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The isolation of yeast ribosomes associated with triose phosphate dehydrogenase
Immunological techniques have been developed for the quantification and isolation of polysomes involved in ovalbumin synthesis.
For the quantification of ovalbumin polysomes, two methods have been developed; one is based on the binding of 125I-anti-ovalbumin, the other is based on the immunoprecipitation of polysomes. There is a good correlation between the relative
rate of ovalbumin synthesis and the extent of 125I-anti-ovalbumin binding to ribosomes or the amount of RNA extracted from polysome immunoprecipitates.
With special care to exclude ribonuclease activity, RNA can be obtained from precipitated polysomes in an undegraded state.
Such RNA can direct the synthesis of complete ovalbumin chains in an in vitro protein-synthesizing system from rabbit reticulocytes. RNA extracted from polysomes not precipitated by anti-ovalbumin antibody
is incapable of directing ovalbumin synthesis. Recovery of messenger RNA activity is essentially quantitative, and the specific
activity of ovalbumin mRNA is increased 2.5-fold over that obtained from total polysomes.
Nascent chains obtained from immunoprecipitated polysomes are of the size of ovalbumin and smaller, in contrast to nascent
chains from total polysomes which contain larger size classes.
The results in total indicate that ovalbumin-synthesizing polysomes can be immunoprecipitated quantitatively in an undegraded
state, and that ovalbumin mRNA can be extracted from such immunoprecipitates. Immunoprecipitation of polysomes may potentially
be a general method for isolation of specific polysomes, including their mRNA content.
1.1. Some properties of the system for the incorporation of amino acids into the cell-free preparation have been studied. The system requires a supply of ATP (and its generating system), and also the presence of ribosomes.2.2. s-RNA has been found to be an essential intermediate in the incorporation of amino acids into protein, but the RNA component in the ribosomal fraction also takes part.3.3. Chloramphenicol, DNAase (EC 3.1.4.5), or the supply of all four ribonucleoside triphosphates has no marked effect on the incorporation, but puromycin and RNAase (EC 2.7.7.16) are potent inhibitors.4.4. The nature of the protein synthesized in this system has been examined using chromatography on DEAE-cellulose columns. Radioactivity was observed only in the protein of the ribosomal fraction and alkali-eluting part, and not in the soluble protein portion.5.Using these results, the characteristics of the present incorporation system were discussed.
Specific binding of antibody directed against MOPC-21 myeloma protein to MOPC-21 polysomes has been demonstrated. It was also shown that this same antibody bound specifically to the ribosomal subunits of these polysomes, suggesting that the antibody is not binding exclusively to the nascent polypeptide chains on the polysomes. The antibody was bound specifically to polysomes of a nonproducing variant XC1 which had been isolated in the presence of MOPC-21 myeloma protein while the antibody was not bound to XC1 polysomes isolated in the absence of myeloma protein. This suggests that the myeloma protein is adsorbed to the polysomes during the isolation procedure.
Ribosomes may be specific in their role in protein biosynthesis. A preliminary study suggests that antisera against specific beef liver proteins selectively precipitate ribosomes to which the specific proteins are bound.
1. (1) The pulse-labeled RNA extracted from enriched polysomal fractions of Saccharomyces carlsbergensis has been characterized by sedimentation analysis, by the effect of actinomycin D on the observed sedimentation patterns, by its sensitivity to ribonuclease action and by determinations of its base composition. It is concluded that this pulse-labeled RNA shows many of the properties which in general are ascribed to messenger RNA.
2. (2) It is shown, that polysomes which are charged with nascent α-glucosidase can be precipitated rather specifically with the aid of a purified rabbit anti-α-glucosidase γ-globulin fraction and a purified anti-rabbit γ-globulin antiserum from the goat.
3. (3) The pulse-labeled RNA extracted from the precipitated polysomes charged with nascent α-glucosidase has a sucrose gradient sedimentation pattern which is quite distinct from that of the pulse-labeled RNA from the total polysomal fraction.
4. (4) From this pattern and from the patterns obtained in pulse-double-labeling experiments with maltose-induced and non-induced protoplasts, it is tentatively concluded that the messenger RNA coding for the inducible enzyme α-glucosidase has a sedimentation coefficient of about 28 S.
A method is described for an immunological fractionation of polysomes, based on the immunological specificity of their nascent chains, with cross contamination usually below 5%. The method was used to study turnover of RNA associated with specific polysome fractions in the chick lens and indicated that during embryonic development such RNA may become more stable. The extent of stabilisation varies between specific ribosome fractions.
1.1. Lymph nodes of hyperimmunized rabbits have been found to contain polyribosomes which are dissociated into monomers by ribonuclease.2.2. By means of an enzyme-antienzyme system, specific inhibition of bacterial α-amylase activity with these ribosomal fractions has been observed.
1.1. To understand more fully the nature of electrostatic binding to ribosomes and to gain greater insight into the effect such binding may have on the structure of these particles, the binding of the oxidized monomer form of cytochrome c to 80-S rat-liver ribosomes and to the 47-S ribosomal subunit was investigated.2.2. The binding to 80-S ribosomes and to the larger ribosomal subunit, 47 S, was found to be similar. The binding constants decreased rapidly from 0.16·106 almost to zero as the ionic strength increased from 0.022 to 0.124. The number of binding sites on the 80-S monomers also decreased with increasing ionic strength perhaps as a result of an increase in compactness at the higher salt concentrations.3.3. In solutions of 0.04 ionic strength there was a slight but continuous decrease in binding strengths as the pH increased from 6.5 to 9.5. Secondary binding, only one-tenth as strong, but also electrostatic in nature, became evident above pH 7.6. At neutral pH, 115 molecules of cytochrome were bound per 80-S ribosome and 93 molecules per 47-S subunit.4.4. The form taken by the complex was a function of pH, ionic strength, temperature, and the ratio of the concentrations of the reactants.
1. We present evidence suggesting a sudden switch from translational control to transcriptional control of protein synthesis in mid‐exponential growth of bacterial batch cultures. At a critical cell density a switch from large to small polysomes occurs during a short period of exponential growth. The profile of specific polysomes engaged in synthesis of a constitutive enzyme, ribitol dehydrogenase, changes at the same point but in an opposite way: a linear profile peaking at monosomes changes to a dome‐shaped profile peaking at about 15 ribosomes/mRNA, which persists into late exponential phase despite a gradual reduction in the total polysome population. The switch in the pattern of protein synthesis is exhibited dramatically by changes in the specific activity of ribitol dehydrogenase in cell extracts at different stages of batch culture. In early exponential phase the specific activity of the enzyme is constant, but it begins to rise suddenly, at the same point at which the polysome profiles change, and continues to increase up to the end of exponential phase. This effect is exhibited by the strains of Klebsiella aerogenes taht are inducible for (in the presence of the inducer), consitutive for, or superproducers of ribitol dehydrogenase, and it appears to be unrelated to catabolite repression.
2. The above results depend on improved techniques for production of large amounts of bacterial polysomes and the ability to label nascent peptides attached to polysomes very specifically with radioactive antibody to ribitol dehydrogenase. Our success was due to the observation that sodium heparin completely abolishes non‐specific interactions of the antibody with the polysomes.
We have reported the rapid incorporation of labeled amino acids into
the proteins of rabbit reticulocytes in vitro and the stimulation of this process
by certain amino acids, iron, fructose-amino acids, glucose, and some,
as yet, unidentified material in the filtrate of boiled plasma. The incorporation
was measured in the mixture of the total proteins of the reticulocytes
(1, 2).
In the study reported here two protein fractions were isolated, the hemoglobin,
which comprises more than 75 per cent of the total, and the water-insoluble
proteins. The hemoglobin was fractionated into heme and globin.
Experiments were carried out with four C^(14)-labeled amino acids and with
different stimulators to ascertain whether heme synthesis and amino acid
incorporation went parallel or not.
CHYMOTRYPSINOGEN SYNTHESIS IN THE EXOCRINE CELL OF THE GUINEA PIG PANCREAS WAS STUDIED UNDER THE FOLLOWING CONDITIONS: Animals fed after a fast of approximately 48 hours received approximately 1 hour after feeding an intravenous injection of DL-leucine-1-C(14). At various time intervals (1 to 45 minutes) after the injection, the glands were removed and fractionated into a series of cell fractions of known cytological significance. Ten to twelve animals were used for each time point. From each cell fraction, the chymotrypsinogen was isolated by acid extraction and purified by (NH(4))(2)SO(4) fractionation, isoelectric precipitation, and chromatography. Because of the minuteness of the quantities involved, chymotrypsinogen amounts were calculated from enzymatic activity figures, and a carrier method was used to precipitate and count the enzyme. The chymotrypsinogen isolated from the attached ribonucleoprotein particles of the microsomal fraction had the highest specific radioactivity at the early time points (1 to 3 minutes). After long intervals (at 15 to 45 minutes), the specific radioactivity of the enzyme increased in the microsomal contents and finally in the zymogen granules. The results are compatible with the view that the chymotrypsinogen is synthesized in or on the attached RNP particles and subsequently transported to other cell compartments.
Ribonucleoprotein (RNP)1 particles isolated by DOC treatment from pancreatic microsomes have a RNA content of 35 to 45 per cent of their dry weight. In the analytical ultracentrifuge about 85 per cent of the material has a sedimentation coefficient of ∼85 S. These particles contain amylase, RNase, and trypsin-activatable proteolytic activities which cannot be washed off or detached by incubation in 0.44 M sucrose. The enzymes are released, however, by incubation in the presence of low concentrations of ATP, PP, or EDTA, and high concentrations of IP and AMP. At the same time, and at the same concentrations, ∼80 per cent of the RNA and ∼25 per cent of the protein of the particles becomes also non-sedimentable. The simultaneous addition of Mg++ to the incubation medium prevents these losses. This finding, together with the observation that all the Mg++ of the particles is released by the same agents, makes it likely that Mg++ holds the particles together, and that its removal by the chelators used causes the particles to disintegrate. These findings are discussed in relation to the molecular structure of the RNP particles.
An increase in aldolase activity has been observed during the incubation of the microsomal fraction of the corn scutellum. This increase is maximal when adenosine triphosphate, amino acids, magnesium, potassium, guanosine triphosphate, and a pH-5.2 protein are present during the incubation period. These cofactors are similar to those that would be required for protein-synthesizing systems. The increase in aldolase is inhibited by -fluorophenylalanine, chloramphenicol and methionine sulfoxide. The latter inhibits only in the early stages. EDTA and ribonuclease are also inhibitory, indicating a role for ribonucleic acid. Ribonuclease releases a “bound” aldolase from the microsomal granule. The amount of this “bound” enzyme increases after approximately a one-hour lag period. Evidence is furnished that the increase in enzyme activity is not merely due to a release of the enzyme from the particulate matter, but rather that there is actually a net increase in the enzyme. The accumulated data suggests that there has been a synthesis of aldolase, but, since it is premature to advanced such a conclusion, the increase activity has been referred to as an “apparent” aldolase synthesis.
Five different methods of preparing extracts from bacterial cells yielded similar distributions of macromolecular constituents as indicated by ultracentrifugal patterns. Extracts from a number of different bacteria showed essentially similar patterns. Three principal components were found, with sedimentation constants of approximately 40, 29, and 5 S. In many extracts, it was evident that the most slowly moving peak consisted of two components, a broad diffuse boundary and a sharp spike containing desoxypentose nucleic acid. Extracts from the photosynthetic bacterium Rhodospirillum rubrum contained, in addition to the above-mentioned components, a component of very large size (sedimentation constant approximately 190 S), with which the entire pigment complement of the cells was associated. Chemical analyses of crudely fractionated extracts showed that the bulk of the pentose nucleic acid of the cells was associated with the particles of sedimentation constants from 20 to 40 S, while the desoxypentose nucleic acid was almost all present in the fraction of lower sedimentation rate.
Immunology and Cell Biology focuses on the general functioning of the immune system in its broadest sense, with a particular emphasis on its cell biology. Areas that are covered include but are not limited to: Cellular immunology, Innate and adaptive immunity, Immune responses to pathogens,Tumour immunology,Immunopathology, Immunotherapy, Immunogenetics, Immunological studies in humans and model organisms (including mouse, rat, Drosophila etc)
Studies have been conducted on the isolation and stability of large particles, with a sedimentation coefficient of 80 S, observed in extracts from yeast. With knowledge of some of the factors which cause the decomposition and aggregation of these particles, it was possible to obtain a solvent mixture in which the particles were stable. Isolation of the 80 S particles showed that they composed at least 1.5% of the wet weight of the cells and about 50% of the ribonucleic acid (RNA). Analyses indicated that the 80 S particles were a ribonucleoprotein containing about 42% RNA and 58% protein. Ultracentrifugation and electron microscopy showed that the particles were almost uniform in size with a diameter of about 21 mμ.
1. A comparison of the sensitivity and precision of the biuret, ultraviolet absorption, Folin-Ciocalteu and ninhydrin methods for the analysis of specific precipitates has been made, and the uses and limitations of each have been discussed.
2. The ultraviolet absorption of samples in 0.25 N acetic acid and the biuret methods were found to be simple and accurate, the limits of 2 S. D. being ±5 to 10%. The former may be used for samples containing as little as 5 μg nitrogen whereas the lower limit of the latter is about 20′ μg nitrogen.
3. The Folin-Ciocalteu method may be used for samples as small as 5 μg nitrogen and 2 S.D. equals ±15%.
4. The ninhydrin method has a sensitivity making it capable of determining as little as one microgram of protein nitrogen but for reproducible results should probably not be employed without the use of a standard.
The intracellular distribution of amylase in rat pancreas has been studied by differential centrifugation of pancreas homogenates. Although the secretory granules showed high activity, at least half the amylase activity was recovered in the microsome and supernatant fluid fractions. In the latter fractions, the proportion of the total enzyme activity and the concentration of amylase activity relative to protein-nitrogen showed consistent variations with changes in the secretory state of the gland. Apparently some of the amylase is associated with the endoplasmic reticulum in the living pancreas cell, and during homogenization in 0.88M sucrose.the endoplasmic reticulum is broken into pieces resembling one another in composition but differing in size. During exposure to 0.25M sucrose some dissociation of the components of this material takes place, but when such dissociation occurs the amylase protein is not preferentially associated with either the ribonucleoprotein granules or the lipoprotein reticulum of the microsome fraction.
1.1. The apparent amylase content of homogenates of pigeon pancreas was increased by disintegration with ballotini beads. Most of this additional amylase was released from the microsome fraction sedimenting at 18,000 g from 0.25 M sucrose. Very little was obtained from the ultramicrosome fraction subsequently spun down at 105,000 g.2.2. On electron microscopy, the microsome fraction was found to contain numerous vesicles which are presumed to originate from the endoplasmic reticulum. When this microsome fraction was stored in water, the inactive amylase became active and this coincided with the disappearance of vesicles from the preparation. It was concluded that the inactive enzyme was present in the interior of the vesicles and was thus rendered inaccessible to the substrate. When the enzyme was activated by rupture of the vesicles in water, it remained attached to the debris. It is therefore likely that the amylase is attached to the interior of the intact vesicle.3.3. Uptake of 14C-glycine by the microsome fraction is high, and especially so by the proteins not associated with ribonucleic acid, that is, the proteins of the vesicle wall. It is thus possible that the site of amylase attachment to the vesicle wall is also its site of formation.
A procedure for purification of alkaline phosphatase from E. coli is described, and several physical-chemical propetiies of the purified enzyme are reported. When the cells are grown under the condition of phosphate deprivation, which is optimal for the synthesis of alkaline phosphatase, about 6% of the total protein synthesized is alkaline phosphatase.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
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