DINTZIS, BORSOOK, AND VINOGRAD 



97 



BIOLOGICAL ACTIVITY OF MICROSOMAL PARTICLES 



In the following experiments rabbit reticulocytes were centrifuged several 

 times in buffer, suspended at 37° in a medium containing amino acids, iron, 

 and other materials necessary to ensure optimal hemoglobin syntheses. A given 

 C 14 labeled amino acid was added, and the living cells were incubated for a 

 definite time period; then several volumes of ice-cold saline were added and the 

 cells centrifuged several times from cold saline to remove extracellular label. 

 The cells were then frozen, and microsomes were prepared as described above. 



When the cells were incubated for various time intervals with carboxyl C 14 

 labeled leucine (15,500 cpm/mg) the resultant specific activities of protein in 

 microsomes and in hemoglobin were as shown in table 1. 



It would appear that a steady-state concentration of labeled amino acids in 

 the microsomes is reached within approximately 10 minutes or less. 



Incubation 

 Time, 

 min. 







1 



5 



15 



60 



240 



Rate of Activity 



Increase in 

 Hemoglobin, 

 cpm/mg/min 



0.11 



0.22 

 0.29 

 0.21 

 0.15 



The labeled amino acid in the purified microsomes is present in some tightly 

 bound form, as shown by the facts that (1) it is not removed at all by dissolv- 

 ing the microsomes in buffer saturated with nonlabeled leucine and (2) little, 

 if any, count is removed by extraction with trichloroacetic acid. 



However, the label is rapidly turned over in the living cell, as shown by the 

 fact that if cells are labeled for 15 minutes in medium containing radioleucine, 

 and then placed in medium containing nonlabeled leucine for 15 minutes, 90 

 per cent of the label is removed from the microsomes. This shows that most 

 of the label present in the microsomal particle is in a dynamic state, and is not 

 a permanent part of the microsome structure. 



In order to obtain information concerning the amino acid composition of the 

 transient material in the microsome, experiments were conducted using various 

 labeled amino acids in the incubation medium. Radioleucine was always run 

 as a control, and the molar ratios of other amino acids to leucine were deter- 

 mined in the hemoglobin and microsomes after 15 minutes of incubation. The 

 results are summarized in table 2, where the labeled amino acid ratios are com- 

 pared with the total amino acid ratios determined by microbiological assay. It 

 can be seen that the leucine-histidine ratio of transient material in the micro- 

 some is compatible with the supposition that this material is hemoglobin pro- 

 tein and not microsome structural protein. The leucine-phenylalanine ratio is 



