Mitochondrial Ribosomes in HeLa Cells

HeLa cell mitochondria contain 60S ribosomes which seem to consist of subunits of 45S and 35S particles. The 16S and 12S RNA components are coded by mitochondrial DNA.     

Activity of Thylakoid-bound Ribosomes in Pea Chloroplasts

Pea (Pisum sativum) chloroplast thylakoid membranes were prepared by washing in hypotonic buffers. These membranes contained bound ribosomes which were active in protein synthesis when supplemented with soluble components from a strain of Escherichia coli low in ribonuclease. After dissolving the membranes by Triton and purification of the ribosomes, sucrose density gradient profiles indicated the presence of polysomal material as well as monomeric ribosomes. Most of the products of protein synthesis remained associated with the thylakoid membranes even after ribosomes were removed completely by high salt concentrations in the absence of Mg²⁺. Of the newly formed products, 50% could be digested by pronase, while the remainder were protected by their association with the thylakoid membranes. The products are likely to be a mixture of intrinsic and extrinsic membrane proteins, with only the former completely protected by the membranes from attack by proteases.     

Ribonuclease Activity Associated With Ribosomes of Zea mays

At pH 6.5, a ribonuclease(s) is associated with ribosomes isolated from corn (Zea mays L.) and cannot be removed by repeated differential centrifugation or by sedimenting through the sucrose gradient. The enzyme is active under conditions favoring the maintenance of integrity of the ribosomes. Little or no latent ribonuclease appears to be present. The activity of the enzyme at pH 5.8 is stimulated by KCl and inhibited by polyvinyl sulfate, zinc, and bentonite. Deoxyribonuclease is also found on the particles.

The enzyme can be removed from ribosomes by adsorption onto bentonite. Ribosomes are also adsorbed but to a much lesser extent at low bentonite concentrations. The enzyme is easily dissociated from ribosomes by raising the pH to 8.5, and readsorbed when the pH is lowered.

The ribonuclease activity on ribosomes shows a sharp increase with cell age that parallels closely the increase in total activity in the homogenate. The ratio of activities of deoxyribonuclease to ribonuclease on ribosomes also changes with cell age and again the changes appear to reflect changes in the homogenate. It is suggested that most of the association of ribonuclease with corn ribosomes may not be meaningful in vivo and occurs only after the cells are ruptured.

     

Synthesis of Chloroplast and Cytosol Ribosomes in Regenerating Acetabularia Cells

The incorporation of ³H-uridine into 80S cytosol and into 70S chloroplast ribosomes has been studied in Acetabularia in order to find out whether or not the nuclei undergo changes in the capability of rRNA formation during differentiation. To reinforce the differentiation process regenerating Acetabularia cells were chosen. Regeneration was induced by amputation of the apical part of the cell. The incorporation into cytosol ribosomes increased as early as 24 h after amputation. The stimulation reaches a maximum three to four days after amputation and does not depend on light at least for three days. It can be prevented, however, by addition of cycloheximide to the culture medium. The stimulation pattern of the uridine incorporation into chloroplast ribosomes is similar to the cytosol ribosomes with the exception that it is delayed by two days. This process is dependent on light and is not inhibited by cycloheximide. The response to amputation seems to be independent of chloroplast energy production as small and large fragments are stimulated to the same degree.     

Viral RNAs Associated with Ribosomes in Sindbis Virus-Infected HeLa Cells

Virus specific RNA ribosome complexes were isolated by sucrose density gradient centrifugation of cytoplasmic extracts from HeLa cells infected at 42 C with an RNA(+) mutant (ts2) of Sindbis virus. Viral RNA-ribosome complexes were accumulated by infected cells treated with sodium fluoride and cycloheximide. The RNA-ribosome complexes were characterized by (i) their sensitivity to the action of ribonuclease or ethylenediaminetetraacetic acid, (ii) their density in cesium chloride gradients, and (iii) presence of host ribosomes and viral RNAs. The viral RNAs were isolated and characterized. The results showed that two species of single-stranded RNAs (a 28s and 18 to 15s species) were associated with the complexes. Base composition analysis of the viral RNAs indicated that both species had a higher adenine content than the 42s or 26s forms of viral RNAs. The RNAs associated with the ribosome complexes were virus specific since they annealed with denatured double-stranded RNAs from the infected cells. Little or no 42S RNA was associated with the RNA-ribosome complexes. The results suggest that the 28s and 18 to 15s forms of RNAs may represent viral messenger RNAs.     

Purification of Ribosomes from Neurospora Crassa

Abstract

A method for preparing ribosomes from Neurospora crassa is described in which che ribosomes prepared are free from the enzymes cytochrome c oxidase and NADPH-cytochrome c reductase, DNA and phospholipid material.     

Inhibition of Protein Synthesis in Cell-Free Systems from Interferon-Treated, Infected Cells: Further Characterization and Effect of Formylmethionyl-tRNA(F)

The translation of encephalomyocarditis virus (EMC) RNA is markedly inhibited in cell-free systems from interferon-treated, vaccinia virus-infected L-cells (10, 11). The polypeptide products synthesized in response to EMC RNA in cell-free systems from these and untreated infected cells have been analyzed by electrophoresis on polyacrylamide gels. Qualitatively, the same EMC-specific polypeptides were synthesized throughout. In experiments using preincubated microsomes from normal Krebs cells to assay cell sap from L-cells which had been exposed to interferon prior to infection, only the amount of the EMC-specific polypeptide products was reduced. This result suggests that there is an inhibition very early in translation in interferon-treated, infected cells. Initiation seems a priori the more attractive site for this inhibition, but an effect shortly after initiation cannot be excluded. With unfractionated cell-free systems from interferon-treated infected L-cells, however, there appeared to be an additional minor inhibitory effect on polypeptide chain elongation, in that the EMC-specific polypeptides synthesized showed not only a reduction in amount but also a bias towards lower molecular weight. The formylated methionyl initiator tRNA (Fmet-tRNA_F) was used as a further probe into the apparent effect on intiation. With this reagent we have confirmed that there is one major initiation site for the translation of EMC RNA in these cell-free systems. In addition, the results have shown that EMC-specific polypeptide chains initiated with Fmet escape the major interferon-mediated inhibition at or shortly after initiation.     

Localization of Glucose-6-phosphate Dehydrogenase Activity on Ribosomes of Granular Endoplasmic Reticulum, in Peroxisomes and Peripheral Cytoplasm of Rat Liver Parenchymal Cells

Glucose-6-phosphate dehydrogenase activity has been localized ultrastructurally in fixed tissues. Activity was found in particular in association with ribosomes of granular endoplasmatic reticulum. Biochemical studies indicated that glucose-6-phosphate dehydrogenase activity is also present in the cytoplasm and in peroxisomes. Fixation may be held responsible for selective inactivation of part of glucose-6-phosphate dehydrogenase activity. In the present study, we applied the ferricyanide method for the demonstration of glucose-6-phosphate dehydrogenase activity in unfixed cryostat sections of rat liver in combination with the semipermeable membrane technique and in isolated rat liver parenchymal cells. Isolated liver parenchymal cells were permeabilized with 0.025% glutaraldehyde after NADP⁺ protection of the active site of glucose-6-phosphate dehydrogenase. This treatment resulted in only slight inactivation of glucose-6-phosphate dehydrogenase activity. The composition of the incubation medium was optimized on the basis of rapid light microscopical analysis of the formation of reddish-brown final reaction product in sections. With the optimized method, electron dense reaction product was observed in cryostat sections on granular endoplasmic reticulum, in mitochondria and at the cell border. However, the ultrastructural morphology was rather poor. In contrast, the morphology of incubated isolated cells was preserved much better. Electron dense precipitate was found on ribosomes of the granular endoplasmic reticulum, in peroxisomes and the cytoplasm, particularly at the periphery of cells. In conclusion, our ultrastructural study clearly demonstrates that it is essential to use mildly-fixed cells to allow detection of glucose-6-phosphate dehydrogenase activity in all cellular compartments where activity is present.     

Exosomal GAPDH from Proximal Tubule Cells Regulate ENaC Activity

Exosomes are nanometer-scale, cell-derived vesicles that contain various molecules including nucleic acids, proteins, and lipids. These vesicles can release their cargo into adjacent or distant cells and mediate intercellular communication and cellular function. Here we examined the regulation of epithelial sodium channels in mpkCCD cells and distal tubule Xenopus 2F3 cells by exosomes isolated from proximal tubule LLC-PK1 cells. Cultured mpkCCD cells were stained with CTX coupled to a green fluorophore in order to label the cell membranes and freshly isolated exosomes from LLC-PK1 cells were labeled with the red lipophilic dye PKH26 in order to visualize uptake of exosomes into the cells. Single-channel patch clamp recordings showed the open probability of ENaC in Xenopus 2F3 cells and in freshly isolated split-open tubules decreased in response to exogenous application of exosomes derived from LLC-PK1 proximal tubule cells. Active GAPDH was identified within exosomes derived from proximal tubule LLC-PK1 cells. The effect on ENaC activity in Xenopus 2F3 cells was blunted after application of exosomes transfected with the GAPDH inhibitor heptelidic acid. Also, we show GAPDH and ENaC subunits associate in mpkCCD cells. These studies examine a potential role for exosomes in the regulation of ENaC activity and examine a possible mechanism for communication from proximal tubule cells to distal tubule and collecting duct cells.     

Characterization of Ribosomes from Dormant Spores of Bacillus cereus

Characterization of ribosomes from dormant spores and vegetative cells of Bacillus cereus strain T has been carried out. Polyuridylic acid binding activity, ribonuclease activity associated with ribosomes, thermal denaturation profile, and sedimentation coefficients are essentially identical for both ribosomal preparations. However, ribosomal protein content of dormant spore ribosomes is about 70% of that of vegetative ribosomes. Polyacrylamide gel electrophoresis of ribosomal proteins shows that some ribosomal proteins are missing from dormant spore ribosomes. Sucrose density gradient centrifugation of ribosomes shows the existence of defective ribosomal subunits, in addition to 30S and 50S subunits, in dormant spore ribosomes. These results indicate that the ribosomes from dormant spores are distinctively different from those of vegetative cells.     

Direct Determination of Phosphatase Activity from Physiological Substrates in Cells

A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min^-1 mg^-1 for PPi, to 56 ± 11 nmol min^-1 mg^-1 for AMP, to 79 ± 23 nmol min^-1 mg^-1 for beta-glycerophosphate and to 73 ± 15 nmol min^-1 mg^-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole – a TNAP inhibitor- served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC₅₀ value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.