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HeLa cells were found to enter and complete mitosis in the presence of puromycin or actinomycin D. Daughter cells formed in the presence of puromycin contained completed nuclear membranes. Daughter cells resumed protein synthesis shortly after completing mitosis however, this resumption of protein synthesis could be prevented by actinomycin D. Data presented here indicate that all cellular constituents necessary for successful initiation and completion of mitosis are produced prior to the onset of mitosis. They also indicate that new messenger RNA synthesis is required for resumption of protein synthesis after mitosis, and that the inhibition of protein synthesis observed in mitotic mammalian cells 18 a result of messer ger RNA depletion. © 1967.

Specific aminoacyl-tRNA's from different mammalian organs and species have been compared by methylated albumin column chromatography. The elution profile of such chromatographs were usually similar, both between tRNA's from different organs in the same animal, and across widely divergent mammalian species. Species differences were found in the elution profile of avian leucyl- and tyrosyl-tRNA's, but not among other tRNA's. In contrast to the similarities of methylated albumin-kieselguhr elution profiles of specific tRNA's from most mammalian organs, the elution profile of Ehrlich ascites tumor cells phenylalanyl-, seryl-, glycyl-and tyrosyl-tRNA differed from normal mouse organ tRNA. An extra peak of tyrosyl-tRNA has also been found in HeLa cells and adeno-7 virus transformed cells, but not in all tumor cells. The tyrosyl-tRNA of mammalian and chick fibroblasts in cell culture, and fibroblasts in the body exhibited a markedly different elution profile from the tyrosyl-tRNA of epithelial cells in culture and of normal organs. The tRNA differences discovered by in vitro charging have all been confirmed in vivo. Possible reasons for these variations are discussed. © 1968.

HeLa cells were found to enter and complete mitosis in the presence of puromycin or actinomycin D. Daughter cells formed in the presence of puromycin contained completed nuclear membranes. Daughter cells resumed protein synthesis shortly after completing mitosis however, this resumption of protein synthesis could be prevented by actinomycin D. Data presented here indicate that all cellular constituents necessary for successful initiation and completion of mitosis are produced prior to the onset of mitosis. They also indicate that new messenger RNA synthesis is required for resumption of protein synthesis after mitosis, and that the inhibition of protein synthesis observed in mitotic mammalian cells 18 a result of messer ger RNA depletion. © 1967.

We have compared the biochemical capacity of cultured cells from a variety of animal sources to replicate the genetic information of two small RNA viruses. Mengovirus is shown to infect and kill cultured cells from many animal species regardless of whether the virus replicated well, moderately, or poorly in any cell type. The production of infectious viral RNA, and of viral protein by any cell type was proportional to the yield of intact virus produced by that cell type. The virus yield per cell was compared in a variety of cells employing two small RNA viruses (mengovirus and bovine enterovirus 1) capable of adsorbing to, penetrating, and infecting all the cells tested. The yields varied over a range of more than a thousandfold with both viruses, but the patterns of cell susceptibility were markedly different for each virus. Both viruses caused profound inhibition of host cell protein synthesis even in cells which replicated the virus very inefficiently. Possible biochemical explanations for such highly specific host cell restriction are discussed. © 1967.

Phenylalanine uptake in Chlorella fusca was measured, using the membrane filter technique. The cells were synchronized, and harvested at specific points of the life cycle. Experiments with autospores showed that the uptake followed saturation kinetics, with a Km= 5 μM. Vmax, was 0.1 nmol/min × 107 cells. The optimum temperature for the uptake was 40°C, and the activation energy was 1700 J/mol. The uptake showed a high specificity towards l‐phenylalanine; presence of the unlabelled stereoisomer did not inhibit the uptake. Uptake of l‐phenylalanine was inhibited in the presence of other analogues or other amino acids, but only if they were present in concentrations considerably higher than that of L‐phenylalanine. Variations in the ratio of Na4+ to K+ in the external solution during uptake experiments did not have any influence upon the uptake rate of l‐phenylalanine. The cells were able to take up the amino acid against a concentration gradient. At pool maximum the ratio between internal and external amino acid concentration was 1000/1. 2,4‐Dinitro‐phenol inhibited the uptake completely. Exchange between internal and external l‐phenylalanine could not be demonstrated. The Km value did not change during the life cycle of the cells. The uptake rate reached a maximum at the end of the light period, and fell to a minimum just before sporulation started. It is concluded that Chlorella fusca cells have a highly specific, active uptake system for l‐phenylalanine. The system is constitutive, independent on the K or Na concentration, and the mechanism of uptake does not change during the life cycle of the cells. [ABSTRACT FROM AUTHOR]

2-Thiocytidine incorporated into tRNAPhe from yeast by tRNA nucleotidyl transferase can be alkylated by iodoacetamide and its derivatives. Using this method a spin label was introduced specifically into tRNAPhe and the product, tRNAPhe-C-(s.l.)s²C-A, can still be aminoacylated by phenylalanyl-tRNA synthetase. Electron paramagnetic resonance spectra obtained between 10° and 70°C for tRNAPhe-C-(s.l.)s²C-A and the pentanucleotide C-A-C-(s.1.)s²C-A (a model compound) were evaluated for the correlation time, τc, of the reorientation of the spin label in its environment and for the motional anisotropy parameter, ε. Spin-labeled pentanucleotide is characterized by a spin label motion with an activation energy, EA, of 1-67 × 10-3 J × mol-1 × K-1 (7.0 kcal × mol-1 × °C-1) ) and a strongly anisotropic motion with a long axis of the rotational ellipsoid along the N-O bond of the radical. This motion is temperature independent. In comparison spin label bound to the tRNA molecule is more immobilised and its motion is more isotropic. In addition, the slope of log τc(T) and the nature of the motion changes at a critical temperature Tcr which, in the absence of magnesium, varies with ionic strength. In the presence of magnesium Tcr is 47.5 °C, whereas the midpoint temperature of the optical melting curve, Tm, is 75 °C. The melting transition monitored by the spin label is a cooperative process associated with the melting of a higher-ordered structure of the tRNAPhe molecule. [ABSTRACT FROM AUTHOR]

The phenylalanine-specific tRNAPhe from 13-day-old chick embryo muscle was purified. Its fluorescent and hydrophobic Y+ base was excised by mild acid treatment and characterized chromatographically and by its ultraviolet absorption and fluorescent spectra. The compound obtained after removal of the Y+ base from chick embryo tRNAPhe was characterized. 1. Chick embryo t RNAPhe looses by the removal of the Y+ base its phenylalanine acceptor capacity. 2. In this state it has a reduced sedimentation coefficient of 2.8 S. a low Tm value (22 °C), only rudimentary hyperchromicity, but unchanged electrophoretic properties. 3. The compound obtained after removal of the Y+ base from chick embryo tRNAPhe regains normal melting behaviour and phenylalanine acceptor capacity when exposed to special renaturation conditions. 4. The Km of the renatured form is 0.25 × 10-6 M and V = 220 pmol × min-1 × ml-1 as compared tO Km = 0.11 × 10-6 M and V = 370 pmol x min-1 × ml-1 for the native chick embryo tRNAPhe. [ABSTRACT FROM AUTHOR]

Phenylalanyl-tRNA synthetase from yeast forms an enzyme · phenylalanyl adenylate complex, which has been isolated from the reaction mixture by gel filtration. The study of this complex leads to the conclusion that there are two binding sites for phenylalanyl adenylate per molecule of enzyme. No complex between enzyme and ATP could be characterised. Mg2+ is required for complex formation, but it can be replaced by monovalent cations in the transfer of phenylalanine from the complex to tRNAPhe. The kinetics of the transfer reaction show evidence that the rate-limiting step is not the release of enzyme-bound Phe-tRNAPhe but the acylation of tRNAPhe after the ternary complex phenylalanyl adenylate · enzyme · tRNAPhe has been formed. [ABSTRACT FROM AUTHOR]

The addition of polyamines to an Escherichia coli cell-free polypeptide synthetic reaction mixture containing dialyzed ribosomes, Sephadex-treated supernatant from 100000 × g centrifugation. and suitable amounts of Mg2+ increased the polyphenylalanine synthesis from either [14C]phenylalanine or [14C]phenylalanyl-tRNA up to twice that synthesized in the same system without polyamines but at optimal Mg2+ concentration. No similar effect was observed with cadaverine, agmatine. histamine. Ca2+ or Mn2+. These results, together with the presence of polyamines in unwashed ribosomes and the rapid incorporation of polyamines into ribosomes incubated with polyamines, suggest that polyamines are essential components of E. coli ribosomes for maximum polypeptide synthesis. The increase of polyphenylalanine synthesis by polyamines in an E. coli cell-free system occurred at the level of aminoacyl-tRNA binding to ribosomes, but not at the levels of peptide bond formation and translocation. In the rat-liver cell-free system, an approximately 1.6-fold increase of polyphenylalanine synthesis was observed by the addition of spermidine. However. the addition of putrescine to the reaction mixture caused only a sparing effect on the Mg2+ requirement for polyphenylalanine synthesis. [ABSTRACT FROM AUTHOR]

When a mixture of the light enzyme of gramicidin S synthetase containing thioester-bound D-[14C]phenylalanine and the heavy enzyme is passed through a Sepharose column containing carboxyl-bound proline (substrate amino acid) as ligand, the two enzymes are retained as the result of the affinity between the heavy enzyme and the ligand and the affinity between the two enzymes. Upon elution and separation of the two enzymes it was demonstrated that the D-[14C]phenylalanyl groups had been transferred to the heavy enzyme. This transfer did not take place when similar experiments were carried out using as ligands valine, ornithine or leucine which are also substrates for the heavy enzyme. Hence binding of proline to the heavy enzyme is required for transfer to take place. Upon incubation of the heavy enzyme containing the transferred D-[14C]phenylalanyl group with the light enzyme, ATP and the five unlabelled substrate amino acids, radioactivity is incorporated into gramicidin S. This suggests that in the biosynthesis of gramicidin S, the D-phenylalanyl group is transferred to an acceptor site on the heavy enzyme and subsequently to the imino group of thioesterbound proline. In view of what is known about gramicidin S synthesis it seems likely that the acceptor site is a thiol site. [ABSTRACT FROM AUTHOR]

The pleuromutilin derivative 14-deoxy-14-[(2-diethylaminoethyl)-mercaptoacetoxy]-mutilin HCl interferes strongly with cell-free poly(U)-directed polyphenylalanine synthesis in an extract from Escherichia coli (supernatant obtained by centrifugation at 30000 x g), The compound is unable to inhibit the assembly of initiation complexes and does not significantly influence Phe-tRNA attachment to the ribosomal A-site catalyzed by elongation factor T. It does, however, reduce the puromycin release of formylmethionine from initiator tRNA bound to the P-site. indicating that either the peptidyl transferase is affected or the binding of puromycin is abolished by this antibiotic. The influence of this agent on protein synthesis performed by E. coli polysomes is less pronounced than the inhibition of polyphenylalanine synthesis. The opposite effect has been reported for chloramphenicol. This mirror-image-like behaviour seems to suggest that both antibotics are related in their mode of action. The formation of the dipeptide fMet-Ala coded for by R-17 phage RNA is also impeded by the pleuromutilin derivative. The binding of AIa-tRNA to the A-site is affected in this reaction. Since attachment of Phe-tRNA to ribosomes with an empty P-site (dependent on elongation factor T) is not hindered, the inhibition of the tRNA binding to initiation complexes seems to indicate that the antibotic requires an occupied P-site in order to prevent binding of aminoacyl-tRNA to the A-site. [ABSTRACT FROM AUTHOR]

It has been shown that the initial rates, v, of the formation of aminoacyl-tRNA, catalyzed by phenylalanine-tRNA synthetase, do not depend on the concentration of the substrates ATP and phenylalanine in a simple way. In contrast a normal Michaelis-Menten behaviour has been observed for the substrate tRNA. The reciprocal plots of v versus v/[S] for ATP and phenylalanine are curved and only approach linearity at low and high concentrations of the varied substrate. From these limiting slopes two Km and two V values corresponding to low and high concentrations of ATP and phenylalanine have been determined. Since the reciprocal plots for tRNA are linear, only one Km and V value has been obtained for this substrate. In order to show that this kinetic behaviour is an intrinsic property of the enzyme and not an artifact, a new procedure for the purification of the enzyme has been devised and the purity and stability of the preparation carefully checked. In addition the influence of ammonium ions and of spermine on the initial rates has been measured to verify that the non-linearity of the reciprocal plots is not the result of the substrate acting as a base. Inhibition experiments with tRNAox (a modified tRNA obtained from native tRNA by periodate oxidation of the 2':3'-diol in the 3'-terminal ribose to the corresponding dialdehyde) at low and high phenylalanine concentrations have shown that it is also not a consequence of two fundamentally different reaction mechanisms being followed at the two concentration ranges. On the basis of this and other evidence it is proposed that two mutually interacting sites are engaged, of which one or both are operative, depending on the concentration of the substrates. Studies of pyrophosphate inhibition at low tRNA concentration have provided evidence that the aminoacylation of tRNA proceeds via two catalytic steps in which aminoacyl-AMP occurs as an intermediate. At high tRNA concentrations it has been found that this substrate is adding prior to the dissociation of pyrophosphate. [ABSTRACT FROM AUTHOR]

Formycinmonophosphate (FMP) has been incorporated in to the 3′-terminus of phenyl- alanine tRNA from yeast by tRNA nucleotidyl transferase, tRNA PhepCpCpF can be amino-acylated with the cognate synthetase exhibiting the same Km and an about 50-times lowered V compared to tRNA PhepCpCpA. Quantum yield of FMP fluorescence in tRNA PhepCpCpF is about four times lower than in the monomer. Comparative studies of tRNA PhepCpCpF and the oligonucleotide CpApCpCpF show that the differences between the fluorescence properties of tRNA PhepCpCpF and the monomeric formycin-nucleotide are due only to stacking interactions and do not arise from the tertiary structure of tRNA. After periodate oxidation of the 3′-terminal ribose of tRNAPhepCpCpF the full fluorescence of formycin is reconstituted. This emphasises the important role of the intact ribose ring for stabilization of stacking units. Periodate oxidation of tRNA PhepCpCpF followed by borohydride reduction leads to a tRNA that acts as competitive inhibitor in the aminoacylation reaction. This together with its high quantum yield makes it a valuable tool in fluorescence spectroscopic studies of the aminoacylation reaction. [ABSTRACT FROM AUTHOR]

Discusses the kinetic analysis of subunit interactions in the enzyme levo phenylalanine ammonia-lyase. Steady state of the reaction; Enzyme inhibitors; Behavior of the free enzyme; Conformation of the unliganded protomer; Enzyme substrate binding.

The article discusses three factors associated with elevated phenylalanine levels on newborn screening tests, low birth weight, race, and tyrosinemia, and its effect to incidence of false positive diagnosis of phenylketonuria. The study revealed that nonwhite infants from lower socioeconomic status had higher incidence of false positives. It also showed that in infants with low birth weight, phenylketonuria and morbidity is associated with neonatal phenylalanine elevations. Other false positives diagnosis are attributed to morbidity, higher protein formulas, drugs, persistent but variant forms of phenylalanine elevations, and laboratory error.

Phenylalanyl-tRNA synthetase from yeast forms an enzyme · phenylalanyl adenylate complex, which has been isolated from the reaction mixture by gel filtration. The study of this complex leads to the conclusion that there are two binding sites for phenylalanyl adenylate per molecule of enzyme. No complex between enzyme and ATP could be characterised. Mg2+ is required for complex formation, but it can be replaced by monovalent cations in the transfer of phenylalanine from the complex to tRNAPhe. The kinetics of the transfer reaction show evidence that the rate-limiting step is not the release of enzyme-bound Phe-tRNAPhe but the acylation of tRNAPhe after the ternary complex phenylalanyl adenylate · enzyme · tRNAPhe has been formed. [ABSTRACT FROM AUTHOR]

The article presents a list of topics discusses in the June 1, 1971 issue of the Clinical & Experimental Immunology. Some of the topics discusses in this issue are: Acid phosphatase of macrophagal lysosomes in delayed-type hypersensitivity, Adjuvant-induced antired blood cell activity in CBA mice, Agar technique, a radioactive antiserum in, for the demonstration of immunoglobulin secretion by lymphoid cells, antiglobulin antibodies, the effect of I-phenylalanine mustard on and antigens of nasopharyngeal carcinoma.

In intact Sinapis alba seedlings the synthetic growth regulator α-chIoro-β-(3-chloro-o-tolyl)propionitrile (PRB-8.) inhibits the phytochrome-mediated anthocyanin synthesis, carotenoid synthesis and L-phenylalanine ammonia-lyase activity. This action was compared with that of other auxins and with that of α-chloro-β-(3-chloro-o-tolyl)propionitrile-homologues. From the results it appeared. that PRB-8 and some other o-tolyl derivatives are more inhibitive than other known auxins. [ABSTRACT FROM AUTHOR]

The reactions leading to cinnamic acids front phenylalanine as only substrate were investigated in organelles from. Quercus pedunculata Ehrh. roots. - "F 10000" fraction, including mitochondria and micro-bodies, catalyses the first reaction, i.e., cinnamate formation by deamination of phenylalanine. - Microsomal fraction catalyses all the steps from phenylalanine to caffeic acid via cinnamate and p-coumarate. These result suggest that microsomes are the intracellular site of the cinnamic units synthesis. The enzymes involved in these reactions, associated in the same cellular compartment, does not form a multienzyme system. The formation of caffeic acid by isolated microsomes is demonstrated for the first time; the reaction may be realised by an enzyme different from phenolase. - The free phenolic acids are the metabolically active forms. [ABSTRACT FROM AUTHOR]

Tobacco rice, carrot and tomato tissue cultures were grown in liquid media containing L-phenylalanine or L-tyrosine, or both together. The addition of these amino acids increased their respective cellular levels (4-20 fold ), but did not lower the level of chorismate mutase, an enzyme in the biosynthetic pathway of phenylalanine and tyrosine. These results indicate that the biosynthesis of phenylalanine and tyrosine in cultured plant cells is not regulated by repression of the synthesis of chorismate mutase by phenylalanine or tyrosine. The four species of tissue cultures studied here had similar enzyme specific activities four days after inoculation into fresh culture medium: tobacco 0.4, rice 0.5, tomato 0.4 and carrot 0.75 μmol prephenate formed in 30 min per mg protein. [ABSTRACT FROM AUTHOR]

Thymectomy has been found to be ineffective as a means of potentiating the immunosuppressive action of Melphalan in adult CBA-p mice challenged with A-strain mammary carcinoma transplants. This was true when the thymectomy was performed 2 days, 4 weeks or 7 weeks prior to injection of a single dose (15 mg/kg body weight) of Melphalan, administered 24 hours prior to tumour transplantation, and also when thymectomy was combined with multiple doses of Melphalan given before and after transplantation. These findings are discussed. [ABSTRACT FROM AUTHOR]

Oral phenylalanine loads were given to 15 mothers of children with cleft lip and palate defects and 19 control women. Four of the 15 mothers exhibited unusual tolerance curves, i.e., the phenylalanine levels did not rise appreciably during four hours nor did the tyrosine levels increase normally. Two of the women studied also showed a deviation from normal in the appearance of tyrosine in the blood after a tyrosine load. Attempts to stress the mothers who exhibited a normal response to the single phenylalanine load by giving them a double load were not successful. The possible implications of these findings are discussed. [ABSTRACT FROM AUTHOR]

No relationship between the phenylalanine content of the feedings prior to the PKU screening test and a positive test result (≥ 4 mg/100 ml) could be demonstrated in a study of hospital records of 68 phenylketonuric, 14 atypical, and 26 hyperphenylalaninemic infants. The mean age at testing was 62.8 hours. The median intake before testing was 383 mg of phenylalanine with a range of 0 in five cases to over 3,000 mg for two late tested infants. One quarter of the infants with positive tests had ingested less than 200 mg of phenylalanine, while only one of the 11 with negative tests had less than 200 mg. The "missed" cases were represented at each intake interval, and intakes ranged from 174 to 1,326 mg phenylalanine. Screening test results ranged from 1 to 40 mg/100 ml (mean of 10.0) with wide variation at all intervals of phenylalanine intake. There was a gradual increase in mean levels from 7.1 mg/100 ml in the group with least intake to 13.0 for those having the most. This increase was attributable mainly to the PKUs whose mean levels were higher than those of the atypicals and hypers at all intake intervals, and went much higher with increased intake. Serial tests on 20 infants who had repeat testing in the first few days of life when feeding was documented demonstrated the rapid rise in serum levels in PKUs as feeding continued in contrast to the atypicals and hypers who showed only slight increases during this period. The authors conclude there is no evidence that missed cases are significantly related to feeding protein or early testing in routine PKU screening of the newborn infant. [ABSTRACT FROM AUTHOR]

Routine Phenistix urine testing in antenatal clinics in Derby (5,000 births annually) revealed an undiagnosed case of phenylketonuria in an otherwise healthy young married woman of I.Q. 65. A synthetic diet containing 30 mg/kg/day of phenylalamine kept her blood level between 2 and 6 mg/100 ml. Pregnancy was terminated at 38 weeks, with the birth of a 1,910 gm girl whose sole physical abnormality, apart from low birth weight, was a single umbilical artery. After a transient tyrosinemia she has developed normally; her growth recovered to about the 25th percentile. Her developmental quotient at 10 months of age was 98. It may be as suggested by Auerbach that positive urine tests for phenylketones in the mother are a better guide to risk of fetal brain damage than arbitrary levels of blond phenylalanine, in that this gives a direct indication of body handling of the raised blond phenylalanine. However, we still do not know if the baby has normal intelligence because of, or in spite of, the diet. [ABSTRACT FROM AUTHOR]

Maternal hyperphenylalaninemia was induced by feeding excess L-phenylalanine to pregnant rhesus monkeys. Animals which received the phenylalanine supplement throughout the entire pregnancy gave birth to infants with low birth weight. Marked elevations of phenylalanine and tyrosine were consistently observed in maternal sera; the levels of phenylalanine and tyrosine in umbilical cord sera were greater than the corresponding maternal value in eight of nine pregnancies. Maternal and umbilical cord levels of 3-CH[sub 3]-histidine were reduced in all pregnancies. Values for the other amino acids, and the cord: maternal ratios for all amino acids, including phenylalanine and tyrosine, were within the control range. Infant monkeys born to mothers with hyperphylalaninemia demonstrated a significant reduction in learning behavior. This experiment indicates that not only is maternal hyperaminoacidemia reflected in the free amino acids of fetal blood, but an added insult is also produced by a normal placental process which functions to maintain higher levels of each of the free amino acids in the fetus than in the maternal organism. [ABSTRACT FROM AUTHOR]

Explains the interpretation of positive findings of elevated blood phenylalanine levels. Guides for the diagnosis of phenylketonuria; Description of situations typically encountered as a result of an initial findings of a positive test for phenylalanine elevation; Recommendations for long-term management of treatment of phenylketonuria.

A comparison was made of the kinetics of polysome breakdown and of the fate of RNA formed during phenylalanine starvation in both relaxed and stringent Escherichia coli cells. In the stringent strain polysome breakdown is completed within 15 min of the onset of starvation as against the 60 min required for this process in relaxed cells. In both strains phenylalanine starvation decreases the quantity of newly-formed RNA in the extract, and a constant percentage of the RNA found m the extract is always connected with the polysomes. Phenylalanine starvation does not affect the distribution of RNA between extract and pelletable membrane fractions prepared from stringent cells in relaxed cells on the other hand, most of the RNA formed during starvation remains attached to the pelletable membrane fraction The RNA released into extracts of starved relaxed cells is mainly 4 S RNA, with small quantities of 16 S RNA. All the 23 S RNA and most of the 16 S RNA formed during starvation remain connected with the pelletable membrane fraction. [ABSTRACT FROM AUTHOR]

1. Polyribosomes were broken down into 77 S monomers either by RNAase action (RNAase ribosomes) or by incubation under conditions of amino acid incorporation at 5 mM Mg2+ ("run-off ribosomes"). Various properties of these two types of monomers were compared. 2. "Run-off ribosomes" showed a high degree of poly(U)-directed phenylalanine incorporation. At 0.5 mM Mg2+ they contained a large number of particles with a sedimentation coefficient of 59 S. 3. When "run-off ribosomes" were incubated under conditions favourable for poly(U)-directed [14C]phenylalanine incorporation and then examined at 0.5 mM Mg2+, radioactivity was associated with 77 S monomers and not with 59 S particles. 4. A preparation of "run-off ribosomes" was isolated which contained a substantial amount of 77 S particles at 0.5 mM. Mg2+ after the phenylalanine incorporation directed by poly(U). Before incubation these "run-off ribosomes" contained a negligible amount of 77 S ribosomes at 0.5 mM Mg2+.. 5. It was concluded that before "run-off ribosomes" had reacted with poly(U), the sedimentation coefficient could be converted from 77 S into 59 S at 0.5 mM Mg2+. After [14C]phenylalanine incorporation directed by poly(U), however, the sedimentation coefficient remained 77 S at 0.5 mM Mg2+. [ABSTRACT FROM AUTHOR]

tRNA and an enzyme fraction which contain aminoacyl-tRNA-synthetases and transformylase, were prepared from Mycoplasma laidlawii A. After chromatography of tRNA on benzoylated-DEAE-cellulose, amino acid acceptor activities were determined. One or more species of tRNA were found for the following amino acids (number of isoaccepting species in brackets): alanine (1), arginine (2), cysteine (1), glycine (1), isoleucine (2), leucine (3), methionine (2), phenylalanine (1), serine (3), tyrosine (1), and valine (3). The methionine specific tRNA was separated into a homologously formylatable and a non-formylatable species. Cross-reactions in heterologous systems from M. laidlawii A, Escherichia coli, and yeast were studied in some detail for methionine and phenylalanine. Aminoacylation was possible in all combinations of methionine specific tRNAs and enzymes. Transformylase of mycoplasma formylates tRNA from E. coli, but none of the methionine specific tRNAs from yeast. The content of minor nucleotides in tRNA from M. laidlawii A was determined mainly from [32P]tRNA by two-dimensional thin layer chromatography. The findings are compared with those reported for other mycoplasma species and discussed in view of the genetic relationship of mycoplasma to other microorganisms. [ABSTRACT FROM AUTHOR]

The influence of the ion and water cloud shell on the structure of polyelectrolytes is very important especially for small angle studies. In the present work the change in the radius of gyration of phenylalanine specific tRNA under the influence of various counterions was investigated. tRNAPhe(yeast) was studied in different solutions at pH 7.5, each containing only one certain kind of cation. As cations lithium, cesium, barium and spermidin were used. The influence of the ions on the radius of gyration of tRNAPhe(yeast) was considerable. The value of R was 23.6 Å with Li+ ions, 26.2 Å with Cs+ ions and 26.3 Å with Ba++ ions; these resets are discussed. [ABSTRACT FROM AUTHOR]

The effect of the nature of the amino acid residue on the acceptor activity of substrates of ribosomal peptidyl transferase was investigated. We tested 2'(3')-O-aminoacyladenosines conraining the following amino acid residues: L-alanine, L-glutamine, glycine, L-3-(1-benzyl-4-imidazolyl)-alanine, L-leucine, L-lysine, L-methionine, L-methionine S-oxide, L-phenylalanine, D-phenylalanine, L-proline, L-serine and L-valine as acceptors of the acylaminoacyl residue transferred from peptidyl-tRNA. The acceptor activity of these compounds was dependent on the nature of the side chain of the amino acid residues bound to adenosine. The acceptor activity was also affected by the nature of the donor tRNA derivative. With L-acetylphenylalanyl-tRNA or L-acetylleucine npetanucleotide fragments derived from L-acetylleucyl-tRNA donating the acetylphenylalanyl and the acetylleucyl residue, respectively, a high acceptor activity was shown by the basic 2'(3')-O-L-lysyladenosine or 2'(3')-O-L-3-(1-benzyL4-imidazolyl)-alanyladenosine which acted only as weak acceptors of lysine peptides from (Lys)n-tRNA. The transfer reaction catalyzed by peptidyl transferase was stereospecific with respect to acceptor substrates. The acylaminoacyl residue was transferred from tRNA to 2'(3')-O-L-phenylalanyladenosine, whereas 2'(3')-O-D-phenylalanyladenosine, containing a D-phenylalanine residue, was completely inactive as acceptor. [ABSTRACT FROM AUTHOR]

The scattered X-ray intensities from dilute solutions of tRNAPhe (yeast) in 0.05 M Tris buffer at pH 7.5 were measured at 17°. The radius of gyration B (24.4 Å), the molecular weight M (26100) and the volume V (41500 Å3) were determined. A comparison of experimental with theoretical scattering curves of different bodies shows that the shape of the molecule cannot be well represented by a simple triaxial body. The calculated scattering curves which fit the measured scattering data best are for an elongated body with two different cross-sections. No significant change of X-ray small angle scattering was observed on increasing the temperature from 17° to 40°. At higher temperatures the radius of gyration is increased with increasing temperature and at 70° the molecule has a random coil conformation. [ABSTRACT FROM AUTHOR]

The Indian plant Glycosmic arborea (Roxb.) DC. (Rutaceae) contains alkaloids of different types. The main alkaloid of the Glycosmis leaves is the 4-quinazolone derivative arborine (VI). Previously it has been shown that labelled anthranilic acid (X) and phenylalanine (XI) are specifically incorporated into this alkaloid. This study was performed in order to elucidate the possible pathway of arborine formation in viva. During these investigations an improved procedure for the isolation of arborine was developed. The following results were obtained: After feeding of [14CH3]methionine the N-methyl group of VI was specifically labelled. The same result was obtained after the application of [14CH]3N- methylanthranilic acid. These results seem to indicate that anthranilic acid gets methylated prior to the condensation with phenylalanine which leads to VI. But on the other hand after feeding of [2-14C]glycosminine (V) labelled arborine was also isolated. However in this case the incorporation rate was much lower compared with those of the two other mentioned precursors. It should be mentioned that V is only sparingly soluble in solvents suitable for feeding experiments. The N-1 atom of VI is provided by the nitrogen of anthranilic acid. The experiment with [3- 14C,15N]phenylalanine shows clearly that with the exception of the carboxyl group the aliphatic side chain of XI is incorporated into arborine, Therefore phenylacetic acid can be excluded as an immediate precursor of VI. [U-³H]anthranoylphenylalanine (XIV) was a very poor precursor of arborine. N-Methyl-N-phenyl- [14CO-CH2] acetylanthranilamid (XV) was transformed with high efficiency in Glycosmis into arborine. However it is doubtful whether the N-phenylacetyl derivative of N-methylanthranilamide is a true precursor in arborine biosynthesis. Surprisingly it could be shown that the transformation of XV →VI takes also place in pea plants. Apparently the ring closure of XV is catalyzed by an enzym or enzymatic system which is unspecific and widely distributed. [ABSTRACT FROM AUTHOR]

Adenosine 5'-O-(1-thiotriphosphate) was incorporated in tRNAPhe-C-C by C-C-A pyrophosphorylase. The resulting tRNAPhe-C-C&Smacr; A could be charged with phenylalanine by aminoacyltRNA synthetase and pyrophosphorolyzed with C-C-A pyrophosphorylase. Degradation with snake venom phosphodiesterase, however, was markedly reduced. Adenosine 5'-O-(1-thiotriphosphate) did not substitute for ATP in the activation of phenylalanine by aminoacyl-tRNA synthetase, but was a competitive inhibitor. [ABSTRACT FROM AUTHOR]

The behavior of N-acetylphenylalanyl-tRNA as an initiator of phenylalanine polymerisation in an Escherichia coli cell-free system shows some analogy with the behavior of formylmethionyl- tRNA during the synthesis of natural proteins. At low Mg++ concentrations, polyphenylalanine chain initiation depends on N-acetylphenylalanyl-tRNA and the same initiation factors which are required for protein chain initiation with formylmethionyl-tRNA. However in a rabbit reticulocyte cell-free system, N-acetylphenylalanine is not incorporated into polyphénylalanine at any Mg++ concentration. In this paper, we showed that : a) N-acetylphenylalanyl-tRNA did bind to reticulocyte ribosomes in the presence of poly U. This binding was GTP dependent and needed a factor which was extracted by washing ribosomes with salt (NH4CI 1 M); b) the bound N-acetylphenylalanyl-tRNA was partially released by puromycin or by addition of phenylalanyl-tRNA. In this last case, incorporation of N-acetylphenylalanine into polyphenyl- alanine was noticed (ratio N-acetylphenylalanine/phenylalanine = 1/20); c) the ribosomal wash fluid also greatly enhanced phenylalanyl-tRNA binding to salt- treated ribosomes, in the presence of poly U and GTP. The maximum binding was obtained with the same concentrations of Mg++ and K+ as for N-acetylphenylalanyl-tRNA binding. It was not possible to separate on a DEAE-cellulose column the factors that were required for phenyl- alanyl-tRNA and acetylphenylalanyl-tRNA binding. On the other hand, a preparation of the binding enzyme from reticulocyte supernatant enhanced both phenylalanyl-tRNA and acetyl- phenylalanyl-tRNA binding to ribosomes; d) the optimum Mg+÷ concentration for phenylalanine polymerisation was higher with salt- washed ribosomes than with sucrose-washed ribosomes. Adding the wash fluid shifted the Mg++ optimum back to the lower value. N-acetylphenylalanyl-tRNA was not required for this shift as noticed in Escherichia coli. One likely explanation of our results with reticulocytes is the possibility that the ‘binding enzyme’ of Schweet et al., which was present in our ribosome wash fluid, could work with both N-acetyl as well as with phenylalanyl-tRNA. Thus, the inability to incorporate N-acetylphenyl- alanine might be due to a competition between these two compounds for the same enzyme and the same ribosomal site. If this is indeed the case, it is in marked contrast to results obtained with Escherichia coti and could reflect a basic difference in the mechanism of protein chain initiation. [ABSTRACT FROM AUTHOR]

The effect of actinomycin D and DL-p-fluorophenylalanine on ribosome formation has been studied in exponentially growing Tetrahymena. cells. Both compounds cause inhibition of ribosome formation. However, ribosome formation is qualitatively very similar in inhibited and uninhibited cells. p-Fluorophenylalanine is incorporated into ribosomal proteins of completed ribosomal particles when used in inhibitor concentration (1 mg/ml). This demonstrates that limited modifications of ribosomal proteins, due to the presence of the amino acid analogue in the ribosomal polypeptide chains still allow the modified proteins to assemble with ribosomal RNA to give intact ribosomal particles. The p-fluorophenylalanine containing ribosomes have normal physical-chemical properties with respect to dissociation into sub-units by dialysis against low Mg++ concentration, sedimentation values in sucrose gradients and buoyant density in isopycnic CsCl gradients. The regulatory aspects of ribosome biosynthesis in the light of the present results are discussed. [ABSTRACT FROM AUTHOR]

The activity of rat liver phenylalanine hydroxylase has been measured in systems which, in addition to the components necessary for hydroxylation, contained ascorbic acid and catalase, 6-Methyl-tetrahydropterine or the corresponding 6,7-dimethyl compound was used as tetrahydropterine cofactor. Simple kinetic experiments lead to the determination of rate constants for the following reactions: the aerobic oxidation of the tetrahydropterine cofactor, the reduction of the dihydropterine cofactor by ascorbic acid and other reductants and the conversion of the dihydropterine cofactor from an active to an inactive form. These rate constants are used for the calculation of the actual concentration of tetrahydropterine cofactor in the hydroxylase system and for correction of the measured rates of hydroxylation for the inactivation of the dihydropterine cofactor. Ascorbic acid, as well as other reductants that may be present, increase the instability of phenylalanine hydroxylase, but this effect is negligible at sufficiently high catalase concentrations. Certain non-reducing inhibitors can also stabilize the enzyme. The initial rate of reaction at 30° is linear during the first 5 or 6 min. In contrast at temperatures of 25° and below there is a lag period, whose duration may be reduced by preincubation of the enzyme with tetrahydropterine cofactor. A similar preincubation of the enzyme with L-phenylalanine seems to activate the hydroxylase briefly. [ABSTRACT FROM AUTHOR]

An investigation has been made of the acid soluble protein fractions of wool that has been reduced with benzylmercaptane and treated with methyliodide. When this modified wool was shaken in dilute formic acid (50 v/v) at room temperature for 1 hour, 7% of a protein material was dissolved. After dialysis and subsequent lyophylisation the protein material was isolated, and shown to be heterogeneous by paper electrophoresis. Fractionation of this protein on Sephadex G-75 in dilute acetic acid gave more than six components of relatively high content of tyrosine and phenylalanine. One of these fractions, obtained in good yield, was purified by rechromatography. For further separation the protein fraction was subjected to ion exchange chromatography on Dowex 1 (×2) columns. By stepwise elution with buffers of decreasing pH-values, containing acetic acid and 8 M urea, four subfractions were obtained. The amino acid compositions of reduced wool, of the protein obtained by formic acid extraction, of the fraction after gel filtration on Sephadex G-75 and of one subfraction on Dowex 1 (×2) are given. In all three fractions an increase in glycine, tyrosine and phenylalanine content was observed with relatively low amounts of basic and acidic amino acids. The subfraction obtained after the final step of separation on Dowex 1 (×2) was further characterised by endgroup determinations. Dnp­glycine was the only N-terminal amino acid with traces of Dnp­alanine as contaminant. Hydrazinolysis resulted in tyrosine on the C-terminus. After digestion of the subfraction by trypsin and chymotrypsin a number of peptides with different chain lengths was obtained, indicating that, tyrosyl residues had accumulated and that larger arginyl­peptides were present. Sedimentation measurements by ultracentrifugation indicated that the subfraction had a weight average of apparent molecular weight in the range of 9,000 to 13,000. This is in agreement with the molecular weight of 16,400 derived from the amount of Dnp­glycine and the calculated one from the amino acid composition. The analytical results show that the subfraction represents a single polypeptide chain containing only one N-terminal residue and about hundred aminoacids of which about fifty are glycine, tyrosine and phenylalanine. [ABSTRACT FROM AUTHOR]

By mild treatment of tRNAyeastPhe with acid (pH 2.9, 37°, 3–4 hours) the base Y+, which according to RajBhandary, Khorana et al. is located right next to the anticodon, can be excised without breaking the chain. Y+ and the acid treated tRNAPhe (=tRNAHClPhe) were characterized and the conversion of tRNAPhe to tRNAHClPhe was investigated in detail. Y+ is a rather hydrophobic compound of unknown structure. Y+ and ribose were found when the nucleoside Y was treated with acid. Some physical and chemical properties of Y+ are reported. Y+ can be isolated easily in pure form from oligonucleotides, tRNAyeastPhe, or even unfractionated tRNAyeast (not tRNAcolt) by mild acid treatment and subsequent extraction into chloroform. tRNAHClPhe and tRNAPhe have very similar molecular sizes and shapes since their Sephadex elution profiles were overlapping. The facile separation of tRNAPhe and tRNAHClPhe by countercurrent distribution, on columns of methylated albumin on Kieselgur or benzoylated DEAE­cellulose is due, at least in part, to the removal of a hydrophobic residue from an exposed region of the tRNAPhe. tRNAHClPhe can be charged with Phe to the same extent as tRNAPhe. Since also the Michaelis constant of phenylalanyl tRNA synthetase is the same for both tRNAs (1.5 × 10-7 M) and the maximal velocity of charging is decreased by only one third in tRNAHClPhe, it can be concluded, that Y and probably the whole anticodon loop are not essential parts of the synthetase recognition region of tRNAPhe in homologous charging reactions. It was found that tRNAHClPhe and tRNAPhe from which the —CpCpA endgroups had been removed do not differ in their acceptor capacities for CTP and ATP. In the ribosome systems, Phe-tRNAHClPhe does not bind to poly (U) or other polynucleotides and does not transfer its amino acid onto growing peptide chains. The complete loss of codon recognition is attributed largely to a change in the conformation of the anticodon loop. The ultraviolet spectra of an Y-containing hexanucleotide and of its acid conversion products are in agreement with the notion of a strong interaction between Y+ and its neighbouring bases, as are the rate differences in the liberation of Y+ from Y [1.5 times faster] and the hexanucleotide, respectively. The rate of tRNAHClPhe formation, as measured by methylated albumin on Kieselgur­column chromatography and binding assays, is the same as the rate of Y+-liberation from the hexa­nucleotide. The acid induced conversion of tRNAPhe to tRNAHClPhe is a completely selective reaction affecting Y as the only nucleoside of tRNAPhe. [ABSTRACT FROM AUTHOR]

We isolated a mixture of glyco­peptidolipids (mycosides C) from a non­photochromogenic atypical strain of Mycobacteria. The main component, mycoside C1217, contains a 3 D-hydroxyacid with 28 carbons, two 6-deoxysugars and four amino compounds. The two sugars are 6-deoxytalose and trimethylrhamnose (a minor component contains 3,4-dimethylrhamnose instead of trimethylrhamnose). The four amino compounds are three aminoacids, phenylalanine, allo­threonine and alanine, plus an aminoalcool identified with alaninol by gas chromatography of the N­trifluoroacetyl derivative. The phenylalanine is N-terminal in the peptide, and linked to the fatty acid by an amide bond. We established, by Β-elimination in alkaline medium, that the deoxytaiose is linked to the hydroxyl of the allo­threonine by a glycosidic bond. After a mild hydrolysis of the mycoside, we isolated a glycopeptide containing allo­threonine, alanine, alaninol and trimethylrhamnose. By use of the dimethylamino-naphtalene sulfonyl method and of Edman degradation, we have shown that the sequence of this glycopeptide is: allo­threonyl­alanyl­alanimol­trimethylrhamnose All these results, along with those obtained from some other reactions, support the following structure for the mycoside C1217, which is in agreement with mass spectrometric results: C25H51-CHOH-CH2-CO-Phe-αThr-Ala-alaninol-O-Me3Rha O-(Ac2-6-deTal) [ABSTRACT FROM AUTHOR]

Poly U-directed stimulation of preincubated calf lens ribosomes revealed that these particles from a mammalian source have a phenylalanine incorporation ability comparable to the Escherichia coli system. A search was made for optimal conditions. After variation of the magnesium concentration during preincubation two levels of maximal incorporation were observed. The region of highest relative stimulation did not coincide with these maxima. Preincubation under conditions of optimal amino acid incorporation yields 80S monomers and to a lesser extent 120S particles. No smaller units or breakdown products were observed in the analytical ultracentrifuge. Such particles could be demonstrated after prolonged dialysis of either polysomes or 80S monomers against 0.1 mM MgC12. Their sedimentation coefficients were 50S and 30S respectively but not 60 and 40 S. [ABSTRACT FROM AUTHOR]

Within the last few years, the quinoline alkaloids viridicatine (I), viridicatol (II) and 3-O-me thyl viridicatine (III) have been isolated from the mycelium and culture medium of Penicillium cyclopium Westling, Penicillium puberulum Bainier and Penicillium viridicatum Westling. Two further alkaloids found in the culture medium of P. cyclopium und P. viridicatum, cyclopenine (IV) and cyctopenol (V) are benzodiazepine derivatives. We have shown, using labelled precursors with P. viridicatum strain V41 and P. cyctopium strain SM72 that cyclopenine, cyclopenol, viridicatine and viridicatol are synthesised from anthranilic acid and phenylalanine. These results, together with the easy conversion of cyclopenine and cyclopenol into viridicatine and viridicatol respectively by means of acid or alkali, suggested that an in vivo relationship could exist between these two groups of alkaloids. in this paper we show that cyclopenine and cyclopenol are intermediates in the formation of viridicatine and viridicatol and that the transformation of the former to the latter is effected by an enzyme which we have named cyctopenase. This transformation occurs with fresh homogenised mycelium with dried lyophilised mycelium and with mycelium free extract, that with the dead mycelium being much faster than with the other preparations. Using the mycelium free extract it was shown that the "agent of activity" depends upon pH, is not dialysable and can be destroyed by heating at 60° for 3 min, thus indicating the enzymatic nature of the transformation. Additional products of the transformation are carbon dioxide and methylamine. Cyclopenase activity is shown to be strongest in the surface cultured mycelium of P. viridicatum, strain V41. In the surface cultured P. cyclopium, strain SM72 mycelium the activity was much tess and in the submerged culture of both strains practically no activity was detected. This agrees with the observation that, under normal conditions only P. viridicatum strain V41 produces an appreciable amount of viridicatine and viridicatol. These experiments, together with previous work, indicate that anthranilic acid and a phenylpropane (e.g. phenylalanine) combine to give the benzodiazepines, cyclopenine and cyclopenol and these are transformed by the enzyme cyclopenase to the quinoline derivatives viridicatine and viridicatol. The detailed mechanism of this transformation is being investigated. [ABSTRACT FROM AUTHOR]

We have studied the repression of the first enzymes of the main branch and the pathways after the branchpoint in the aromatic biosynthesis of Saccharomyces cerevisiae. Anthranilate synthetase is repressed by L-tryptophan and prephenate dehydratase by L-phenylalanine. 3-Deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP) synthetases are not repressed by L-phenylalanine, L-tyrosine and L-tryptophan, chorismate mutases are repressed neither by L-phenylalanine nor by L-tyrosine, and prephenate dehydrogenase shows no repression by L-tyrosine. L-Tryptophan exhibits an induction of the chorismate mutase. Besides chorismate mutase is activated by L-tryptophan. L-Phenylalanine exhibits an induction and also an activation of the prephenate dehydrogenase. By simultaneous addition of L-tryptophan, L-phenylalanine, and L-tyrosine p-aminobenzoic acid was accumulated and the production of p-aminobenzoate synthetase was induced. The tyrosine-sensitive and phenylalanine-sensitive isoenzymes of DAHP synthetase belong to the V-system of the allosteric enzymes. Chorismate mutase and prephenate dehydrogenase are members of the K-system. In an earlier publication we have discussed the feedback inhibition of the described enzymes. In this paper we discuss the whole events of the regulation phenomena in Saccharomyces cerevisiae. We also considered the degradation of L-tryptophan, L-phenylalanine, and L-tyrosine to tryptophol, phenylethanol, and tyrosol respectively in connection with the regulatory events. [ABSTRACT FROM AUTHOR]

A synergistic coupling was observed for ligands binding to the specific sites for L-phenyl-alanine and ATP. Dissociation constants were measured by the use of fluorescence titration and equilibrium dialysis techniques. It is found that L-phenylalaninol, L-phenylalanine methylester, and phenylethylamine bind with an increased affinity when ATP, ADP, AMP or adenosine were present. With L-phenylalanine, synergistic binding was only observed for adenosine; ATP has probably no effect. Results of competition experiments, the stoichiometry of complex formation and the magnitude of dissociation constants indicate that synergistic coupling involves the binding sites for ATP and amino acid at the active site. [ABSTRACT FROM AUTHOR]

C-peptide from the duck was isolated and its ammo acid sequence determined. The duck C-peptide contains 26 amino acids and differs substantially from the known mammalian C-peptides. It is the first C-peptide shown to contain the amino acids histidine, tyrosine and phenylalanine, The sequence was given. A comparison of duck C-peptide with the known mammalian C-peptides shows that the terminals are homologous and that the five invariable positions of the nine known mammalian C-peptides can be retained by means of four deletions in the duck C-peptide. [ABSTRACT FROM AUTHOR]

The physical and biological properties of two new poly(U) analogs, poly(2-thiouridylic acid) or poly(s2U) and poly(2,4-dithiouridylic acid) or poly(s2s4U) have been investigated. Ultraviolet absorption and circular dichroism spectra of poly(s2U) and poly(s2s4U) as well as those of the corresponding monomers were measured. Absorption-temperature and absorption-pH profiles of both poly(s2U) and poly(s2s4U) displayed sharp cooperative transitions. The phase transition diagrams of poly(s2U) and poly(s2s4U) show the high stability of their secondary structures. Neither poly(s2U) nor poly(s2s4U) formed helical complexes with poly(A). It was concluded from the corresponding phase transition diagrams that the reason for this behaviour is the larger thermodynamical stability of the poly(s2U) or poly(s2s4U) helices over the hypothetical complexes poly(s2U) · poly(A) or poly(s2s4U · poly(A). Sedimentation velocity analysis of poly(s2U) and poly(s2s4U) revealed sedimentation coefficients s20, w of 28 S and 12 S, respectively. A more detailed analysis of poly(s2U) lead to the conclusion that poly(s2U) exists as an intra-molecular associate of two anti-parallel poly(s2U) strands in the form of a large hairpin. Consistent with their physical properties, poly(s2U and poly(s2s4U) are not able to replace poly(U) as messengers in the cell-free synthesis of poly(phenylalanine). Poly(s2U) is only poorly, poly(s2s4U) not at all phosphorolyzed by polynucleotide phosphorylase. [ABSTRACT FROM AUTHOR]

1. A cell-free protein-synthesising system has been prepared from the liver of adult male Xenopus laevis which shows several adaptive changes following induction of egg-yolk protein synthesis by administration of oestradiol-17β in vivo, 2. The product of the cell-free system from oestrogen-treated animals is characterised by elevated incorporation of radioactive serine relative to phenylalanine. This is taken as an indication of the synthesis of phosvitin, which has a high serine content, in vitro. 3. Increased protein-synthetic activity of Xenopus liver ribosomes following oestrogen treatment can be demonstrated and is maintained after the particles are washed in 0.5 M KCl. This effect of the hormone is also apparent when the ribosomes are first stripped of endogenous mRNA by preincubation and then added to a fresh cell-free system containing poly (U). It is concluded that oestrogen treatment produces an intrinsic activation of synthetic ability of ribosomes in the liver. 4. Increased ability of ribosomes to translate poly(U) does not result from changes in their ability to bind the polynucleotide, either before or after preincubation. 5. Cell sap from liver of oestrogen-treated animals shows increased ability to support amino acid incorporation by ribosomes from control or treated animals in vitro. This is not due to enhanced initiation of peptide synthesis or to increased availability of endogenous aminoacyl- tRNA in vitro. 6. The effects of oestradiol on Xenopus liver are considered in relation to the translational control of protein synthesis by hormones and other agents in a variety of systems. [ABSTRACT FROM AUTHOR]

On oxidizing wool by performic acid a soluble protein fraction is obtained containing high amounts of tyrosine, phenylalanine and glycine. This fraction, termed “δkeratose”, is extremely heterogeneous as shown by solubility characteristics, electrophoresis and end-group determination. Anion-exchange chromatography allows isolation of subfractions with different amino-acid compositions. This process, however, involves a marked loss of tyrosine-rich proteins absorbed irreversibly. Repeated preparative electrophoresis on polyvinylchloride layers permits isolation of many subfractions on a preparative scale without loss of aromatic amino acids. The aminoacid composition of these subfractions is investigated. In spite of the wide differences among them some common features for all fractions can be established. It is shown that possible contamination by other proteins is not responsible for the heterogeneity of δ-keratose but rather due to a wide variation in the anfino-acid composition among the demonstrated tyrosine-rich proteins. [ABSTRACT FROM AUTHOR]

The purification of a tyrosinase from cell-free extracts of Streptomyces glaucescens is described. The enzyme obtained is homogeneous according to the criteria of ultracentrifuge analysis and polyacrylamide gel electrophoresis. An average molecular weight of 29100 was determined by dodecylsulfate-gel electrophoresis and by sedimentation equilibrium analysis in the ultracentrifuge. The amino acid composition of the pure enzyme is also reported. The enzyme contains one atom of copper in the cuprous form per molecule mid is strongly inhibited by KCN and 4-nitro-catechol. The enzyme shows optimal activity at pH 6.8; it has an isoelectric point at pH 6.95 and is most stable at pH 8.0. Variations of Km values between pH 5.0 and 8.9 when L-tyrosine methylester and 3,4-dihydroxy-L-phenylalanine, were used as substrate was interpreted by assuming that different ionizable groups are involved in the oxidation of these two substrates. The Michaelis-Menten constants and kcat are strongly dependent on the oxygen concentration for 3,4-dihydroxy-L-phenylalanine and almost, independent, for L-tyrosine methyl ester. Kinetic parameters obtained for a series of mono- and o-diphenol substrates are also reported. [ABSTRACT FROM AUTHOR]

We have designed a method for the large scale isolation of pure and highly active subunits from yeast cytoplasmic ribosomes. The method consists of the incubation of yeast, protoplasts in the presence of 1 mM NaN3. This causes the disappearance of polysomes and the formation of 80-S ribosomes that can be dissociated in 0.5 M KCl. In this way over 90% of the ribosomes can be dissociated into subunits. The subunits are isolated by centrifugation in a B-XV zonal rotor using an equivolumetric sucrose gradient. When this separation is performed at 5 °C, dimerization of 40-S particles to 65-S particles occurs and no pure subunits can be obtained, At 10 °C, there is no dimerization and the 60-S + 40-S subunits obtained are less than 1% cross-contaminated. In poly(U)-directed protein synthesis one subunit pair polymerizes phenylalanine residues at a rate of 1.7 residue per min. About 70% of the subunits were estimated to participate in this synthesis. Subunits prepared in the absence of dithiothreitol still bound phenylalanyltRNA, but, were inactive in phenylalanine polymerization. The 60-S subunits prepared without dithiothreitol were as active as those prepared in the presence of dithiothreitol when their peptidyltransferase activity was tested. [ABSTRACT FROM AUTHOR]