Induction of translational errors (misreading) by tuberactinomycins and capreomycins

The well characterized translocation inhibitor viomycin (=tuberactinomycin B) promotes translational errors (misreading) in an in vitro system from Escherichia coli. It strongly stimulates both the binding of noncognate Tyr-tRNA to poly(U)-programmed ribosomes and the subsequent synthesis of acPhe(Tyr)_n-tRNA ($\text{n}\text{?20}$). The closely related antibiotics capreomycin and tuberactinomycins A,N and O also inhibit translocation and induce misreading.     

Effects of the peptide antibiotics tyrocidine and the linear gramicidin on RNA SYNTHESIS AND SPORULATION OF Bacillus brevis.

Effects of the two peptide antibiotics tyrocidine and the linear gramicidin on exponential growing cultures of $\text{Bacillus brevis}$ (ATCC 8185) were studied. Both antibiotics are produced by this bacterial strain. Our results demonstrate that the addition of tyrocidine leads to inhibition of RNA synthesis followed by a cessation of growth. In contrast, gramicidin does not affect net RNA synthesis and the cells continue to grow. However, sporulation is inhibited by gramicidin. The addition of a mixture of tyrocidine and gramicidin prevents partially the inhibitory effect of tyrocidine on RNA synthesis. The results presented are essentially in agreement with $\text{in vitro}$ results described previously (8,9).     

Effects of N,N′-dicyclohexylcarbodiimide on isolated and reconstituted cytochrome b-c1 complex from bovine heart mitochondria

N,N′-Dicyclohexylcarbodiimide (DCCD) inhibits the activity of ubiquinol-cytochrome c reductase in the isolated and reconstitued mitochondrial cytochrome b-c₁ complex. DCCD inhibits equally electron flow and proton translocation (i.e., the $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ ratio is not affected) catalysed by the enzyme reconstituted into phospholipid vesicles. The inhibitory effects are accompanied by structural alterations in the polypeptide pattern of both isolated and reconstituted enzyme. Cross-linking was observed between subunits V (iron-sulfur protein) and VII, indicating that these polypeptides are in close proximity. A clear correlation was found between the kinetics of inhibition of enzymic activity and the cross-linking, suggesting that the two phenomena may be coupled. Binding of [¹⁴C]DCCD was also observed, to all subunits with the isolated enzyme and preferentially to cytochrome b with the reconstituted vesicles; in both cases, however, it was not correlated kinetically with the inhibition of the enzymic activity.     

Lactate dehydrogenase-C mRNA: Its isolation and invitro translation

Lactate dehydrogenase-C (LDH-C) mRNA was purified from $\text{DBA}\text{2}$ mouse testes and translated $\text{in}\text{vitro}$. First, the LDH-C synthesizing polysomes were isolated by double immunoprecipitation using specific anti-LDH-C and anti-horse immunoglobulin antibodies. Extraction of mRNA was made from the isolated polysomes using hot sodium dodecyl sulfate-phenol method at alkaline pH. In a wheat germ cell-free translation system, the mRNA coded for a polypeptide chain that could be immunoprecipitated with specific anti LDH-C antibody and comigrated with authentic LDH-C in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Titles of related papers in other sections

Electrophoretic analysis by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that the light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complex of barley thylakoids contains only one polypeptide of apparent molecular weight 26 000. The barley mutant, deficient in chlorophyll b and this light-harvesting complex, lacks this polypeptide.The addition of a nonionic detergent, Triton X-100, to the sodium dodecyl solubilization buffer prior to SDS polyacrylamide tube gel electrophoresis, allowed separation of a relatively stable complex, characterized as an oligomeric form of the light-harvesting complex. The oligomer also contained a polypeptide with an apparent molecular weight of 26 000. The absorption and fluorescence spectral properties of the oligomer are similar to those of the monomer. It is suggested that the oligomer of the light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ is closer to the in vivo form rather than the monomer.     

Lack of involvement of lipoic acid in membrane-associated energy transduction in Escherichia coli.

Oxidative phosphorylation, active transport of proline, aerobic- and ATP-driven proton translocation and transhydrogenation of NADP⁺ by NADH, occurred in lipoic acid-deficient cells or vesicles of a lipoic acid auxotroph of $\text{E. coli}$, W1485 lip 2. Addition of lipoic acid had little effect on these processes. Tributyltin chloride, which has been proposed to inhibit oxidative phosphorylation by reaction with lipoic acid (Cain $\text{et al.}$, Biochem. J. (1977) $\text{166}$, 593), was an effective inhibitor of aerobic and ATP-dependent proton translocation and transhydrogenation in lipoic acid-deficient vesicles from this organism. Our results do not support the proposal of Partis $\text{et al.}$ (FEBS Lett. (1977) $\text{75}$, 47) that lipoic acid is involved in the energy transducing processes associated with the membrane of $\text{E. coli}$.     

Inhibition of initiation of DNA synthesis by aminoglycoside antibiotics

In a $\text{dna}\text{C}{}^{\mathrm{ts}}$ mutant of $\text{E. coli}$, the reinitiation of DNA synthesis, which occurred by the shift of the culture from a restrictive temperature to a permissive temperature, was markedly prevented by habakacin, dibekacin, kanamycin, and gentamicin. On the contrary, chloramphenicol did not inhibit the reinitiation synthesis for 30 min. In a parallel experiment, leucine uptake into protein was profoundly blocked by chloramphenicol, but only slightly by habekacin. Habekacin did not significantly affect DNA elongation of the cells at a restrictive temperature. We propose that inhibition of initiation of replication by aminoglycoside antibiotics is related to their lethality.     

Trypsin inhibitory activity of a polypeptide isolated from red kidney beans, that also enhances lymphocyte stimulation.

A polypeptide isolated from red kidney beans, $\text{Phaseolus}\text{vulgaris}$, which has previously been shown to stimulate RNA synthesis in cultures of mouse spleen lymphocytes and plasmolyzed $\text{E.}\text{coli}$, is here shown to be a potent inhibitor of trypsin and α-chymotrypsin. This polypeptide is compared with commercially available trypsin inhibitors with regard to their capacity to inhibit some proteolytic enzymes and to stimulate $\text{in}\text{vitro}$ cultures of lymphocytes. Similar to FV the lima been trypsin inhibitor was found to possess a stimulating effect on the RNA as well as the DNA synthesis in lymphocyte cultures.     

Major intrinsic polypeptide of lens membrane: Biochemical and immunological characterization of the major cyanogen bromide fragment

A protein of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 has been shown to be the major component of eye-lens junctions, which are similar but not identical to the gap junctions of liver and other tissues. Cyanogen bromide cleavage of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 polypeptide from bovine lenses yields a major fragment of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 15 000 (fragment 1). However, if the junctions are first treated with trypsin or carboxypeptidase Y, cyanogen bromide treatment yields a fragment of reduced molecular weight. Since protease treatment has been shown to cleave residues almost exclusively from the carboxy-terminal end of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 polypeptide, it follows that fragment 1 represents the carboxy-terminal half of this molecule, part of which is exposed to proteolytic attack outside the membrane. This latter result is corroborated by the fact that antisera which recognize both the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 polypeptide and fragment 1 fail to do so after preadsorption with intact membranes. In addition, comparative amino acid and partial sequence analyses of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 polypeptide and fragment 1 indicate that fragment 1 is more hydrophilic in character, suggesting that much of the amino-terminal half of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 26 000 polypeptide is buried within the lipid bilayer.     

Resolution of the light-harvesting chlorophyll ab-protein of Vicia faba chloroplasts into two different chlorophyll-protein complexes

Thylakoids of Vicia faba chloroplasts disaggregated by sodium dodecyl sulfate were separated by means of different electrophoretic systems. Under the conditions of a high resolving gel system the chlorophyll containing zone previously termed chlorophyll-protein complex II or light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ was found to be inhomogeneous. It represents a mixture of two distinct chlorophyll-proteins characterized by different spectral properties and different apoproteins. One chlorophyll-protein exhibits a chlorophyll $\text{a}\text{b}$ ratio of 0.9 and is associated with polypeptides of 24 000 and 23 000 daltons. The 24 000 dalton band is proved to bind chlorophyll and has a light-harvesting function. The function of the 23 000 dalton band is unknown. The second chlorophyll-protein has a chlorophyll $\text{a}\text{b}$ ratio of 2.1 and an additional absorption maximum in the position of 637 nm. It is associated with only one polypeptide which has an apparent molecular weight of 23 000. The two 23 000 dalton polypeptides occurring in both complexes are not identical.     

On the mechanism of the glucagon-induced inhibition of hepatic protein synthesis.

The intraperitoneal administration of glucagon (200 μg) to rats produced a transient increase of the hepatic polypeptide chain completion time, the increase being maximum at 5 min returning to control values at 20 min. This inhibitory effect was sustained when glucagon was constantly supplied by continuous infusion. Postmitochondrial supernatants from livers of the control group or rats treated with glucagon for 5 min showed no difference in their protein synthetic activity. After 20 min of intraperitoneal administration of the hormone, that is, when the effect on protein synthesis had vanished, the levels of cAMP were still 40% above those of the control group, and the ribosomal proteins were 110% more phosphorylated. These results suggest that the observed effect of glucagon is not due to its direct action on the protein synthesis machinery. On the other hand, the variations in the hepatic amino acid content brought about by glucagon do not appear to be quantitatively significant to account for the observed inhibition of protein synthesis. The effect of glucagon was always paralleled by a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio which may be responsible for the observed decrease in the rates of elongation and/or termination steps of protein synthesis. Glucagon also produced a rise in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio in both cellular compartments, cytosol and mitochondria, as reflected by the rise in the lactate to pyruvate and the β-hydroxybutyrate to acetoacetate ratios. This shift of the NAD⁺ couple to a more reduced state seems to be the result of an increased mobilization and oxidation of fatty acids brought about by the hormone. It is postulated then that the primary effect of glucagon leading to a decrease in protein synthesis is probably to increase the state of reduction of the hepatic nicotinamide nucleotide system. This point of view is supported by the fact that the nicotinamide and adenine nucleotide systems in rat liver are in equilibrium through cytosolic equilibrium reactions, so that a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio brought about by glucagon may be secondary to the increase in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio. This hypothesis is supported by the fact that glucagon was not effective in inhibiting hepatic protein synthesis in rats pretreated with a drug, 2-benzene-sulfonamido-5-(β-methoxy-ethoxy)pyrimidine, that prevents fatty acid mobilization and the subsequent changes in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ and $\text{[ATP]}\text{[ADP]}$ ratios. Furthermore, the administration of exogenous fatty acid brings about an inhibition of the rate of hepatic protein synthesis accompanied by a decrease in the ATP levels and an increase in the state of reduction of the NAD⁺ system.     

Pancreatic polypeptide response to ethanol in humans and dogs

We studied the effect of a drink of various concentrations of pure ethanol and several commonly ingested alcoholic beverages on plasma levels of immunoreactive pancreatic polypeptide in six healthy human volunteers and compared the results to a protein-rich meal. A drink of distilled water (250 ml) and of pure ethanol (250 ml or 125 ml in the case of 40% v/v ethanol) in concentrations (4, 10, 20, and 40%, v/v) normally present in beer, wine, liquor and whisky did not stimulate plasma pancreatic polypeptide levels above basal. Neither beer, red and white wine (250 ml each) nor whisky (125 ml) caused an increase in basal plasma pancreatic polypeptide levels. The 90-min integrated plasma pancreatic polypeptide response to the protein-rich meal was significantly reduced by an additional drink of 250 ml of white wine ($\text{5987 ± 1315}$ versus $\text{4126 ± 809}\text{pmol · min}{}^{\mathrm{?1}}\text{· 1}{}^{\mathrm{?1}}$). An intravenous infusion of ethanol (300 mg · kg^?1 over 30 min) did not increase plasma pancreatic polypeptide levels above basal.In six dogs with gastric and duodenal fistulas the infusion of pure ethanol into a peripheral vein, into the stomach or into the duodenum did not alter plasma pancreatic polypeptide levels. When ethanol (200 ml of either 1.8, 10 or 40%, v/v) was given as an intragastric bolus injection, only 40% ethanol caused an increase in the mean 90-min integrated plasma pancreatic polypeptide response which was only one-twelfth of the pancreatic polypeptide response to an oral mixed meat meal (35 g · kg^?1). We conclude that in man neither an intravenous infusion nor a drink of ethanol in concentrations normally present in beer, wine and whisky, release pancreatic polypeptide. Also, beer, red and white wine and whisky have no effect on plasma pancreatic polypeptide concentrations. In dogs, a large amount of intragastric ethanol was needed to produce a very small rise in plasma pancreatic polypeptide levels. These results do not favour the hypothesis that, in man and dog, pancreatic polypeptide is the hormonal mediator of the ethanol induced inhibition of exocrine pancreatic secretion.     

Fatty acid utilization and purine nucleotide binding in brown adipose tissue of genetically obese (ob/ob) mice

The activities of the main enzymes involved in fatty acid utilization i.e. palmitoyl CoA synthetase as well as peroxisomal and mitochondrial β-oxidation were measured in brown adipose tissue homogenates of lean and $\text{ob}$/$\text{ob}$ mice kept at 23°C or acclimated at 4°C. The proton conductance pathway, i.e. the number of purine nucleotide (GDP) binding sites and the percentage of 32,000 polypeptide in brown adipose tissue mitochondria were also measured. In the if$\text{ob}$/$\text{ob}$ mice at 23° C, the specific activities of the palmitoyl CoA synthetase and of the β-oxidation as well as the number of GDB binding sites were lower than in the lean mice by 26%, 43% and 37%, respectively. The percentage of 32, 000 polypeptide, however, was the same in both groups. In the $\text{ob}$/$\text{ob}$ mice at 23° C, the lower homogenate β-oxidation specific activity was due to the fact that the peroxisomal and mitochondrial specific activities were 44% and 37% lower, respectively. Cold acclimation at 4° C was found to cause an increase of the palmitoyl CoA synthetase specific activity, of the palmitoyl CoA synthetase and peroxisomal β-oxidation total activities and of the number of GDP binding sites, in both lean and $\text{ob}$/$\text{ob}$ mice. Cold acclimation increased the percentage of 32,000 polypeptide in the $\text{ob}$/$\text{ob}$ mice only.     

Cytochrome P-450 purified to apparent homogeneity from phenobarbital-induced rabbit liver microsomes: catalytic activity and other properties

Cytochrome P-450 was purified to a content of over 17 nmoles per mg of protein from liver microsomes of phenobarbital-treated rabbits by fractionation with polyethylene glycol 6000, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography in the presence of Renex 690, a nonionic detergent. The purified preparation exhibited a single polypeptide band (molecular weight, 49,000 daltons) when submitted to SDS-polyacrylamide gel electrophoresis. Cytochromes P-420 and $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase were absent. The reconstituted system containing purified cytochrome P-450, reductase, and phosphatidylcholine catalyzed the hydroxylation of benzphetamine, cyclohexane, aniline, and laurate.     

Androgen on the estrogen receptor I — Binding and in vivo nuclear translocation

In the immature rat uterus, high concentrations of androgens competed specifically with estradiol on the estrogen receptor (RE). This competition was stereospecific for C₁₉ steroids bearing a 17β and/or 3 hydroxyl group. Very low affinity ligands, such as testosterone, could not compete with estradiol at equilibrium but decreased the association rate of estradiol on its receptor. High doses (> 0.4mg) of 5 α aihydrotestosterone provoked $\text{in vivo}$ as $\text{in vitro}$ the nuclear translocation of RE. The nuclear receptor thus formed displayed the same 5.2 S sedimentation constant as that induced by estradiol. We conclude that the weak affinity binding of androgens to the estrogen receptor is sufficient to induce its nuclear translocation $\text{in vivo}$ provided androgen concentration is high enough in uterus to occupy the estradiol binding site. Conversely, progesterone which does not bind RE could not provoke its nuclear translocation.     

Mitochondrial heterogeneity in foetal and suckling rat liver: differential leucine incorporation into the proteins of two mitochondrial populations.

Iodination of intact mitochondria with ¹²⁵I results in the labeling of essentially one polypeptide with an approximate MW of 30 000. This polypeptide seems to be a component of the inner boundary membrane as it can not be removed from the mitochondria by procedures which destroy the outer membrane (e.g. incubation with digitonin). The amount of the radio-active label which can be bound to this polypeptide is determined by ADP, atractylate, and bongkrekate, components which act on the functional state and the position of the $\text{ADP}\text{ATP}$ carrier in the membrane. [1,2]     

Amino acid uptake by yeasts: IV. Effect of thiol reagents on l-leucine transport in Saccharomyces cerevisiae

(1) $\text{N-}\text{Ethylmaleimide}$ (a penetrating SH- reagent) inactivated l-[¹⁴C]leucine entrance (binding and translocation) into Saccharomyces cerevisiae, the extent of inhibition depending on the time of preincubation with $\text{N-}\text{ethylmaleimide}$, $\text{N-}\text{ethylmaleimide}$ concentration, the amino acid external and internal concentration, and the energization state of the yeast cells. With d-glucose-energized yeast, $\text{N-}\text{ethylmaleimide}$ inhibited l-[¹⁴C]leucine entrance in all the assayed experimental conditions, but with starved yeast and low (0.1 mM) amino acid concentration, it did not inhibit l-[¹⁴C]leucine binding, except when the cells were preincubated with l-leucine. With the rho^? respiratory-deficient mutant (energized cells), $\text{N-}\text{ethylmaleimide}$ inhibited l[¹⁴C]leucine entrance as with the energized wild-type, though to a lesser extent. (2) Analysis of the $\text{N-}\text{ethylmaleimide}$ effect as a function of l-[¹⁴C]leucine concentration showed a significant decrease of $\text{J}{}_{\mathrm{<mtext>max</mtext>}}$ values of the high- (S₁) and low- (S₂) affinity amino acid transport systems, but $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ values were not significantly modified. (3) When assayed in the presence of d-glucose, $\text{N-}\text{ethylmaleimide}$ inhibition of d-glucose uptake and respiration contributed significantly to inactivation of l-[¹⁴C]leucine entrance. Pretreatment of yeast cells with 2,4-dinitrophenol enhanced the effect of l-[¹⁴C]leucine binding and translocation. (4) Bromoacetylsulfanilic acid and bromoacetylaminoisophthalic acid, two non-penetrating SH- reagents, did not inactivate l-[¹⁴C]leucine entrance, while $\text{p-}\text{chloromercuribenzoate}$, a slowly penetrating SH- reagent, inactivated it to a limited extent. When compared with the effect of $\text{N-}\text{ethylmaleimide}$, these negative results indicate that thiol groups of the l-[¹⁴C]leucine carrier were not exposed on the outer surface of the yeast cell permeability barrier.     

Detection of proteins which recognize and bind oriC sequences

Extracts from $\text{E.}$$\text{coli}$ capable of supporting $\text{in}$$\text{vitro}$$\text{oriC}$-dependent DNA replication have been examined with a protein blotting protocol to identify DNA-binding proteins. Four polypeptide chains with apparent affinity for $\text{oriC}$ DNA were detected.