically in a 1 : 1 ratio with $\text{P-700}{}^{\mathrm{+}}$. The Photosystem II transient is formed in less than 100 μs and decays via first-order kinetics with a halftime of 400–900 μs. Additionally, the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for radical decay is temperature independent between 20 and 4 °C; however, below 4 °C the transient signal exhibits Arrhenius behavior with an activation energy of approx. 10 kcal · mol^?1. Inhibition of electron transport through Photosystem II by $\text{o-}\text{phenanthroline}$, 3-(3,4-dichlorophenyl)-1,1-dimethylurea or reduced $\text{2,5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isopropyl}\text{-p-}\text{benzoquinone}$ suppresses the formation of the light-induced transient. At low concentrations (0.2 mM), $\text{2,5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isopropyl}\text{-p-}\text{benzoquinone}$ partially inhibits the free radical formation, however, the decay kinetics are unaltered. High concentrations of $\text{2,5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isopropyl}\text{-p-}\text{benzoquinone}$ (1–5 mM) restore both the transient signal and electron flow through Photosystem II. These findings suggest that this “quinoidal” type ESR transient functions as the physiological donor to the oxidized reaction center chlorophyll, $\text{P-680}{}^{\mathrm{+}}$.     

Targeting of the antiviral protein from Phytolaccaamericana with an antibody

The antiviral protein (PAP) of $\text{Phytolacca}\text{americana}$ was conjugated with the Fab' fragment of IgG from a rabbit antiserum against murine leukemia L1210 cells via a disulfide bond employing $\text{N}$-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as the coupling agent. The conjugate showed a potent $\text{in}\text{vitro}$ cytotoxicity against L1210 cells which was competitively blocked by F(ab′)₂ directed against L1210 cells. PAP itself did not exhibit the cytotoxicity at the concentration corresponding to the PAP content in the conjugate concurrently tested.     

Bromoacetylcholine as an affinity label of the acetylcholine receptor from Torpedo californica

Bromoacetyl[methyl-³H]choline is a highly specific label for the reduced acetylcholine binding site on the acetylcholine receptor from $\text{Torpedo californica}$. Only one of two binding sites per receptor monomer is susceptible to labeling. The labeled site is on the α chain of the receptor.     

Sodium dependence of maximum flux, JM, and Km of amino acid transport in Ehrlich ascites cells

The Michaelis-Menten parameters, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$ and $\text{K}{}_{\mathrm{m}}$ of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na⁺ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$, decreased with decrease in extracellular Na⁺ for both α-aminoisobutyric acid and phenylalanine but the $\text{K}{}_{\mathrm{m}}$ for α-aminoisobutyric acid increased markedly as the Na⁺ concentration fell whereas the $\text{K}{}_{\mathrm{m}}$ for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na⁺-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na⁺-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na⁺-independent but it has a high $\text{K}{}_{\mathrm{m}}$ and is not additive with the Na⁺-dependent flux.     

In. vitro evaluation of corpus luteum function of cycling and pregnant rhesus monkeys: Progesterone production by dispersed luteal cells

Corpus luteum function in the cycling and the pregnant rhesus monkey (Macaca mulatta) was evaluated through short term $\text{in vitro}$ studies of progesterone production by suspensions of collagenase-dispersed luteal cells in the presence and absence of exogenous gonadotropin (human chortonic gonadotropin, HCG). Cells from mid-luteal phase of the menstrual cycle secreted progesterone, as measured by accumulation of this hormone in the incubation medium, and responded to the addition of 100 ng HCG/ml with a marked increase in progesterone secretion above basal level ($\text{63.7 ± 13.1}\text{versus}\text{24.7 ± 5.5}\text{ng progesterone}\text{/}\text{ml}\text{/5 × 10}{}^4\text{cells}\text{/ 3}\text{hr}\text{,}\text{X}\text{± S.E.,}\text{n}\text{= 6;}\text{p}\text{< 0.05}$). However, luteal cells from early pregnancy (23–26 days after fertilization) secreted significantly less progesterone than cells of the non-fertile menstrual cycle (3.6 ± 2.4 versus 24.7 ± 5.5 ng/ml/5 × 10⁴ cells/3 hr, n = 3; p < 0.05) and did not respond to HCG with enhanced secretion. By mid-pregnancy (108–118 days gestation) luteal cells exhibited partially renewed function, and near the time of parturition (163–166 days gestation) basal and HCG-stimulated progesterone secretion (30.2 ± 5.6 and 63.0 ± 13.0 ng/ml/5 × 10⁴ cells/3 hr, respectively; n = 3) was equivalent to that of cells from the luteal phase of the non-fertile menstrual cycle. The data suggest that following a period around the fourth week of gestation, when steroidogenic activity is markedly diminished, the corpus luteum of pregnancy progressively reacquires its functional capacity and at term exhibits gonadotropin-sensitive steroidogenesis similar to that of the corpus luteum of the menstrual cycle.