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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]

The cytoplasm of mammalian cells undoubtedly contain a number of different ribonuclease activities, few if any of which have been well characterized. We describe the purification of an exoribonuclease from rabbit reticulocytes which is able to degrade capped RNAs in a 5' to 3' manner. The purified enzyme contains polypeptides of 62 and 58 kDa and may contain an additional polypeptide of 54 kDa. It behaves as a complex of 150 kDa when analyzed by HPLC gel retardation on Superdex 200HR. It is heat-labile, dependent upon divalent cations (Mg2+) for activity, resistant to placental ribonuclease inhibitor, and active over a broad range (10-200 mM) of monovalent cation (K+) concentrations. The enzyme requires a polynucleotide chain of at least 10 bases for activity and cleaves oligonucleotides, up to an octamer long, from the 5' end of an appropriate substrate. In the case of a capped RNA substrate, product analysis by TLC and PAGE indicates that a capped trinucleotide or tetranucleotide or both is produced. Examination of the kinetics of the enzyme with capped and triphosphate-terminated substrates shows that that the cap structure inhibits the action of the enzyme. Furthermore, the data suggest that the rate-limiting step involves the positioning of the enzyme at the 5' end of the substrate and/or the cleavage of the first intemucleotide bond. [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]

A highly active and soluble glucose-6-phosphatase has been purified to near homogeneity from rat liver. Successful purification has been initiated by covalent labeling of the enzyme in native rat liver microsomes with pyridoxal 5′-phosphate and NaBH4, followed by solubilization of the microsomes with Triton X-100, chromatography on phenyl-Sepharose, hydroxyapatite, DEAE-Sephacel and a second chromatography step on hydroxyapatite. The final enzyme preparation obtained was approximately 700-fold purified over the activity of starting microsomes. As judged by SDS/PAGE the purified glucose-6-phosphatase is composed of a single protein with a molecular mass of 35 kDa. The present work demonstrates that the purified glucose-6-phosphatase must be arranged in the native microsomal membrane so that it is accessible to pyridoxal 5′-phosphate from the cytoplasmic side. [ABSTRACT FROM AUTHOR]

A study is presented on the role of F0 and F1 subunits in oligomycin-sensitive H+ conduction and energy transfer reactions of bovine heart mitochondrial F0F1 H+-ATP synthase. Mild treatment with azodicarboxylic acid bis(dimethylamide) (diamide) enhanced oligomycinsensitive H+ conduction in submitochondrial particles containing F1 attached to F0. This effect was associated with stimulation of the ATPase activity, with no effect on its inhibition by oligomycin, and depression of the 32P1 - ATP exchange. The stimulatory effect of diamide on H+ conduction decreased in particles from which F1 subunits were partially removed by urea. The stimulatory effect exerted by diamide in the submitochondrial particles with F1 attached to Fo was directly correlated with a decrease of the original electrophoretic bands of a subunit of F0 (F0I-PVP protein) and the y subunit of F1, with corresponding formation of their cross-linking product. In F0 liposomes, devoid of γ subunit, diamide failed to stimulate H+ conduction and to cause disappearance of F0I-PVP protein, unless purified γ subunit was added back. The addition to Fo liposomes of γ subunit, but not that of α and β subunits, caused per se inhibition of H+ conduction. It is concluded that F0I-PVP and γ subunits are directly involved in the gate of the F0F1 H+ ATP synthase. Data are also presented indicating contribution to the gate of oligomycin-sensitivity conferral protein and of another protein subunit of F0, F6. [ABSTRACT FROM AUTHOR]

Spinach leaf mitochondrial F0F1 ATPase has been purified and is shown to consist of twelve polypeptides. Five of the polypeptides constitute the F1 part of the enzyme. The remaining polypeptides, with molecular masses of 28 kDa, 2.1 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa, belong to the F0 part of the enzyme. This is the first report concerning identification of the subunits of the plant mitochondrial F0. The identification of the components is achieved on the basis of the N-terminal amino acid sequence analysis and Western blot technique using monospecific antibodies against proteins characterized in other sources. The 28-kDa protein crossreacts with antibodies against the subunit of bovine heart ATPase with N-terminal Pro-Val-Pro- which corresponds to subunit Fob of Escherichia coli FQFI. Sequence analysis of the N-terminal 32 amino acids of the 23-kDa protein reveals that this protein is similar to mammalian oligomycin-sensitivity-conferring protein and corresponds to the F1δ subunit of the chloroplast and E. coli ATPases. The 18.5-kDa protein crossreacts with antibodies against subunit 6 of the beef heart F0 and its N-terminal sequence of 14 amino acids shows a high degree of sequence similarity to the conserved regions at N-terminus of the ATPase subunits 6 from different sources. ATPase subunit 6 corresponds to subunit F0a of the E. coli enzyme. The 15-kDa protein and the 10.5-kDa protein crossreact with antibodies against F6 and the endogenous ATPase inhibitor protein of beef heart F0F1-ATPase, respectively. The 9.5-kDa protein is an N,N′-dicyclohexylcarbodiimide-binding protein corresponding to subunit F0c of the E. coli enzyme. The 8.5-kDa protein is of unknown identity. The isolated spinach mitochondrial F0F1 ATPase catalyzes oligomycin-sensitive ATPase activity of 3.5 μmol · mg-1 · min-1. The enzyme catalyzes also hydrolysis of GTP (7.5 μmol · mg-1 · min-1) and ITP (4.4 μmol · mg-1 · min-1). Hydrolysis of ATP was stimulated fivefold in the presence of amphiphilic detergents, however the hydrolysis of other nucleotides could not be stimulated by these agents. These results show that the plant mitochondrial F0F1 ATPase complex differs in composition from the other mitochondrial, chloroplast and bacterial ATPases. The enzyme is, however, more closely related to the yeast mitochondrial ATPase and to the animal mitochondrial ATPase than to the chloroplast enzyme. The plant mitochondrial enzyme, however, exhibits catalytic properties which are characteristic for the chloroplast enzyme. [ABSTRACT FROM AUTHOR]

Cathepsin J has been partially purified [Liao, J. C. R. & Lenney, J. F. (1984) Biochem. Biophys. Res. Commun. 124, 909-916], but its detailed properties are still unknown. In this study, we have purified cathepsin J completely and characterized it. It was purified to homogeneity from the mitochondrial-lysosomal fraction of rat liver by acid treatment, followed by ammonium sulfate precipitation (20-65%), and chromatographies on S-Sepharose, ConA-Sepharose, Affi-gel 501, HPLC DEAE-SPW and HPLC TSK G3000SW. Cathepsin J was found to be a lysosomal high-molecularmass cysteine protease of about 160 kDa consisted of two different subunits. One subunit (α subunit) was a glycoprotein with a molecular mass of 19- 24 kDa which was reduced to 19 kDa by treatment with endoglycosidase F. It has the amino acid sequence LPESWDWRNVR at its N-terminus, which was very similar to those at the N-termini of rat cathepsins B, H and L. The other subunit (β subunit) was a glycoprotein with a molecular mass of 17 kDa, which was reduced to 14 kDa by treatment with endoglycosidase F. It had DTPANETYPDLLG at its N-terminus, which had no similarity with the N-terminal sequences of other cathepsins. Cathepsin J showed strong affinity for synthetic substrates such as N-benzyloxycarbonyl-phenylalanyl-arginine 4-methyl-coumaryl-7-amide and glycyl-arginine β-naphthylamide. It was activated by thiol reagents and chloride ion and was inhibited by cysteine protease inhibitors. However, its initial inhibition constant Ki(initital) by N-(L-3-transcarboxyoxirane-2-carbonyl)-L-leucine-3-methylbutylamide (E-64-c) was 1800 nM, which was 100500 times those of cathepsins B and L. Many properties of cathepsin J were similar to those of cathepsin C (dipeptidylaminopeptidase I) reported as a lysosomal cysteine protease with dipeptidylaminopeptidase activity [McDonald, J. K., Reilly. T. J. & Ellis, S. (1964) Biochem. Biophys. Res. Commun. 16, 135-140]. Furthermore, antiserum against rat liver cathepsin C reacted with rat liver cathepsin J. These findings suggested that cathepsin J is identical with cathepsin C. [ABSTRACT FROM AUTHOR]

1. As detailed below, we have been able to reproduce observations of time-dependent changes in the activity of acyl-CoA:cholesterol acyl transferase (ACAT) in rat liver microsomes, that were suggested to represent evidence of a role for reversible phosphorylation in the regulation of cholesterol ester formation. 2. ACAT in washed rat liver microsomes was inactivated in a time-dependent manner in the presence of Mg2+. However, this effect of Mg2+ appears to be caused by aggregation of microsomal vesicles rather than dephosphorylation, since it could be abolished by rehomogenization, and was mimicked by Ca2+, another agent which causes aggregation. Fluoride did not prevent this effect of Mg2+, but masked it by causing a rapid activation that appeared to be a non-specific effect of increased ionic strength. 3. Under conditions where other proteins were rapidly dephosphorylated, microsomal ACAT activity from rat liver was not affected by incubation with the purified catalytic subunits of protein phosphatases 1, 2A or 2C. Similar results were obtained using protein phosphatases I or 2A on microsomes from a macrophage cell line (J774.2 cells). Incubation of cultured J774.2 cells with a cell permeable inhibitor of these two protein phosphatases, okadaic acid, also had no effect on cholesterol ester formation. 4. A high-speed-centrifugation supernatant fraction (S303) from rat liver activated ACAT in the presence of MgATP. This effect was not abolished by prior heat-treatment of the fraction, and the supernatant fraction could not be replaced by purified AMP-activated protein kinase or a variety of other protein kinases. 5. The results above were obtained using assays involving endogenous cholesterol as the substrate. The MgATP-dependent activation by S303 was reduced or abolished when the assays were carried out in the presence of the detergent Triton WR-1339 plus cholesterol, or detergent alone. 6. These results do not support the idea that ACAT is regulated by reversible phosphorylation. The most likely explanation for the effect of S303 is that it is an artefact caused by changes in the availability of endogenous cholesterol to the enzyme. [ABSTRACT FROM AUTHOR]

C155 and E252 are respiratory-defective mutants of Saccharomyces cerevisiae, previously assigned to complementation groups G37 and G142, respectively. The following evidence suggested that both mutants were likely to have lesions in components of the mitochondrial translational machinery: C155 and E252 display a pleiotropic deficiency in cytochromes α, α3 b; both strains are severly limited in their ability to incorporate radioactive methionine into the mitochondrial translation products and, in addition, display a tendency to loose wild-type mitochondrial DNA. This set of characteristics is commonly found in strains affected in mitochondrial protein synthesis. To identify the biochemical lesions, each mutant was transformed with a wild-type yeast genomic library and clones complemented for the respiratory defect were selected for growth on a non-fermentable substrate. Analysis of the cloned genes revealed that C155 has a mutation in a protein which has high sequence similarity to bacterial elongation factor G and that E252 has a mutation in a protein homologous to bacterial initiation factor 2. Disruption of the chromosomal copy of each gene in a wild-type haploid yeast induced a phenotype analogous to that of the original mutants, but does not affect cell viability. These results indicate that both gene products function exclusively in mitochondrial protein synthesis. Subcloning of the IFM1 gene, coding for the mitochondrial initiation factor, indicates that the amino-terminal 423 residues of the protein are sufficient to promote peptide-chain initiation in vivo. [ABSTRACT FROM AUTHOR]

A homogeneous preparation of casein kinase II has been isolated from the ribosome-free extracts of Rana temporaria oocytes by means of chromatography on heparin-Sepharose, phosphocellulose and mono Q. The enzyme consists of three subunits with molecular mass of 43 kDa, 41 kDa and 29 kDa. The protein kinase was labelled with radioactive iodine and injected back into oocytes. As shown by histoautoradiography the enzyme forms a diffuse ring around the nucleus in the oocyte cytoplasm. A part of casein kinase II is found in informosomes. During oocytes maturation casein kinase II activity increases 7 h after progesterone administration and at the final stages of maturation (20-23 h). Cycloheximide blocks the second augmentation of kinase activity and does not influence the first one. [ABSTRACT FROM AUTHOR]

Stigmatellin and its derivatives represent a third class of Qo site inhibitors besides the hydroxyquinone derivatives and the E-β-methoxyacrylate (MOA) inhibitors [von Jagow and Link (1986) Methods Enzymol. 126, 253-271]. The stigmatellins consist of a chromone ring system connected to an substituted alkenyl side chain. Alterations in the side chain, i.e. saturation of the C=C double bonds, shift of a methoxy group or loss of the methyl groups, specifically affect the binding characteristics. Besides changing the red shift spectrum of reduced cytochrome b566 and the EPR spectrum of the Rieske iron-sulfur cluster, the side chain alterations diminish the binding affinity and the extent of the midpoint potential shift of the iron-sulfur protein. Thus, the side chain of the molecule makes an essential contribution to the binding energy and is not necessary solely for partitioning the molecule into the hydrophobic phase, as assumed so far. [ABSTRACT FROM AUTHOR]

Microsomes containing 12-15 U/mg phosphorylase phosphatase were obtained from skeletal muscle glycogen particles following glycogen digestion and differential centrifugation. The phosphatase associated with the membranes is in an inhibited state; dilution induces dissociation and deinhibition of the enzyme. Phosphatase-depleted membranes can rebind purified phosphatase catalytic subunit but not the complex between catalytic subunit and inhibitor 2. Binding involves a receptor, deduced from saturation phenomena, which is responsible for inhibition of the bound enzyme and which is a protein, since trypsin treatment releases all bound enzyme and prevents rebinding. The phosphatase extracted from the membranes is of type 1 and is a mixture of complexes, the major ones displaying a Mr of 300 000 and 70 000. From these complexes the 35-kDa catalytic subunit can be obtained either by trypsin treatment or by acetone precipitation. Purification to homogeneity involves chromatography on polylysine and FPLC chromatography on Mono Q and Polyanion SI columns. The purified enzyme exhibits a specific activity of 26 800 U/mg (27 900 U/mg after trypsin treatment) and consists of a major protein of 38 kDa (SDS gel electrophoresis). A minor component of 33 kDa, which may represent either a proteolytic product or an isozyme, can be separated. Both 38-kDa and 33-kDa catalytic subunits form a 70-kDa inactive complex with inhibitor 2 and upon incubation of the complexes the catalytic subunit is slowly converted to the inactive conformation which can then be reactivated by either the kinase FΛ or trypsin and Mn2+. Alternatively the inactive catalytic subunit is reactivated by Mn2+ alone once it has been isolated by FPLC chromatography on SI. The observation that the same catalytic subunit is present at various cell locations (namely cytosol, glycogen particles and microsomes), though in different conformations, is in favour of the hypothesis that displacement of the catalytic subunit from one cell site to the other may represent a new mechanism for phosphatase regulation in skeletal muscle. [ABSTRACT FROM AUTHOR]

Experimental evidence is presented showing the existence of an NADH-consuming enzyme in heart mitochondria, in addition to the NADH-ubiquinone oxidase of complex I. In contrast to the latter, the novel enzyme is accessible from the extramitochondrial space. Removal of the outer membranes from intact mitochondria had no influence on exogenous NADH consumption, indicating its location at the cytosolic face of the inner membrane. The enzyme could be solubilized from this membrane and purified by sedimentation through preformed sucrose gradients. Liver mitochondria exhibited no oxidation of external NADH, suggesting that the enzyme is organo-specific. The "exogenous NADH dehydrogenase" of heart mitochondria was found to introduce reducing equivalents into the respiratory chain before the rotenone block, indicating that the enzyme is associated with complex I. The enzyme was also demonstrated to be involved in electron flow from the respiratory chain to exogenous electron acceptors, including NAD+. This permitted us to elicit the existence of an energy-dependent reversed electron flow from complex II to complex I. The redox shuttle established by the novel enzyme could be of significance for the regulation of cellular NADH and the metabolic activation of foreign compounds such as adriamycin. [ABSTRACT FROM AUTHOR]

The import of cytochrome c into mitochondria can be resolved into a number of discrete steps. Here we report on the covalent attachment of heine to apocytochrome c by the enzyme cytochrome c heme lyase in mitochondria from Neurospora crassa. A new method was developed to measure directly the linkage of heine to apocytochrome c. This method is independent of conformational changes in the protein accompanying heine attachment. Tryptic peptides of [35S]cysteine-labelled apocytochrome c, and of enzymatically formed holocytochrome c, were resolved by reverse-phase HPLC. The cysteine-containing peptide to which heine was attached eluted later than the corresponding peptide from apocytochrome c and could be quantified by counting 35S radioactivity as a measure of holocytochrome c formation. Using this procedure, the covalent attachment of heme to apocytochrome c, which is dependent on the enzyme cytochrome c heme lyase, could be measured. Activity required berne (as heroin) and could be reversibly inhibited by the analogue deuterohemin. Holocytochrome c formation was stimulated 5 –10- fold by NADH > NADPH > glutathione and was independent of a potential across the inner mitochondrial membrane. NADH was not required for the binding of apocytochrome c to mitochondria and was not involved in the reduction of the cysteine thiols prior to berne attachment. Holocytochrome c formation was also dependent on a cytosolic factor that was necessary for the heine attaching step of cytochrome c import. The factor was a heat-stable, protease-insensitive, low-molecular-mass component of unknown function. Cytochrome c heme lyase appeared to be a soluble protein located in the mitochondrial intermembrane space and was distinct from the previously identified apocytochrome c binding protein having a similar location. A model is presented in which the covalent attachment of heine by cytochrome c heme lyase also plays an essential role in the import pathway of cytochrome c. [ABSTRACT FROM AUTHOR]

The primary structure of the cytochrome c1 subunit of ubiquinol-cytochrome-c reductase from mitochondria of Neurospora crassa was determined by sequencing the cDNA of a bank cloned in Escherichia coli. From the coding region the sequence of 332 amino acids, corresponding to the molecular mass of 36496 Da, was derived for the precursor protein. The mature protein, the N terminus of which was previously sequenced [Tsugita et al. (1979) in Cytochrome oxidase (King, T. E. et al., eds) pp. 67–77, Elsevier, New York], consists of 262 amino acids and has the molecular mass of 29908 Da including the heme. The sequence contains an N-terminal hydrophilic part of 211 residues, which carries the heme, a hydrophobic stretch of 15 residues, which is assumed to anchor the protein to the membrane, and a C-terminal hydrophilic part of 36 residues. The N-terminal presequence of 70 amino acids contains 9 positive charges but only 1 negative charge and is characterized by a stretch of 20 uncharged residues. [ABSTRACT FROM AUTHOR]

Rat liver 60S ribosomal subunits were irradiated with 254-nm ultraviolet light (1.26 × 104 quanta/subunit), under conditions which preserved their functional activity. Cross-linked RNA-protein complexes were recovered after unreacted proteins had been removed by repeated acetic acid extractions. Proteins linked to the whole rRNA, to 5S RNA and to 28–5.8S RNAs were identified by two-dimensional gel electrophoresis after RNA hydrolysis by ribonucleases T1 and A. Our results showed that numerous proteins interact with rRNAs (at least ten with 28–5.8S RNA, eight with 5S RNA and among these three are common to both) and have been discussed in the light of all the available data. [ABSTRACT FROM AUTHOR]

Poly(A) polymerase has been purified to near homogeneity from the cytoplasm of Artemia salina Cryptobiotic gastrulae by ion-exchange chromatography on DEAE-Sepharose. DEAE-Sepharose CL-6B and phosphocellulose P11, gel filtration on CL-Sepharose 6B, affinity chromatography on poIy(A) Sepharose 4B and ATP-agarose. The enzyme is fully dependent on exogenous oligo(riboadenylic acid) and is free of any nuclease or other enzyme activities. In standard assay conditions the enzyme preparation has aspecific activity of 56 μmol AMP h-1 (mg protein)-1. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis reveals the presence of only two proteins with Mr 94000 and 70000. The Mr-70000 protein has been identified as poly(A) polymerase. The enzymes exclusively activated by Mn2+. Addition of Ca2+, Mg2+, Zn2+ , NH4+, K+ or Na+ inhibits the enzymatic reaction. The activity is specific for ATP and competitive inhibition is observed in the presence of other ribonucleoside 5' triphosphates AMP incorporation is time-dependent and is increased non-linearly with protein and primer concentration. [ABSTRACT FROM AUTHOR]

Administration of estradiol to ovariectomized mature rats for 1 h induces a transient increase in the peptide elongation rate on uterine ribosomes. An inhibitor of the peptide elongation rate, which appears to be regulated by estrogen treatment in vivo, can be extracted from ribosomes of estrogen-deprived rats. The extracted inhibitor or a native inhibitor-ribosome complex affects the rate of the peptide elongation reaction in a uterine cell-free protein synthesis system by inhibiting the ability of selected tRNAs in the assay to be charged with amino acids by their respective aminoacyl-tRNA synthetases. The degree of inhibition of charging of the affected tRNAs ranges from 22% to 78%, the order of inhibition being Pro > Val > Arg > Try > Leu > Glu > Ile > Gly > His > Ser > Lys. Inhibition results from a specific dose-dependent, and presumably reversible, effect of the inhibitor on tRNA, but not on the aminoacyl-tRNA synthetase. The effect does not result from removal of A- C-C terminal nucleotides from the 3′ end of tRNA, but does inhibit the ability of selected tRNAs to bind to the aminoacyl-tRNA synthetases. We propose that regulation of the peptide elongation rate on uterine ribosomes by estradiol occurs through the estradiol-induced inactivation of a ribosome-associated inhibitor, which causes a reversible alteration to selected tRNAs. The modified tRNAs are unable to bind to their respective aminoacyl- tRNA synthetase to become charged with an amino acid thus causing the availability of selected aminoacyl- tRNAs to become rate-limiting in the sequential elongation of peptides. [ABSTRACT FROM AUTHOR]

70S ribosomes and 30S and 50S ribosomal subunits from Escherichia coli were modified under non-denaturing conditions with the chemical reagent dimethylsulfate. The ribosomal 23S and 16S RNAs were isolated after the reaction and the last 200 nucleotides from the 3' ends were analyzed for differences in the chemical modification. A number of accessibility changes could be detected for 23S and 16S RNA when 70S ribosomes as opposed to the isolated subunits were modified. In addition to a number of sites which were protected from modification several guanosines showed enhanced reactivities, indicating conformational changes in the ribosomal RNA structures when 30S and 50S subunits associate to a 70S particle. Most of the accessibility changes can be localized in double- helical regions within the secondary structures of the two RNAs. The results confirm the importance of the ribosomal RNAs for ribosomal functions and help to define the RNA domains which constitute the subunit interface of E. coli ribosomes. [ABSTRACT FROM AUTHOR]

To investigate the mechanism for the transfer of ribosomal proteins from cytoplasm into nuclei and nucleoli, the uptake of ³H-labelled ribosomal proteins by the isolated rat liver nuclei was investigated in the system containing ATP, GTP, CTP, UTP, and an energy-regenerating system. 1. In the presence of cell sap, the transfer became temperature-dependent. The concentration of ribosomal proteins was also very important for their specific transfer and 10-15 µg of ribosomal proteins/ml were suitable in the presence of 107 nuclei. 2. Removal of one of the four nucleoside triphosphates from the complete system containing cell sap, especially that of CTP or UTP, resulted in a marked decrease of the uptake. A low concentration of actinomycin D inhibited significantly the transfer of ribosomal proteins, while α-amanitin to a less extent. The results indicate that the uptake of ribosomal proteins by liver nuclei is largely dependent on RNA synthesis especially rRNA synthesis. 3. The transfer was enhanced to some extent by ATP alone. Other nucleoside triphosphates were less effective. Non-hydrolyzable ATP analogues, adenosine 5'-[β-γ-methylene]triphosphate and adenosine 5'-[α,β-methylene]-triphosphate were only slightly stimulative. Although ATP enhanced the transfer into the extranucleolar fraction to some extent, the maximal transfer not only into nucleoli but also into the extranucleolar fraction was dependent on the rRNA synthesis. 4. Sedimentation analyses of the nucleolar fraction of rat liver nuclei incubated with [³H]GTP or ³H-labelled ribosomal proteins, showed that small but distinct amounts of the both labeled compounds were incorporated into 60S preribosomal particles although most of them were found in ribonucleoprotein particles below 30S which were previously shown to be degraded products containing larger rRNA precursors [Tsurugi, K., Morita, T., and Ogata, K. (1972) Eur. J. Biochem. 25, 117-128]. [ABSTRACT FROM AUTHOR]

ATP-dependent Ca2+ uptake by brain microsomes was classified into two fractions according to the sensitivity to saponin. Properties of each fraction of Ca2+ uptake were examined and compared with those of inside-out membrane vesicles of erythrocyte and cardiac sarcoplasmic reticulum. The concentration of saponin for 50% inhibition (IC50) of major saponin-sensitive Ca2+ uptake was 11 µg/ml, and this uptake was enhanced by calmodulin. The minosr saponin-insensitive Ca2+ uptake fraction (IC50; 90 µg/ml) was not affected by calmodulin but was enhanced by oxalate or 0.1 M KCl. The IC50 of saponin for inside-out membrane vesicles of erythrocyte and cardiac sarcoplasmic reticulum was 11.3 and 114.8 µg/ml, respectively. A characteristic ring-like saponin-cholesterol micellar structure was observed electron microscopically in most membrane vesicles of brain microsomes and erythrocyte membrane vesicles but not in the cardiac sarcoplasmic reticulum. These observations indicate that saponin-sensitive and insensitive Ca2+ uptake was derived from plasma membranes and endoplasmic reticulum, respectively. Saponin proved useful for distinguishing the Ca2+ transport activity of plasma membrane from the Ca2+ uptake of other cellular organelles in the membrane preparations. [ABSTRACT FROM AUTHOR]

The activation of docosahexaenoic acid by rat brain microsomes was studied using an assay method based on the extraction of unreacted [1-14C]docosahexanoic acid and the insolubility of [1-14C]docosahexaenoyl-CoA in heptane. This reaction showed a requirement for ATP, CoA, and MgCl2 and exhibited optimal activity at pH 8.0 in the presence of dithiothreitol and when incubated at 45 °C. The apparent Km values for ATP (185 µM), CoA (4.88 µM), MgCl2 (555 µM) and [1-14C]docosahexaenoic acid (26 µM) were determined. The presence of bovine serum albumin or Triton X-100 in the incubation medium caused a significant decrease in the Km and Vm values for [1-14C]docosahexaenoic acid. The enzyme was labile at 45 °C (t1/2 : 3.3 min) and 37 °C (t1/2 : 26..5 min) and lost 36% of its activity after freezing and thawing. The transition temperature (Tc) obtained from Arrhenius plot was 27 °C with the activation energies of 74 kJ/mol between 0 °C and 27 °C and 30 kJ/mol between 27 °C and 45 °C. [1-14C]Palmitic acid activation in rat brain and liver microsomes showed apparent Km values of 25 µM and 29 µM respectively, with V values of 13 and 46 nmol x min-1. The presence of Triton X-100 (0.05%) in the incubation medium enhanced the V value of the liver enzyme fourfold without affecting the Km value. Brain palmitoyl-CoA synthetase, on the other hand, showed a decreased Km value in the presence of Triton X-100 with unchanged V. The Tc obtained were 25 °C and 28 °C for brain and liver enzyme with an apparent activation energy of 109 and 24 kJ/mol below and above Tc for brain enzyme and 86 and 3.3 kJ/mol for liver enzyme. The similar results obtained for the activation of docosahexaenoate and palmitate in brain microsomes suggest the possible existence of a single long-chain acyl-CoA synthetase. The differences observed in the activation of palmitate between brain and liver microsomes may be due to organ differences. Fatty acid competition studies showed a greater inhibition of labeled docosahexaenoic and palmitic acid activation in the presence of unlabeled unsaturated fatty acids. The Ki values for unlabeled docosahexaenoate and arachidonate were 38 µM and 19 µM respectively for the activation of [1-14C]docosahexaenoate. In contrast, the activation of unlabeled saturated fatty acids for activation of labeled docosahexaenoate is much less than that for activation of labeled palmitate. The preferential inhibition by unsaturated fatty acids, especially the polyunsaturated fatty acids, of the activation of docosahexaenoic and palmitic acids, as compared to inhibition by saturated fatty acids may be the results of the higher affinity or ready accessibility of these more unsaturated substrates to the membrane-bound enzyme. [ABSTRACT FROM AUTHOR]

Hyperproduction of phosphate-binding protein, PhoS, in strains carrying a multicopy plasmic containing the phoS gene, resulted in saturation of export sites. As a consequence, pre-PhoS was accumulated both in the inner membrane and in the cytoplasm. This was evidenced both in electron-microscopy and after cell fractionation. Only the membrane-associated precursor could be matured and exported. The signal sequence of the cytoplasmic pre-PhoS was slowly degraded. It was first cleaved about in its middle and then completely removed. Electron microscope studies demonstrated that the cytoplasmic pre-PhoS cannot be exported post-translationally. [ABSTRACT FROM AUTHOR]

The basic assumption in this paper is that the secondary structure era 5-S ribosomal RNA cannot be represented by a single model. We propose that the molecule can adopt, at least within the ribosome, a series of slightly different structures of nearly equal stability. The different structures arise from the existence of ambiguous base-pairing opportunities in bulged helices and the adjacent interior loops. In cubacterial 5 S RNAs there is one such an area, in eukaryotic 5-S RNAs two such areas that can give rise to structural switches. We explain how a change in secondary structure in these areas may influence the relative orientation of the surrounding helices, in other words how bulges and interior loops may serve as articulations and give rise to a flexible tertiary structure. [ABSTRACT FROM AUTHOR]

The puromycin-induced stimulation of [3H]dihydrorosaramicin binding is due to a twofold increase in affinity of the macrolide antibiotic, with no change in the number of binding sites. Conversely, the binding of [3H]puromycin (A site) is stimulated by rosaramicin. The synergistic effect observed between the two antibiotics can be explained by a conformational change with positive effect, which occurs at the level of their binding sites. Various effectors of [3H]dihydrorosaramicin binding have been tested. Adenosine and dimethyladenosine stimulate the binding; phenylalanine, uridine and gougerotin (A site) have no effect whereas AMP, ADP, ATP, GTP, puromycin 5″-phosphate and lincomycin (P site) are inhibitors. These results point to the importance of the purine moiety in the stimulatory effect and of the phosphate function in reversing this effect. It is concluded that rosaramicin binds to the ribosomal P site and that the synergism observed between rosaramicin and puromycin may be related to interactions between the A and P sites. [ABSTRACT FROM AUTHOR]

1. The isolated and liposome-reconstituted mitochondrial phosphate carrier exhibits a sigmoidal inhibition curve by mersalyl, similar to that found with intact mitochondria. In contrast a hyperbolic inhibition curve is found (a) by titration of the soluble carrier with mersalyl before reconstitution in liposomes and (b) by titration of the reconstituted carrier with mersalyl after successively pretreatment of the mitochondria with low, non-inhibitory concentrations of mersalyl, excess N-ethylmaleimide and dithiothreitol. 2. The inhibition of the reconstituted, but not of the soluble, phosphate carrier by mersalyl can be reversed by dithiothreitol. 3. Cupric di(1,10-phenanthroline) inhibits the soluble but not the reconstituted phosphate carrier. The inhibited phosphate carrier can be reactivated by dithiothreitol in the soluble state but not after reconstitution in liposomes. 4. The data support the previously suggested model of the phosphate carrier, assuming a dinner of two identical subunits for the active unit. [ABSTRACT FROM AUTHOR]

Decrease in the rate of cyclic electron transport (JE) measured from the absorbance changes associated with reaction centre bacteriochlorophyll led to a less than proportionate decrease in the membrane potential (&Lamda;ψ) measured by electrochromism. In principle this result can be explained either by a &Lamda;ψ-dependent slip in the H+/e- coupling ration (nE) or by a &Lamda;ψ-dependent chance in the membrane ionic conductance. Simultaneous measurement of the membrane ionic current (JDIS/&Lamda;ψ) did not reveal any significant changes in the H+/e- ratio (JDIS/JE) showed that conductance changes (JDIS/&Lamda;ψ) account quantitatively for the curved dependance of &Lamda;ψ on JE. Simultaneous recordings of JDIS and the extravesicular pH from cresol-red absorbance changes, suggest that protons are the main current-carrying species across the chromatophore membrane at high values of &Lamda;%psi; in the presence and absence of Fo-ATPase inhibitor. At reduced &Lamda;ψ the flux of other ions outweighs the hydrogen ion current. [ABSTRACT FROM AUTHOR]

CD spectroscopy reveals that mitochondrial malate dehydrogenase in 3M guanidinium chloride shows little residual secondary structure. Refolding of the denatured protein by dilution with buffer of pH 7.5 does not restore the CD spectrum of the native enzyme. A partially folded intermediate, possessing 25% of the α-helix content of the native enzyme, is formed upon dilution. The partially folded intermediate binds the extrinsic probe 1-anilinonphtalene-8-sulfonate, and the increase in fluorescene (tenfold) is accompanied by a blue shift in the band position of the emission spectrum. Partially folded malate dehydrogenae is devoid of catalytic activity. In vitro refolding of the denatured protein takes place in the presence of dithiotreitol and thioredioxin. In the presence of micromolar concentrations of thioredoxin, a recovery of approximately 70% of the catalytic activity was observed. Emission-anisotropy titrations of oxidized thioredoxin, tagged with a fluorescent probe, revealed that the oxidoreductase recognizes partially folded intermediates of malate dehydrogenase with a dissociation constant pf 6 µM. Moreover, a covalently linked complex formed by thioredoxin and monomeric malate dehydrogenase was detected by SDS/PAGE. A general mechanism is postulated for the reactivation of denatured proteins by thioredoxin. [ABSTRACT FROM AUTHOR]

The nuclear ABC1 gene was isolated as a multicopy suppressor of a cytochrome b mRNA translation defect. Its inactivation leads to a respiratory deficiency suggesting a block in the bc1 segment of the respiratory chain [Bousquet, I., Dujardin, G. & Slonimski, P.P. (1991) EMBO J. 10, 2023-2031]. In the present study, we established that deleting the ABC1 chromosomal gene from Saccharomyces cerevisiae does not prevent the assembly of the bc1 complex (complex III) but markedly impairs the kinetics of its high-potential electron transfer pathway occurring on the positive, outer, side of the membrane, which results in reduced activity of the bc1 complex. In addition, the activity of complex II and its cytochrome b560 decrease drastically and complex IV activity is halved. It is also observed that the binding of the quinol to the bc1 complex ubiquinol oxidation site is affected and that adding exogenous quinones partially compensats for the respiratory deficiency in vitro, although the quinone content of mutant and wild-type mitochondria are similar. Lastly, complexes II, III and IV are found to be thermosensitive and the bc1 complex exhibits greater sensitivity than the wild-type strain to center N and P inhibitors, suggesting that the three multisubunit complexes have undergone structural modifications. The data suggest that the ABC1 gene product acts as a chaperone-like protein essential for the proper conformation and efficient functioning of the bc1 complex and the effects of the Abc1 protein on the complexes II and IV might result from interactions with the modified bc1 complex. [ABSTRACT FROM AUTHOR]

The increased proton permeability induced by the addition of a synthetic proton translocator to non-respiring hamster brown-fat mitochondria is unaffected by purine nucleotide addition. In contrast the permeability induced by fatty acids is inhibited by nucleotide, indicating that fatty acids act at the 32000-Mr uncoupling protein. Fatty acids lower the affinity of nucleotide binding to the 32000-Mr protein, but not sufficiently to explain their uncoupling ation. The sensitivity of the fatty acid modulation of permeability is dependent on chain length, extent of unsaturation and pH. There is a requirement for an unesterified carboxyl group. In respiring mitochondria fatty acids act in the presence of nucleotide by lowering the ‘break-point’ potential at which the conductance of the 32000-Mr protein increases. Fatty acids have no effect on the chloride uniport activity of the 32000-Mr protein, but decouple the interference between chloride and protons when the simultaneous transport of both ions is attempted. [ABSTRACT FROM AUTHOR]

The enzyme alkyiglycerol monooxygenase was solubilized with 2 % of Triton X100 and partially purified approximately to 83-fold with a 36 % yield after a procedure which included 6-aminohexyl-Sepharose and DEAE- cellulose column chromatography, followed by a sucrose density gradient centrifugation. The molecular weight of the native form was estimated to be approximately 400000 as judged by Sepharose 6B column chromatography in the presence of Triton X-100. The enzyme had a pH optimum near 8.5 and a Km of 0.66 mM for 1O-hexadecylglyceroI. The microsomal alkyiglycerol monooxygenase was stimulated by Triton X-100, but did not affect kinetically the initial velocity except to maintain it for a much longer period of time. The purified enzyme required absolutely both molecular oxygen and reduced pteridine as an electron donor. Furthermore, reduced glutathione and phospholipids were necessary for expression of full enzyme activity. N- Ethylmaleimidc and p-chloromercuribenzoate significantly inhibited the enzyme activity, suggesting that alkylglycerol monooxygenase is a -SH enzyme. [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 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 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 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]