THE BIOSYNTHESIS OF ALKALINE PHOSPHATASE WITH A PARTICULATE FRACTION OF ESCHERICHIA COLI

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Abstract
By digitonin lysis of penicillin spheroplasts of E. coli a particulate fraction-P1 supported the sustained synthesis of alkaline phosphatase when supplied with amino-acids, nucleotide triphosphates and other cofactors. This P1 fraction, when subjected to mild ultrasonic treatment in the presence of sucrose and Mg2+, yielded the P1 (S) fraction, consisting of integrated particulate subcellular particles containing deoxyribonucleic-acid (DNA) and ribonucleic-acid (RNA). The P1 (S) fraction from E. coli K 10 wild type (R+1 R+2 P+) grown under repressed conditions supported the immediate synthesis of alkaline phosphatase in-vitro. The synthesis occurred in phases. The first was followed by a lag, and then a subsequent linear rapid phase that continued for at least 3 hr. Actinomycin-D inhibited the appearance of the second phase. The particles synthesize enzyme even when prepared from repressed cells. Synthesis of the specific messenger RNA for alkaline phosphatase in-vivo was not inhibited when the bacteria were grown in an excess of inorganic phosphate. Phosphate inhibited synthesis of enzyme to the same extent with the P1 (S) fractions of 2 constitutive strains as with the P1 (S) fraction of the wild-type strain. Inorganic phosphate inhibited amino-acid incorporation with the P1 (S) fraction and also inhibited enzyme synthesis in-vitro. The effect on amino-acid incorporation could be partially overcome by adding Mn2+ to the incubation mixtures, However, Mn2+ inhibited the synthesis of alkaline phosphatase. Inhibition of the incorporation of [P32] CTP into RNA was overcome by Mn2+. The effect of phosphate on amino-acid uptake was probably due to a phos-phorolysis of RNA by polynucleotide phosphorylase, also present in the P1 (S) fraction. This phosphorolysis may account for the instability of messenger RNA in-vitro and in-vivo. Phosphate specifically inhibited the formation of alkaline phosphatase, since it did not markedly affect the induced formation of [beta]-galactosidase by the same P1 (S) fraction. The specific effect is attributed to the prevention of formation of the enzymically active dimer from precursors, a Zn2+-dependent reaction. The repression of the synthesis of alkaline phosphatase in-vivo in the wild-type strain may be the sum of these 2 effects.