Fritz Lipmann

The interrelationship between guanosine triphosphatase and amino acid polymerization

Abstract The purified complement, the G factor of Nishizuka and Lipmann (1), and salt-washed ribosomes were, by themselves, virtually free of guanosine triphosphatase (GTPase) activity. By saturation with one component, GTP hydrolysis was linearly dependent upon the amount added of the other. The K m for GTP for the maximally active system was 2 × 10 −6 m , and the K i for guanosine diphosphate was 7.6 × 10 −6 m . Addition of poly U and other polynucleotides and of sRNA stimulated GTPase. The GTPase was inhibited by sulfhydryl-blocking agents. The amino acid polymerization and GTPase functions of the ribosome were dissociated by brief heating to 55 °, which “uncoupled” by abolishing the synthetic but enhancing the hydrolytic effect. The function of GTP in amino acid polymerization is discussed.

Comparison of amino acid polymerization factors isolated from rat liver and rabbit reticulocytes

Abstract Using preliminary ammonium sulfate fractionation followed by calcium phosphate adsorption and elution, two complementary fractions, T 1 and T 2 , analogous to Schweets TF-1 and TF-2, were obtained from liver as well as reticulocytes. In both cases, a further separation of the two fractions was achieved by DEAE-cellulose column adsorption and elution with a potassium chloride gradient. The prepurified liver fractions were also identified as T 1 and T 2 , respectively, after Sephadex G-200 chromatography. Cross complementation was obtained between T 1 and T 2 derived from either source. A ribosome-linked GTPase partly coincided with the T 2 fraction but did not respond to the addition of template or aminoacyl-sRNA. The T 1 fraction catalyzed GTP-linked binding of aminoacyl-sRNA to template-charged ribosomes, and GTP hydrolysis was observed coincident with the T 1 -linked binding of aminoacyl-sRNA to the ribosome but exceeded the binding reaction about 20-fold. The binding assay was performed in the absence of a sulfhydryl compound to prevent polymerization. The T 2 function was not studied in detail, except that it caused polymerization when added to the T 1 system together with a sulfhydryl compound. A common nomenclature is proposed, and a separation procedure that is generally applicable to mammalian cell extractions is presented.

Bacterial Production of Antibiotic Polypeptides by Thiol-Linked Synthesis on Protein Templates

Publisher Summary This chapter deals with the discussion of bacterial production of antibiotic polypeptides by thiol-linked synthesis on protein templates. Gramicidin S and tyrocidine are the two polypeptides for which the detail has been obtained with regard to fractionation of the participating enzymes and identification of the pantetheine-linked protein present in all polyenzymes. For their synthesis, the polyenzymes for gramicidin S and tyrocidine have to be combined with a mono-enzyme that activates and racemizes phenylalaninethat, in the D-form, initiates the process in both cases. The best method for surveying the contents of the different enzyme fractions is to separate the components in the supernatant of a crude bacterial extract chromatographically on Sephadex G-200. This aids in isolating the polyenzymes because they are involved in sequential synthesis. It was found that, in addition to an amino acid-dependent ATP-PP i exchange, an adenosine triphosphate–adenosine monophosphate (ATP–AMP) exchange, most pronounced with phenylalanine, also occurred. Studies on the thio-ester-linked biosynthesis of both gramicidin S and tyrocidine in the laboratories were followed by observations that other antibiotics are synthesized in an analogous manner.

Fusidic Acid: Inhibition of Factor T2 in Reticulocyte Protein Synthesis

The steroid antibiotic fusidic acid inhibits reticulocyte protein synthesis. This inhibition appears to be due to interference with the activity of the T2 supernatant fraction, and strengthens the proposition that T2 is functionally analogous to the G-factor of bacterial protein synthesis, which is also specifically inhibited by this antibiotic.

Messenger Ribonucleic Acid

Publisher Summary This chapter reviews the relatively new appearance of a variety of RNAs that are functionally distinct are closely interrelated. Ribosomal RNA seems to stand apart with only a passive participation in information transfer. Its relationship to messenger ribonucleic acid (mRNA) is unsettled, one group favoring a “melting” of mRNA into the ribosome, while others favor separate paths of synthesis from a common pool of mononucleotides. The general mode of biosynthesis of sequence patterns has proven to be a one-by-one addition and the differences of sequence pattern between ribosomal and the template carrying mRNA are difficult to explain. The chapter discusses that the rapid turnover of mRNA was originally thought to be a characteristic of this class of material. Such turnover now seems rather to be a feature of microbial mRNA during rapid growth. It is related to a rapid rate of protein synthesis and the need for fast replacement in view of an apparently rapidly changing mRNA population with a probably sequential translating of DNA into protein synthesis during the growth cycle. Thus mRNA may be defined, as of now, as a sequence-determining template that combines with ribosomes for catalytic functioning in protein synthesis.

Role of divalent ions in poly U-directed phenylalanine polymerization*

The activity of different divalent cations in the poly U-directed transfer of phenylalanine from phenylalanyl sRNA to polyphenylalanine was investigated. Highly purified protein fractions (ribosome-dependent GTPase and the protein fraction which complements with ribosome-dependent GTPase in phenylalanine polymerization) and ribosomes were used. The order of activities Ca2+ > Mg2+ > Mn2+ was found. The products were confirmed as polyphenylalanine by paper chromatography. The related ribosome-dependent GTPase reaction was also investigated. If ammonium ions were present, the order of activities was Mg2+ > Mn2+ > Ca2+. In the absence of ammonium ions, this order was reversed. The requirements of both polymerization and GTPase reactions were studied in solutions containing magnesium and calcium ions. Considerable polymerization was found in solution containing calcium ions in the absence of added ribosome-dependent GTPase. The requirements of the polymerization reaction were investigated in greater detail by chromatography of the products. Significant synthesis of smaller peptides in the absence of added ribosome-dependent GTPase was then detected in solutions containing magnesium ions. We have studied the effect of removal of residual magnesium ions from the calcium ion system by previous dialysis of the ribosomes against a calcium ion solution. These ribosomes were still active, but small amounts of magnesium ion were stimulatory. The ribosomes were inactive after dialysis in the absence of divalent ions.

Enzyme-bound phosphopantetheine in tyrocidine biosynthesis.

Abstract Phosphopantetheine has been demonstrated to be an enzyme-bound cofactor in one of the three enzyme fractions (light, intermediate, and heavy) required for tyrocidine biosynthesis. The enzyme-bound pantothenate had to be liberated by heating with alkali and treating the product with alkaline phosphatase. One mole of cofactor was estimated to be present per mole of heavy enzyme. It was absent from the light and intermediate enzyme fractions.

Ribosome specificity for the formation of guanosine polyphosphates

Abstract Ribosomes obtained from Bacillus brevis (ATCC 8185) were slightly active in synthesizing guanosine polyphosphates, which activity was greatly stimulated by addition of Escherichia coli stringent factor. Chlamydomonas reinhardtii chloroplast ribosomes did not produce guanosine polyphosphates on incubation but responded with abundant synthesis to addition of the stringent factor from E. coli . In contrast, cytoplasmic ribosomes from the same organism did not respond. Interchange experiments between either subunit from chloroplasts with the E. coli counterparts showed good activity. When the small subunit of cytoplasmic Chlamydomonas ribosomes was combined with the large subunit of E. coli or of chloroplasts, a small but definite response was obtained.

Polypeptide biosynthesis from thioesters of amino acids

Abstract Each of the two enzyme fractions for gramicidin S (GS) biosynthesis and the three for tyrocidine (Ty) biosynthesis form complexes with the corresponding substrate amino acids. These complexes, isolated by Sephadex G-50 gel filtration, contain equivalent amounts of aminoacyl adenylate and amino acid bound as thioester. As shown previously by denaturation through trichloroacetic acid (TCA) precipitation, (NH 4 ) 2 SO 4 precipitation of these enzyme-amino acid complexes also discharges the noncovalently bound aminoacyl adenylate, retaining only the amino acids covalently bound as thioester, but without loss of activity. Combination of the thus prepared GS light enzyme charged with phenylalanine thioester and GS heavy enzyme charged with proline, valine, ornithine, and leucine thioester results in GS biosynthesis in the absence of aminoacyl adenylate. Moreover, the d -phenylalanine thioester of N -acetylcysteine or of thiophenol replaces the natural donor of activated amino acid, aminoacyl-AMP, produced by the reaction of ATP and amino acid, further substantiating the key role of thioester intermediates in antibiotic polypeptide biosynthesis.

Studies on the biosynthesis of valinomycin.

For some time, this laboratory has been exploring the in vitro synthesis of antibiotic polypeptides on polyenzymes; the latter catalyze the reaction between amino acids t ATP yielding aminoacyl adenylates. Eventually, the amino acid residues are transferred to a thioester linkage and a pantetheine carrier protein attached to each polyenzyme collects the amino acids into polypeptides [ 1,2] . In view of these results, we proposed approaching the biosynthesis of valinomycin, a cyclododecadepsipeptide, which contains the thricerepeating sequence of the tetradepsipeptide: LlactylL-valyl-D-a-hydroxyisovaleryl-D-valyl. Earlier reports on its biosynthesis in vegetative cells by MacDonald and Slater [3] suggested that D-cr-hydroxyisovaleric acid is the direct precursor of the D-o-hydroxyisovaleric acid moiety in valinomycin. The Dand L-valyl paits of valinomycin were shown to be derived from L-valine. In 1974, Ristow et al. [4] published briefly on the cell-free synthesis of valinomycin from L-valine using L-threonine or Lalanine as the precursors of lactic acid. We will describe here experiments where, under appropriate conditions, radioactively marked lactic acid is easily, but alanine poorly, incorporated into .valinomycin by living cells. Since we were unable, using the same organism as Ristow et al. [4], to obtain in vitro synthesis in our cell homogenates, after disruption of the bacteria by means of a sonitier or a French-press, we prepared protoplasts which showed a considerably greater activity in forming valinomycin