Amino Acid-incorporating Activity Research Articles

Correlation between Polyribosome Level and the Ability to Induce Nitrate Reductase in Dark-grown Corn Seedlings

Nitrate reductase can be induced in excised shoots of 3-day-old dark-grown Zea mays (var. WF9 x M14) seedlings in the absence of light. In contrast, leaves of 10-day-old dark-grown seedlings require a light treatment in order to induce enzymatic activity. Leaves of 10-day-old dark-grown seedlings contain a very low level of polyribosomes while 3-day-old shoots contain a very high level of polyribosomes. There is a gradual loss of polyribosomes from 3 to 10 days and a gradual loss of in vitro protein synthetic activity of the ribosome preparations. The loss of polyribosomes and decrease in their amino acid-incorporating activity correlate positively with the loss of ability to induce nitrate reducase activity as leaves of dark-grown corn seedlings age. These results corroborate and extend our previous results, in that light is not required for nitrate reductase induction per se in leaves of dark-grown seedlings but is required to reactivate the protein synthetic apparatus of older leaves.

Amino acid incorporation activity of lettuce seed ribosomes during germination

Ribosomes were prepared from lettuce seeds (whose germination is light-dependent) at different stages of germination and after various light treatments. The amino acid incorporation activity of these ribosomes in the absence and in the presence of poly U was compared. The results show that during the first three hours of germination, incorporation activity of ribosomes in the absence of synthetic mRNA increases. This increase is independent on illumination. The endogenous amino acid incorporation activity of ribosomes, prepared from illuminated seeds, continues to increase, first slowly till the 18th hour and more rapidly from the 18th till the 24th hour. In contrast the endogenous amino acid incorporation activity of ribosomes obtained from unilluminated seeds decreased slowly after the three hours of imbibition. No difference was found in the capacity to incorporate [14 C] phe-tRNA into polypeptide in the presence of poly U with the different ribosomal preparations.

Protein synthesis during germination of Peronospora tabacina (Adam) conidia

The characteristics of an in vitro amino acid-incorporating system prepared from germinated Peronospora tabacina conidia are described. Material sedimenting at 20,000 g and at 117,000 g was found to possess significant amino acid-incorporating activity. Although germination is accompanied by an increase in the activity of both these fractions, if RNA synthesis is inhibited during germination, activity increases only in the 20,000 g fraction. Since germ tube formation occurs in the absence of RNA synthesis, it is suggested that the protein required for germ tube formation is largely synthesized by this 20,000 g fraction. The increase in amino acid-incorporating activity in this fraction begins during the preparation of conidia at 0o) and prior to the start of incubation at 15 o and may result from the removal of some factor during washing.

Ribosomes liés à un système membranaire chez E. coli. Étude des liaisons ribosomes - membrane

Bound Ribosomes, sedimenting with the Cell-debris at 30 000 g, have a higher aminoacid-incorporation activity than the free ribosomes. Two kinds of bonds, to cytoplasmic membrane or to a membrane system, are present in the isolated subcellular fraction (C30).o1)Ribosomes are attached on newly synthetized mRNA chains which are transcripted from DNA bound to cytoplasmic membrane (the cells are harvested in early exponential phase). They are released from Cell debris after the action of DNase.2)Ribosomes are directly bound to Cell membrane; they are not affected by DNase and are released from Cell debris by action of detergents like DOC or Triton X100. Ribosomes are attached on newly synthetized mRNA chains which are transcripted from DNA bound to cytoplasmic membrane (the cells are harvested in early exponential phase). They are released from Cell debris after the action of DNase. Ribosomes are directly bound to Cell membrane; they are not affected by DNase and are released from Cell debris by action of detergents like DOC or Triton X100. The higher activity of aminoacid incorporation in a cell free system can be explained by the cytoplasmic membrane protection or by the continuous transcription of mRNA on DNA template located on the Cytoplasmic membrane. Après avoir montré qu'il existe des ribosomes liés sédimentant à 30 000 g avec les débris cellulaires nous rapportons dans ce travail les résultats d'expériences montrant que ces ribosomes sont attachés à un système membranaire de deux façons différentes:u— Les uns par l'intermédiaire du RNA messager transcrit au niveau de la membrane cytoplasmique contenant du DNA. Ces ribosomes peuvent être détachés par action de la DNase sans que leur activité soit détruite.— Les autres sont directement liés aux débris cellulaires. Ils sont détachés par action de détergents tels que le désoxycholate de sodium ou le triton X100. — Les uns par l'intermédiaire du RNA messager transcrit au niveau de la membrane cytoplasmique contenant du DNA. Ces ribosomes peuvent être détachés par action de la DNase sans que leur activité soit détruite. — Les autres sont directement liés aux débris cellulaires. Ils sont détachés par action de détergents tels que le désoxycholate de sodium ou le triton X100. L'augmentation de l'activité d'incorporation de l'ensemble de ces ribosomes peut s'expliquer soit par un effet protecteur de la membrane cytoplasmique, soit encore par la transcription continue de RNA messager. Nach Isolierung von Ribosomen die mit der Zellwand sedimentieren (30 000 g), geben wir hier die Ergebnisse von Experimenten die zeigen dass diese Ribosomen auf zweierlei Arten an die Zellmembrane gebunden sind.o1)Die einen sind durch neu-synthetisierte RNS Ketten an die Zellmembran gebunden. Sie können durch DNase von den Zellsedimenten getrennt werden und besitzen dann die gleichen Eigenschaften wie die freien Ribosomen, die vom Cytoplasma extraktiert werden.2)Die anderen sind direkt an die Zellmembrane gebunden. Diese werden durch Natrium Desoxycholat oder Triton X100 von den Zellsedimenten getrennt, aber nicht mit DNase. Die einen sind durch neu-synthetisierte RNS Ketten an die Zellmembran gebunden. Sie können durch DNase von den Zellsedimenten getrennt werden und besitzen dann die gleichen Eigenschaften wie die freien Ribosomen, die vom Cytoplasma extraktiert werden. Die anderen sind direkt an die Zellmembrane gebunden. Diese werden durch Natrium Desoxycholat oder Triton X100 von den Zellsedimenten getrennt, aber nicht mit DNase. Die hohe Aktivität in Aminosäure einbau kann entweder durch Protektion der Zellmembrane oder durch beständige Transkription von RNS an der Zellwand erklärt werden.

Enhancement of Protein Synthesis in Isolated Chloroplasts by Irradiation of Fern Gametophytes with Blue Light

Irradiation of the gametophytes of Pteridium aquilinum with blue light led to a nearly 5-fold increase in the amino acid-incorporating activity of isolated chloroplasts. The blue light effect was not due to increased synthesis of ATP or other energy donors by the chloroplasts but was probably related to an increased production of chlorophyll and photosynthetic enzymes.

Protein synthesis in cell-free systems: an effect of interferon.

The activity of ribosome and cell-sap fractions from interferon-treated and control chick embryo fibroblasts was compared in mixed chick-mouse and purely chick cell-free systems capable of the synthesis of viral polypeptide(s) in response to viral ribonucleic acid (RNA). Interferon treatment of cells did not affect the intrinsic amino acid incorporation activity of these systems or their response to polyuridylic acid. With encephalomyocarditis (EMC) virus RNA as messenger, however, a fraction of the ribosomes from interferon-treated cells appeared less active than parallel controls. The results obtained with the corresponding cell-sap fractions were variable. Although competition between endogenous and added messengers cannot be excluded in these systems, a reduced level of translation of EMC RNA with interferon-treated cell ribosomes was also suggested by the results of analyses of tryptic digests of the products formed in response to the RNA. In addition, these analyses showed that this reduced activity must reflect a reduction in the rate or frequency of translation rather than a decrease in the length of the EMC RNA translated, for the same polypeptides were synthesized in response to the RNA with material from interferon-treated and control cells. Interferon added directly to the cell-free system was without effect. Although suggestive, these results do not provide definitive evidence for or against the hypothesis that virus protein synthesis is inhibited at the translational level in the interferon-treated cell. Possible alternative interpretations of the data are discussed.

Studies on the mechanism of the inhibition of protein synthesis induced by intracellular ATP depletion

The deleterious effect of intracellular ATP depletion on the protein-synthesizing system was studied in rabbit reticulocytes and Landschütz ascites tumor cells. The progressive decline of the ATP content of the cells induced by anaerobic incubation in glucose-free Krebs-Ringer salt solution was attended by a prompt inhibition of protein synthesis. The onset of the arrest of protein synthesis in the course of incubation under nitrogen, coincident with a 20–30 % decrease of the ATP level, entailed no impairment of the cell-free amino acid-incorporating activity and was readily reversible by reaeration of the cells. This early inhibition showed no correlation with the degree of ATP deprivation but appeared to be determined by the inhibitory effect of the accumulating ADP and AMP on the process of peptide chain elongation. Extensive ATP depletion following prolonged anaerobic incubation gave rise to a lesion characterized by dissociation of the polysomes and a concomitant decline of the cell-free amino acid-incorporating activity. However, the expression of this lesion was masked by factors interfering with the peptide chain elongation. Therefore, drastic dissociation of the polysomes within the cells occurred only during the brief anaerobic-aerobic transition period following readmission of air when the block of the chain elongation by the products of ATP splitting had been relieved by partial resynthesis of ATP. On prolonging the aerobic reincubation of the cells, progressive reversal of the derangement of protein synthesis took place. It was concluded that the lesion concerned was localized at the stage of peptide initiation. The initiation defect caused by severe ATP deficiency was shown to reside exclusively in the cell-sap fraction and to differ essentially from the ribosomal lesion induced by NaF. In addition, the detrimental effect of NaF on protein synthesis proved to be entirely unrelated to the inhibitory action of this poison on the energy metabolism. An inhibitory pattern essentially similar to the one above was obtained when ATP depletion was brought about by incubating the cells in the presence of 2,4-dinitrophenol. Furthermore, the alterations induced by the uncoupling action of dinitrophenol on the oxidative phosphorylation were completely reversible by washing away the uncoupling agent. The mechanism underlying the above alterations of the protein-synthesizing system are discussed in light of the pertinent data, and the possible regulatory implication of the inhibitory influence of the products of ATP breakdown on peptide chain elongation is considered.

Regulation of polyribosome formation and protein synthesis in the uterus. Effect of administration of graded doses of oestradiol-17-beta and actinomycin D on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of polyribosomes.

1. The effects of graded doses of oestradiol-17beta and actinomycin D, administered separately or together, on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of uterine polyribosomes are described. Preparations of polyribosomes isolated from uteri of ovariectomized adult rats were determined for cytoplasmic concentration in vivo and assayed for [(14)C]leucine-incorporation activity in the cell-free system, exactly as described by Teng & Hamilton (1967b). 2. A minimal dose of 10mug of oestradiol-17beta administered for 10h was found to increase, by about 100%, both the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of the polyribosomes. A minimal dose of 250mug of actinomycin D administered for 10h was found to inhibit, by about 50%, the incorporation activity in vitro of the polyribosomes. All doses of the inhibitor administered for 10h failed to alter the cytoplasmic concentration in vivo of the polyribosomes. 3. A dose of 10mug of oestradiol-17beta restored to the control value the inhibitory effect of a dose of either 50 or 125mug of actinomycin D on the activity in vitro of the polyribosomes, at 10h after treatment with the inhibitor and the hormone. In these experiments, there was an increase of 60-100% in the cytoplasmic concentration in vivo of the polyribosomes. 4. A dose of 125mug of actinomycin D, administered to animals along with 10mug of oestradiol-17beta for 6-36h, abolished the hormone-induced enhancement of the incorporation activity in vitro, but did not prevent an increase of about 200% in the cytoplasmic concentration in vivo of the polyribosomes. However, treatment with 750mug of the inhibitor abolished both stimulatory effects of the hormone. 5. The results reported indicate that the stimulatory effects of oestradiol-17beta in vivo on the number and activity of the cytoplasmic polyribosomes in the uterus of the ovariectomized rat have different sensitivities to actinomycin D, but the primary molecular mechanisms responsible for the results are unknown. The major conclusion drawn is that the formation and appearance in the cytoplasm of newly formed polyribosomes are important features of the early action of oestrogen in the uterus.

Incorporation of N-formylmethionine into peptides by Pseudomonas aeruginosa extracts

A cell-free system capable of carrying out amino acid incorporation into peptides was developed from exponentially growing cultures of Pseudomonas aeruginosa. The amino acid incorporation activity required an ATP energy generating system, Mg 2+, NH 4 +, tRNA and an amino acid mixture for maximum activity. Ribonuclease, puromycin and chloramphenicol inhibited protein synthesis. N-Formylmethionyl-tRNA was synthesized with a P. aeruginosa enzyme system, methionine, P. aeruginosa tRNA, formate and tetrahydrofolic acid. N-formylmethionine was incorporated into peptides by the P. aeruginosa cell-free system. These results indicate that P. aeruginosa extracts contain methionyl-tRNA transformylase and the complete system for the incorporation of N-formylmethionine into peptides.

Protein Synthesis in the Early Prereplicative Phase of Isoproterenol-stimulated Synthesis of Deoxyribonucleic Acid

Abstract The stimulation of DNA synthesis which occurs in mouse salivary gland 20 to 30 hours after a single administration of isoproterenol is inhibited by drugs that inhibit protein synthesis. Both cycloheximide and puromycin are most effective in inhibiting isoproterenol-stimulated DNA synthesis when given 1 hour after the isoproterenol. At this time, the aminonucleoside of puromycin has no effect on the subsequent onset of DNA synthesis. Although cycloheximide has only a temporary effect on protein synthesis in mouse salivary gland, its effect on isoproterenol-stimulated DNA synthesis, when given 1 hour after isoproterenol, consists not in a mere delay but in a true inhibition. The free ribosome fraction of mouse salivary gland shows an increase in protein synthesis 1 hour after isoproterenol, when the stimulated gland is incubated in vitro. In addition, ribosomes isolated from salivary glands of mice killed 1 hour after the administration of isoproterenol have a higher amino acid-incorporating activity in a cell-free system than free ribosomes isolated from control salivary glands. This increased capacity for protein synthesis is accompanied by an increase in the number of free polysomes in the stimulated gland. These results indicate that isoproterenol causes an early stimulation of protein synthesis in mouse salivary gland and that this stimulation is relevant to the subsequent onset of DNA synthesis.

Dissociation of ribosomes from dormant cysts of Artemia salina in potassium-free media

Ribosomes from dormant cysts of Artemia salina have a low endogenous activity of amino acid incorporation, but readily polymerize phenylalanine in the presence of polyU. Their activity of polyU-dependent phenylalanine incorporation is rapidly lost in K + or Mg 2+ deficient media. Increasing the MgCl 2 concentration from 1.5 mM to 5–10 mM does not effectively protect the ribosomes against inactiyation through K + deficiency. The endogenous amino acid incorporation by ribosomes from developing embryos is not markedly affected by K + deficiency under comparable conditions. The inactivation of the ribosomes from unincubated cysts in K + or Mg 2+ deficient media is correlated with a dissociation of the particles into subunits. The dissociation by K + deficiency is somewhat delayed, but not prevented by increasing the MgCl 2 concentration from 1.5 mM to 5–10 mM; 5–10 mM NH 4Cl is highly protective, but NaCl less effective. Subunits are formed in KCl-containining media already at moderately reduced MgCl 2 concentrations. However, their stability is less satisfactory, apparently due to K +-stimulated activation of latent ribosomal nucleases. The experiments indicate that the ribosomal monomers characteristic of early Artemia embryos are selectively inactivated and dissociated in K +-deficient media. Analogous conditions may occur in other cell materials with low endogenous ribosomal activity.

Changes in the polysomal pattern after fertilization in Fucus eggs

At high potassium concentrations it is possible to isolate largely intact polysomes from fertilized and unfertilized Fucus vesiculosus eggs, and to study the distribution pattern of ribosomal particles in a sucrose gradient. The changes in the ribosomal pattern before and after fertilization reflect the time course of protein synthesis. In the unfertilized egg there is a pool of inactive light polysomes which are fairly resistant to ribonuclease treatment, whilst a heavier polysomal fraction shows some amino-acid incorporation activity. 2 hr after fertilization, the heavy polysome fraction is strongly increased and highly active; the light polysomes disappear. 6 hr after fertilization, when protein synthesis is depressed, new monomeric and possibly dimeric ribosomes are formed; these ribosomes have a very low incorporation activity. The rearrangement of ribosomal particles does not appear to be necessarily linked with the synthesis of new RNA.

Functional and Structural Studies of Membrane-bound and Free Ribosomes from Rat Spleen

Abstract The functional and structural properties of sodium deoxycholate-liberated bound and free ribosomes from rat spleen have been compared in vitro. The endogenous amino acid-incorporating activity of bound ribosomes relative to that of free ribosomes (B:F) varies only slightly (71 to 103%) under a variety of isolation and incubation conditions. However, the net exogenous activity for 14C-phenylalanine with the use of polyuridylic acid (poly U) as messenger varies widely (B:F, 11 to 75%) and is greatly dependent on experimental conditions. Minimal translation of poly U and the lowest B:F ratio are seen when the source of supernatant factors is a spleen pH 5 fraction. Maximal translation of poly U and the highest B:F ratio are achieved with a liver pH 5 fraction, isolation of ribosomes in the presence of a rat liver supernatant, the omission of centrifugation through 2 m sucrose, and the addition of a 0.5 m NH4Cl wash. Relative to the minimal system, incorporation is enhanced 54-fold for bound ribosomes and 8-fold for free ribosomes when these optimal conditions are used. Rat spleen bound and free ribosomes are immunologically identical, have similar sedimentation coefficients, similar contents of RNA and nucleotide bases, and virtually identical patterns of polyribosomes and structural and rapidly labeled RNAs. These results caution that different ribosome populations may seem more or less alike functionally, depending upon the particular conditions of isolation and incubation.

The ribosomes of encysted embryos of Artemia salina during cryptobiosis and resumption of development

Summary Commercially availableArtemia salina cysts contain embryos arrested at the gastrula stage. A correlated biochemical and electron optical study was made of the ribosomes from such cysts and from rehydrated cysts during their resumption and completion of embryonic development. 1. Protein synthesis increased slowly and continuously from shortly after hydration throughout the period studied (until more than 50% of the larvae had hatched). 2. Electron micrographs from sectioned cysts and cyst fractions and UV absorption data from cyst fractions showed that the cytoplasm of unincubated cysts was packed with ribosomes, virtually all of which were monomeric and not attached to membranes. 3. During development the proportion of polysomes increased at the expense of the monosomes. However, at all incubation times the great majority of ribosomes existed as free monosomes. 4. The failure of detergent or trypsin treatments to increase incorporation activity significantly in the nonincubated material led to the conclusion that the initiation of protein synthesis inArtemia is due not to the release of preprogrammed particles, but to a gradual programming of free monosomes. 5. That the monosomes constituted a pool of unprogrammed ribosomes was demonstrated by their very low amino acid incorporation activity and by the marked stimulation of this activity by artificial mRNA (polyuridylic acid). These studies, together with electron optical studies of negatively stained preparations of ribosomes and data on the K+ and Mg++ optima of the ribosomes indicate that the structure and conditions for function in these particles apparently are the same as those for ribosomes from most other materials. Therefore, commercially availableArtemia cysts provide a convenient source for unprogrammed ribosomes for studying messenger functions.

The effect of ribonuclease on rat-liver ribosomes.

1. Rat-liver ribosomes lose about 50% of their amino acid-incorporating activity when preincubated with ribonuclease. 2. This preincubation results also in loss of about 50% of the original protein content and 75% of the RNA. 3. Ribosomes sedimented by ultracentrifugation, after preincubation with ribonuclease, show negligible contamination by crystalline enzyme. 4. Washing of ribosomes treated with ribonuclease releases further protein, restoring the original RNA/protein ratio. 5. The washed particle is again capable of promoting amino acid incorporation. 6. Examination of ribosomes treated with ribonuclease in the analytical ultracentrifuge reveals destruction of ribosomes, disappearance of dimers and a decrease in the sedimentation coefficient of monomers. 7. Washed ribosomes consist of even smaller particles with a sedimentation coefficient 60s.

Regulation of polyribosome formation and protein synthesis in the uterus. Effect of ovariectomy and administration of oestradiol-17β on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of polyribosomal preparation

1. The hormonal regulation of cytoplasmic protein synthesis in the uterus is described. Polyribosomal preparation from uteri of normal or ovariectomized rats was isolated by procedure 3 and assayed for [(14)C]leucine-incorporation activity in the cell-free system, as described by Teng & Hamilton (1967). 2. Ovariectomy of normal animals caused, 3 weeks after surgery, a 50-60% increase in the amino acid-incorporation activity in vitro of uterine polyribosomal preparation, but a 90-95% decrease in the cytoplasmic concentration in vivo of the preparation. 3. Administration of 10mug. of oestradiol-17beta to ovariectomized rats at zero time caused, 10-12hr. later, a 100% stimulation in amino acid-incorporation activity in vitro of the uterine polyribosomal preparation. From 12hr. to 36hr. after hormone administration, the activity in vitro of the preparation decreased. If a second dose of hormone was administered at 36hr., the activity in vitro of the preparation continued to decrease, and approached at 48hr. and 72hr. the lower activity observed for the preparation from normal animals. The cytoplasmic concentration of polyribosomal preparation increased by 600-700% under these experimental conditions. If a second dose of oestradiol-17beta was not administered at 36hr., the initially elevated cytoplasmic concentration of the preparation decreased by 50% from 36hr. to 72hr., and the activity in vitro of the preparation was not fully depressed to the ;normal' value. 4. Pretreatment of ovariectomized animals with actinomycin D or cycloheximide abolished 80-90% of the stimulatory effects of hormone treatment on the amino acid-incorporation activity in vitro and cytoplasmic concentration in vivo of uterine polyribosomal preparation. 5. Two major conclusions are drawn from the results reported: that during early oestrogen action new polyribosomes having amino acid-incorporation properties different from those of the old ones appear and accumulate in the cytoplasm of the uterus; and that the regulation of cytoplasmic protein synthesis in the organ by oestrogen is of an indirect nature, with dual effects of the hormone on genetic transcription resulting in turn in a regulation of the rate and amount of genetic translation.

Effects of growth hormone and tri-iodothyronine on amino acid incorporation by microsomal subfractions from rat liver.

A two-step procedure without the use of detergents for the separation of smooth and rough endoplasmic reticulum of rat-liver and the fractionation of the latter into “light rough membranes”“heavy rough membranes” and “free polysomes” is described. The distribution of RNA, protein and phospholipid in these fractions as well as their amino acid incorporation characteristics are also described. The heavy rough membrane fraction incorporated 14C-labelled phenylalanine, isoleucine and lysine into protein at a rate 3–5 times greater than that exhibited by the light rough membranes and free polysomes. Stimulation of the incorporation of [14C]phenylalanine and [14C]-isoleucine by poly U and poly UA showed that, per mg ribosomal RNA, the heavy rough membrane fraction was also more active in its response to synthetic messenger RNAs than the other two fractions. Hypophysectomy caused a marked reduction in the amount of ribosomes and phospholipids, the amino acid incorporation capacity and the response to poly U and poly UA in all three rough microsomal subfractions. These effects were reversed in 3–4 days by a combined treatment of hypophysectomized rats with human growth hormone and 3,3′,5-tri-iodo-l-thyronine. Although the amino acid incorporation activity in the presence of endogenous and synthetic template RNA was affected in all the subfractions, the effect of hormone deprivation and replacement was most marked in the heavy rough membrane fraction. The relative stimulation of the incorporation of [14C]phenylalanine and [14C]isoleucine by poly U and poly UA, respectively, by free and membrane-bound polysomes was only slightly lowered in a few cases after hormonal treatment of hypophysectomized rats. However, per mg ribosomal RNA, hormone treatment led to a 2–4-fold enhancement of the incorporation of phenylalanine and isoleucine in response to the synthetic polyribonucleotides. Treatment of heavy rough membranes with sodium deoxycholate showed that much of the hormonal effect on amino acid incorporation was manifested in the ribosomes but that the maximal hormonal effect was detected when these were associated with the membranes. It is concluded that growth hormone and thyroid hormone together control both the amount and amino acid incorporation activity of free and membrane-associated polyribosomes of rat-liver.

A critical study of amino acid incorporation into protein by isolated liver mitochondria from adult rats.

1. Mitochondria were isolated from rat liver in a way that kept bacterial contamination at a minimum. 2. The activity of oxidative phosphorylation was unchanged under these conditions, whereas the ability of the preparations to incorporate amino acids into protein was insignificant, though it could be enhanced somewhat by the presence of EDTA. This enhancement was sensitive to ribonuclease. 3. The active time of incorporation did not exceed 15min. at 30 degrees . 4. Microsomal contamination, as measured by glucose 6-phosphatase activity, was about 5%. 5. The ability of isolated bacteria to incorporate amino acids into protein was greatly enhanced by the addition of mitochondria or heat-inactivated mitochondria. 6. A correlation was found between the growth rate of bacteria and the amino acid-incorporating activity. 7. Amino acid incorporation by combined mitochondrial-bacterial systems was inhibited by 2,4-dinitrophenol. 8. The results confirm and extend the earlier findings made in our Laboratory that isolated liver mitochondria, when free from contaminating bacteria and obtained from adult rats, are not able to catalyse the incorporation of amino acids into protein at a measurable rate. 9. The results are discussed with special emphasis on the validity of these findings.

The effect of a single intraperitoneal dose of the hepatocarcinogen 4-dimethylaminoazobenzene on the rough-surfaced endoplasmic reticulum of the liver of the rat

1. Changes in the structure and function of the rough-surfaced endoplasmic reticulum of the rat liver as deduced by electron microscopy, polysome analysis and the amino acid-incorporating activity of microsome fractions have been followed at various time-intervals after a single intraperitoneal dose of the hepatocarcinogen 4-dimethylaminoazobenzene. 2. The earliest effect observed was detachment of polysomes and disorganization and vesiculation of the cisternae of the rough-surfaced endoplasmic reticulum. This occurred after 6hr. 3. Subsequent to this was a phase of polysome disaggregation accompanied by impaired amino acid-incorporating activity by microsomes together with a much enhanced stimulating effect of polyuridylic acid on amino acid uptake. This reached a maximum at 24hr. 4. By 40hr. polysomes had re-formed and the amino acid-incorporating activity, together with the polyuridylic acid effect, were similar to those in controls. 5. It was not until 112hr. that something like the normal structure of the rough-surfaced endoplasmic reticulum was re-established. 6. It is not yet possible to relate these changes specifically with the process of azo-dye carcinogenesis.

Studies on the Removal of Endogenous Messenger Ribonucleic Acid Activity from Rat Liver Microsomes: EFFECT OF REMOVAL ON POLYURIDYLIC ACID-DIRECTED INCORPORATION OF PHENYLALANINE

Abstract Rat liver microsomes preincubated for 12 min with all the factors necessary for amino acid incorporation do not incorporate phenylalanine in the absence of polyuridylic acid. In the presence of polyuridylic acid, preincubated microsomes show a 3- to 4-fold greater polyuridylic acid-directed phenylalanine incorporation than nonpreincubated microsomes. By varying preincubation time, temperature, and magnesium ion, adenosine triphosphate, or guanosine triphosphate concentration, the amount of phenylalanine incorporated during preincubation and the phenylalanine-incorporating activity remaining after preincubation are altered. Under these various conditions the response of preincubated microsomes to polyuridylic acid is shown to be directly related to the amount of amino acid incorporated during preincubation and inversely related to the amino acid-incorporating activity remaining after preincubation. Preincubated microsomes are more sensitive to low levels of polyuridylic acid and require higher levels of polyuridylic acid to saturate their capacity to incorporate phenylalanine. Furthermore, preincubated microsomes from which endogenous messenger ribonucleic acid activity has been removed bind greater amounts of polyuridylic acid.