432 A. I. OP A RI N 



cell structure. In the experiments we used cultures of Micrococcus lysodeikticus 

 No. 2665, Fleming strain, grown for 24 hours on meat peptone agar. The 

 lysozyme was obtained by the method of Alderton, Ward & Fcvold [4]. The 

 mixture in which the lysis was conducted consisted of 3-53 mg NaCl, 12-84 i"g 

 succinic acid neutralized with o-i"„ NaOH solution to pH 6-5, 0-05 mg MgSO.i, 

 0-4 mg of lysozyme, and sucrose solution, the concentration of which varied 

 according to the conditions of the experiment. The protoplasts were obtained 

 at the following molar concentrations of sucrose: 084; 074; 0-64; 0-54; 0-44; 

 0-34 and 0-24. The total volume of the reaction mixture was 2 ml. The weight 

 of bacterial mass for the experiments on incorporation of labelled amino acids 

 was 54 mg; for experiments on the biosynthesis of protein and for the deter- 

 mination of phosphate fractions it amoimted to 150 mg per sample. Lysis lasted 

 for 30 minutes at a temperature of 37°. Photographs taken in phase contrast 

 and with dark-field illumination testify that the protoplasts obtained after lysis 

 were completely homogenous structures, sharply distinct from intact cells, and 

 contained no admixture of the latter (Fig. 2). The micrographs of the proto- 

 plasts illustrate the process of the gradual breakdown of the cell with varying 

 concentrations of sucrose present in the medium during lysis (Fig. 3). Lysis 

 of bacteria in different concentrations of sucrose led to the passage of intra- 

 cellular substance into the surroimding solution; this can be estimated by 

 measuring the absorption at 260 my.. The breakdown of the structure greatly 

 influences the course of various processes. We were interested primarily in the 

 integrated multistage processes of respiration and protein synthesis in which 

 particularly marked co-ordinating effects of cell structure could be expected. We 

 succeeded [5] in finding the threshold of preservation of the 'structure' below 

 which the mentioned processes are sharply inhibited. This threshold proved to 

 be different for respiration and for the incorporation of labelled amino acids. As 

 shown by the following graph lowering of the sucrose concentration in the 

 medium to less than 0-64 m results in a decrease of the uptake of oxygen by the 

 protoplasts associated with alteration of the respiratory quotient (o-85-o-5o). The 

 latter phenomenon provides evidence that there occurs not only a decrease in in- 

 tensity of the process but also a failure of the co-ordination of its Hnks (Fig. 4). 

 Experiments on the incorporation of labelled amino acids were carried on with 

 [i^C]glycine, added at a level of 150,000 counts rnin ml of reaction mixture. 

 After incubation the reaction was stopped with trichloroacetic acid. The preci- 

 pitate was then washed in the centrifuge (25 times), dried and assayed for radio- 

 activity. From the radiometric data given in Fig. 4, it can be seen that within the 

 limits of 0-84 to 044 M-sucrose concentration the rates of incorporation are 

 approximately equal (1200-1000 counts/min/io mg of dry weight of the proto- 

 plasts). On lowering the sucrose concentration below the level indicated, the 

 incorporation was abruptly inhibited. In the absence of sucrose there is prac- 

 tically no incorporation, in accordance with the findings of other authors. The 

 incorporation of glycine in the protoplasts is completely suppressed by biomycin 

 (200 /xg/ml of reaction mixture) ; considerable inhibition (of the order of 90- 

 95"/,,) is caused by 2 : 4-dinitrophenol, potassium cyanide, sodium azide, or 

 sodium fluoride. It may be concluded from these data that the incorporation 



