Allan J. Morris

The selective inhibition of protein assembly by gougerotin

Abstract 1. 1. The mechanism by which the antibiotic gougerotin inhibits the biosynthesis of protein has been investigated. Gougerotin has been shown to inhibit selectively the incorporation of amino acids into protein but not the release of completed protein chains from ribosomes. In this way, inhibition by gougerotin allows a distinction to be drawn between these two stages of protein synthesis. 2. 2. Gougerotin has been shown to exert a competitive inhibition upon the action of puromycin in the cell-free system. These studies have demonstrated the locus of action of gougerotin to be the enzyme which forms the peptide bond during the polymerization of amino acids into protein. 3. 3. The action of puromycin, which involves formation of a peptide bond between an incomplete polypeptide chain and the puromycin molecule, does not require the presence of GTP. 4. 4. Structural similarities of gougerotin, like puromycin, to aminoacyl soluble RNA have been proposed as the basis for the observed action of gougerotin.

Nonuniform size distribution of nascent peptides: The effect of messenger RNA structure upon the rate of translation

Abstract The size distribution of bacteriophage MS2 coat protein nascent chains purified from MS2-infected Escherichia coli has been determined. Accumulations of nascent chains of discrete sizes were observed, providing evidence that the rate of chain elongation during coat protein biosynthesis is not uniform. A correlation of the size of nascent peptides which accumulate during MS2 coat protein biosynthesis and the position on the MS2 coat protein mRNA of the ribosome carrying those lengths of nascent peptides may be made. This correlation leads to the hypothesis that regions of mRNA secondary structure impede the movement of ribosomes during chain elongation and thus serve as the origin of the accumulation of nascent chains of discrete sizes.

The distribution of nucleoside triphosphate pyrophosphohydrolase in the tissues of the rabbit

Abstract Nucleoside triphosphate pyrophosphohydrolase activity has been detected in a number of tissues of the rabbit. These tissues include brain, pancreas, kidney, thymus, spleen, lung, heart muscle, skeletal muscle, bone marrow, and liver. Brain and liver have been found to contain the highest concentrations of nucleoside triphosphate pyrophosphohydrolase activity of the tissues tested. This enzyme has been purified some 680-fold from rabbit liver. The substrate specificity, pH optimum, electrophoretic mobility in polyacrylamide gels, and apparent molecular weight have been studied and compared to the enzyme which was originally purified and characterized from rabbit red cells (1). A mechanism has been suggested for the biological role of nucleoside triphosphate pyrophosphohydrolase.

Inhibition of peptide bond formation by cytidyl derivatives of puromycin

Abstract Preparation of cytidine -2′(3′)-P-5′- puromycin mixed isomers as well as cytidine 3′-P-5′- puromycin and cytidine -2′-P-5′- puromycin are described. The ability of these substances to inhibit amino acid incorporation in a cell-free protein-synthesizing system from rabbit reticulocytes has been determined over a wide range of concentration. On the basis of the concentration required to produce 50 % inhibition of amino acid incorporation, cytidine -3′-P-5′- puromycin was approximately 7 times more potent than puromycin itself. Cytidine -2′-P-5′- puromycin , on the other hand, was 22 times less effective than puromycin. The implications of these results regarding the substrate specificity of peptidyl transferase and the ribosomal acceptor site are discussed.

Individual Variation of Nucleoside Triphosphate Pyrophosphohydrolase Activity in Human Erythrocytes, Granulocytes, Lymphocytes, and Platelets

Analysis of the nucleoside triphosphate pyrophosphohydrolase specific activity of red cells obtained from a random Caucasian population indicated at least two subclasses. The specific activity of 18% of the population ranged from undetectable activity to 27.5 nmol ITP cleaved/20 min/mg hemoglobin. The remainder of the population had higher activity, 27.5–125 nmoles ITP cleaved/20 min/mg hemoglobin. The variation of NTPH activity evident in the red cells of an individual is reflected in granulocytes, lymphocytes, and platelets of that individual. Erythrocyte activity ranges from 0.7 to 21 units (nmol of ITP cleaved in 20 min)/107 cells, granulocytes have 17–201 units/107 cells, lymphocytes have 91–462 units/107 cells, and platelets have 1.1–7.1 units/107 platelets. These cell differences are discussed with respect to the hypothesis that NTPH prevents incorporation of ITP or dITP into nucleic acids.

Isolation of functional ribosomes and polysomes from lyophilized fungi

Abstract Functional monosomes and polysomes were readily isolated from lyophilized mycelia and spores of the tetrapolar basidiomycetes Schizophyllum commune and Coprinus lagopus. 1. 1. Monosomes having sedimentation properties similar to rabbit reticulocyte monosomes predominate in these ribosomal preparations. 2. 2. Polysomes of dimer, trimer, and tetramer size are consistently present. The polysomes are stable at low Mg2+ concentrations and are sensitive to ribonuclease treatment. 3. 3. The ribosomes are functional in a cell-free protein synthesizing system containing no exogenous mRNA. The isolation of intact polysomes from the material lyophilized prior to grinding is considered significant because of the advantages of this technique to molecular studies on organisms with resistant cell walls and because of the indication that lyophilization has little effect on the functional integrity of the polysome-mRNA complex.

Nonuniform size distribution of nascent peptides: The role of messenger RNA

Abstract The origin of the nonuniform size distribution of nascent rabbit globin peptides has been investigated in the reticulocyte lysate and wheat germ cell-free protein synthesis systems. Increasing the concentrations of the cellular components involved in protein synthesis failed to alter the elution pattern observed upon chromatographic analysis of reticulocyte lysate nascent chains. Nascent chains isolated from a globin messenger RNA-directed wheat germ cell-free system showed a nonuniform size distribution of nascent peptides similar to that of the rabbit reticulocyte nascent chains. These observations indicate that the nonuniformity of the globin nascent chains arises from a unique property of the messenger RNA being translated and not from limiting concentrations of a component or components of the reticulocyte protein synthesis system.

Interaction of globin and heme during hemoglobin biosynthesis

Abstract Conditions for the biosynthesis of labeled protoporphyrin IX and labeled heme from δ-aminolevulinic-14C acid have been established in a broken-cell system prepared from rabbit reticulocytes. Labeled soluble hemoglobin and labeled nascent globin were also synthesized in this reaction mixture as indicated by leucine-3H incorporation. Examination of the 3H- and 14C-labeled materials present in the ribosomepolysome fraction, high-speed supernatant fraction, and purified soluble hemoglobin fraction were performed. The absence of porphyrin-14C in the nascent globyl-sRNA fraction, examined by Sephadex G-200 chromatography in the presence of sodium dodecyl sulfate, as contrasted to the large amounts of 14C-labeled heme and 3H-labeled globin in the hemoglobin of the high-speed supernatant fraction, supports the conclusion that the interaction of heme and globin occurs after completion of the primary structure of the globin chains.

Thermolability characteristics and inheritance of human red cell nucleoside triphosphate pyrophosphohydrolase

Biochemical and genetic data which serve to define further the variation of nucleoside triphosphate pyrophosphohydrolase (NTPH) activity in red cells are presented [see also Verhoef, V. L., Fuller, S. A., and Morris, A. J. (1980). Biochem. Genet.18:235; Soder, C., Henderson, J. F., Zombor, G., McCoy, E. E., Verhoef, V., and Morris, A. J. (1976). Can. J. Biochem.54:843]. Examination of the in vivo stability of red cell NTPH via separation of cells by density (age) reveals that loss of NTPH activity during the lifetime of the erythrocytes is the same in individuals with high and low NTPH specific activity. However, when the thermolability of lysate NTPH was measured, three phenotypes could be distinguished. In order of decreasing thermolability, these correspond to NTPH specific activities of 0–5, 12–25, and greater than 25. These data provide the first evidence of physical differences in the molecules of NTPH associated with the various specific activities of NTPH present in the human population. Family data are also presented which show that the mode of inheritance of differences in NTPH specific activity cannot be ascribed to a simple autosomal one gene-two allele system. We propose alternatively that the family data conform well to an hypothesis of three alleles at one NTPH locus controlling NTPH activity.

Analyses of globin release from reticulocyte ribosomes

Abstract Investigations of the biosynthetic processes which constitute the respective stages of protein synthesis indicate the presence of a specific mechanism for the release of completed protein from the biosynthetic template (Morris, 1964) . It was demonstrated early in the studies of protein biosynthesis that cell-free systems prepared from rabbit reticulocytes synthesize hemoglobin molecules which are no longer attached to the ribosomal template and are chromatographically indistinguishable from carrier hemoglobin (Schweet et al , 1958) . The details of the process by which completed protein chains are liberated to a nonparticulate state have been a subject of previous publications from this laboratory. It is the purpose of this communication to present the results of further studies which indicate the release of globin molecules from reticulocyte ribosomes involves cleavage of the globyl-sRNA bond at the ribosomal level and that the first soluble product of the release reaction is the protein molecule, free from soluble RNA. A mechanism for the termination and release of completed protein chains from the ribosomal template is proposed.