Assembly Of Polypeptide Chains Research Articles

Identification of fibrillogenic regions in human triosephosphate isomerase.

Background. Amyloid secondary structure relies on the intermolecular assembly of polypeptide chains through main-chain interaction. According to this, all proteins have the potential to form amyloid structure, nevertheless, in nature only few proteins aggregate into toxic or functional amyloids. Structural characteristics differ greatly among amyloid proteins reported, so it has been difficult to link the fibrillogenic propensity with structural topology. However, there are ubiquitous topologies not represented in the amyloidome that could be considered as amyloid-resistant attributable to structural features, such is the case of TIM barrel topology.Methods. This work was aimed to study the fibrillogenic propensity of human triosephosphate isomerase (HsTPI) as a model of TIM barrels. In order to do so, aggregation of HsTPI was evaluated under native-like and destabilizing conditions. Fibrillogenic regions were identified by bioinformatics approaches, protein fragmentation and peptide aggregation.Results. We identified four fibrillogenic regions in the HsTPI corresponding to the β3, β6, β7 y α8 of the TIM barrel. From these, the β3-strand region (residues 59–66) was highly fibrillogenic. In aggregation assays, HsTPI under native-like conditions led to amorphous assemblies while under partially denaturing conditions (urea 3.2 M) formed more structured aggregates. This slightly structured aggregates exhibited residual cross-β structure, as demonstrated by the recognition of the WO1 antibody and ATR-FTIR analysis.Discussion. Despite the fibrillogenic regions present in HsTPI, the enzyme maintained under native-favoring conditions displayed low fibrillogenic propensity. This amyloid-resistance can be attributed to the three-dimensional arrangement of the protein, where β-strands, susceptible to aggregation, are protected in the core of the molecule. Destabilization of the protein structure may expose inner regions promoting β-aggregation, as well as the formation of hydrophobic disordered aggregates. Being this last pathway kinetically favored over the thermodynamically more stable fibril aggregation pathway.

Conformational Parameters for Amino Acids in Helical, β-Sheet, and Random Coil Regions Calculated from Proteins: After 40 Years

Forty years ago, Peter Y. Chou and Gerald D. Fasman (1974a), relying on the information from fifteen proteins calculated α helix, β-sheet, and coil conformational parameters, Pα,Pβ,and Pc,for the 20 naturally occurring amino acids from the frequency of occurrence of each amino acid residue in the α, β, and coil conformations. Secondary structure of these 15 proteins had been determined by X-ray crystallography. Although the accuracy could not go over to the level of 60% too much, these values utilized for a long time to provide a simple procedure, devoid of complex computer calculations, to predict the secondary structure of proteins from their known amino acid sequences. In the same article of Peter Y. Chou and Gerald D. Fasman, a detailed analysis of the helix and β-sheet boundary residues in proteins provided amino acid frequencies at the N-and C-terminal ends which were used to delineate helical and β regions. Charged residues are found with the greatest frequency at both helical ends, but they were mostly absent in β-sheet regions. In the same article a mechanism of protein folding was proposed, whereby helix nucleation starts at the centers of the helix where the Pα values are highest, and propagates in both directions, until strong helix breakers where Pα values are lowest, terminate the growth at both ends. Similarly, residues with the highest Pβ values will initiate β regions and residues with the lowest Pβ values will terminate β regions. The helical region with the largest Pα was proposed as the site of the first fold during protein renaturation. The mechanism whereby proteins fold into their native conformation, capable of biological activity, has been a long sought after goal. With the elucidation of the three-dimensional structure of many proteins through X-ray crystallography, a new momentum has been given to understanding the factors governing this complex assembly of polypeptide chains. In this paper, using similar statistics from 20 347 proteins, the level of reliability of formerly found results is discussed.

Phase diagram of polypeptide chains

We use a coarse grained protein model that enables us to determine the equilibrium phase diagram of natively folded α-helical and unfolded β-sheet forming peptides. The phase diagram shows that there are only two thermodynamically stable peptide phases, the peptide solution and the bulk fibrillar phase. In addition, it reveals the existence of various metastable peptide phases. The liquidlike oligomeric phases are metastable with respect to the fibrillar phases, and there is a hierarchy of metastability. The presented phase diagram provides a solid basis for understanding the assembly of polypeptide chains into the phases formed in their natively folded and unfolded conformations.

Assembly of the Asparagine- and Glutamine-Rich Yeast Prions into Protein Fibrils

The proteins Ure2, Sup35 and Rnq1 from the baker's yeast have infectious properties, termed prions, at the origin of heritable and transmissible phenotypic changes. It is widely believed that prion properties arise from the assembly of Ure2p, Sup35p and Rnq1p into insoluble fibrils. Yeast prions possess regions crucial for their propagation that can be either N- or C-terminal. These regions have unusual amino acid composition. They are very rich in glutamine and asparagine residues and resemble in that to huntingtin, a protein involved in the neurodegenerative Huntington's disease. Yeast prions assembly process has been hypothesized to be the consequence of the properties of glutamines and asparagines to engage in polar protein-protein interactions, termed polar-zippers. While this can certainly occur under certain conditions, glutamine and asparagine residues can establish other kinds of interactions with a variety of amino acid residues thus mediating protein-protein interactions involved in the assembly of polypeptide chains into high molecular weight oligomers. This review details the interactions that can be established by glutamine and asparagine residues that may allow a better understanding of their role in mediating protein-protein interactions and prion propagation.

Liquid-phase peptide synthesis on polyethylene glycol (PEG) supports using strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group: application of PEGylated peptides in biochemical assays.

Stepwise synthetic assembly of polypeptide chains reversibly linked to polyethylene glycol represents a hybrid between traditional solution and solid-phase chemistries and combines the inherent advantages of both approaches. The technical simplicity and scalability of the liquid-phase peptide synthesis method renders it particularly attractive for multiple parallel syntheses, combinatorial approaches and the large-scale preparation of peptides. The versatile protection strategy based on the N alpha-fluorenylmethoxycarbonyl group commonly used in solid-phase peptide synthesis was adapted to the liquid-phase approach. Fluoride ions were used rather than the conventional organic base piperidine for the repetitive amino-deprotection step. Using a range of acid- and base-labile linkers between the polymer and the peptide, it was shown that free and fully side-chain protected peptides can be obtained using our version of the liquid-phase peptide synthesis method. Protocols for simultaneous multiple syntheses requiring a minimum of equipment are presented and the use of polyethylene glycol-bound peptides in biochemical binding and functional assay systems is demonstrated.

Coupling Endoplasmic Reticulum Stress to the Cell Death Program

The endoplasmic reticulum (ER) is the site of assembly of polypeptide chains destined for secretion or routing into various subcellular compartments. It also regulates cellular responses to stress and intracellular Ca(2+) levels. A variety of toxic insults can result in ER stress that ultimately leads to apoptosis. Apoptosis is initiated by the activation of members of the caspase family and serves as a central mechanism in the cell death process. The present study was carried out to determine the role of caspases in triggering ER stress-induced cell death. Treatment of cells with ER stress inducers such as brefeldin-A or thapsigargin induces the expression of caspase-12 protein and also leads to translocation of cytosolic caspase-7 to the ER surface. Caspase-12, like most other members of the caspase family, requires cleavage of the prodomain to activate its proapoptotic form. Caspase-7 associates with caspase-12 and cleaves the prodomain to generate active caspase-12, resulting in increased cell death. We propose that any cellular insult that causes prolonged ER stress may induce apoptosis through caspase-7-mediated caspase-12 activation. The data underscore the involvement of ER and caspases associated with it in the ER stress-induced apoptotic process.

The Role of ERp57 in Disulfide Bond Formation during the Assembly of Major Histocompatibility Complex Class I in a Synchronized Semipermeabilized Cell Translation System

We have established a semipermeabilized cell system that reproduces the folding and assembly of a major histocompatibility complex (MHC) class I complex as it would occur in the intact cell. The translation of the MHC class I heavy chain (HLA-B27) in this system was synchronized allowing the folding and assembly of polypeptide chains synthesized within a short time frame to be analyzed. This has enabled us to dissect the time course of interaction of both disulfide and nondisulfide-bonded heavy chain with various molecular chaperones during its assembly in a functionally intact endoplasmic reticulum. The results demonstrate that unassembled, nondisulfide-bonded forms of heavy chain interact initially with calnexin. A later and more prolonged interaction of calreticulin, specifically with assembled, disulfide-bonded heavy chain, highlights distinct differences in the roles of these two proteins in the assembly of MHC class I molecules. We also demonstrate that the thiol-dependent reductase ERp57 initially interacts with nondisulfide-bonded heavy chain, but this rapidly becomes disulfide-bonded and indicates that heavy chain folding occurs during its interaction with ERp57. In addition, we also confirm a direct interaction between MHC class I heavy chain and tapasin, emphasizing the role that this protein plays in the later stages of MHC class I assembly.

Kinetics of Folding of Leucine Zipper Domains

Leucine zippers are short coiled coils frequently found in transcription factors where they serve as dimerization domains. The basic features contributing to the thermodynamic stability of leucine zippers are well understood, but very little is known about their folding kinetics. Leucine zippers have a simple and well defined structure and are, therefore, excellent models for the study of the concerted folding and assembly of polypeptide chains. Here we report on a fluorescence stopped flow investigation of the kinetics of association and dissociation of a series of model leucine zippers based on the common sequence Xzero-EYEALEKKLAAX1EAKX2QALEKKLEALEHG-amide (Xzero = N alpha-acetyl, N alpha-fluorescein-GGG, or N alpha-dimethylaminocoumarin-GGG; Xl = Leu or Ala; X2 = Leu, Ala, or Asn). When Xzero is fluorescein, self-quenching between adjacent fluorophores leads to a decrease in fluorescence emission intensity whereas unfolding of the coiled coil leads to an increase. In a heteromeric coiled coil containing both fluorophores, resonance energy transfer between the donor coumarin and the acceptor fluorescein is observed, and the mixing of labeled and nonlabeled peptides allows the measurement of the rates of strand exchange between leucine zippers. Exchange rates do not depend on peptide concentration, indicating that strand exchange is governed by the rate of dissociation of the coiled coil. Strand exchange between leucine zippers with X1 and X2 = Leu occurs with a half-time of approximately 30 min. A single Leu/Ala substitution at X1 or X2 decreases the half-time to approximately 1 s. Folding was also studied in a relaxation experiment in which a preexisting equilibrium between monomeric chains and coiled coils was rapidly disturbed by dilution with buffer, and the relaxation to the new equilibrium was followed by the increase in fluorescence. In peptides with X1, X2 = Ala or X1 = Ala, X2 = Asn the folding process can be described by a simple two-state monomer<-->dimer equilibrium with k(on) approximately 4 x 10(6) M-1 s-1 and k(off) approximately 10 s-1. Kd = k(off)/k(on) approximately 2.5 microM is in good agreement with the value of Kd obtained from equilibrium measurements. The peptides with a single Ala at X1 or X2 exhibit biphasic folding kinetics. One phase is concentration dependent and the other apparently concentration independent. This behavior can be interpreted as a monomer<-->dimer equilibrium coupled to an equilibrium between different conformational isomers. Leu to Ala and Leu to Asn substitutions in the hydrophobic core alter the folding kinetics in a position-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of age and restricted feeding on polypeptide chain assembly kinetics in liver protein synthesis in vivo

Polypeptide assembly rates during in vivo hepatic protein synthesis were studied as a function of age and restricted feeding in male rats. With ageing the time to assemble the average peptide in the liver of fully-fed rats significantly increased. In young rats maintained on a restricted feeding regime known to retard ageing, the time to assemble the average polypeptide was increased 2.5 times. With ageing the rate of peptide elongation increased so that at 2 years of age the underfed animals assembled peptides at a significantly faster rate than their age-matched controls. The rate of elongation of peptides during hepatic protein synthesis was shown to be directly dependent upon circulating T 3 levels rather than the dietary status of the animal. On refeeding young diet restricted rats, polypeptide assembly kinetics did not immediately return to control values although the rate of protein synthesis was significantly increased. Total liver RNA content increased significantly in refed animasl allowing for a greater rate of chain intiation to offset the slow rate of chain elongation. A period of 28 days of ad libitum feeding was required before assembly kinetics returned to control values and is probably indicative of a persistent impaired monodeiodination of T 4 to T 3.

L-leucine transport in liver of Antarctic fish in vivo at 0 degrees C.

Uptake of L-[14C]leucine by liver of an endemic Antarctic fish, Trematomus hansoni, was studied by a single injection technique with [3H]inulin as a reference. Rate constants for leucine influx and efflux and incorporation into liver protein were determined by analysis of isotope distribution in the free and protein-bound compartments of liver and in blood draining the liver at various times after injection. Transport rates were slower than in temperate fish at 20 degrees C, but saturation properties and ability to accumulate leucine in liver were comparable. Kinetic analysis indicated that 30% of uptake at 0 degrees C was due to active transport, similar to that in toadfish at 20 degrees C. This contrasts with the absence of this component in toadfish cooled to 10 degrees C. Average polypeptide chain assembly time was 19 min at 0 degrees C. Transport functions were maintained in fish warmed to 10 degrees C; however, protein synthesis declined at temperatures above 5 degrees C. The results indicate this system is adapted to function at extremely low temperatures in a manner qualitatively similar to organisms adapted to much higher temperatures. Transport and synthetic rates, however, were low, consistent with a normal temperature dependency (Q10 about 2.5) for biological reaction rates.

EFFECTS OF TEMPERATURE EXTREMES ON PROTEIN SYNTHESIS IN LIVER OF TOADFISH,OPSANUS TAU, IN VIVO

Amino acid incorporation and polypeptide chain elongation rates were determined in toadfish at the upper and lower ends of their range of temperature tolerance. The method was based on pulse injection of radioactive amino acids into the hepatic portal vein and analysis of ribosome-bound and completed chains at various times after injection. Elongation rates at low temperatures were obtained from the rate of completion and release of polypeptide chains pre-labeled at 20°C. Average polypeptide chain assembly time at 4°C was 6 h; that at 37°C was 1 min. Comparison with rates of total protein synthesis obtained in previous studies indicate a coordinated slowing of all protein synthetic reactions at low temperatures. A 10-fold decline in elongation rate from 7°C to 4°C suggests a specific temperature sensitivity in elongation factors or in formation of aminoacyl-tRNA. At high temperatures (32°-40°C) protein synthetic reactions show a loss of coordination, with elongation rate increasing normally (Q10 about 2) ...

Changes in the Polypeptide Assembly of Guinea Pig Thyroglobulin Induced by Thyrotropin-Regulated Thyroid Activity*

We have previously reported that the relative proportion of three polypeptide chains in guinea pig thyroglobulin is closely related to the iodine content of the protein. The present work demonstrates that it is not the iodine content per se but, rather, TSH-regulated thyroid activity which modulates the substructure of thyroglobulin. In a first set of experiments, the impact of TSH stimulation on sodium dodecyl sulfate (SDS)-induced dissociation of 19S thyroglobulin into 12S subunits was compared to that of iodination. While in control animals the ratio of 12S to 19S thyroglobulin was 48:52, it changed to 35:65 in glands strongly stimulated with TSH and blocked with MMI. This rise in the relative proportion of 19S thyroglobulin occurred despite a simultaneous drop of iodine content from 0.6% to 0.24%. It was only after TSH suppression that the well known inverse correlation between the level of iodination and dissociability reappeared. In a second set of experiments, SDS-treated thyroglobulin was fully reduced by splitting disulfide bonds with mercaptoethanol. In addition to the previously described three polypeptide chains, A, B, and C, a hitherto neglected nonreducible fraction comigrated with 19S thyroglobulin on polyacrylamide gels. Native thyroglobulin with widely varying iodine contents was obtained from unstimulated glands and from glands strongly stimulated with TSH. Drastic changes in the polypeptide chain assembly, depending on the degree of TSH stimulation but entirely independent of iodination, were observed. There was a strong negative correlation between the nonreducible 19S thyroglobulin fraction and both the B and C polypeptide chains with all experimental manipulations. We conclude that thyroglobulin substructure is highly dependent on the degree of TSH stimulation of the thyroid. TSH, through stimulation of unknown metabolic pathways, is a more important determinant of thyroglobulin substructure than the degree of iodination of the protein.

The effect of protein depletion on the rate of protein synthesis in rat liver

In order to understand the mechanism of decreased protein synthesis in the liver of rats fed a protein-free diet, the average polypeptide chain assembly time ( t c ) was measured by the method of Mathews et al. (J. Biol. Chem. (1973) 248, 1329). For rats fed a normal diet, t c in liver in vivo was 1.28 min. A 10-day period of protein depletion led to a value of t c = 2.08 min , corresponding to a 38% depression in polypeptide elongation rate. Protein depletion caused an extensive breakdown of hepatic polysomes and refeeding of a complete mixture of amino acids resulted in rapid recovery of polysomal profile. But t c in the liver of the refed animals gave still depressed value of 1.95 min. The amount and size distribution of poly(A)-containing mRNA in the liver, as determined by [ 3H]poly(U) hybridization, were the same for normal and depleted groups. These results suggest that both initiation and elongation steps of protein synthesis are depressed in the liver of protein-depleted rats. Refeeding of amino acid mixture rapidly restores initiation but not elongation activity.

KINETIC STUDIES ON AMINO ACID UPTAKE AND PROTEIN SYNTHESIS IN LIVER OF TEMPERATURE ACCLIMATED TOADFISH

1. A new method is described for measurement of intracellular uptake of amino acids by liver in toadfish (Opsanus tau) in vivo with simultaneous determination of incorporation into aminoacyl transfer RNA and of polypeptide chain assembly time, a kinetic parameter for protein synthesis.2. Mixed L-amino acids or L-leucine supplied by a pulse injection into the hepatic portal vein are strongly concentrated by liver (up to 75% of dose) compared with D-amino acid or mannitol markers. Uptake occurs against an apparent concentration gradient with halftimes of 15 seconds at 24° and 30 seconds at 11°.3. The uptake of pulse-injected L-amino acids into intracellular space of liver and the equilibration of the aminoacyl-tRNA pool are rapid compared with the kinetics of polypeptide chain assembly and thus do not interfere with assembly time determination.4. Polypeptide chain assembly times were 4.0 and 2.5 minutes for warm- and cold-acclimated fish, respectively, at 24° and were 17 and 11.5 minutes for the same groups...

On the rate of ribosome translocation anisotropy and ribosome saturation in the polysome

This study examines the rate of ribosome translocation in the mammalian polysome engaged in protein synthesis by utilizing our knowledge of the hydrodynamic behavior of the rat liver polysomes, sedimenting in a linear sucrose density gradient. The average distance between adjacent ribosomes in the polysome was estimated assuming an extended linear configuration of the polysomes during sedimentation. Based on this estimate, the velocity of ribosome movement along the messenger RNA appears to be non-uniform and inversely related to the ribosome content of the polysome. Such non-uniformity prevails at stages of translation prior to ribosome “saturation” of the polysome. A correlation has been made between the results reported herein and previously published evidence on the rate of polypeptide chain synthesis. The steady-state condition for the polypeptide chain assembly is viewed as representing the state of ribosome “saturation”, characterized by a minimal ribosome velocity and a maximum density of ribosome distribution, both functions being uniform throughout the entire length of the polysome.

Effect of Thyroid Hormone on Polypeptide Chain Assembly Kinetics in Liver Protein Synthesis in Vivo

Abstract The time constant for assembly and release of polypeptide chains in the process of protein synthesis in rat liver in vivo has been determined by a new technique which is independent of most sources of variation and error in amino acid incorporation experiments. In this method the ratio of incorporation of 14C-amino acids into completed soluble proteins to that in total protein (including ribosome-bound chains) is measured as a function of short incubation periods (15 to 105 s) after hepatic portal vein injection to obtain tc, the average polypeptide chain assembly time. For normal male Long-Evans rats, ages 2 to 2½ months, tc in liver in vivo is 1.16 ± 0.16 min. Induction of hyperthyroidism by three injections of triiodothyronine (40 µg/100 g body weight) over 72 hours led to a value of tc = 0.92 ± 0.08 min, representing a 27% increase in synthetic rate. Surgically thyroidectomized rats showed a markedly longer time for polypeptide chain assembly, tc = 1.92 ± 0.22 min, corresponding to a 39% depression in protein synthetic output compared with sham-operated and normal controls of the same age. Almost complete recovery of protein synthetic rate in thyroidectomized rats was achieved by replacement doses of triiodothyronine (20 µg/100 g body weight) given daily for 2 days prior to assay. The results are discussed in relation to the mechanism of control of the protein synthetic pathway, the anabolic action of the thyroid hormones, and other methodology which has been used in attempts to estimate protein synthetic capacity.

Mechanism of action of specific soluble inhibitors of protein synthesis in differentiated cells

We have previously isolated from reticulocyte pH 5 fraction a soluble protein which inhibited serumalbumin cell free synthesis and from liver pH 5 fraction a protein which inhibited hemoglobin synthesis. None of these proteins act on the homologous system. These proteins acted neither on aminoacyl tRNA synthesis nor on the release and assembly of polypeptide chains. They are devoid of any non-specific nuclease activity. When reticulocyte RNA was incubated with liver inhibitor and purified, it retained its specific stimulatory activity on hemoglobin synthesis. This excludes the possibility that the inhibitor has acted by a specific nuclease activity on messenger RNA. An evidence was given of a binding of reticulocyte inhibitor with liver RNAs and preferentially with RNAs which sediment in the region of the messenger RNAs. Some binding was also observed with a viral RNA. These inhibitors may play a role in the selection of messenger RNAs in differentiated cells in which certain genes are transcribed but not translated into proteins. Nous avons isolé de la fraction pH 5 de réticulocyte une protéine soluble qui inhibe la synthèse acellulaire de sérumalbumine et de la fraction pH 5 de foie une protéien qui inhibe la synthèse acellulaire d'hémoglobine. Aucune de ces deux protéines n'agit sur le système homologue. Ces inhibiteurs n'agissent ni sur la synthèse des aminoacyl tRNA synthétases, ni sur la libération et l'union des chaînes polypeptidiques. Ils sont dépourvus d'activité ribonucléasiques non spécifique. Si l'on incube du RNA de réticulocyte avec l'inhibiteur de foie et qu'on purifie cet RNA, on constate qu'il a conservé le pouvoir de stimuler spécifiquement la synthèse de l'hémoglobine. Ceci permet d'exclure tout mécanisme impliquant une action nucléasique des inhibiteurs sur les RNA messangers. La démonstration directe d'une liaison entre l'inhibiteur de réticulocyte et le RNA de foie a été donnée. Cette liaison se fait préférentiellement avec les RNA qui sédimentent comme les RNA messagers. Une combinaison avec du RNA viral a également été observée. Ces inhibiteurs peuvent jouer un rôle par la sélection des RNA messagers dans les cellules différenciées chez lesquelles certains gènes sont transcrits mais non traduits en protéines. Wir haben aus der pH 5 — Fraktion der Retikulozyten ein lösliches Protein getrennt, welches die zellenlose Synthese des Serumalbumins hemmt, und aus der pH 5 — Fraktion der Leber ein Protein isoliert, welches die zellenlose Synthese des Hämoglobins inhibiert. Keines dieser beiden Proteine wirkt auf das homologe System. Diese Inhibitoren wirken weder auf die Synthese der Aminoacyl-tRNA-Synthetasen noch auf das Freisetzen und die Kopplung der Polypeptidketten. Sie weisen keine aspezifische Ribonuklease-Aktivität auf Wenn man die Retikulozyt-RNA mit dem Leberinhibitor inkubiert und wenn man diese RNA reinigt, so beobachtet man, dass sie das spezifische Anregungsvermögen in der Hämoglobinsynthese beibehalten hat. Dadurch wird jeder Mechanismus, der auf einer Nukleasewirkung der Inhibitoren auf die Messenger-RNA begründet wäre, ausgeschlossen. Der direkte Nachweis einer Bindung zwischen dem Retikulozytinhibitor und der Leber-RNA ist gegeben worden. Diese Bindung erfolgt vorzugsweise mit den RNA, welche wie die Messenger-RNA sedimentieren. Eine Bindung mit Viren-RNA ist ebenfalls beobachtet worden. Diese Inhibitoren können eine Rolle in der Selektion der Messenger-RNA in differenzierten Zellen, bei welchen gewisse Gene « umgeschriebenaber nicht zu Proteinen « übersetztwerden, spielen.

Effects of phenothiazines on in vitro cerebral protein synthesis

1. (1) Promazine, at concentrations above 0·2 mM, strongly inhibits the incorporation of 14C-amino acids into protein by rat brain ribosomal systems but not by liver systems. 2. (2) PolyU-directed incorporation of 14C-phenylalanine by preincubated rat brain ribosomal systems is inhibited by about 48 per cent by 1 mM promazine. 3. (3) Promazine does not inhibit the incorporation of 14C-phenylalanine into 14C-phenylalanyl tRNA by rat brain enzymes. 4. (4) The inhibitory effect of promazine apparently occurs at some step in the sequence of reactions leading to the assembly of polypeptide chains on the ribosomes since the transfer of 14C-valine from 14C-valyl tRNA by rat brain systems is inhibited. 5. (5) Evidence is presented to suggest that promazine inhibition results from an interaction between the drug and the soluble brain transferases or some factor(s) present in the transferase fraction.

Effect of thyroid hormones on metabolism IV. Comparative aspects of enzyme responses.

ARTICLESEffect of thyroid hormones on metabolism IV. Comparative aspects of enzyme responsesB Szepsi, and RA FreedlandB Szepsi, and RA FreedlandPublished Online:01 May 1969https://doi.org/10.1152/ajplegacy.1969.216.5.1054MoreSectionsPDF (646 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByCapillarity and oxygen diffusion distances of the soleus muscle of guinea pigs and rats. Effects of hyperthyroidismComparative Biochemistry and Physiology Part A: Physiology, Vol. 82, No. 2Sucrose diet induced enzymatic and hormonal responses affecting carbohydrate, lipid and energy metabolism in two species differing in insulin availability: Spiny and ob/ob miceInternational Journal of Biochemistry, Vol. 16, No. 4Changes in the rates of synthesis and messenger RNA levels of hepatic glucose-6-phosphate and 6-phosphogluconate dehydrogenases following induction by diet or thyroid hormone.Journal of Biological Chemistry, Vol. 257, No. 19METABOLIC ASPECTS OF THE CALORIGENIC EFFECT OF THYROID HORMONE IN MAMMALSClinical Endocrinology, Vol. 13, No. 5Enzyme changes associated with mitochondrial malic enzyme deficiency in miceBiochimica et Biophysica Acta (BBA) - General Subjects, Vol. 587, No. 3Enhancement of renal gluconeogenesis by clofibrateBiochemical Pharmacology, Vol. 27, No. 18Effects of L-Thyroxine in acute renal failureResearch in Experimental Medicine, Vol. 168, No. 2Beeinflussung der quantitativen Relation von proteingebundenen Sulfhydril- zu Bisulfidgruppen durch ThyroxinResearch in Experimental Medicine, Vol. 165, No. 1Effect of thyroid extract and triiodothyronine, with or without the addition of theophylline, on regeneration of the liver in ratsBulletin of Experimental Biology and Medicine, Vol. 78, No. 2Effect of Thyroid Hormone on Polypeptide Chain Assembly Kinetics in Liver Protein Synthesis in VivoJournal of Biological Chemistry, Vol. 248, No. 4Effect of riboflavin deficiency on metabolism of the rat in hyperthyroid and euthyroid stateBiochimica et Biophysica Acta (BBA) - Enzymology, Vol. 268, No. 3Enzyme Response to Thyrotoxicosis and Hypothyroidism in Human Liver and Muscle: Comparative AspectsEuropean Journal of Clinical Investigation, Vol. 2, No. 3 More from this issue > Volume 216Issue 5May 1969Pages 1054-1056 Copyright & PermissionsCopyright © 1969 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1969.216.5.1054PubMed5768051History Published online 1 May 1969 Published in print 1 May 1969 Metrics

The number of growing polypeptide chains on reticulocyte polyribosomes

Reticulocyte polyribosomes synthesizing hemoglobin can be separated physically from ribosomes inactive in protein synthesis. After labeling with [ 14 C]leucine or phenylalanine, the specific activity of the polyribosomes was determined. By measuring the intra-cellular specific activity of the amino acids, the number of leucine or phenylalanine residues per ribosome was calculated. The results are consistent with the conclusion that there is a single growing polypeptide chain per ribosome in the polyribosomes. This calculation is based upon the assumption that all of the reticulocyte polyribosomes are active in protein synthesis. Experiments with cell fractionation and varying magnesium ion concentrations support this assumption. The effect of a non-uniform rate of polypeptide chain assembly is discussed.