Isolation of 80S ribosomes from spores of Aspergillus fumigatus and their antigenicity in rabbits

Ribosomes were obtained from spores of Aspergillus fumigatus by mechanical disruption and differential centrifugation. The initial preparation (crude ribosomes) contained spore components which appeared to be broken fragments of plasmalemma with or without organelles. Purified ribosomes free of membranous material were prepared by gel filtration chromatography on Sepharose CL-4B. Monomeric 80S ribosomes consisting of 40% protein and 60% RNA, and morphologically characteristic of fungal ribosomes were isolated.Serological reactions in sera from rabbits injected with crude or purified ribosomes were similar indicating that the purification process did not change or eliminate antigens that stimulated antibody detectable either by indirect hemagglutination (IHA) or in gel precipitin tests.     

Ultrastructural changes in the spore and mycelia of Ascosphaera apis after treatment with benomyl (Benlate 50 W)

In Ascosphaera apis, after 8 days growth in darkness at 28° C, numerous sporocysts were observed, within which mature spores were seen aggregated into a spore ball. The mature spore of A. apis had a thick spore wall with an electron-opaque outer layer, a spore membrane with many depressions, and sporoplasm containing numerous ribosomes and mitochondria. In the cytoplasm of the mycelium, mitochondria with well-defined cristae and numerous ribosomes were observed. At a concentration of 1 g/ml of culture medium, benomyl appeared to inhibit colony growth of A. apis, but some sporocysts containing deformed spores were found. Deformed spores possessed a thick spore wall with a grainy matrix, and depressions were no longer detected in the spore membrane. Ribosomes were lacking in the sporoplasm and mitochondria appeared degenerate. The mycelium from the treated culture contained mitochondria with an electron-lucid matrix and no well defined cristae, while ribosomes were completely depleted. The significance of these observations in relation to the use of benomyl to control chalkbrood disease in the honey bee is discussed.     

Genetics of ribosomal protein methylation in Escherichia coli. I. A mutant deficient in methylation of protein L11.

Summary Several thousand mutagenized clones of Escherichia coli were screened for methyl group incorporation into protein in crude extracts, in order to isolate mutants lacking the full complement of methyl groups in ribosomal proteins. One mutant isolated by this method and designated prm-1 incorporated 6–7 methyl groups per ribosome upon incubation of its ribosomes with a partially purified enzyme preparation from E. coli wild-type. The methyl groups were located exclusively in the 50S particle and for the most part (85%) in protein L11. Three methylated amino acids were detected: -N-trimethyllysine, -N-monomethyllysine, and an uncharacterized amino acid. These accounted respectively for 4.6, 1.3 and 0.9 methyl groups per ribosome. These results indicate that protein L11 in wild-type contains a stoichiometric amount of these methylated amino acids which are absent in mutant prm-1. Since this mutant is fully viable, its methylation deficiency does not result in a major defect in ribosome assembly or functioning.     

STUDIES ON THE FUNCTION OF INTRACELLULAR RIBONUCLEASES : IV. Some Observations on the Properties of Ribosomes and Polysomes from Rat Liver and Hepatomas

Polysome and ribosome preparations from normal rat liver and from a series of transplantable rat hepatomas of different growth rates were compared. All the hepatomas had a significantly higher percentage of RNA in a polysome preparation than did the normal liver, and the polysome preparations from the tumors, with the exception of the Dunning hepatoma which has a high lipid content, gave a greater yield of RNA and protein per gram of wet tissue than the liver did. Heavier polysomes were considerably less prevalent in the tumors than in the liver, and the tumors contained a larger proportion of monomer and dimer ribosomes than the liver did. Evidence is presented that the increased monomer and dimer ribosome population of the hepatomas studied is not an artifact of preparation, but represents the true intracellular distribution. Ribosomes from normal liver and Morris 5123-D hepatoma were readily dissociated by 20 min'' treatment with 1.0 mM EDTA, but ribosomes from the Dunning, Novikoff ascites, and McCoy MDAB hepatomas were little affected by such treatment. With higher concentrations of EDTA, the ribosomes from the Novikoff ascites and McCoy MDAB hepatomas broke down and did not form specific subunits as did ribosomes from liver and the Morris 5123-D hepatoma but rather gave rise to a variety of small degradation products. This behavior is ascribed to a higher RNase content of the Novikoff and McCoy MDAB hepatomas. Dunning hepatoma ribosomes were resistant to 4 mM EDTA.     

Effect of various methods of preparation on the apparent protein composition of eukaryotic ribosomes. An essential preliminary to stoicheiometric measurements.

1. We investigated whether there is any change in the relative amounts of ribosomal proteins during the isolation or extraction of the ribosomes by different methods, or during electrophoresis of the proteins. 2. To see whether proteins are lost (or gained) during the preparation of the ribosome we compared the two-dimensional protein pattern of three preparations: (a) ribosomes conventionally prepared by ultracentrifugation; (b) crude ribosomes obtained by pH5 precipitation; (c) crude ribosomes prepared by gel filtration. 3. To see whether proteins were lost during protein extraction we compared the two-dimensional pattern of ribosomes by using three different extraction methods (LiCl/urea, acetic acid and guanidine hydrochloride). 4. In all experiments listed above the relative amounts of the great majority of the proteins remained unchanged. We interpret this as showing that the relative amounts of ribosomal proteins (as we observed them on a two-dimensional gel) correspond to the proportions existing in the particle in vivo.     

Isolation of the major intrinsic transmembrane protein of the human erythrocyte membrane

Summary The major intrinsic protein of the human erythrocyte membrane commonly referred to as Band 3, was isolated by a multi-step procedure. Extraction of ghost membranes in dilute solutions of lithium diiodosalicylate removed most of the proteins considered to be extrinsic to the membrane. The resulting membrane fragments were solubilized in sodium dodecyl sulfate, and the major sialoglycoprotein (glycophorin A) was removed by wheat germ agglutinin-Sepharose affinity chromatography. Gel filtration in sodium dodecyl sulfate was used as the final step to yield the band 3 polypeptide in electrophoretically homogeneous form.     

Multiple effects of kanamycin on translational accuracy

Summary We have studied the effects of kanamycin on the accuracy of translation in vitro by wild-type and mutant ribosomes from Escherichia coli. Kanamycin stimulates the leucine missense error of poly(U) translation by wild-type, Ram, and streptomycin-resistant ribosomes in characteristic ways; in particular, the streptomycin-resistant ribosomes are significantly less error-prone than wild-type or Ram ribosomes at all concentrations of the antibiotic. Kinetic analysis of the effects of kanamycin on the translational accuracy of wild-type ribosomes reveals a different concentration dependence for the perturbation of the initial selectivity and for the proofreading. Furthermore, the initial selectivity of streptomycin-resistant ribosomes is not affected by kanamycin; the drug enhances only the error of proofreading by this mutant ribosome. We suggest that the multiple effects of kanamycin on the errors of translation are due to separate effects at different ribosomal sites.Abbreviations N-AcPhe N-acetylphenylalanine - Km Kanamycin (used in the Figures and Tables only) - Str streptomycin (-'-) - EF elongation factor - TCA trichloroacetic acid - Ram ribosome ambiguity mutant     

Characterization of proteins from mitochondrial ribosomes of Locusta migratoria by three different two-dimensional gel electrophoretic methods and comparison with cytoplasmic ribosomal proteins

• 1.1. Proteins were isolated from subunits of mitochondrial and cytoplasmic ribosomes of Locusta migratoria and were analyzed by means of two-dimensional gel electrophoreses using three different electrophoresis systems.
• 2.2. Using the system of Czempiel et al. (1976) proteins from whole locust mitochondrial ribosomes (combined subunits) were separated into 72 spots; proteins from the large and small subunits resulted in 48 and 29 spots respectively.
• 3.3. The mol. wt distribution of mitochondrial ribosome proteins was estimated by using the electrophoresis system of O'Farrell (1975). These mol. wts are in the range of 11,000–56,000, the average mol. wt is about 29,500. Assuming one copy of protein per ribosome this gives a total mol. wt for the protein part of mitochondrial ribosomes of ca. 2.1 x 10⁶.
• 4.4. Parallel separation of cytoplasmic and mitochondrial ribosome proteins was achieved using the system of Geyl et al. (1981). Cytoplasmic ribosome proteins produced 65 spots and revealed a more alkaline character than mitochondrial ribosome proteins.

     

Biogenesis of mitochondria 51

Summary An examination of the effect of the aminoglycoside antibiotics paromomycin and neomycin on mitochondrial ribosome function in yeast has been made. Both antibiotics are potent inhibitors of protein synthesis in isolated mitochondria. With isolated mitochondrial ribosomes programmed with polyuridylic acid (poly U), the drugs are shown to inhibit polyphenylalanine synthesis at moderately high concentrations (above 100 g/ml). At lower concentrations (about 10 g/ml), paromomycin and neomycin cause a 2–3 fold stimulation in the extent of misreading of the UUU codons in poly U, over and above the significant level of misreading catalyzed by the ribosomes in the absence of drugs.Comparative studies have been made between a paromomycin sensitive strain D585-11C and a mutant strain 4810P carrying the parl-r mutation in mtDNA, which leads tohigh resistance to both paromomycin and neomycin in vivo. A high level of resistance to these antibiotics is observed in strain 4810P at the level of mitochondrial protein synthesis in vitro. Whilst the degree of resistance of isolated mitochondrial ribosomes from strain 4810P judged by the inhibition of polyphenylalanine synthesis by paromomycin and neomycin is not extensive, studies on misreading of the poly U message promoted by these drugs demonstrate convincingly the altered properties of mitochondrial ribosomes from the mutant strain 4810P. These ribosomes show resistance to the stimulation of misreading of the codon UUU brought about by paromomycin and neomycin in wild-type mitochondrial ribosomes. Although strain 4810P was originally isolated as being resistant to paromomycin, in all the in vitro amino acid incorporation systems tested here, the 4810P mitochondrial ribosomes show a higher degree of resistance to neomycin than to paromomycin.It is concluded that the parl-r mutation in strain 4810P affects a component of the mitochondrial ribosome, possibly by altering the 15S rRNA or a protein of the small ribosomal subunit. The further elucidation of the functions in the ribosomes that are modified by the parl-r mutation was hampered by the inability of current preparations of yeast mitochondrial ribosomes to translate efficiently natural messenger RNAs from the several sources tested.     

Morphometric and autoradiographic analysis of crude synaptosomal preparations from rat cerebral cortex

Morphometric and autoradiographic studies have been made of a crude synaptosomal preparation, which has been used extensively for membrane transport studies. When filters are used to separate membrane bound structures from incubation medium, the structures which survive filtration are those that are entrapped within the matrix of the filter structure. The population of membrane bound structures differs when one compares pellets of the preparation to sections of loaded 0.45 and 0.65 m pore size filters. Both the relative numbers of synaptosomes, mitochondria, and other membrane bound structures (OMBS) and the mean size of each of the structures differ for pellet, 0.45 m, and 0.65 m filters. The percentage of total membrane bound volume attributable to synaptosomes increases from 28 in the crude preparation to 40 in 0.45 m filters and 61 in 0.65 m filters. The total volume of synaptosomes entrapped by differing pore size filters roughly correlates with the amount of substrate uptake. Neither mitochondrial volume nor the volume of other membrane bound structures was found to correlate with uptake. These results indicated that only the synaptosomes contribute measurably to this function. Autoradiographic studies confirm this conclusion. EM autoradiography following loading of the synaptosomal preparation with tritiated glutamate or GABA showed about 81% of the grains to be associated with synaptosomes. It is concluded that crude synaptosomal preparations may be used without further purification for membrane transport studies with unambiguous results.     

The isolation and properties of cardiac ribosomes and polysomes

1. A method is described by which good yields of ribosomes and polysomes free of contamination by submitochondrial fragments can be prepared from rat cardiac muscle. These preparations are capable of incorporation of amino acids into protein in vitro. 2. The ribosome preparation consists of 32% of monomeric ribosomes and 68% of ribosomal aggregates or polysomes. The polysome preparation has a decreased monomeric content. Dimers, trimers, tetramers, pentamers and larger components can be differentiated. 3. The polysome aggregate structure is degraded to monomeric ribosomes on incubation with small amounts of ribonuclease or by preparation in the absence of Mg²⁺ ions. The degradation in the absence of Mg²⁺ ions was not reversible and drastically decreased the incorporation of amino acids in vitro. 4. The cardiac ribosomes contained two major RNA species sedimenting at 19s and 28s in a 1:2·4 ratio. 5. The RNA/protein ratio of cardiac ribosomes and polysomes was consistently lower than that of similar preparations from liver. The concentrations of Na⁺ and K⁺ ions present during preparation had a great effect on the RNA/protein ratio. 6. Optimum conditions for the incorporation of amino acids into protein in vitro are reported. Cardiac ribosomes have a lower rate of incorporation of amino acids in vitro than liver ribosomes. 7. Heart cell sap is less active than liver cell sap: evidence is presented that a factor, present in liver cell sap and concerned with stimulating the synthesis of the peptide chain, is lacking in heart cell sap. 8. Pulse-labelling of perfused hearts followed by examination of the subcellular structures showed that the ribosomal fraction was the most active in the incorporation of amino acids in vitro.     

Decay and synthesis of ribosomal proteins during Dictyostelium discoideum development

Summary Ribosome turnover is a prominent process during cell differentiation in Dictyostelium discoideum. At the end of 24 h of development on filters, the cells contain only 30% of the ribosome content of vegetatively growing cells. We determined the relative rates of synthesis and decay of each of the ribosomal proteins during this period. Approximately 80% of the total vegetative cell ribosomal proteins were degraded during the course of fruiting body construction. Ribosomal RNA and protein degradation apparently occurred coordinately during development. Although all ribosomal proteins decayed during development, some were more stable and a few less stable than the average. In addition, all the ribosomal proteins were synthesized during this period. Most ribosomal proteins were synthesized at the same rate as other cellular proteins, although a number were made at lower or higher rates. It was estimated that about 35% of the ribosomes in developed cells represented those, that were made during cell differentiation. Differential decay and/or synthesis of ribosomal proteins could account for the observed difference in protein content of ribosomes from growing amoebae and late development cells and spores.Paper No. 4 in the series, Studies on Ribosomal Proteins in Dictyostelium discoideum. Paper No. 3 is Ramagopal and Ennis (1982)     

Preparation of a human lung purified plasma membrane fraction: Confirmation by enzyme markers,electron microscopy,and histamine H1 receptor binding

Summary A simple and rapid method of isolating plasma membranes from human peripheral lung tissue is described. The method involves homogenization of tissue in 0.25m sucrose-buffered medium followed by differential and sucrose density gradient centrifugation. Enzymatic and morphological characterization of the plasma membrane fraction revealed minimal contamination by nonplasma membrane fragments. The isolated plasma membranes showed an 18-fold purification of 5-nucleotidase activity compared to the original homogenate. Electronmicroscopic studies of the plasma membrane fraction revealed the presence of small membrane vesicles having a trilaminar membrane structure. To further examine the purity of the plasma membrane preparation, the binding of the H₁ receptor antagonist,³H pyrilamine, to the plasma membrane-enriched fraction was compared to the binding to crude membrane preparations. Both the plasma membrane-enriched fraction and the crude membrane preparation had similar K_d's for the histamine antagonist, but the plasma membrane-enriched fraction had a threefold greater binding capacity, reflecting the relative enrichment of plasma membranes of the preparation. Thus, a method has been developed for the isolation of plasma membranes from human peripheral lung which should provide material for a variety of biochemical and pharmacological studies.     

Purification of α-amylase from Bacillus lentus cultures

Acetone fractionation of Bacillus lentus culture filtrate yielded the highest -amylase activity and the 66.6% fraction reached 13-fold that of the crude enzyme preparation. Gel filtration and ion exchange chromatography afforded a pure -amylase (relative molecular mass, 42 000). The pure enzyme was highly active on starch and dextrin. It produced a mixture of oligosaccharides as major products of starch hydrolysis. Maximal activity was reached at 70° C and pH 6.1. Ca²⁺, Na⁺, K⁺ and Sr²⁺ ions stabilized or slightly stimulated the enzyme whereas Ag⁺, Co²⁺, Hg²⁺, Zn²⁺, Cd²⁺ and Fe³⁺ ions strongly inhibited the activity. The enzyme contained 16 amino acids, of which aspartic and glutamic acids were present in the highest proportions. Correspondence to: S. H. Omar     

Regulators of Cell Division in Plant Tissues

The cytokinins, 6-benzylaminopurine and kinetin, markedly enhanced the yield of both free and membrane-bound 80S ribosomes per unit weight of radish (Raphanus sativus) cotyledon tissue. The response was observed only after the induction of growth by cytokinin; during the lag period preceding cytokinin-induced growth, ribosome yields from both control and cytokinin-treated cotyledons were below detectable levels. Mannitol depressed both growth and ribosome yield to the same degree. The enhanced ribosome yield appeared to be an indirect effect of cytokinin and was probably a consequence of cytokinin-induced growth. The effect of 6-benzylaminopurine on ribosome yield was not reflected in enhanced levels of cytoplasmic ribosomal RNA, while recently synthesized ribosomes were found to be more readily recovered from cytokinin-treated tissue than from control tissue. It was concluded that cytokinin-enhanced ribosome yield resulted from enhanced ribosome recovery or extractability and that ribosome yield is an unreliable indication of ribosome level in plant tissue.     

Protein crystalloids in ribosome-deficient plastids of Aeonium domesticum cv. variegatum (Crassulaceae)

Protein crystalloids are typical constituents of Aeonium domesticum plastids. They are composed of hexagonally arranged tube-like elements situated in the stroma without a bordering membrane. The single tubule has an external diameter of about 20 nm and an internal one of about 10 nm. The green-white-green mesochimera Ae. domesticum cv. variegatum contains normal chloroplasts in the green tissue and colourless plastids in the pale tissue. The defective plastids have a double-layered envelope, scarce internal membrane structures and contain, in the mature stage, a large vacuole. Plastid ribosomes can be detected only rarely in proplastids. They lose their ribosome complement entirely in the course of development. Polyacrylamide gel electrophoresis of total nucleic acids extracted from white tissue revealed the absence of the 23S and 16S rRNA normally present in plastids. Despite the loss of ribosomes, the plastids contain large protein crystalloids, which are structurally identical with those of normal green chloroplasts. Consequences concerning problems of encoding and transport of crystalloid protein(s) are briefly discussed.Abbreviations CAM crassulacean acid metabolism - FIP fraction I protein - L I epidermis - L II subepidermal layer - L III leaf core - SPC succulent protein crystalloid This is the first part of a series on the crystalloid-forming succulent protein     

The stoichiometry of the ribosomal proteins of Escherichia coli

Summary A ribosome preparation from E. coli made without stringent washing procedures has been shown to contain the same relative amounts of nearly all the ribosomal proteins as ribosomes in intact cells. Stoichiometric measurements on all the proteins of this preparation except for L8, L20, L31 and L34 have been made using an isotope dilution technique. When the scatter of the values obtained, the uncertainty in the molecular weights, and the losses occurring during extraction are taken into account, none of the proteins except L7/L12 is present at a level significantly different from one molecule per ribosome. There are multiple copies of L7/L12. These data suggest that the ribosomes of Escherichia coli are homogeneous in vivo.     

Purification of apo-3-D-(-) hydroxybutyrate dehydrogenase from rat liver mitochondria

Summary 3-D-(-) hydroxybutyrate dehydrogenase (EC 1.1.1.30) from rat-liver mitochondria was purified in the form of the soluble, phospholipid-free apoenzyme by a procedure involving: (1) solubilization of the membrane bound enzyme by controlled digestion of membrane phospholipids with porcine pancreas phospholipase A₂; (2) stabilization and separation of the released apoenzyme as a complex with egg-lecithin by gel filtration on Sephadex G-100; and (3) specific displacement of the apoenzyme from the enzyme-lecithin complex by treatment withBothrops atrox venom phospholipase A₂ (in the absence of Ca²⁺ ions) and subsequent separation of the displaced apoenzyme by gel filtration on Sephadex G-100. The method described is adequate for samples containing about 40 mg of mitochondrial protein. The yield in activity is 42% of that present in mitochondria and the degree of purification of the apodehydrogenase is about 170 fold. The purified apodehydrogenase shows one single sharp band when submitted to SDS polyacrylamide gel electrophoresis, with a mobility corresponding to a molecular weight of 38000 daltons. Gel filtration of the apoenzyme on Sephadex G-100 shows two active peaks with molecular weights of 76000 and 38500 daltons, indicating two different states of aggregation, namely, monomer and dimer. The corresponding diffusion coefficients are 7.73 (monomer) and 5.70 (dimer) × 10^–7. The apodehydrogenase preparation is devoid of phospholipids and is catalytically inactive. It can be reactivated by addition of egg lecithin or phospholipid mixtures containing lecithin in a suitable physical state. Reactivation occurs after formation of an active apodehydrogenase phospholipid complex.Abbreviations HBD 3-D-(-) hydroxybutyrate dehydrogenase - apoHBD 3-D-(-) hydroxybutyrate dehydrogenase apoenzyme - SMP submitochondrial particles - DFP diisopropylfluorophosphate - BSA bovine serum albumin - MPL mitochondrial phospholipids - L-diC₁₄ 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine - lysoC₁₄ 1-myristoyl-sn, glycero-3-phosphorylcholine - D-diC₁₀ 2.3-didecanoyl-sn-glycero-1-phosphorylcholine - tlc thin layer chromatography - SDS sodium dodecylsulfate Dedicated to ProfessorLuis F. Leloir on the occasion of his 70th birthday.