Intermediate reactions in protein synthesis by the isolated cytoplasmic-membrane fraction of Bacillus megaterium

Abstract

The conditions necessary to obtain high and reproducible levels of incorporation of [C14] amino acids into the protein fraction of the isolated cytoplasmic membrane of B. megaterium were studied. The labelled protein obtained was degraded by partial and total acid hydrolysis. In all cases investigated the added [C14] amino acid had been incorporated unchanged into peptides. Peptide-bond formation in the membrane system was inhibited by chloramphenicol to exactly the same extent as protein synthesis in the whole cell. The cytoplasmic-membrane fraction contained amino acid-dependent enzymes catalysing the exchange of P32 between [p32]pyrophosphate and adenosine tri-phosphate. These enzymes are not inhibited by chloramphenicol. The membrane fraction may be split into 2 fractions (soluble and sedimented) by treatment with ultrasonic vibrations. The soluble fraction contains the amino acid-activating enzymes and also the ribonucleic acid in an extractable form. When the membranes are labelled with [C14] -amino acids, the amount of radioactivity in the protein of the soluble fraction increases much more rapidly with time than that of the sedimented fraction. The ribonucleic acid of the soluble fraction carries bound radioactivity in a form not precipitable with hot trichloroacetic acid. It is readily removed by mild treatments at alkaline pH or by ribonuclease. The possible role of the ribonucleic acid in the amino acid-incorporation process is discussed. Evidence was also obtained indicating that lipids may play a part in protein synthesis.