Ribosomes, fractionation

A wheat germ, cell-free, translation extract was fractionated into three concentrated parts using ammonium sulfate the 0 - 40 % saturated fraction, the 40 - 60 % saturated fraction, and the ribosome fraction. These fractions were tested for their ability to enhance the translational activity of the wheat germ, cell-free extract for dihydrofolate reductase. The fortified cell-free system supplemented with the 0 - 40 % ammonium sulfate fraction enhanced the efficiency of protein synthesis by 50 %. 

SCX column on H PLC), the eluates were directly transferred onto a reversed-phase HPLC column, and subsequently the eluates from the second column were validated by tandem MS. Of 78 existing proteins, 75 were correctly identified. The analysis of an enriched yeast ribosomal fraction identified over 80% of the subunits in the complex (Lui, 2002). According to the described method of validation and optimization, the potential of this method lies within practical application due to automation and up-scaling. 

Ribosomes from Escherichia coli Asymmetrical Separation of ribosomes, their subunits, and t-RNA/low-MW protein mixture in samples collected at different protein production phases and in the presence of antibiotics, specific genes, and proteins calculation of a ribosome number per cell and a ribosome fraction using peak area [6]... 

A similar observation was made with ribosomal fractions from boron-suflBcient and boron-deficient sunflower plants (54). The disturbed... 

In summary, the nature of the chylomicron protein is undetermined. It is possible that during fat absorption a protein is formed in the cells of intestinal mucosa, perhaps in the ribosomal fraction, at the same time that triglyceride synthesis occurs. The newly formed protein or proteins may be unrelated to plasma lipoproteins and perhaps constitute the structural chylomicron protein corrresponding to the unidentified fraction in the studies by Rodbell et al. (1 9) and Scanu and Page (1959). Upon entering the lymph and then the blood, chylomicrons may acquire additional protein at the expense of circulating hpoproteins of hepatic derivation (Scanu and Page, 1959). 

Employing [ Cjuridine and following the breakdown and synthesis of RNA in experiments similar to those described above gave results comparable to those obtained for protein (Klein et ah, 1974). The loss of radioactivity from subcellular fractions has also been determined in prelabeled explants cultured under conditions of protein deprivation (Hickey and Klein, 1973). As might be expected, from the observations on total RNA, most of the radioactivity lost from the sensitive regions (brain and neural tube) was derived from the ribosomal fraction. 

The enzyme has been found ubiquitously in the nuclei of a great variety of eukaryotic cells, including human [11] and plant [12]. However, three previous studies have presented evidence that the enzyme is also localized in the cytoplasm. Roberts et al. [13] have found the enzyme associated with polysomes and ribosomes of HeLa cells. A poly(ADP-ribose) synthetase was also reported to occur in rat liver mitochondria [14]. More recently we found a significant level of enzymatic activity associated with testis mitochondria [15, 16]. The present paper is a follow-up of this study and it presents evidence that after the nuclear fraction, the most important level of poly(ADP-ribose) synthetase in spermatogenic cells is associated with the micro-somal-ribosomal fraction. 

Table 1 shows the amount of enzyme units as well as the specific activity of poly-(ADP-ribose) synthetase in the different cell fractions of rat testis. These results show that a large proportion of enzyme is localized in the nuclear fraction and in the microsomal-ribosomal fraction. However, the specific activity of the enzyme was larger in the latter fraction. In contrast, the specific activity of the enzyme in the microsomal fraction of rat liver, brain, and kidney was negligible (Table 2). 

Electron microscopic studies of the microsomal-ribosomal fraction revealed the presence of abundant small vesicles and large aggregates of ribosomes (Fig. 1 A). Care-... 

Fig. 2. Effect of DNAase I and RNAase on the activity of poly(ADP-ribose) synthetase. The microsomal-ribosomal fraction was preincubated at 25° C alone ( ), with RNAase (0 250 jug ml" ) or with DNAase I 250 jug ml" ). At the times indicated, aliquots were assayed for enzymatic activity...

The activity of the enzyme associated with the microsomal-ribosomal fraction of the rat testis can be demonstrated by the absence of DNA in the assay. In the presence of DNA, the enzyme was stimulated three to five times. Treatment of the microsomal-ribosomal fraction with DNAase I at 25°C induced a considerable decrease in the enzymatic activity (Fig. 2). After a 2 h preincubation, less than 20% of the initial activity was detected. On the other hand, the samples preincubated in the presence... 

At the age indicated, the microsomal-ribosomal fraction was prepared from two different pools of testis. Hence, the activities shown are the average of two determinations... 

Qermont and Perey [26] have shown that after birth, there is a good chronological relationship between the age of the rat and the type of spermatogenic cells present in the seminiferous tubules. This particular situation can be used to determine the type of spermatogenic cell in which poly(ADP-ribose) synthetase appears associated with the microsomal-ribosomal fraction. Accordingly, this cellular fraction was isolated from testis of rats at different time intervals after birth and the activity of the enzyme was measured. As shown in Table 4, the peak of activity was obtained with rats of 24- to 30-days-old. Thereafter, the specific activity of the enzyme reached the lower adult level. 

These results indicate that the maximal activity of poly(ADP-ribose) synthetase associated with the microsomal-ribosomal fraction of the testis was attained in rats... 

Our results demonstrate that by using cell fractionation procedures, it is possible to measure quite a significant level of poly(ADP-ribose) synthetase in the microsomal-ribosomal fraction of the testis and not in other somatic tissues. However, a major criticism to this experimental approach, is that the activity detected might well be due to a contamination with the nuclear enzyme. One fact that supports this criticism is that the nuclei contain a considerable amount of enzyme. On the other hand, no matter which procedure one uses to homogenize the tissue, the possibiUty of damaging the nucleus and consequently releasing some amount of enzyme, is difficult to discard. 

However, arguments suggesting that this activity is unlikely due to contamination with the nuclear enzyme were also presented. First, the apparent K , for NAD was 105 liM for the microsomal activity, compared with a value of 210 (jM for the nuclear enzyme [15, 16]. Second, the maximal specific activity of the enzyme associated with the microsomal-ribosomal fraction was found in testis of 24- to 30-days-old rats. If contamination with the nuclear enzyme is likely, one should expect that the activity in this fraction would parallel the activity in the nuclear fraction. On the contrary, developmental studies carried out by Momii and Koide [27] demonstrated that the activity of the enzyme in isolated nuclei increases with age and reaches a maximal value in the adult. 

The enzyme associated with the microsomal-ribosomal fraction of the testis showed a considerable level of activity in the absence of added DNA. However, if calf-thymus DNA was added to the assay mixture, three to four times more stimulation of the activity was observed. Furthermore, the experiments carried out with DNAase 1 strongly suggest that the activity observed in this cytoplasmic fraction was due to the presence of DNA. Therefore, a pertinent question remains as to the nature of this cytoplasmic DNA responsible for the residual activity of the enzyme. To answer this, further work will be necessary. 

A cytoplasmic poly(ADPR) polymerase activity was first detected by Roberts et al. in 1975 [1]. The enzyme associated with ribosomes and polysomes in HeLa cells requires both DNA and histones for activity. A mitochondrial enzyme has been described in rat liver and in testis (for review see [2, 3]). Finally, the existence of an active poly-(ADPR) polymerase associated with ribonucleoproteins in the microsomal-ribosomal fraction of testis has been reported. The enzyme shows a considerable activity without exogenous DNA and is insensitive to RNase A [3]. 

Table 1 summarizes the poly(ADPR) polymerase activities measured in free mRNP isolated from mouse plasmacytoma, Krebs II ascites-tumor cells or rat liver. In contrast to the tumoral cells, the activity associated to rat liver free mRNP is very low nevertheless, it is twelvefold higher than the activity associated with the microsomal-ribosomal fraction reported by Burzio et al. [3]. The addition of calf thymus DNA induces only an increase of about 20% in enzymatic activity. 

ADP-Ribosylation in Cytoplasmic Ribonucleoprotein Particles Which Contain Silent mRNA. We demonstrated that ADP-ribosyl transferase activity is associated with cytoplasmic free mRNP isolated from a variety of organs and tumor cells mouse plasmacytoma, rat liver, rat brain, Krebs n cell rat brain cultured neurons and astrocytes (25-28). Table 1 summarizes the mRNP poly(ADP-ribose) polymerase activity. On a protein basis, in contrast to the tumor cells, the activity associated with rat liver and whole rat brain free mRNP is very low. Nevertheless, it is 12 fold higher than the activity associated with the microsomal ribosomal fraction reported by Burizo et al. (19). In brain mRNP and mainly in neuronal mRNP die activity is much higher than in the rat liver mRNP on a DNA basis (not shown). 

Poly(ADP-ribosyl)ation is generally described as a nuclear event. However, extranuclear poly(ADP-ribose) polymerase activities have been detected in the cytosol of baby hamster kidney ceUs (1), the ribosomal fraction of HeLa cells (2) and rat testis (3). The activities looked like the nuclear enzyme in diat they totally or partially depended on DNA. We have previously reported the association of a poly(ADP-ribose) polymerase with a specific ribonucleoprotein complex, namely free messenger ribonucleoprotein particles (free mRNP) (4-6). The enzyme has the particularity to be DNA-independent. In diis paper, we have b n interested in the proteins which are poly(ADP-ribosyl)ated in free mRNP. We also present evidence of the existence of a poly(ADP-ribose) glycohydrolase in free mRNP. 

In a comparison of supernatant and ribosomal tRNA populations, Wettstein [55] had found that Leu-tRNAi, which accounts for more than 50% of coli Leu-tRNA, is present much less frequently in ribosomal fractions than is the second fraction (MAK peak 2) and that, in T4-infected cells, it is largely excluded from the ribosomal fraction. In an extension of this work, Sueoka and Kano-Sueoka [56] obtained polysomal, ribosomal, and supernatant fractions from noninfected cells, as well as from infected cells, 1.5, 3, 6, and 10 minutes after T2 infection. Transfer RNA was then isolated from the various fractions, deacylated, and reacylated with radioactive leucine. The proportion of the various Leu-tRNA species in each preparation was analyzed by MAK chromatography. As shown in Table I, the Leu-tRNAi content of the polysomal fraction, which amounted to 20% of total Leu-tRNA before infection, fell to 6% within 1.5 minutes after infection and then remained essentially constant up to 10 minutes, suggesting a reduced usage of this species and consequently of the CpUpG codon. 

In vitro protein synthesis in the presence of added ribosomes and mRNA (or polysomes) and ATP/GTP can be supported by supernatant (cytoplasmic, or post-ribosomal) fractions obtained from dry peanut seeds (Table 5.6) and dry wheat embryos [76]. Consequently, components of the cytoplasm essential for protein synthesis (e.g. initiation and elongation factors, tRNA, amino acids, and aminoacyl tRNA-synthesizing enzymes—synthetases) must be present in the dry seed, presumably in sufficient quantities to permit resumption of protein synthesis in the seed upon imbibition. 

Fig. 5.13. Sucrose density gradient of the ribosomal fraction from endosperms of castor bean seeds imbibed from 0 to 72 h. — zero time, o—o 24 h after the start of...

DiflFerences may exist between tissues in the sites of initiation of carbohydrate prosthetic groups, e.g., no glucosamine- C incorporation was observed into the ribosome fraction of either thyroid tissue (Bouchilloux and Cheftel, 1966 Chef tel and Bouchilloux, 1968) or Ehrlich ascites cells (Cook et al, 1965 Eylar and Cook, 1965). Although it has been clearly demonstrated that some initiation occurs at the ribosomal level in rat liver, it cannot be ruled out that this reaction also occurs after release of polypeptide from the ribosome. 

To determine if glycosylation occurs on ribosomes and at the level of nascent polypeptides, we pulse-labeled whole cells with radioactive mannose, stopped the labeling with cycloheximide to freeze the polysomes and then prepared protoplasts from the labeled cells. A significant amount of radioactivity was found in the isolated ribosomal fraction. 

At this time, Mahlon Hoagland returned to our laboratories after a period with Lipmann and Linderstrom-Lang, and undertook to find out whether the ATP-requiring step involved enzyme(s) in the soluble protein fraction, or in the ribosomal fraction. His discovery of the first step in protein synthesis followed. ... 

Still in connection with the structure of ribosomes, Cohen and Lichtenstein (51) made the interesting observation that in E. coli about 15% of the total polyamines (putrescine and spermidine) of the bacterium are found in the ribosome fraction and that these ribosomal polyamines do not exchange with those in the medium. Moreover, it was found that spermidine acted in conjunction with Mg++ in preserving the structural integrity of the particles. 

While the role df lipids in protein s mthesis has not been clarified, there have been reports of a number of different amino acid-lipid complexes. Hendler 93) has investigated in further detail the metabolically active amino acid-lipid compounds which are rapidly formed in intact cells of hen oviduct. By means of countercurrent distribution and chromatography on columns of aliunina-silica and of silicic acid, he has been able to demonstrate a large number of these compounds. These, furthermore, show a distribution pattern which can be altered in a typical fashion by inhibitors, such as dinitrophenol. Lipid-amino acid complexes similar to those in hen oviduct were also found to be present in the membranes and crude ribosome fraction of E. coli. 

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