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Cytosolic 5′-nucleotidase/phosphotransferase specific for 6-hydroxypurine monophosphate derivatives (cN-II), belongs to a class of phosphohydrolases that act through the formation of an enzyme–phosphate intermediate. Sequence alignment with members of the P-type ATPases/L-2-haloacid dehalogenase superfamily identified three highly conserved motifs in cN-II and other cytosolic nucleotidases. Mutagenesis studies at specific amino acids occurring in cN-II conserved motifs were performed. The modification of the measured kinetic parameters, caused by conservative and nonconservative substitutions, suggested that motif I is involved in the formation and stabilization of the covalent enzyme–phosphate intermediate. Similarly, T249 in motif II as well as K292 in motif III also contribute to stabilize the phospho–enzyme adduct. Finally, D351 and D356 in motif III coordinate magnesium ion, which is required for catalysis. These findings were consistent with data already determined for P-type ATPases, haloacid dehalogenases and phosphotransferases, thus suggesting that cN-II and other mammalian 5′-nucleotidases are characterized by a 3D arrangement related to the 2-haloacid dehalogenase superfold. Structural determinants involved in differential regulation by nonprotein ligands and redox reagents of the two naturally occurring cN-II forms generated by proteolysis were ascertained by combined biochemical and mass spectrometric investigations. These experiments indicated that the C-terminal region of cN-II contains a cysteine prone to form a disulfide bond, thereby inactivating the enzyme. Proteolysis events that generate the observed cN-II forms, eliminating this C-terminal portion, may prevent loss of enzymic activity and can be regarded as regulatory phenomena. [ABSTRACT FROM AUTHOR]

Poly(β-l-malate) (PMLA) has been reported as an unconventional, physiologically important biopolymer in plasmodia of myxomycetes, and has been proposed to function in the storage and transport of nuclear proteins by mimicking the phospho(deoxy)ribose backbone of nucleic acids. It is distributed in the cytoplasm and especially in the nuclei of these giant, multinucleate cells. We report here for the first time an increase in growth rate and a shortening of the cell cycle after the injection of purified PMLA. By comparing two strains ofPhysarum polycephalumthat differed in their production levels of PMLA, it was found that growth activation and cell cycle shortening correlated with the relative increases of PMLA levels in the cytoplasm or the nuclei. Growth rates of a low PMLA producer strain (LU897 × LU898) were increased by 40–50% while those of a high producer strain (M3CVIII) were increased by only 0–17% in comparison with controls. In both strains, shortening of the cell cycle occurred to a similar extent (7.2–9.5%), and this was associated with similar increases in nuclear PMLA levels. The effects showed saturation dependences with regard to the amount of injected PMLA. A steep rise of intracellular PMLA shortly after injection was followed by the appearance of histone H1 in the cytoplasm. The increase in growth rate, the shortening of the cell cycle duration and the appearance of H1 in the cytoplasm suggest that PMLA competes with nucleic acids in binding to proteins that control translation and/or transcription. Thus, PMLA could play an important role in the coordination of molecular pathways that are responsible for the synchronous functioning of the multinucleate plasmodium. [ABSTRACT FROM AUTHOR]

In Klebsiella pneumoniae, the nif specific transcriptional activator NifA is inhibited by NifL in response to molecular oxygen and ammonium. Here, we demonstrate complex formation between NifL and NifA (approximately 1 : 1 ratio), when synthesized in the presence of oxygen and/or ammonium. Under simultaneous oxygen- and nitrogen-limitation, significant but fewer NifL–NifA complexes (approximately 1%) were formed in the cytoplasm as a majority of NifL was sequestered to the cytoplasmic membrane. These findings indicate that inhibition of NifA in the presence of oxygen and/or ammonium occurs via direct NifL interaction and formation of those inhibitory NifL–NifA complexes appears to be directly and exclusively dependent on the localization of NifL in the cytoplasm. We further observed evidence that the nitrogen sensory protein GlnK forms a trimeric complex with NifL and NifA under nitrogen limitation. Binding of GlnK to NifL–NifA was specific; however the amount of GlnK within these complexes was small. Finally, two lines of evidence were obtained that under anaerobic conditions but in the presence of ammonium additional NtrC-independent GlnK synthesis inhibited the formation of stable inhibitory NifL–NifA complexes. Thus, we propose that the NifL–NifA–GlnK complex reflects a transitional structure and hypothesize that under nitrogen-limitation, GlnK interacts with the inhibitory NifL–NifA complex, resulting in its dissociation. [ABSTRACT FROM AUTHOR]

Transport of presecretory proteins into the mammalian rough endoplasmic reticulum involves a protein translocase that comprises the integral membrane proteins Sec61αp, Sec61βp, and Sec61γp as core components. Electron microscopic analysis of protein translocase in rough microsomal membranes suggested that between three and four heterotrimeric Sec61p complexes form the central unit of protein translocase. Here we analyzed the stoichiometry of heterotrimeric Sec61p complexes present in cotranslationally active protein translocases of canine pancreatic microsomes and various other lumenal and membrane components believed to be subunits of protein translocase and to be involved in covalent modifications. Based on these numbers, the capacity for cotranslational transport was estimated for the endoplasmic reticulum of the human pancreas. [ABSTRACT FROM AUTHOR]

Escherichia coli 70-S ribosomes contain a third site for tRNA binding, additional to the A and P sites. This conclusion is based on several findings. Direct measurements showed that in the presence of poly(U), when both A and P sites are occupied by Ac[14C]Phe-tRNAPhe, ribosomes are capable of binding additionally deacylated non-cognate [³H]tRNA. If ribosomes in the preparation are active enough, the total binding of labeled ligands amounted to 2.5 mol/mol ribosomes. In the absence of poly(U), when the A site can not bind, the P site and the ‘additional’ site can be filled simultaneously with Ac[14C]Phe-tRNAPhe and deacylated [³H]tRNA, or with [³H]tRNA alone; the total binding exceeds in this case 1.5 mol/mol ribosomes. The binding at the ‘additional’ site is not sensitive to the template. [³H]tRNA bound there is able to exchange rapidly for unlabeled tRNA in solution. Deacylated tRNA is prefered to the aminoacylated one. The binding of AcPhe-tRNAPhe was not observed there at all. The 3′-end adenosine is essential for the affinity. The function of the ‘additional’ site is not known, but its existence has to be considered when tRNA · ribosome complexes are studied. [ABSTRACT FROM AUTHOR]

The procedure of isolation and renaturation of all ribosomal proteins from the 30-S subunit of Escherichia coli ribosomes is described. Absorption spectra of these proteins in the near-ultraviolet region have been measured and molar absorption coefficients have been determined on the basis of nitrogen content. Molar absorption coefficients have been calculated for 20 proteins with a known amino acid sequence and the calculated values have been compared with the experimentally determined ones. The absorption spectra obtained allow an easy, precise and highly reproducible spectrophotometric determination of the concentration of individual ribosomal proteins. Circular dichroic spectra of 21 individual proteins from the 30-S subunit of E. coli ribosomes were measured in the range 184–310 nm. The secondary structure of the proteins studied was calculated from the spectra in the range 190–240 nm. Almost all proteins (except proteins S12. S17, S18 and S19) are characterized by a high content of secondary structure. Circular dichroic spectra in the near-ultraviolet region (240–310 nm) indicate that the side groups of aromatic amino acids are fixed in the tertiary structure of the proteins studied. Some internal characteristics (independent of the measurement conditions) of the circular dichroic spectrum in the far-ultraviolet region were proposed as a measure of the resemblance to the native state of ribosomal proteins; these characteristics may be useful for comparison of protein preparations obtained by different methods in laboratories. [ABSTRACT FROM AUTHOR]

The synthesis of ribosomal proteins has been examined during growth and encystment of Acanthamoeba castellani.. Cells have been radiolabelled with [35S]methionine and ribosomal proteins have been extracted either from ribosomes or from total cell extracts. The results show that there is less synthesis of ribosomal proteins relative to total cell proteins soon after transfer of growing cells into non-nutrient medium, suggesting that the synthesis of all ribosomal proteins is under coordinate control. Furthermore, synthesis of ribosomal RNA and ribosomal proteins seems to be regulated coordinately, since within 1 h there is 70–80% decline in synthesis of both. Translation of total RNA in vitro indicates that the relative decrease in the synthesis of ribosomal proteins during development is not only due to a relative decrease in the content of ribosomal protein mRNA. However, a considerable (about 10-fold) and coordinate decrease in the relative content of ribosomal protein mRNA occurs during encystment. These data suggest a difference in the control of the synthesis of total proteins and ribosomal proteins as well as in the regulation of the content of total mRNA and ribosomal protein mRNA. The comparison of patterns of ribosomal proteins indicates that during development no qualitative changes occur. The content of one protein of the small ribosomal subunit is low in growing cells and increases during encystation. Since the differences in the concentration of this protein are neither due to changing levels of the corresponding mRNA nor to changing rates of synthesis, changing affinities of this protein to ribosomes in vivo are suggested. [ABSTRACT FROM AUTHOR]

The temperature dependence of the adenine nucleotide exchange in mitochondria has been determined by employing a rapid mixing, quenching and sampling apparatus and the inhibitor quench-back exchange method. Thus the exchange is resolved down to 0.1 s. Rates are evaluated from accumulating the time-dependent progress at about 10 points. The exchange rate in liver mitochondria was determined from -10deg;C; to +10°C; in the presence of 20% glycol, from 0°C to 25°C. and from 20°C to 40°C under partial inhibition by carboxyatractylate. The total range between -10°C to +40°C has only one temperature break at 13°C. From the Arrhenius plot between -10°C to +13°C, EA ≈ 140 kJ and above 13°C, EA ≈ 56 kJ is evaluated, corresponding to a Q10 of 8 and 2 respectively. In beef heart mitochondria the exchange rate was measured between 0°C and 20°C, and between 15°C and 30°C under partial inhibition with carboxyalractylate. There is a temperature break around 14°C with EA ≈ 143 kJ between 0°C and 14°C and EA 60 kJ from 15°C to 30°C. The extrapolated translocation rates at 37 °C are 500 and 1800 µmol min&&sup-1; (g protein)&sup-1; for rat liver and for beef heart mitochondria respectively. The temperature break is suggested to reflect a conformation change since there is no reversed break at low temperature, the temperature break changes in sonic particles and no lipid phase transition at 14°C in mitochondria has been reported. [ABSTRACT FROM AUTHOR]

A putative precursor of carbamoyl-phosphate synthase was isolated from a microsomal wash fraction and purified by high-pressure liquid chromatography. Autolytic degradation and limited proteolysis were used to characterize the putative precursor of carbamoyl-phosphate synthase and to show its similarity to the processed enzyme. The carbamoyl-phosphate synthase precursor underwent a time-dependent and concentration-dependent conversion into a dimeric or polymeric form. When labelled with 125I and incubated with foetal rat liver mitochondria the precursor was bound to the mitochondria and about 30% of the label was imported into the matrix space. This labelling required the presence of ATP and was time-dependent. Mitoplasts also imported the carbamoyl-phosphate synthase precursor. After import of the precursor, increases in carbamoyl-phosphate synthase activity could be demonstrated in foetal rat liver mitochondria. [ABSTRACT FROM AUTHOR]

[³H]Tetracycline was covalently incorporated into rat liver ribosomes and isolated 40-S and 60-S subunits on irradiation at 254 nm. The antibiotic was almost exclusively incorporated into ribosomal proteins. At least some of these proteins are assumed to be involved in ribosomal function, since photoincorporated tetracycline was found to inhibit the activity of 40-S and 60-S subunits in the poly(U)-directed protein-synthesizing system as well as that of the 40-S subunit in the poly(U)-mediated [14C]Phe-tRNA binding. The results from simultaneous one-dimensional and two-dimensional gel electrophoreses showed a small distribution of label among ribosomal proteins in 60-S subunits and in 80-S ribosomes, L10 being the most radioactive protein. As non-acylated tRNA partly competed with this labeling, it is likely that tetracycline interaction with these proteins occurred at a functional site. L10 has already been found to interact with puromycin [Reboud, A. M., Dubost, S., Buisson, M. & Reboud, J. P. (1981) Biochemistry, 20, 5281-5288]. In the case of free 40-S subunits the label distribution was wider among ribosomal proteins. No particular role has yet been found for the most labeled protein, S12, but protein S3a, which was also highly labeled, has already been reported to be involved in subunit function. [ABSTRACT FROM AUTHOR]

Anti-(P1-450) and anti-(P-448) are two antibodies developed against distinctly different Forms of 3-methyl- cholanthrene-induced cytochrome P-450 in C57BL/6N mouse liver microsomes [Negishi and Nebert, J. Blot C/win. 254, 11015-11023 (1979)]. The effects of these antibodies on long-Evans and Sprague-Dawley rat liver microsomes and rat `minimal deviation' hepatoma 7777 microsomes were studied. Rat microsomal aryl hydrocarbon hydroxylase activity, induced by polycyclic aromatic compounds, is preferentially inhibited by anti-(Pi-450) more so than anti-(P-448). Polycyclic-aromatic-induced rat liver microsomal acetanilide 4-hydroxylase activity is preferentially inhibited by anti-(P-448) more so than anti-(P,-450). Anti (P1 -450) precipitates a 56-kDa moiety in polycyclic aromatic-induced rat liver microsomes. Anti-(P-448) precipitates a 55-kDa moiety in control, phenobarbital-treated, or polycyclic-aromatic-induced rat liver microsomes. A heterogeneity was found amongst individual Morris `minimal deviation' hepatomas 7777. indicating the 3-metliylcholanthrene-induced aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase activities must represent different forms of rat P1-450. Clone 46 DNA, a portion of the mouse cloned P1-450 structural gene. hybridizes to one gene (or a small number of genes) in rat liver and to 3-niethylcholanthrene-induced rat 23-S P1-450 mRNA. As has already been shown in the mouse [Tukey, Nebert and Negishi, J. Biol. Chem. 256, 6969-6974 (1981)], rat liver P1-450 induction is under transcriptional control, and there is no evidence for gene amplification or some gross form of DNA rearrangement. These data demonstrate striking similarities between rat and mouse for the P1-450 structural gene, the P1-450 mRNA, the P1-450 protein and the P-448 protein. Polycyclic-aronlatic-induced P1-450, and its association with polycyclic-aromatic-induced aryl hydrocarbon hydroxylase activity, therefore does not appear to be closely correlated with polycyclic-aromatic-induced P-448 in either rat or mouse. [ABSTRACT FROM AUTHOR]

Transport of apocytochrome c across the outer mitochondrial membrane and conversion to bolocytochrome c were studied in vitro. Apocytochrome c was synthesized in a cell-free homogenate from Neurospora erassa. Transfer in vitro was accomplished in a reconstituted system consisting of the postribosomal supernatant of the cell-free homogenate and of isolated and purified mitochondria from Neurospora. The reconstituted system has the following characteristics: 1. Apocytochrome c is rapidly cleared from the supernatant and holocytochrome c appears in the mitochondria with the same kinetics. More than 80 % of the apocytochrome c employed is converted to holocytochrome c. No transient accumulation of apocytochrome c is found in mitochondria. 2. The heme group becomes covalently linked to apocytochrome c in the reconstituted system as demonstrated by analysis of tryptic peptide maps of the apoprotein and holoprotein. 3. Deuterobemin added to the reconstituted system but not deuteroporphyrin inhibits the formation of holo- cytochrome c. This inhibition is reversed by protohemin. 4. In the presence of deuterohemin about half of the apocytoclirome c remains in the supernatant; the other half becomes associated with the mitochondria. The latter portion is tightly bound and is specifically released upon incubation of the mitochondria with excess apocytochrome c. It is converted to holocytochrome c after addition of protohemin. We conclude from these observations that apocytochrome c is transported across the outer mitochondrial membrane via receptor sites. In the presence of the heme analogue deuterohemin, binding to the receptor sites on the cytoplasmic surface of the outer mitochondrial membrane still takes place but translocation does not. The latter step is apparently coupled to the covalent linkage of the heme group. We suggest that the formation of the thioether bonds between apoprotein and heme is catalysed by an enzyme in the intermembrane space and that deuterohemin can compete with protohemin for binding to the enzyme. Finally, the data indicate that it is the heme group and not the porphyrin group which is coupled to the apoprotein. [ABSTRACT FROM AUTHOR]

The function of protein L1 in protein biosynthesis has been examined using ribosomes from two independently derived mutants of Escherichia coli. both of which lacked this protein. In systems in vitro with phage MS2 RNA or poly(U) as message, the mutant ribosomes showed from 40% to 60% of the activity of wild-type ribosomes. The reduction in activity was apparent in the kinetics of [14C]phenylalanine incorporation throughout the incubation period. The activities were restored fully to the wild-type level by the addition of purified protein L1. These results show on the one hand that protein L1 is not essential for protein biosynthesis but, on the other, its presence can significantly increase the overall rate of this process. The data further indicate it as likely that protein L1 exerts its effect at the elongation step. [ABSTRACT FROM AUTHOR]

In order to study the functional role of the spectral species of cytochrome b-565 observed in mitochondria, genetically manipulated strains of Saccharomyces cerevisiae have been used. Strains have been found which are devoid of cytochrome h-565 under certain conditions and which nevertheless are able to grow on a respirable substrate. Two different methods have been used to determine the cytochrome b-565 content: anaerobic titrations and antimycin-A-induced reduction of cytochrome b-565. Both yield the same results. [ABSTRACT FROM AUTHOR]

A wild-type strain of Saccharomyces cerevisiae and temperature-dependent revertants of isonuclear box mutants partially or completely devoid of cytochrome b-565, were used to study the role of this cytochrome in oxidative phosphorylation. At the mitochondrial level, the phosphorylating data and the succinate oxidase activity at three different temperatures (16°C, 28°C and 36 °C) were measured in these strains showing various cytochrome b-565 contents. It is concluded that this cytochrome b-565 is not in the main pathway of the electron transfer chain. At the optimum growth temperature (28 °C), the measurements of the P/0 ratio for the wild phenotype strains, with ethanol as substrate, led to the conclusions that two phosphorylation sites of the respiratory chain are functional in these strains. The growth yields for wild phenotype strains and revertants grown in vivo in complex media, with ethanol or galactose as the energy source (at 16°C, 28 °C and 36 °C), were compared with the cytochrome b-565 content. The growth yields showed small variations if compared to the reference strain, when the cytochrome b-565 content was greatly diminished or absent. Thus the ATP production at site II is independent of the cytochrome b-565 content. Cytochrome b-565 does not play an essential role in oxidative phosphorylation. [ABSTRACT FROM AUTHOR]

The physicochemical properties of a long-chain-acyl-CoA hydrolase (EC 3.1.2.2) of rat liver microsomes have been studied. The hydrolase sedimented as a homogenous species with a sedimentation coefficient, s20, w, of 4.1 S and the molecular weight of 59000 was obtained. Amino acid composition of the enzyme was determined. The specific absorption coefficient, A280 nm1 % was estimated as 9.8 (1 cm cell length) and the circular dichroic data suggested 50–60% alpha-helical structure. The enzyme contained three sulphydryl groups, and one of these was exposed to the environment. The hydrolase was inhibited by the serine-directed reagent phenylmethylsulfonyl fluoride, as well as p-hydroxymercuribenzoate, N-ethylmaleimide and 5.5′-dithiobis(2-nitrobenzoic acid). Reactivation by means of dithiothreitol was never complete with phenylmethylsulfonyl fluoride and p-hydroxymercuribenzoate as inhibitors. [ABSTRACT FROM AUTHOR]

The effect of inorganic phosphate on Ca2+ retention has been investigated using phosphate-depleted liver mitochondria. Phosphate induces the release of Ca2+ through an efflux route insensitive to ruthenium red. This effect is not due to functional or structural damage, since mitochondria maintain their membrane potential during phosphate-induced Ca2+ efflux. Direct enzymatic measurement of mitochondrial pyridine nucleotides has established that changes in their redox state (i.e. increased oxidation) do not play a role in the phosphate-effect. The phosphate-induced Ca2+ efflux requires transport of phosphate out of mitochondria. However, the fluxes of Ca2+ and phosphate do not coincide: the release of phosphate preceeds that of Ca2+. [ABSTRACT FROM AUTHOR]

In leaves of rye seedlings (Secale cereale L.) grown at 32°C the formation of plastid (70-S) ribosomes is specifically prevented. The resulting plastid-ribosome-deficient leaves can be used as a suitable system to identify chloroplast proteins which are translation products of cytosolic (80-S) ribosomes. The ribosome deficiency in plastids is accompanied by a bleaching of the leaves in light. In experiments aimed at finding the primary heat-sensitive event leading to ribosome deficiency the DNA of rye chloroplasts has been identified. Its properties are similar to those of chloroplast DNAs from other higher plants. The ribosome-deficient plastids isolated from heat-bleached rye leaves contained a DNA species which was indistinguishable from that of chloroplasts with regard to buoyant density in CsCl equilibrium gradients, reassociation properties and fragment patterns obtained upon cleavage by restriction endonucleases. Its quantity was comparable to that of chloroplast DNA of green leaves grown at a permissive temperature (22°C). These results suggest that, unlike the effect in heat-bleached Euglena strains, lack of chloroplast DNA cannot be considered as the reason for the primary effect of high temperature on rye leaves but steps in the biosynthetic pathway of plastid ribosomes themselves must be affected more directly. [ABSTRACT FROM AUTHOR]

The fluorescent probe 9-aminoacridine interacts with the membranes of chromatophores by an energy-independent binding and by two light-energy-dependent processes: a nigericin-insensitive interaction and a nigericin-sensitive interaction. This has been demonstrated by measurements of the medium concentration of 9-aminoacridine by flow dialysis and by conventional fluorescence measurements. The energy-independent and the actinic-light-induced nigericin-sensitive quenching occur according to the Freundlich equation indicating adsorption of the probe to the membrane. The actinic-light-induced nigericin-insensitive quenching cannot simply be described by an adsorption process. This quenching appears to be dependent on the number of probe molecules bound to the membrane independently of energy. The light-induced nigericin-sensitive quenching appears to be dependent on the pH-gradient across the membrane. The observation that this quenching is the result of an adsorption excludes quantitative ΔpH determinations which are based on the assumption that the probe is entrapped inside the chromatophores. The values calculated for the ΔpH based on this assumption are dependent on protein concentration and two-three-fold larger than those determined from the uptake of [14C]methylamine as measured with the flow dialysis method. [ABSTRACT FROM AUTHOR]

The synthesis by yeast microsomes of compounds that are probably dolichol-pyrophosphate derivatives containing N,N'-diacetylchitobiose and several mannose residues is described. However, the presence of monosaccharide residues other than N-acetylglucosamine and mannose has not been ruled out. The amount of the lipid derivatives synthesized was enhanced by the addition to the incubation mixture of an organic-solvent-soluble extract from rat liver known to contain dolicholpyrophosphate oligosaccharides. Incubation of these derivatives with yeast microsomes led to the transfer of about fifteen percent of their saccharide moieties to endogenous proteins. The oligosaccharides released from the dolichol derivatives by mild acid hydrolysis could serve as primers for the synthesis of a polysaccharide having the characteristics of mannan outer-chain. It is suggested that the dolichol-pyrophosphate derivatives described in this paper are intermediates in the synthesis of mannan inner core. [ABSTRACT FROM AUTHOR]

We have studied the steps taking place in initiation of protein synthesis in rabbit reticulocyte cell-free systems by using native 40-S subunits. Binding of the initiator Met-tRNAf to the native 40-S subunits took place without additions of initiation factors, in the presence or absence of poly(A,G,U) but GTP is required. There is no requirement of mRNA for the binding reaction, suggesting the presence of eIF-2 and eIF-3 in native 40-S subunits. Polydextran sulphate and N-ethylmaleimide block the binding of Met-tRNAf to native 40-S subunits, whereas pactamycin and edeine A1 enhance the binding. Aurintricarboxylic acid inhibits the poly(A,G,U)-dependent stimulation of the reaction and releases some initiation factor(s) from native 40-S subunits. Addition of 60-S subunits to the native 40-S · Met-tRNAf complex in the absence of eIF-5 leads to a 80-S. Met-f complex, reactive with puromycin, suggesting that this initiation factor was present in the native 40-S subunits. Formation of methionyl-puromycin with the 80-S · Met-tRNAf complex is blocked by inhibitors of peptide bond formation, such as anisomycin, trichodermin, trichothecin, bruceantin and harringtonine. Pactamycin, edeine A1, aurintricarboxylic acid, pyrocatechol violet and adrenochrome do. not inhibit peptide bond formation but lead to the formation of an unusual 40-S-Met-tRNAf complex which, after the joining of the 60-S subunit, is not reactive in the puromycin reaction. [ABSTRACT FROM AUTHOR]

The swelling of rat liver mitochondria has been studied in isotonic ammonium salts of phosphate, thiophosphate, monofluorphosphate, difluorphosphate or in isotonic ammonium malate induced by low concentrations of the mentioned anions. The data lead to the following conclusions. 1. Phosphate, thiophosphate, and monofluorphosphate are taken up by the mitochondria, whereas difluorphosphate is not, indicating that the substrate of the transporter represents the divalent anion HPO2-4 and not H2PO&4macr;. 2. Thiophosphate, monofluorphosphate and phosphate, but not difluorphosphate induce the swelling of mitochondria in isotonic ammonium malate, only after a lag phase. The length of the lag phase depends on the concentration of inducing anion and pH. This phenomena cannot be explained by the current concept of two independent phosphate-transporting systems in mitochondria. [ABSTRACT FROM AUTHOR]

Addition of ruthenium red to mitochondria isolated from brain, adrenal cortex, parotid gland and skeletal muscle inhibits the further uptake of Ca2+ by these mitochondria but induces little or no net Ca2+ afflux; the further addition of Na+, however, induces rapid afflux of Ca2+. The velocity of the Na+-induced afflux of Ca2+ from these mitochondria exhibits a sigmoidal dependence on the [Na+]. Addition of Na+ to mitochondria exhibiting the most active Na+-dependent afflux of Ca2+ (brain and adrenal cortex) also releases Ca2+ in the absence of ruthenium red and, under these conditions, the mitochondria become uncoupled. It is concluded that the afflux of Ca2+ from these mitochondria occurs via a Na+-dependent pathway, possibly a Na+-Ca2+ antiporter, that is distinct from the ruthenium-red-sensitive carrier that catalyses energy-linked Ca2+ influx. The possible role of the Na+-dependent afflux process in the distribution of Ca2+ between the mitochondria and the cytosol is discussed. In contrast, mitochondria from liver, kidney, lung, uterus muscle and ileum muscle exhibit no Na+-dependent afflux of Ca2+. [ABSTRACT FROM AUTHOR]

Initial velocities of energy-dependent Ca2+ uptake in cardiac mitochondria were measured with the ethyleneglycol bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid/ruthenium red quenching technique. Accurate concentrations of free Ca2+ between 1 and 15 µM were generated with ATP as a Ca2+ buffer system. Using the method of iterative approximation, the accurate value of the initial velocity of ATP-driven 45Ca uptake, and the time (t0.5) for half-maximal uptake can be calculated. The rate of initial Ca2+ uptake increased in response to the rising free Ca2+ concentrations in the medium (in the presence of 0.6-5.0 mM ATP). A maximal rate was obtained between 10 and 15 µM of free Ca2+. For different ATP concentrations (0.6-5.0 mM), the half-maximal rate of Ca2+ transport (Km) was observed between 4.1 and 7.4 µM Ca2+ (with a mean Km of 5.6 ± 0.6 µM Ca2+; n = 5). Hill plots of the data yield straight lines with an average slope n of 1.93 ± 0.29, indicating the existence of two binding sites for Ca2+ in the transmembrane transport system. Keeping the free Ca2+ concentration on a constant level (6 µM), the rate of initial Ca2+ uptake also increased continually with rising ATP concentrations (maximum velocity υ = 7.1 nmol of Ca2+ × mg protein-1 × s-1.). From energy-independent calcium-ion binding studies with cardiac mitochondria, a dissociation constant (Kd) of 26.8 × 10-6M and a total number of external binding sites (n) of 12.6 nmol of Ca2+/mg of protein was obtained. The data are discussed with regard to the physiological role which cardiac mitochondria may play as a reversible storage site during the contraction relaxation cycle of the heart. [ABSTRACT FROM AUTHOR]

This paper describes three types of experiments which indicate that the binding sites for elongation factor 1 (EF-1) and elongation factor 2 (EF-2) on ascites cell ribosomes are not identical and perhaps not even overlapping. The experimental evidence presented includes direct competitive binding of labeled elongation factors to ribosomes as well as the influence of pokeweed antiviral protein and Escherichia coli anti-L7/L12 proteins on the binding and function of the two factors. It is further shown that EF-1β from Artemia salina does not function in displacing EF-1 from mouse ascites tumor cell ribosomes. These results also support our recently proposed model that EF-1 remains bound to the ribosome during the peptide chain elongation cycle. [ABSTRACT FROM AUTHOR]

By means of exchange studies, in which 3H-labelled 50-S subunits and unlabelled 70-S ribosomes from Escherichia coli MRE 600 were used, it has been demonstrated that the 30-S subunit is the only target for IF-3 in the dissociation of 70-S ribosomes. The interference of IF-3 with the dynamic equilibrium of 70-S in &equilibrium; 50-S + 30-S occurs by binding of the factor to the 30-S subunit. The 30-S-IF-3 complex in impaired in the association reaction, which implies that IF-3 is acting as an anti-association factor. The action of IF-1 is two-fold. Firstly IF-1 increases the rate of exhcange of the ribosomal subparticles in the 70-S ribosome without changing the position of the equilibrium. Thus the spontaneous equilibrium is attained more rapidly in the presence of IF-1. This kinetic effect of IF-1 is also demonstrated in the IF-3-mediated dissociation of 70-S ribosomes. Secondly IF-1 is able to increase the IF-3-mediated dissociation. It seems likely that the explanation for the latter phenomenon must be sought in the binding of IF-1 to 70-S ribosomes, resulting in a loosening of the ribosomes structure, as well as to 30-S. IF-3 complex, thaereby slowing down the association reactions of the subunits [ABSTRACT FROM AUTHOR]

Age-related changes in the activities of microsomal and mitochondrial elongating systems have been determined in mouse brain from birth to maturity. In microsomes, the components necessary for behenyl-CoA (docosanoly-CoA) elongation have been found to be NADPH and malonyl-CoA. In mitochondria, both NADH and NADPH are used and acetyl-CoA is the only donor of two-carbon-atoms unit. The synthesised fatty acids were identified by thin-layer and gas chromatography. The specific activity is higher in microsomes than in mitochondria. In microsomes, the specific activity for malonyl-Co-A incorporation reached a maximum at 15 - 20 days of age; this peak was not obtained in the Quaking and Jimpy mutants. The increase in enzyme activity (specific activity and total activity per brain) paralleled the myelin deposition. The activity of the mitochondrial system increases regularly during development: it is not correlated to myelination and it is not affected in the Quaking mutant. The interplay between microsomal and mitochondrial elongation systems is studied [ABSTRACT FROM AUTHOR]

1 A different biochemical behaviour has been previously shown during several stages of the development of the insect Ceratitis capitata; thus, the acyl transferase activity has a different participation in the pathways of synthesis of triacylglycerols and phosphoglycerides by either larval or pharate adult stages of development. The results reported here are concerned with the behavior of mitochondria and microsomes from larvae and pharate adults for the synthesis of triacylglycerols and phosphoglycerides. In all preparations of either mitochondrial or microsomal fractions the optimum protein amount, previously determined, has been used. 2. The highest incorporation of [14C]palmitate into triacylglycerols and phosphoglycerides was achieved by the microsomal fraction from the larval stage of development of the insect. However, mitochondrial fractions from pharate adults utilized higher levels of [14C]palmitate than the microsomal preparation. These differences were not correlated with the palmitoyl-CoA synthetase activity. 3. Glycerol 3-phosphate influenced the fatty acid incorporation in a different manner depending on the stage of development of the insect. Increasing concentrations of glycerol 3-phosphate stimulated the synthesis of triacylglycerols by microsomal and mitochondrial preparations. This effect was not exhibited by the subcellular preparations of pharate adults. 4. Double-label experiments using [14C]glycerol 3-phosphate and[3H]palmitate showed qualitative differences in the synthesis of triacylglycerols by mitochondrial preparations from either larvae of pharate adults. Incorporation of the labelled fatty acids to endogenous triglycerides was preferentially shown by the mitochondria larval preparations. The pathways of synthesis of triacylglycerols and phosphoglycerides by microsomal preparations showed only quantitative differences depending on the stage of development of the insect. 5. Acylation of 14C-labelled lyso-phosphatidylcholine and 14C-labelled lyso-phosphatidylethanolamine by [3H]olate was assayed in the presence of miochondrial and microsomal preparations from the different stages of development of the insect. Microsomal fractions showed an efficient acyl transferase activity mainly when lyso-phosphatidyl-ethanolamine was used as a substrate [ABSTRACT FROM AUTHOR]

1. Solubility of mitochondrial membranes in various solvent systems was determined quantitatively. The most effective agent was the anionic detergent, sodium dodecylsulphate, which solubilizes 90% of the protein at the concentration of 0.1% followed by Triton X-100 (70%), sodium deoxycholate (60%), Brij 56 (50%), and guanidine hydrochloride (40%) at a concentration of 2M. 2. Affinity chromatography of a clear 0.1% sodium dodecylsulphate solution of digitonized mitochondria on Sepharose 4B containing carboxyatractylate always resulted in the separation of two fractions, one of which was not retained by the column and the other which could be obtained after elution with 2% sodium dodecylsulphate. 3. The retained protein showed a high binding specificity for ATP and [³H]atractylate when compared with the unretained fraction. The amount of bound [³H]atractylate or carboxyatractylate-sensitive binding of ATP was 10.5 ± 4 nmol/mg protein, and 22 ± 8 nmol/mg protein, respectively. 4. The major component within the retained fraction, comprising 85% of the total weight, was protein, followed by phospholipids (14%) and approximately 1% triglycerides. Sodium dodecyl- sulphate-polyacrylamide gel electrophoresis revealed a major (95%) and a minor (5 %) component with an apparent molecular weight of 26000 ± 1000 and 8300 ± 400, respectively. The gels did not stain for carbohydrates. Ultracentrifugal analysis showed a single, symmetrical boundary. [ABSTRACT FROM AUTHOR]

Oxidative phosphorylation in rat liver mitochondria, the mitoplast or inner membrane-matrix fraction, and inverted inner membrane vesicles was studied with firefly luciferase luminescence All preparations showed relatively high ATP/O ratios under initial reaction conditions in the presence of Mg2+, but only intact mitochondria demonstrated significant respiratory control. Mitochondria and mitoplasts, but not vesicles, catalyzed nearly complete conversion of ADP to ATP. Vesicles converted between 50% and 70% of added ADP to ATP establishing a phosphate potential of 9.8 to 10.5 kcal/mol (43.9 kJ/mol), In "loosely coupled" submitochondrial systems which do not display respiratory control, the phosphate potential appears to be a useful parameter of the integrity of the phosphorylating membrane, while ATP/O ratios may be dependent on the relative concentrations of ADP and ATP in the reaction medium. The rapid kinetics of oxidative synthesis of ATP by inverted inner membrane vesicles was investigated in stopped-flow rapid-mixing experiments. After an oxygen pulse to reduced vesicles, ATP synthesis commenced within 20 ms. In antimycin-inhibited vesicles, ATP synthesis resulting from the rapid oxidation of the terminal portion of the respiratory chain commenced within 20 ms and took approximately 100 ms for half completion. Since half oxidation of the terminal portion of the respiratory chain occurs within a few milliseconds, ATP synthesis resulting from such redox reactions must occur over a significantly longer time course than the redox reactions themselves. [ABSTRACT FROM AUTHOR]

Rat liver microsomes contain phosphatidate phosphatases which split phosphatidic acid into inorganic phosphate and diacylglycerol and a system of phospholipases and lipases, which split phosphatidic acid into free fatty acids, glycerol and inorganic phosphate. In the presence of ATP, CoA and [1-14C]palmitate, part of the monoacyl-sn-glycerol 3-phosphate formed by phospholipase action is reesterified, yielding radioactive phosphatidic acid. The sum of di- and triacylglycerols formed from phosphatidic acid in the presence of ATP and CoA exceeded the amount of diacylglycerol formed in their absence. The yield of neutral lipids from sn-glycerol 3-phosphate and monoacyl-sn-glycerol 3-phosphate markedly exceeded that from phosphatidic acid. Comparison of the yields of di- and triacylglycerols from glycerol-labelled and fatty-acid-labelled phosphatidic acid was used to establish the extent of deacylation and reacylation. About 60% of the diacylglycerol was formed by direct dephosphorylation. The triacylglycerols, on the other hand. were formed almost exclusively from recycled phosphatidic acid. [ABSTRACT FROM AUTHOR]

Analyses are described of three types of ribosomal fragment, all derived from the well-characterized ribonucleoprotein species consisting of proteins S7, S9, S10, S14 and S19, together with RNA from sections O′-D-E′-K-P-P′-E-A of the 16-S sequence. I. When 30-S subunits were hydrolysed with ribonuclease T1 in the presence of deoxycholate in addition to the components previously described, a fragment could be isolated which contained only proteins S7 and S 19~ together with minor amounts of S 13 or S 14. Oligonucleotide analysis of this fragment showed that it contained RNA from sections O′-E′ and P-A of the 16-S RNA~ but that section K (from the middle of this area) was missing. 2. It has previously been shown that when 30-S subunits are irradiated with ultraviolet light, protein S 7 is the primary target of cross-linking of protein to RNA. By making use of this reaction, ribonucleoprotein fragments were isolated from irradiated 30-S subunits, the unbound proteins were removed, and RNA fragments containing covalently linked protein S 7 were identified. It was possible to demonstrate that the site of cross-linking lies within the O′-A region of the 16-S RNA, and more precise experiments showed that this site almost certainly is in the P-A region. 3, When the parent five-protein ribonucleoprotein fragment (above) was deproteinized under very mild conditions, RNA complexes could be isolated which consisted of non-contiguous sequences~ but which migrated as a single species into a polyacrylamide gel. Analysis of one of these complexes showed that it contained sequences from sections O′-E′ and P-A, but that section K was missing (cf. first paragraph above). This demonstrated that these two separate regions of RNA interact within the 30-S subunit, independently of the presence of protein. [ABSTRACT FROM AUTHOR]

Two forms of serine transhydroxyrnethylase, separable by TEAE-cellutose chromatography, are present in wild-type extracts of Saccharomyces cerevisiae. One of these forms is mitochondrial and the other one cytoplasmic. The mitochondrial form is absent in the mutant tmp3. [ABSTRACT FROM AUTHOR]

Ribosomes from stringent strains of bacteria generate (p)ppGpp if incubated with uncharged tRNA, a ribosomal wash fraction, GTP and ATP. By contrast, an analogous system from rat liver does not transform GTP to (p)ppGpp but degrades it to guanine. The reaction requires the ribosomal subunits, a 40 000-Mr and a 60 000-Mr microsomal wash protein factor and is inhibited if the ribosomal A-site is charged with aminoacyl tRNA. The degradation of GTP to guanine occurs in the following four distinct reaction steps: (a) hydrolysis of GTP to GDP + Pi, (b) hydrolysis of GDP to GMP + Pi, (c) hydrolysis of GMP to guanosine + Pi, (d) hydrolysis of guanosine to guanine + ribose. The reaction step (a) is inhibited by fusidic acid, cycloheximide, emetine, tetracycline and puromycin. The hydrolysis of GDP is inhibited strongly by fusidic acid, emetine and tetracycline. A putative physiological significance of this ribosome-dependent pathway in the processes of growth control of animal cells under conditions of amino acid deprivation is discussed. [ABSTRACT FROM AUTHOR]

Yeast mitochondrial DNA labelled in vitro by incubation of isolated mitochondria with DNA precursors exhibits skewed profiles on isopycnic CsCl gradients. The skew is not due to nuclear DNA nor to single-stranded mitochondrial DNA in the product labelled in vitro. Simultaneous labelling with [³H]dTTP and [14C]dGTP in vitro indicates a gradient of base composition in the DNA labelled in vitro. Thus, selective degradation of mitochondrial DNA occurs during incubation, converting large molecules having the mean density of mitochondrial DNA into smaller molecules of higher mean density and with higher G:T ratio. Similarly skewed distributions can also be produced by incubation of mitochondrial DNA labelled in vivo with the yeast mitochondriaI fraction or with micrococcal endonuclease, an enzyme known to selectively hydrolyse (A + T)-rich regions of DNA [ABSTRACT FROM AUTHOR]

The reactivity of the sulfhydryl groups of Escherichia coli ribosome has been investigated using a fluorescent label. Under denaturing conditions, all the -SH groups can be titrated. In the native form of the ribosome, six and less than one labels can be respectively conjugated to the 30-S and 50-S subparticles without loss of activity for poly(U)-dependent polyphenylalanine synthesis. The most reactive thiol groups belong to proteins S1, S12, S18, S21. The binding of mRNA plus tRNA to the 70-S ribosomes affects the spectroscopic properties of the labels showing that conformational changes are induced by these interactions. Furthermore, the treatment of these complexes by the labelling agent demonstrates that the -SH group belonging to protein Si is partially protected whereas other thiol groups located on the 50-S subparticle become reactive. [ABSTRACT FROM AUTHOR]

Ribosomal binding complexes directed by poly(U) or T4 mRNA were formed with aminoacyl-tRNA or its derivatives bound to predominantly the P or A binding site. The defined binding complexes were reacted with colicin E3 and the reaction was assessed by the ability of the complexes to proceed with polypeptide synthesis. The results indicated that only one of the four complexes tested was completely resistant to colicin E3-induced inactivation: that of Phe-tRNA bound in the presence of poly(U) to the A-site. The poly(U)-directed complex of AcPhe-tRNA and the T4-mRNAdirected complex at the A-site appeared slightly resistant, while the T4 mRNA initiation complex was inactivated by colicin E3 in a manner similar to non-complexed ribosomes. Colicin E3 added to ribosomes after protein synthesis had been initiated affected the subsequent polymerization in a manner corresponding to the response of the hinding complexes. Thus, poly(U)- translating ribosomes were less affected than ribosomes translating the viral mRNA. The vulnerability of natural-mRNA-directed binding complexes to inactivation by colicin E3 is in accord with the mode of inactivation by the colicin in vivo. [ABSTRACT FROM AUTHOR]

Globin mRNA can be translated with relatively high efficiency in a fractionated cell-free system containing ribosomes prepared from cysts of Artemia salina. These ribosomes have unusually low endogenous activity for peptide synthesis in the absence of added mRNA. The system requires components from the postribosomal supernatant and from the 0.5 M KCl ribosomal wash fraction. Both these fractions were derived from either rabbit reticulocytes or unstimulated Friend leukemia cells that produce little or no hemoglobin. The activity of mRNA and enzyme fractions from rabbit reticulocytes and Friend leukemia cells were tested in this system in vitro for their ability to direct the synthesis of the a and j8 chains of globin. The α:β chain ratio synthesized from mRNA in the rabbit reticulocyte salt wash fraction was 4:1. The corresponding value for the 9-S mRNA fraction from the salt-washed reticulocyte ribosomes was 1:4, thus these two fractions appear to provide sources enriched in either α or β globin mRNA. Under all conditions tested, the ratio and amounts of peptides formed in vitro appear to reflect mRNA composition. Globin mRNA from dimethylsulfoxide-stimulated Friend leukemia cells when translated in vitro produced a and p chains in a ratio of 1:1. These peptides are formed in the same ratio in the intact cells. [ABSTRACT FROM AUTHOR]

Liver microsomal UDP-glucuronyltransferase (1-naphthol as substrate) is activated about 10-fotd by treatment of native microsomes in vitro with Triton X-100. Following pretreatment of rats with 3-methylcholanthrene or phenobarbital the specific activities of detergent-activated microsomes were increased by about 300% and 40%, respectively, while the corresponding increases in native microsomes were only about 70% and 10%, respectively .In isolated liver perfused with 0.5 mM 1-naphthol, the rate of glururonidation of naphthol, as determined by the appearance of naphthol glucuronide in the perfusate, bile and liver tissue, was 0.1 μmol × min-1 × g liver-1. This rate of glucuronidation was increased by 70% in rats pretreated with 3-methylcholanthrene and by 140% following phenobarbital treatment, in contrast to the changes observed in microsomes. A UDP-glucuronic acid level of 0.3 μmol/g liver was determined in perfused liver from control rats. This level remained unchanged during perfusion with 0.5 mM 1-naphthoI. In the perfused liver from phenobarbital or 3-methylcholanthrene-treated rats, the UDP-glucuronate level increased to 0.5 μmol/g liver. In control, 3-methylcholanthrene or phenobarbital-treated rats, rates of glucuronidation of 1-naphthol in the perfused liver system correspond best with the UDP-glucuronyltransferase activities determined in native microsomes when the different UDP-glucuronate levels, microsomal protein contents and liver weights are taken into account. The results suggest that the enzyme is mostly latent in the microsomal membrane and that this latency is increased after pretreatment of rats with 3-methylcholanthrene despite the marked enzyme induction. [ABSTRACT FROM AUTHOR]

1. Using sedimentation equilibrium studies, the molecular weight, of pig liver esterase was found to be 163000. Sedimentation velocity experiments yielded a sedimentation coefficient s20,w0 of 7.9 S; the diffusion coefficient was found to be 40.7 µm² · s-1 by ultracentrifugation and 40.3 µm² · s-1 by analytical gel filtration. From these data a molecular weight of 181000 was calculated. By analytical disc electrophoresis a molecular weight of 183000 was estimated. 2. Disc electrophoresis of ox liver esterase revealed three enzymatically active bands. The molecular weight of two of them was 54500 and that of the third 1,87000. Extrapolation of the sedimentation coefficients measured at very low enzyme concentrations to infinite dilution yielded a minimum value of 20,w0 = 4.0 S. Based on gel filtration results a diffusion coefficient of 66.0 µm² · s-1 was calculated. From these data a molecular weight, of 57300 was obtained. Sedimentation equilibrium experiments of the ox enzyme showed a non-linear plot of ln absorbance versus r² indicating heterogeneity with respect to molecular weight. The smallest species present had a molecular weight of 59000. 3. By sedimentation equilibrium experiments in the presence of 6 M guanidine-HCl and 0.1 M 2-mercaptoethanol, the subunit weight of pig liver esterase was found to be 58000. For the maleylated enzyme, values of 52500 (sedimentation equilibrium) and 56500 (from s20,w and D20,w) were obtained. 4. By covalent cross linking of the subunits with dimethyl suberimidate and subsequent polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, three bands of molecular weights 60000, 120000 and 180000 were found. All these results give strong evidence for a three subunit structure of pig liver carboxylesterase. [ABSTRACT FROM AUTHOR]

Two initiation factors of protein synthesis designated here as IF-S1 and IF-S2 have been isolated from rat liver cytoplasm. The factors are separated by gel filtration of a supernatant, obtained at 150000 × on Sephadex G-200 from one another and are purified further by chromatography on hydroxyapatite and DEAE-cellulose columns. Both factors stimulate poly(phenylalanine) synthesis at low Mg2+ concentrations in the presence of elongation factors EF-1 and EF-2. The factor EF-S1 stimulates the binding of AcPhe-tRNA and fMet-tRNA to the small ribosomal subunit. This reaction is dependent on GTP; Β,γ-methylene-guanosine triphosphate can replace GTP. IF-S2 is active in the coupling of 40-S and 60-S subunit to active 80-S initiation complexes. This activity depends on poly(U), on Phe-tRNA and partially on GTP. Similarities and possible analogies between the initiation factors described here with those described by other authors are discussed. [ABSTRACT FROM AUTHOR]

An RNA protein complex containing ribosomal 5-S RNA has been isolated by treatment of rat liver 60-S ribosomal subunits with EDTA. The protein moiety of this complex shows one band in dodecylsulfate gel electrophoresis whose mobility suggests a molecular weight of about 32000. The complex exhibits both GTPase and ATPase activities. The optimal conditions for both enzymic activities have been studied. Kinetic experiments revealed that, GTPase and ATPase both had a Km of 3.7 µM, whereas V values for GTPase and ATPase were 5.6 and 2.2 pmol/min, respectively. Inhibition studies have shown that, two different enzymatic sites are involved in both reactions. [ABSTRACT FROM AUTHOR]

Discusses the effects of cerulenin analogues on the fatty acid synthesis activity and on the elongation systems of leek-seedling microsomes. Assays of fatty acid synthesis; Incubation of microsomes with inhibitors; Effect of the side-chain structure on the inhibition.

RNA editing in trypanosomes is the process of insertion and deletion of U residues at specific sites of mitochondrial transcripts mediated by short guide RNAs (gRNAs) that have a 3′ oligo(U) extension. Here we describe the identification by UV-cross-linking of proteins present in mitochondrial extracts from Crithidia fasciculata with a high affinity for gRNAs, and the characterization of the binding specificity. A 65-kDa protein binds to gRNAs provided they are equipped with a U tail, to post-transcriptionally labelled mitoribosomal 9S and 12S RNAs that also possess a 3′ terminal stretch of U residues, and to free oligo(U) sequences with a minimal length of 23–29 nucleotides. It does not bind to a number of control RNAs, one of which has an internal U stretch of 13 residues. Poly(U), but not poly(C) or total yeast RNA, efficiently competes for binding to gRNA. Proteins of 88 kDa and 30 kDa also bind to gRNAs with a U tail, to miochondrial ribosomal RNAs and to oligo(U). These proteins, however, require longer oligo(U) for binding (> 39 nucleotides) and they also have an affinity for other U-rich RNAs and poly(C). For comparison, part of the analysis was also carried out with a mitochondrial extract from Trypanosoma brucei. In this organism, gRNA-binding proteins of 83 kDa and 64 kDa were found with the same preference for 3′-terminal oligomeric U stretches as the C. fasciculata 65-kDa protein, whereas the binding specificity of a 26-kDa protein resembled that of the C. fasciculata 88-kDa and 30-kDa proteins. The possible involvement of the proteins in the editing process is discussed. [ABSTRACT FROM AUTHOR]

Acridones (9-azaanthracen-10-ones), especially halogen-substituted acridone-carboxylic acids are efficient inhibitors of [14C]ADP binding to the bovine mitochondrial ADP/ATP-carrier protein. 7-Iodo-acridone-4-carboxylic acid displaces [14C]ADP in a competitive manner, which indicates that both compounds share an identical binding site. Upon ultraviolet illumination, 7-azido-4-[1′,2′-³H]isopropyl-acridone binds covalently to the ADP/ATP-carrier protein. By sequencing of its proteolytic fragments, radioactivity was detected in Cys159. This amino acid is located close to Lys162, a target of 2-azido-[α-32P]ADP [Dalbon, P., Brandolin, G., Boulay, F., Hoppe, J. & Vignais, P. V. (1988) Biochemistry 27, 5141–5149]. [ABSTRACT FROM AUTHOR]

A 50-kDa protein has been purified to homogeneity from free mRNP of rabbit reticulocytes. This protein, designated as p50, is present within both free mRNP (approximately 4 mol protein/mol globin mRNA) and polyribosomal mRNP (approximately 2 mol protein/mol globin mRNA). p50 is a basic protein (pI ≈ 9.5) and is characterized by a high glycine content oF approximately 20%. Nitrocellulose-filter analysis has shown that p50 interacts with globin mRNA with an association constant of approximately 2.5 × 108 M-1 (100 mM KAc, 4 °C). Various RNA and polyribonucleotides have the following relative affinity for p50; poly(G) > poly(U) > globin mRNA ≈ 16S rRNA > poly(A) > poly(C). p50 can be phosphorylated both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Hydrolysis of exogenous phosphatidylcholine (PtdCho) to 1,2-diacylglycerol by rat liver plasma membranes was stimulated by oleate concentrations as low as 0.1 mM. In the presence of 75 mM ethanol, the fatty acid also enhanced phosphatidylethanol (PtdEtOH) formation from PtdCho. These effects were also observed with linoleate and arachidonate, but not with saturated fatty acids or detergents, and were minimal in microsomes or mitochondria. Release of [³H]choline from exogenous Ptd[³H]Cho was stimulated by oleate, whereas phosphoryl[³H]choline formation was inhibited. Oleate and other unsaturated, but not saturated, fatty acids also stimulated the conversion of exogenous [14C]phosphatidic acid to [14C]diacylglycerol. These data are consistent with stimulatory effects of these fatty acids on both phospholipase D and phosphatidate phosphohydrolase in liver plasma membranes. The stimulatory effect of guanosine 5'-O-[3-thio]triphosphate) (20 µM) on PtdEtOH and diacylglycerol formation from PtdCho was enhanced by low concentrations of oleate. Phospholipase A2 also stimulated PtdEtOH and diacylglycerol formation from exogenous PtdCho. It is proposed that unsaturated fatty acids may play a physiological role in the regulation of diacylglycerol production through activation of phospholipase D and phosphatidate phosphohydrolase. [ABSTRACT FROM AUTHOR]

1. The component fatty acids of the endogenous phospholipids of microsomal preparations of Mucor, when shaken at 30°C, increased in both chain length and in degree of unsaturation. The net effect was the production of γ-1inolenic acid which, over 2 h, increased from 17% to 32% of total fatty acids present. No further significant changes occurred after this time. 2. The major site for desaturation/elongation reactions was at the sn-2 position ofPtdIns. PtdCho and PtdEtn were not implicated. 3. Of numerous metabolites and cofactors added to the microsomes, only malate could prolong the elongation/desaturation reactions for up to 6 h. This effect was shown to be due to a membrane-associated malic enzyme [malate dehydrogenase (decarboxylating) NADP+] with the NADPH produced being used in fatty-acid desaturation. 4. Kinetic analysis of cytosolic and microsomal enzymes [both in 0.1% (mass/vol.) Chaps] could not distinguish between them. However, when the microsomal malic enzyme was dialysed to remove Chaps, it lost 90% of activity, although the cytosolic malic enzyme lost only 20% activity. 5. The structural analogue of malate, tartronic acid, which is an inhibitor of malic enzyme, also inhibited the malate-induced stimulation of fatty-acyl group desaturation and elongation in the microsomal membranes. 6. It is concluded that two distinct malic enzymes exist, one soluble and one membrane bound, with similar active sites. Both have different roles in the production of NADPH, for lipid metabolism. The former will produce NADPH for fatty-acid biosynthesis whilst the latter produces NADPH for fatty-acid desaturation. [ABSTRACT FROM AUTHOR]

The redox potential of the Rieske [2Fe-2S] cluster of the bc1complex from bovine heart mitochondria was determined by cyclic voltammetry of a water-soluble fragment of the iron/sulfur protein. At the nitric-acid-treated bare glassy-carbon electrode, the fragment gave an immediate and stable quasi- reversible response. The midpoint potential at pH 7.2, 25°C and I of 0.01 M was Em = +312 ± 3 mV. This value corresponds within 20 mV to results of an EPR-monitored dye-mediated redox titration. With increasing ionic strength, the midpoint potential decreased linearly with √I up to I = 2.5 M. From the cathodic-to-anodic peak separation, the heterogeneous rate constant, k°, was calculated to be approximately 2 × 10-3 cm/s at low ionic strength; the rate constant increased with increasing ionic strength. From the temperature dependence of the midpoint potential, the standard reaction entropy was calculated as ΔS° = - 155 J · K-1 · mol-1. The pH dependence of the midpoint potential was followed over pH 5.5–10. Above pH 7, redox-state-dependent pK changes were observed. The slope of the curve, -120 mV/pH above pH 9, indicated two deprotonations of the oxidized protein. The pKa values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pKa,ox of approximately 7.5 could also be observed in the optical spectrum of the oxidized protein. Redox-dependent pK values of the iron/sulfur protein are considered to be essential for semiquinone oxidation at the Qo center of the bc1 complex. [ABSTRACT FROM AUTHOR]

We report that compartmentalisation of the stimulatory guanine-nucleotide-binding regulatory protein (Gs) exists in S49 lymphoma cells. In addition to the previously reported cytosolic form of the α subunit of Gs (G,cα) [Ransnäs, L. A., Svoboda P., Jasper, J. R. & Insel, P. A. (1989) Proc. Natl Acad. Sci. USA 86, 7900–7903], three membrane-bound forms of Gsα were identified through rate- zonal centrifugation in sucrose density gradients, Gsα-specific anti-peptide serum and an adenylate cyclase complementation assay. The sedimentation profile of the first pool of Gsα in the high-density portion of the gradient (1.13–1.16 g/cm3) is identical with that of β-adrenergic-receptor binding, Na/K-ATPase and adenylate cyclase activity, and may therefore be identified as plasma-membrane fragments. The second pool, which was recovered in the middle portion of the gradient (1.09–1.11 g/ cm3), contains a much lower total amount of Gsα and correlates with the endoplasmic reticulum (microsomal) enzyme markers, NADPH-cytochrome-c reductase and glucose-6-phosphatase. The identity of the third pool of Gsα located at the top of the gradient (1.06–1.08 g/cm3), is unknown. The Golgi apparatus marker, UDPgalactose: N-acetylglucosamine glycosyltransferase, was partially recovered in this area; however, this enzyme was also present in the high-density portion of the gradient. Complete absence of specific adenylate cyclase and Na/K-ATPase activity indicates that this low-density (light) membrane form of Gsα is distinct from any plasma-membrane fragments. Furthermore, sedimentation at 100000 × g proves its particulate (membrane) character. The light membrane form of Gsα subunit is functionally active in an adenylate cyclase complementation assay using cyc- membranes devoid of Gsα. Overall, our data indicates that a substantial portion of Gsα is localized in membrane pools other than plasma membrane. [ABSTRACT FROM AUTHOR]