Dioxygen and temperature dependence of ubiquinone formation in Escherichia coli: studies of cells charged with 2-octaprenyl phenol.

The multiple aromatic auxotroph Escherichia coli K-12 strain AB 2847 (aroB-) was conditioned for efficient ubiquinone-8 formation. Resting cells readily convert 4-hydroxy[U-14C]benzoate into ubiquinone-8 (60 nmol per g wet weight). Under argon this processing stops at the stage of 2-octaprenyl phenol. Only upon admission of air is the pool of 2-octaprenyl phenol converted to ubiquinone-8. This reaction occurs in the cytoplasmic membrane and is significantly inhibited by cytochrome P-450 inhibitors. The rate for 2-octaprenyl phenol conversion is strongly dependent on temperature. The Arrhenius plot shows inflection points at 32 degrees C and 16 degrees C. Enzymes for ubiquinone-8 synthesis are absent from anaerobically grown E. coli. Processing of 4-hydroxy[U-14C]benzoate by these cells starts only when protein synthesis is permitted under aerobic conditions.     

Catabolite repression of the formation of precursors of electron transfer complexes III and IV in adapting bakers' yeast: dependence upon a mitochondrially translated repressor.

When bakers' yeast cells which had been grown anaerobically in galactose were aerated in the presence of 10% glucose, they showed a 40% decrease in $\text{in}\text{vivo}$ [¹⁴C]-leucine incorporation into a washed mitochondrial membrane fraction compared with cells which had been aerated in a low glucose medium. The observed catabolite repression of membrane protein synthesis was primarily due to a decrease in cytoplasmic translational activity, but this repression was entirely dependent upon concomitant mitochondrial translation. The inductions of reduced coenzyme Q cytochrome $\text{c}$ reductase (complex III) and of cytochrome $\text{c}$ oxidase (complex IV) activities were repressed 30 and 60%, respectively, by aeration of the cells for 8 hours in 10% glucose. The catabolite repression of the formation of these two inner membrane complexes was again shown to be dependent upon concomitant mitochondrial translation. Both the amino acid incorporation and enzyme induction data suggest that catabolite repression of both cytoplasmically and mitochondrially translated mitochondrial membrane proteins is mediated through a mitochondrially translated repressor.     

Membrane-associated reactions in ubiquinone biosynthesis: 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone methyltransferase

The O-methylation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone, which has been previously postulated to be the final reaction in the biosynthesis of ubiquinone was demonstrated in vitro using cell extracts of Escherichia coli. $\text{S-}\text{Adenosyl}\text{-}\text{l}\text{-}\text{methionine}$ was active as the methyl donor for the reaction. The enzyme concerned, S-adenosyl-l-methionine: 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone-O- methyltransferase, was partially purified and shown to have a molecular weight of about 50 000 and to require a divalent metal and dithiothreitol for optimal acitivity in vitro. The methyltransferase was absent from extracts from ubiG^? mutants suggesting that the ubiG gene is the structural gene coding for the methyltransferase. The enzyme, although not firmly membrane-bound, showed some affinity for the cell membrane in broken cell preparations and could utilize the benzoquinone substrate when the latter was free or bound to the cell membrane, with about equal efficiency. It is concluded that in vivo, the methyltransferase reaction probably occurs at the internal surface of the cytoplasmic membrane.     

The effect of antiestrogens on the nuclear binding of the estrogen receptor

Experiments were designed to determine whether or not various antiestrogens in direct competition with estradiol-17β (E₂) would inhibit the translocation of the estrogen receptor complex from the cytoplasm to nuclei in rat uterine tissue. Incubation of the antiestrogens CI-628, $\text{cis}$-clomiphene, U-11,100A and MER-25 with rat uteri caused the nuclear uptake of the antiestrogen receptor complex which was greatest for most antiestrogens at concentrations of 1 × 10^?6 to 1 × 10^?5M. At higher concentrations of CI-628, $\text{cis}$-clomiphene, and U-11,100A the nuclear binding of the antiestrogen receptor complex was greatly decreased. Incubation of the antiestrogens with E₂ resulted in a dramatic inhibition of the nuclear uptake of the estrogen receptor. $\text{Trans}$-clomiphene, a weak estrogen, did not inhibit the movement of the uterine cytoplasmic receptor into the nuclear fraction.     

Adenyl cyclase of oxyntic cells its association with different cellular membranes

The adenyl cyclase of the oxyntic, or acid-secreting, cells of bullfrog gastric mucosa has been found to be a membrane-bound enzyme. A method has been developed to isolate the adenyl cyclase rich membrane fractions in a hypotonic medium containing dithiothreitol, which has been found to protect the hormonal resposivenes of the adenyl cyclase.Highest specific activity of adenyl cyclase was localized in a light membrane fraction which also had abundant K⁺-stimulated ATPase and K⁺-stimulated $\text{p-}\text{nitrophenyl}$ phophatase and very low cytochrome $\text{c}$ oxidase activty. The three gastric secretagogues tested, namely histamine, pentagastrin and methylcholine, significantly stimulated the adenyl cyclase activity of the light membrane fraction.After treatment with 10 mM Mg⁺ further subfractionation of the light membrane fraction on a sucrose density gradient yielded light membrane subfraction 1, light membrane subfraction 2 and light membrane subfraction 3 in order of increasing densities. The three subfractions had different enzymatic and chemical properties. Adenyl cyclase activity has been found to be distributed in all three subfractions. However, the hormonal responsiveness of the three fractions was quite different. Light membrane subfraction 2 could be stimulated by all three secretagogues, light membrane subfraction 1 by histamine and methylcholine, while light membrane subfraction 3 was refractory to all three secretagogues. On the basis of the cholesterol to phospholipid molar ratio, RNA content, glycoprotein content and the enzymatic data it is suggested that light membrane subfraction 1 and light membrane subfraction 2 are of general plasma-membrane type, while light membrane subfraction 3 is largely of cytoplasmic origin.     

Structural properties of the membrane of intact human spermotozoa: A study with fluorescent probes

The properties of the membrane of intact, metabolically active, human persmatozoa have been studied by the use of 1-anilino-8-napthalene sulfonate (ANS). By fluorescence microscopy it was found that at neutral pH ANS is bound exclusively to the membrane of the entire sperm with some preferential binding to the midpiece, while at low pH some preferential binding to the aerosome was observed. By spectrofluorimetry, fluorescence was found to be enhanced 48-fold on binding of ANS to the spermatozoal membrane, with a 50-nm shift in the emission spectrum of the bound dye. $\text{2.47 ± 0.02}$ nmoles of ANS were bound per 10⁶ spermatozoa ($\text{K=2.3–10}{}^{\mathrm{?5}}\text{M}$). Scatchard plots indicate that all the binding sites on the spermatozoal membrane have similar binding characteristics with aZ value of 84.8. Energy transfer with an efficiency of 7% was found for recently ejaculated spermatozoa. The fluorescence of bound ANS depends on the pH of the medium and possibly on the metabolic state of the cell, since addition of succinate or fructose produces an enhancement of fluorescence, while addition of glucose results in a decrease of this parameter. These changes are inhibited by the presence of cyanide.     

Effect of E. coli endotoxin on the structure-function of fatty acid synthetase lipoprotein

The lipoprotein structure of the fatty acid synthetase complex from Ceratitis capitata has been used as a model to $\text{val}\text{i}$ date the claim that phospholipids from membranes assume a $\text{signif}\text{i}$ cant role in the cell-endotoxin interactions. The enzyme-complex was exposed to a ¹⁴C-lipopolysaccharide preparation and the $\text{inte}\text{r}$ action was followed by a) circular dichroism spectra, b) enzyme activity and c) gel filtration chromatography. It should be $\text{emph}\text{a}$ sized that the E. coli endotoxin modifies all these properties of the enzyme complex and that a model involving phospholipids and phase transitions has been proposed to account for these $\text{intera}\text{c}$ tions.     

Errata

Optimal conditions for activation of adenylate cyclase in membrane particles were studied. Enzyme activation with serotonin (5-hydroxytryptamine), NaF, and guanosine $\text{5′-(3-O-}\text{thio}\text{)-}\text{triphosphate}$ (GTPγS) was time- and temperature-dependent. Mg²⁺ was required for enzyme activation. Adenylate cyclase that was activated by NaF or GTPγS was gradually inhibited by $\text{N-}\text{methylmaleimide}$ while enzyme activated with serotonin and GTP responded faster to inhibition by the same sulfhydryl reagent. The enzyme responded in a similar fashion to a spin-labeled $\text{N-}\text{methylmaleimide}$ analog 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrolidinyloxyl (i.e., $\text{N-}\text{methylmaleimide}$ nitroxide). Binding of the spin label was enhanced following enzyme activation by serotonin, NaF, or GTPγS in the presence of Mg²⁺. Activation of the enzyme was accompanied by an increase in the strong immobilization peaks in the EPR spectra. Both effects, the increase in binding and in the strong immobilization peaks, can be induced by Mg²⁺ alone. The results indicate that a general conformational change induced by Mg²⁺ may be essential for adenylate cyclase activation.     

Stimulation of ornithine decarboxylase synthesis in the rat thyroid

High titer antiserum to hepatic ornithine decarboxylase was prepared by employing enzyme·monospecific antibody complex as the immunizing antigen. This new antiserum preparation was successfully labeled with ¹²⁵I and was found to retain its specific immune properties. Iodinated antiserum was used to precipitate thyroid ornithine decarboxylase induced by a mixture of thyroid stimulating hormone and methyl xanthine in rat thyroids $\text{in vitro}$. ¹²⁵I-labeled antibody incorporation into the enzyme antibody complex after induction $\text{in vitro}$ showed an increase which paralleled the increase in enzymatic activity and thus suggested $\text{de novo}$ synthesis of thyroid enzyme protein.     

Studies of odd bases in yeast mitochondrial tRNA: III. Characterization of the tRNA methylases associated with the mitochondria

Whereas m¹G, m²G, m₂²G, m⁷G, T, m¹A, m⁵C and Cm methylase activities were found in total cell enzyme of $\text{Saccharomyces cerevisiae}$ using under-methylated $\text{E. coli}$ tRNA and $\text{E. coli}$ B tRNA in reaction with or without Mg⁺⁺, only m¹G, m²G, m₂²G and T methylases occurred in mitochondria. Mitochondrial and cytoplasmic tRNA cannot be methylated by their homologous enzymes; only mitochondrial tRNA can be methylated in a heterologous reaction by total cell enzyme with formation of T, m⁵C, m¹A and low amounts of m²G and m₂²G.     

Picosecond kinetics in reaction centers of Rps. sphaeroides and the effects of ubiquinone extraction and reconstitution.

Following picosecond light activation, the bacteriochlorophyll and bacteriopheophytin complement of $\text{Rps.}\text{sphaeroides}$ reaction centers depleted of ubiquinone behaves as though it has no primary electron acceptor; the excited intermediary $\text{BChl}\text{BPh}{}^2$ state formed in <10 ps lasts >1 ns. Addition of ubiquinone-10 reconstitutes the very rapid electron transfer rates from the excited intermediary $\text{BCh}\text{BPh}$ state to ubiquinone; the kinetics and rate are similar to that encountered in the untreated reaction centers. Interpretation of the data presented suggests that ubiquinone is the immediate electron acceptor from BPh^?. This is consistent with the model for the primary reactions leading to [(BChl)₂^?BPh]Q^?.     

ESR determinations of membrane permeability in a yeast sterol mutant

Yeast sterol mutants were subjected to ESR analysis in an attempt to elucidate how altered sterol composition correlates with membrane permeability. The technique requires spin labeling the intact yeast cells with a small, water-soluble nitroxide probe (2,2,5,5 tetramethyl-3-pyrrolin-1-oxyl-3-carboxylic acid, PCA), suspending cells in a NiCl₂ solution, and measuring the extent of Ni²⁺ entry through the membrane by its magnetic dipolar line broadening effect on the PCA signal. The wild type, A184D, was found to be impermeable to Ni²⁺ during all growth phases while the sterol mutant $\text{erg}\text{6}\text{2}$ was readily permeable to Ni²⁺. Other sources of line broadening such as increased rotational correlation time and cell nonviability are shown to be negligible. Internal Ni²⁺ concentrations for $\text{erg}\text{6}\text{2}$ and kinetics of Ni²⁺ entry were determined.     

Lipid phase transitions in cytoplasmic and outer membranes of Escherichia coli

The cytoplasmic and outer membranes containing either trans-Δ⁹-octadecenoate, trans-Δ⁹-hexadecenoate or cis-Δ⁹-octadecenoate as predominant unsaturated fatty acid residues in the phospholipids were prepared from a fatty acid auxotroph, Escherichia coli strain K1062. Order-disorder transitions of the phospholipids were revealed in both fractions of the cell envelope by fluorescent probing or wide angle X-ray diffraction. The mid-transition temperatures, $\text{T}\text{t}$, and the range of the transition, $\text{ΔT}$, are similar in the outer and cytoplasmic membrane. Relative to the corresponding extracted lipids, 60–80% of the hydrocarbon chains take part in the transition in the cytoplasmic membrane whereas in the outer membrane only 25–40% of the chains become ordered. The results suggest that in the outer membrane part of the lipids form fluid domains in the form of mono- and/or bilayers.     

Inhibition of (Na+ + K+)-ATPase by ouabain: Involvement of calcium and membrane proteins

Treatment of plasma membrane isolated from murine plasmocytoma MOPC 173 with an EDTA-containing buffer resulted in a 300-fold increase in sensitivity of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$ Mg²⁺-ATPase to ouabain. This phenomenon was associated with the solubilization by EDTA of phospholipid free proteins (approx. 30 000–34 000 daltons) from the cytoplasmic face of the plasma membrane and with removal of about 90% of the membrane bound Ca²⁺. The recovery of the original resistance to ouabain required specifically Ca²⁺ and was associated with a binding of the solubilized proteins to the membrane.     

Synexin protein is non-selective in its ability to increase Ca2+-dependent aggregation of biological and artificial membranes

Synexin, a soluble protein which increases the specificity of Ca²⁺ to aggregate isolated bovine chromaffin granules was prepared from bovine adrenal medullary tissue by the method of Creutz, Pazoles and Pollard (J. Biol. Chem. $\text{253}$, 2858–2866, 1978). We also find that synexin increases both the initial rate and final amplitude of Ca²⁺-promoted aggregation of granule membranes. This effect is Ca²⁺-specific. However in contrast to Creutz $\text{et}\text{al}$, we find that synexin also potentiates aggregation of adrenal medulla and liver mitochondria and microsomes as well as phosphatidylserine vesicles. This lack of membrane specificity argues against the suggestion of Creutz $\text{et}\text{al}$ that synexin specifically binds the granule to the plasma membrane prior to exocytosis $\text{in}\text{vivo}$.     

Selenium binding proteins in rat tissues

The 100,000 × g extracts of rat intestine and colon were incubated $\text{in}\text{vitro}$ with Na₂[⁷⁵Se]O₃. Chromatography of this material on a Sephadex G-100 column produced three radioactive peaks corresponding to molecular weights of 17,000, 68,000 and > 90,000. The 17,000 peak corresponded to a protein which sedimented in the 2S region of a 5–20% ($\text{w}\text{v}$) linear sucrose density gradient. Selenium binding to this protein was specific, stable and sensitive to thiol inhibitors such as p-chloromercuriphenylsulfonic acid (1 mM) and iodoacetamide (2 mM). Chromatography of rat serum - [⁷⁵Se] complex on Sephadex G-100 yielded only two radioactive peaks that corresponded to molecular weights of 68,000 and > 90,000. The 2S selenium binding protein of intestine and colon may mediate the biological functions of selenium in those tissues.     

The DCCD-binding polypeptide is close to the F1 ATPase-binding site on the cytoplasmic surface of the cell membrane of Escherichia coli

Removal of the F₁ ATPase from membrane vesicles of $\text{Escherichia}\text{coli}$ resulted in leakage of protons across the membrane through the F_O portion of the ATPase complex. The leakage of protons was prevented by antiserum to the N,N′-dicyclohexylcarbodiimide (DCCD)-binding polypeptide in everted but not in “right-side out” membrane vesicles. The antiserum prevented the rebinding of F₁ ATPase to F₁-stripped everted membrane vesicles. It is concluded that in F₁-depleted vesicles the DCCD-binding polypeptide is exposed on the cytoplasmic surface of the cell membrane at or close to the binding site of the F₁ ATPase.     

Study of the protein-ubiquinone interaction in succinate-cytochrome C reductase with azido-ubiquinone derivatives

Several azido-ubiquinones have been synthesized for the study of protein-ubiquinone interaction in succinate-cytochrome $\text{c}$ reductase. In the absence of light, azido-ubiquinones are partially effective in restoring enzymatic activity to ubiquinone- and phospholipid-depleted reductase and the binding of azido-ubiquinones can be partially reversed by 5-(10-bromodecyl)-ubiquinone. When 2-azido-3-methoxy-5-geranyl-6-methyl-1,4-benzoquinone reactivated reductase is illuminated with long wavelength UV light, a complete and irreversible inhibition is observed. This specific photo-inactivation, exerted only by 2-azido-3-methoxy-5-geranyl-6-methyl-1,4-benzoquinone, and not by other azido-ubiquinone derivatives, is evidence for the existence of a specific benzoquinone ring binding site in the enzyme.     

Characterization of a membrane-bound nitrate reductase from Azotobacter chroococcum.

Nitrate reductase from the aerobic bacterium $\text{Azotobacter chroococcum}$ is a soluble enzyme with the characteristic features of Pichinoty's type B nitrate reductase. When cell suspensions of $\text{A. chroococcum}$ are repeatedly subcultured in liquid medium with nitrate as the nitrogen source, most of the nitrate-reducing activity is incorporated into the cytoplasmic membrane. The properties of the particulate nitrate reductase closely resemble Pichinoty's type A enzyme.     

N-6-methyl-adenosine in adenovirus type 2 nuclear RNA is conserved in the formation of messenger RNA

In the biogenesis of adenovirus type 2 messenger RNAs, methylation occurs at the 5′ end (cap) and to internal adenosine residues to yield N⁶-methyl-adenosine (m⁶A) (Sommer et al., 1976; Moss &; Koczot, 1976; Wold et al., 1976). The kinetics of accumulation of ³H from methyl-labeled methionine and ¹⁴C from uridine into Ad-2²-specific RNA was measured late in Ad-2 infection. As reported previously (Nevins &; Darnell, 1978a), the rate of accumulation of [¹⁴C]uridine label in nuclear RNA was approximately four- to fivefold faster than in the cytoplasmic RNA, indicating a conservation of about 20% for the total RNA. The initial rates of [³H]methyl label in m⁶A in nuclear RNA and in the cytoplasmic RNA were approximately equal, suggesting a complete (or nearly complete) conservation of m⁶A.In accord with the accumulation kinetics, the ratio of ³H to ¹⁴C was higher in cytoplasmic RNA than in nuclear RNA that hybridized to equivalent regions of the Ad-2 DNA.A mathematical model was designed to evaluate the accumulation of methyl label in m⁶A, taking into consideration the three major parameters that affect the accumulation curves: equilibration of the S-adenosyl-methionine pool, the nuclear dwell time of sequences destined to be mRNA, and the cytoplasmic stability of mRNA. The half-time ($\text{t}\text{1}\text{2}$) for pool equilibration was determined experimentally to be 22 minutes and the nuclear dwell time and the mean life-time of cytoplasmic mRNA were estimated from ¹⁴C label to be about 30 and 70 minutes, respectively.The model gave an excellent fit to the data when the $\text{t}\text{1}\text{2}$ for pool equilibration time of 24 ± 2 minutes, a nuclear dwell time of 25 ± 10 minutes, and a mean cytoplasmic mRNA life-time of 75 ± 30 minutes were used to evaluate accumulation curves. Even when data from a restricted region of the genome, $\text{40.5–52.6}$, which encodes the main portion of at least five 3′ co-terminal mRNAs whose spliced junction with the tripartite leader sequence varies from $\text{38, 40, 43, 45}$, and $\text{48}$ was analyzed, it appeared that m⁶A was conserved.Finally, m⁶A was found to be added in a brief label (3.5 min) mainly to nuclear molecules that were longer than any cytoplasmic RNA. The conservation of m⁶A and its addition prior to splicing raise the possibility that internal methylations are involved, in the formation of mRNA.