Biogenesis of mitochondria 48

Summary Commercial preparations of mikamycin have been shown to act as both inhibitors of mitochondrial protein synthesis and respiration. These preparations are shown to consist of two major streptogramin components (mikamycin A and mikamycin B) and a number of minor components. The major streptogramin components which inhibit mitochondrial protein synthesis in vitro are without effect in vivo due to whole cell impermeability to these compounds.A minor antimycin A-like component is the active compound in mikamycin preparations which inhibits growth of yeast cells on ethanol. The site of this inhibition is at the level of respiratory Complex III.The mitochondrial [mik 1-r] mutation confers resistance to this minor growth inhibitory component and cross resistance to antimycin A. For clarity the designation mik 1 has therefore been renamed ana1 to denote the mitochondrial determinant conferring resistance to antimycin A. Genetic and physical mapping studies localise the ana1 determinant in the region of mitochondrial DNA specifying cytochrome b. It is proposed that the ana1 locus is part of a gene specifying a membrane component of Complex III.     

Adenylate kinase and GDP-kinase activity of rod outer segments in the frog retina. Possible functional role of the T beta subunit of transducin

Pure frog retina rod outer segments (ROS) preparations (A280/A500 = 2,1-2,3) catalyze the synthesis of ATP from ADP in the presence of Mg2+. Adenylate kinase (AK) (ATP:AMP phosphotransferase, EC 2.7.4.3) specific activities for ROS preparations are within the range 2-4 mumole per hour for mg protein. The enzymatic activity of investigated preparations is due to intact, but not destroyed ROS. The component which possesses AK is found in water-soluble, but not in membranous ROS fractions and seems to be a part of the predominant ROS plasma protein--GTP-binding complex of transducin. It has been shown, that this component is the T beta subunit of transducin and its enzymatic activity is controlled by other subunits of the transducin complex. The obtained results indicate that GDP kinase (ATP:GDP phosphotransferase, EC 2.7.4.6) activity of transducin depends on the work of both of T beta and T alpha subunits. It has been shown that in the ROS preparations synthesis of the ATP from ADP and GDP phosphorylation are stimulated by a lowering of Ca2+ concentration (less than 10(-5)-10(-7) M). T beta component is suggested to play the role of phosphotransferase which phosphorylates GDP associated with the T alpha subunits and it leads to formation of a complex T alpha X GTP which is an activator of vertebrate retina ROS phosphodiesterase.     

Regulation of Prenatal and Postnatal Protein Synthesis in Mouse Brain

Abstract: Regulation of protein synthesis during prenatal and postnatal brain development was examined using postmitochondrial supernatant (PMS) fractions and isolated ribosome-pH 5 enzyme systems from fetal, neonatal, and adult neural tissue. The rate of polyuridylic acid (poly-U)-dependent protein synthetic activity was inversely proportional to the endogenous rate of protein synthesis in either the PMS fractions or ribosomal preparations. A careful analysis of the kinetics of the poly-U-dependent polypeptide synthesis revealed that there was a lag in the time at which certain of the PMS preparations could begin to utilize the poly-U template as sole source of mRNA. The lag period was dependent upon the developmental age of the neural tissue used and the Mg²⁺ concentration of the protein synthesis reaction. Since previous work reported that the observed developmental decrease in the rate of polypeptide synthesis utilizing a poly-U template could not be measured in a purified ribosomal-pH 5 enzyme system, ribosomes were obtained by several isolation techniques to determine if the purification procedure might have affected the ribosomes in some manner by removing a specific protein(s) involved in ribosome-cytosol interactions. At 6 mM-Mg²⁺ the rate of poly-U-dependent protein synthesis was inversely proportional to the rate of endogenous synthesis and depended upon the method used to isolate the ribosomes: microsomes ∼Triton X-100-treated < DOC-treated < KCl-treated. However, there was no age-dependent effect with any of the ribosomal preparations. The data suggest that there is a developmental modulating effect of ribosomal activity in PMS preparations which is not found in association with the isolated ribosome-pH 5 enzyme protein synthesizing system.     

Demethylation of ribosomal RNA by hepatocyte microsomal preparations

Previous work has shown that hypomethylation of rRNA is an important control for protein synthesis in rat hepatocytes, and that the net hypomethylation appears to arise from cytoplasmic events. Here we show that demethylation of rRNA is catalyzed by microsomal preparations. The rRNA demethylation is dependent upon NADPH and is almost completely inhibited by carbon monoxide. Demethylation appears to increase following galactosamine intoxication, a hepatotoxin which induces hypomethylation of rRNA and inhibition of protein synthesis.     

Purification of the gene 43, 44, 45, and 62 proteins of the bacteriophage T4 DNA replication apparatus.

A procedure has been developed which allows the T4 bacteriophage proteins corresponding to the products of genes 43, 44, 45, and 62 to be purified to near homogeneity from a single T4-infected cell lysate (greater than 90% single species as judged by sodium dodecyl sulfate polyacrylamide elctrophoresis). In these preparations, the major problem of removing all contaminating nucleases has been overcome. Each of the above proteins is known from genetic analysis to be essential for phage DNA replication. The protein product of gene 43 is T4 DNA polymerase, and its recovery can be monitored using a standard DNA polymerase assay. The other three gene products have been designated as "polymerase accessory proteins," since they directly enhance polymerase function on both single- and double-stranded DNA templates. Their activities were monitored by an "in vitro complementation assay," which measures the stimulation of DNA synthesis observed in a concentrated lysate of T4 mutant-infected Escherichia coli cells when the missing T4 wild type protein is added. Starting from 300 g of infected cell paste, we obtained 9.3 mg of gene 43 protein, 21 mg of gene 45 protein, and 70 mg of a tight complex made up of 44 and 62 proteins; final yields were estimated at 30%, 14%, and 28%, respectively, of the initial activity present in the lysate. When the above purified proteins are incubated with preparations of two other T4 DNA replication proteins (gene 41 and gene 32 proteins) plus deoxyribonucleoside and ribonucleoside triphosphates, extensive DNA synthesis occurs on both single- and double-stranded DNA templates. As reported elsewhere, this synthesis mimicks that catalyzed by the T4 DNA replication apparatus in vivo.     

The relationship between protein synthesis and heat shock proteins levels in rabbit reticulocyte lysates.

Besides heme deficiency, protein synthesis in rabbit reticulocyte lysates becomes inhibited upon exposure to a variety of agents that mimic conditions which induce the heat shock response in cells. This inhibition has been demonstrated to be due primarily to the activation of the heme-regulated eIF-2 alpha kinase (HRI) which causes an arrest in the initiation of translation. In this report, the sensitivity of protein synthesis in hemin-supplemented lysates to inhibition by Hg2+, GSSG, methylene blue, and heat shock was examined in six different reticulocyte lysate preparations. The extent to which translation was inhibited in response to Hg2+, GSSG, methylene blue, and heat shock correlated inversely with the relative levels of the 70-kDa heat shock proteins (hsp 70) and a 56-kDa protein (p56) present in the lysates determined by Western blotting. The ability of hemin to restore protein synthesis upon addition to heme-deficient lysates was also examined. While the restoration of protein synthesis correlated roughly with the levels of hsp 90 present, the results also suggest that the heme regulation of HRI probably involves the interaction of HRI with several factors present in the lysate besides hsp 90. A comparison of two lysate preparations, which had a 2-fold difference in their protein synthesis rates, indicated that the slower translational rate of the one lysate could be accounted for by its low level of constitutive eIF-2 alpha phosphorylation, with its accompanying decrease in the eIF-2B activity and lower level of polyribosome loading. The present study supports the notion that the previously demonstrated interaction of HRI with hsp 90, hsp 70, and p56 in reticulocyte lysates may play a direct role in regulating HRI activation or activity. We hypothesize that the competition of denatured protein and HRI for the binding of hsp 70 may be a molecular signal that triggers the activation of HRI in reticulocyte lysates in response to stress. Possible functions for p56 in the regulation of HRI activity are also discussed.     

Interaction of dnaA46 protein with a stimulatory protein in replication from the Escherichia coli chromosomal origin

Highly purified preparations of dnaA46 protein have permitted its biochemical characterization in comparison with the activities of wild type dnaA protein. We have determined that dnaA46 protein was reduced in its ability to bind to DNA fragments containing oriC. This mutant protein was also defective in binding ATP and was inactive for replication of oriC-containing plasmids in purified enzyme systems. In contrast, dnaA46 protein was active for oriC plasmid replication when added to reactions containing a crude enzyme fraction deficient in dnaA protein. One or more proteins have been identified which appear to interact with dnaA46 protein prior to DNA synthesis. These studies suggest that this interaction is thermolabile. Stimulation of dnaA46 protein activity resulted in a reduction of the prolonged lag prior to DNA synthesis.     

Factors controlling amino acid incorporation by ribosomes from Krebs II mouse ascites-tumour cells

1. A ribosome-cell sap system capable of supporting the incorporation of (14)C-labelled amino acids into protein has been prepared from Krebs II mouse ascites-tumour cells. The requirements of this system for optimum activity and response to added messenger RNA have been investigated. One such system has been obtained for which amino acid incorporation is almost wholly dependent on the addition of suitable messenger RNA. 2. Ribosomes of widely different but predictable activities in the cell-free system have been prepared from Krebs cells pretreated in a variety of ways. The factors in the pretreatment of the cells responsible for these differences have been investigated. 3. The structural and functional properties of these different ribosome preparations and their response to exogenous messenger RNA have been examined and are discussed in the light of modern concepts of the control of protein synthesis.     

Ecdysteroid synthesis by the ring gland of Drosophila melanogaster during late-larval,prepupal and pupal development

Radioimmunoassay has been used to determine the characteristics of ecdysteroid synthesis by ring glands and brain-ring gland preparations from late 3rd-instar larvae of Drosophila melanogaster cultured in vitro. The rate of synthesis and secretion is linear for at least 4 hr in culture. Using a 4-hr culture period, variation in the rate of ecdysteroid synthesis by brain-ring gland preparations during larval, prepupal and pupal development has been examined. The rate of synthesis and secretion is highest in late 3rd-instar larvae and decreases after puparium formation. During pupal development, at a time when the endogenous ecdysteroid titre is again increasing, the rate of ecdysteroid synthesis by brain-ring gland preparations remains low and is only 10% of that prior to puparium formation. It is, therefore, likely that the ring gland is not a major source of ecdysteroids during this period.     

Nuclease activity in preparations of creatine phosphokinase: effect on mRNA stability

Creatine phosphokinase is used to generate ATP with creatine phosphate for $\text{in vitro}$ protein synthesis. Some preparations of this enzyme contain nuclease activity, which can be demonstrated by a sensitive assay of the cleavage of poly(A)-containing RNA. These preparations of creatine phosphokinase support protein synthesis poorly in a cell-free system prepared from HeLa cells. Poly(A)-containing RNA is quite stable in this cell-free system when the phosphorylated sugar fructose 1,6-bisphosphate with no addition of enzyme is used to generate ATP.     

Cell-free translation of polyribosomes from detached pumpkin cotyledons: Effects of starvation and cytokinin

The effects of 6-benzylaminopurine (BAP, 5.10^?5M) treatment of pumpkin cotyledons and their starvation after excision upon polysome/monosome ratio and translational capacity of polysomes in cell-free system were studied. It has been found that starvation causes a progressive polysome degradation. Polysome translation in a wheat germ cell-free proteinsynthesizing system reveals that the translation capacity of polysome preparations decreases with the time after cotyledon excision much more sharply than polysome/monosome ratio. This indicates the starvation damage in elongation steps of protein synthesis. The decrease of postribosomal supernatants activity in the system of poly(U)-directed polyphenylalanine synthesis confirms this conclusion. BAP treatment brings about a very rapid monosome mobilization into polysomes and activation of cell-free translation of ribosome preparations which is however closely parallel to the polysome percentage in them. That means that during this initial period of BAP action only protein synthesis initiation is under BAP control. The experiments with aurintricarboxylic acid (ATA) support this idea.     

Regulation of protein synthesis in rabbit reticulocyte lysates: the hemeregulated protein kinase (HRI) and double stranded RNA induced protein kinase (dRI) phosphorylate the same site(s) on initiation factor eIF-2

Double stranded RNA (dsRNA) induced inhibitor (dRI) has been partially purified (80–100 fold). The dRI inhibits protein synthesis in rabbit reticulocyte lysates; the inhibition is overcome by the initiation factor eIF-2. The dRI preparations phosphorylate the 38,000-dalton subunit of eIF-2. Heme-deficiency in rabbit reticulocyte lysates also induces a translational inhibitor (HRI) which inhibits protein chain initiation by specifically phosphorylating the 38,000-dalton subunit of eIF-2. To establish correlation of the mechanism of inhibition of protein synthesis by dRI and HRI, the phosphopeptide patterns of eIF-2 phosphorylated by using HRI or dRI are compared. Treatment with various proteases of eIF-2 phosphorylated by HRI or dRI yield identical phosphopeptide patterns. This finding suggests that HRI and dRI phosphorylate the same site(s) of the 38,000-dalton subunit of eIF-2 and raises the possibility that dRI may also inhibit protein chain initiation by the mechanism similar to that of HRI.     

Activation of the alarm response of Escherichia by antibiotics

The data on the effect of antibiotics suppressing the synthesis of protein on the activation of the SOS-system are presented. The action of tetracycline, chloramphenicol rifampicin and nalidixic acid, a well-known activator of SOS-response, has been studied. The short-term action of inhibitory concentrations and the prolonged action of subinhibitory concentrations of these preparations on the activity of genes rec A and sul A and the induction of the synthesis of phage lambda have been considered. Chloramphenicol and tetracycline, as well as nalidixic acid, have been shown to be capable of activating genes rec A, sul A and synthesis of the phage. The induction of SOS-response has been found to be more pronounced in the short-term action of inhibitory concentrations of antibiotics on bacteria than in the prolonged subinhibitory concentrations.     

Inhibition of protein synthesis in reticulocyte lysates by a double-stranded RNA component in HeLa mRNA

Double-stranded RNA (dsRNA) inhibits protein synthesis initiation in rabbit reticulocyte lysates by the activation of a latent dsRNA-dependent cAMP-independent protein kinase which phosphorylates the α-subunit of the eukaryotic initiation factor eIF-2. In this study, we describe a dsRNA-like component which is present in preparations of HeLa mRNA (poly A⁺) isolated from total cytoplasmic RNA. The inhibitory species in the HeLa cytoplasmic mRNA was detected by (a) its ability to inhibit protein synthesis with biphasic kinetics in reticulocyte lysates translating endogenous globin mRNA, and (b) by the inefficient translation of HeLa cytoplasmic mRNA in a nuclease-treated mRNA-dependent reticulocyte lysate. The inhibitory component was characterized as dsRNA by several criteria including (i) the ability to activate the lysate dsRNA-dependent eIF-2α kinase (dsI); (ii) the prevention of both dsI activation and inhibition of protein synthesis by high levels of dsRNA or cAMP; (iii) the reversal of inhibition by eIF-2; and (iv) the inability to inhibit protein synthesis in wheat germ extracts which lack latent dsI. By the same criteria, the putative dsRNA component(s) appears to be absent from preparations of HeLa mRNA isolated exclusively from polyribosomes.     

Regulation of lipoprotein lipase synthesis by recombinant tumor necrosis factor--the primary regulatory role of the hormone in 3T3-L1 adipocytes

Tumor necrosis factor (TNF), a protein homologous to cachectin, has been implicated in mediating cachexia. This effect at least in part has been suggested to occur through the influence of the hormone on adipose tissue metabolism. Using fully differentiated 3T3-L1 adipocytes as a model system, we have been investigating the effects of recombinant TNF (rTNF) on key features of adipocyte metabolism. Exposure of fully differentiated 3T3-L1 adipocytes to recombinant tumor necrosis factor resulted in a dose and time-dependent suppression of the activity of lipoprotein lipase. The loss in activity results from an effect on the synthesis of the enzyme, as determined by a decreased incorporation of [35S]methionine into immunoprecipitable lipoprotein lipase. No effect of rTNF on the half-life of the enzyme was observed. General protein synthesis, as judged by [35S]methionine incorporation into acid-insoluble protein, was minimally affected by exposure of the cells to rTNF; this was further confirmed by sodium dodecyl sulfate-polyacrylamide gel analysis of total cellular protein. As opposed to our previously reported results with crude preparations of TNF, no effect on either the ability of the adipocytes to synthesize and store or mobilize triacylglycerol was observed. Our results are consistent with the hypothesis that other hormones present in crude preparations of TNF acting either alone or synergistically with TNF play a major role in the further metabolic derangements associated with adipose tissue during cachexia.     

Macromolecule synthesis in yeast spheroplasts

Conditions have been established for the preparation of spheroplasts of Saccharomyces cerevisiae which are able to increase their net content of protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA), several-fold upon incubation in a medium stabilized with 1 m sorbitol. The rate of RNA and protein synthesis in the spheroplasts is nearly the same as that occurring in whole cells incubated under the same conditions; DNA synthesis occurs at about half the whole cell rate. The spheroplasts synthesize transfer RNA and ribosomal RNA. The newly synthesized ribosomal RNA is incorporated into ribosomes and polysomes. The polysomes are the site of protein synthesis in these spheroplasts. Greater than 90% of the total RNA can be solubilized by treatment of the spheroplasts with sodium dodecyl sulfate or sodium deoxycholate. These spheroplast preparations appear to be a useful subject for the study of RNA metabolism in yeast.     

The Influence of Axis Removal on Protein Metabolism in Cotyledons of Pisum sativum L

The protein metabolism of cotyledons attached to the embryonic axis has been compared with that in cotyledons removed from the axis at the initiation of a 6-day imbibition. Total protein declined in the attached but not in the detached cotyledons. Concurrent with the decline in protein level in the intact cotyledons there was an increased capacity to incorporate exogenously supplied leucine into protein. In contrast, detached cotyledons showed a restricted capacity for protein synthesis. It was demonstrated that ribosomal preparations from cotyledons of intact seedlings contained an increasing proportion of polyribosomes as germination progressed and such ribosomes were active in in vitro amino acid incorporation. Ribosomal preparations from detached cotyledons contained few polyribosomes and had a restricted capacity to incorporate amino acids in vitro. The in vitro incorporation of phenylalanine was stimulated by polyuridylic acid with the stimulation being greatest in ribosomal preparations from detached cotyledons. The results suggest that an axis component may regulate the availability of messenger RNA in the cotyledons during germination.     

Exploiting features of adenovirus replication to support mammalian kinase production

Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products. We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system. Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity. The use of mammalian cells in combination with adenovirus generated a variety of active enzymes which could not be produced in Escherichia coli or baculovirus-infected insect cells. Thus, the utility of adenovirus-mediated enzyme expression as a versatile alternative to established protein production technologies is demonstrated.     

Distinct isoforms of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm

This paper addresses the controversial idea that ADPglucose pyrophosphorylase may be located in the cytosol in some non-photosynthetic plant organs. The intracellular location of the enzyme in developing barley endosperm has been investigated by isolation of intact amyloplasts. Amyloplast preparations contained 13–17% of the total endosperm activity of two plastidial marker enzymes, and less than 0.5% of the total endosperm activity of two cytosolic marker enzymes. Amyloplast preparations contained about 2.5% of the ADPglucose pyrophosphorylase activity, indicating that approximately 15% of the ADPglucose pyrophosphorylase activity in young endosperms is plastidial. Immunoblotting of gels of endosperm and amyloplast extracts also indicated that the enzyme is both inside and outside the amyloplast. Antibodies to the small subunits of the enzyme from barley and maize revealed two bands of protein of different sizes, one of which was located inside and the other outside the amyloplast. The plastidial protein was of the same size as a protein in the chloroplasts of barley leaves which was also recognized by these antibodies. It is suggested that the barley plant contains two distinct isoforms of ADPglucose pyrophosphorylase: one located in plastids (chloroplasts and amyloplasts) and the other in the cytosol of the endosperm. The role of the cytosolic ADPglucose pyrophosphorylase is unknown. Although it may contribute ADPglucose to starch synthesis, the total activity of ADPglucose pyrophosphorylase in the endosperm is far in excess of the rate of starch synthesis and the plastidial isoform is probably capable of catalysing the entire flux of carbon to starch.