George C. Webster

Activation of amino acids and amides by cell-free preparations

Abstract Cell-free extracts of pig liver, yeast, and pea seeds catalyze a pyrophosphate-ATP exchange which is promoted to varying degrees by every amino acid and amide normally occurring in protein. Attainment of this exchange is markedly dependent upon the methods for preparation and assay of the enzymes. The preparations also catalyze the formation of amino acid hydroxamates and of apparent amino acid polynucleotide compounds. The amino acid-polynucleotide compounds are able to transfer their amino acid to the protein of isolated ribonucleoprotein particles in every case examined. The protein-bound amino acid can be removed by incubation with non-labeled amino acid-polynucleotide, but not with the free amino acid.

Protein synthesis by isolated nucleoprotein particles

Isolated ribonucleoprotein particles from peas catalyze a net synthesis of “soluble” protein in the presence of adenosine triphosphate, guanosine triphosphate, manganese ions, phosphoglycerate, soluble polynucleotide, and a mixture of the 18 amino acids and two amides usually found in protein. The protein thus formed exhibits more than one electrophoretic component, and has enzymic activity. If protein synthesis proceeds in the presence of C14-labeled amino acids, the protein formed has a specific activity close to that of the free amino acids. The enzyme system is rather unstable.

Isolation of an alanine-activating enzyme from pig liver.

Abstract An alanine-activating enzyme has been purified about 3800-fold from extracts of pig-liver cytoplasm. It exhibits a single component upon electrophoresis in three different buffer systems between pH 6.8 and 8.6. The enzyme catalyzes both the synthesis of alanyl-s-RNA and an alanine-dependent exchange of pyrophosphate with adenosine-5′-triphosphate. The enzyme is specific for l -alanine, and requires either cobalt or magnesium ions. The rate of the reaction is enhanced by dichlorophenolindophenol, and is strongly inhibited by substances containing sulfhydryl groups.

Effects of nucleotides on neurulation in amphibian embryos

Abstract Conditions have been found under which treatments of frog embryos with solutions of nucleotides promote closure of neural tubes many hours ahead of controls. Under optimum conditions, growth of the whole embryo is apparently enhanced. Adenine nucleotides were more effective than other nucleotides in promoting neurulation, and the nucleotide triphosphates were more effective than the diphosphates. Adenosine-3′-monophosphate is much less effective, and adenosine-5′-monophosphate is inhibitory. The effects were dependent upon the concentrations of nucleotides, duration of treatment, time of application, and pH. The most effective time of treatment was at the start of neural tube formation. The neurulation effect was not related to the extent of penetration of a nucleotide into the cells. Adenosine-3′-monophosphate, the least effective promoting material, penetrates ten times as fast as ADP or ATP. The pH dependence of morphological effects was not related to penetration. C14-ADP at pH 7.2 does not promote neurulation, but penetrates to about the same extent as at pH 5.6, which does promote neurulation. Slight positive effects on neurulation were found with nicotinamide or with DPN (NAD) at pH 5.6 and with yeast RNA at both pH 5.6 and 7.2. Adenine and adenosine had no effect. Radioautography studies showed that the C14-labeled material was limited principally to the neural and chordamesodermal tissues.

Protein synthesis from amino acids associated with ribonucleic acid

Abstract The polynucleotide (sRNA) which reacts with amino acids in the presence of amino acid-activating enzymes and adenosine triphosphate has been purified considerably by ethanol fractionation. Maximal transfer of amino acids from this purified sRNA to the protein of isolated ribonucleoprotein particles requires rather sharply defined optimal conditions. Under such conditions, however, the amino acids bound to sRNA are converted into “soluble” protein more efficiently than are free amino acids.

Inhibition of polynucleotide metabolism by inhibitors of protein synthesis

Abstract Ribonucleoprotein particles isolated from pea seedlings incorporate the carbons of carbon dioxide, formate, glycine, adenine, and uracil, as well as orthophosphate, into their ribonucleic acid. The incorporation requires the presence of a mitochondrial preparation, an oxidizable substrate, ribose 5-phosphate, adenosine triphosphate, and magnesium ions. Incorporation of precursors into polynucleotide is promoted by a mixture of amino acids, and inhibited by various amino acid analogs, by hydroxylamine, and by high concentrations of chloramphenicol. The inhibitors do not inhibit the incorporation of precursors into soluble nucleotides.

Factors affecting the microsomal incorporation of amino acids into non-particulate proteins

Abstract Although the formation of non-particulate proteins by isolated rat-liver microsomes can be increased by employment of a suitable reaction system, synthesis stops after about 20 min at 37°. This cessation of synthesis does not appear to be due to a lack of the activating or transfer enzymes necessary for the incorporation of every amino acid into protein, or to the formation of an inhibitor. Instead, the cessation seems to be due to the inactivation of two components of the reaction system: a rapid, incorporation-linked inactivation of the microsome, and a slower inactivation or consumption of some non-particulate factor necessary for the release of completed proteins from the microsome. Synthesis of non-particulate proteins can be increased considerably by substituting ribosomes for microsomes and by adding fresh non-particulate factor at intervals during the reaction.

Effect of nucleotides on ribonucleic acid formation during development of the amphibian embryo

Abstract 1. 1. Adenine nucleotide treatments that promote neural tube closure in Rana pipiens embryos also produce an increase in RNA synthesis. 2. 2. The order of effectiveness of treatment materials on total RNA synthesis, on 14C nucleotide incorporation into RNA, and on the morphological advance is the same: greatest with ATP, next with ADP, least with adenosine 3′-monophosphate. 3. 3. The differences in effectiveness between adenine nucleotides on morphogenesis and on RNA synthesis is not related to penetration. Adenosine 3′monophosphate, the least effective material, penetrates 10 times as fast as ATP or ADP. 4. 4. Rapid penetration of adenosine 3′monophosphate is not simply related to its level of phosphorylation. Adenosine 3′monophosphate also penetrates 10 times as fast as adenosine 5′monophosphate. 5. 5. Synthesis of RNA alone does not ensure advanced morphological development. ADP at pH 7.2 in R. pipiens does not promote neural tube closure, and adenosine 5′-monophosphate delays tube closure. In both cases excess RNA synthesis is promoted.