DISCUSSION 7AL 
next 5 minutes there was a gradually upswinging curve of incorporation into released ferritin. 
At the end of 5, or actually at the end of 6 minutes the rate of release of radioactive ferritin cor- 
responded exactly with the known rate of ferritin synthesis. In other words, at the end of 6 
minutes every valine in the newly released ferritin had been derived from the pool which was 
established at zero time, and we had, in fact, established the speed with which the pool reached 
its steady state activity. 
L. Mricer: I would like to have a few more facts in connection with this matter of differential 
labeling and its interpretation. I think one consideration almost conspicuous by its absence, 
although some workers in the field have briefly alluded to it, has to do with the relationship 
between what one might call the molecular generation time of a protein and the rate of change 
of specific activity with respect to that molecular generation time. 
If the molecular generation time of the protein is exceedingly short, then obviously it seems 
almost impossible to design an experiment to pick up changes in specific activity of different parts 
of a protein molecule even though they may exist in terms of changes per micro-second, let us 
say. On the other hand, if you have a protein molecule with a sufficiently long molecular genera- 
tion time, then it becomes altogether practical to examine this question critically. That is, can 
one change the specific activity fast enough between the time when the first amino acid residue is 
laid down and the time when the last one is laid down, to see changes that would make or break 
the argument. I do not know whether this has really been examined critically by anyone. 
LoFTFIELD: Well again, in the case of our ferritin experiment we did establish that we changed 
the specific activity of the intracellular leucine or valine from zero to a relatively steady position 
within 15 seconds after the intravenous injection, and that the molecular generation time, to use 
your word, was about 6 minutes. Dintzis found that he had what amounts to a molecular genera- 
tion time of about a minute and a half, and that the amino acid got in extraordinarily fast, 
within ro seconds or something on that order, very much as Prof. Borsooxk has mentioned. 
E. RosBerts: Shouldn’t one be concerned in all such studies with whether there is a good net 
synthesis of protein or whether such a small net synthesis of protein is taking place that the pro- 
tein isolated after the experiment will include an appreciable amount of protein made from 
partially filled templates. In the latter case one would end up with different specific activities in 
different positions. If there is a good net synthesis, so that protein made from originally partially 
filled templates represents only a very small proportion of the total, uniform labeling would be 
observed. 
LoFtTFieLp: I think that this is the case in both the experiments I have cited. In our case we 
found that after ro minutes or 80 minutes the ferritin was substantially homogeneously labeled, 
and Drntzis did exactly the same thing in his most recent and more elegant experiment. During 
the first minute and a half he was getting unequally labeled protein, and by the time he had 
gone 10 minutes, and this is substantially an iv vivo situation since these were intact reticulocytes, 
there had been the complete synthesis of three, four, or five molecules of hemoglobin. Accordingly, 
the specific activity was leveling off for all positions. 
EaG te: I think it is pertinent to point out that this does not explain KRUH’s experiment. 
LoFTFIELD: That’s right. 
MILLER: Are you referring to the im vivo ones or the im vitvo ones? 
EaGte: In this instance to the 7m vityo ones. 
H. RosENBERG: We have always been worried about experiments in which one has to base 
one’s deductions on time intervals of 5 or to seconds after intravenous injections. For instance, 
if you inject isotope into a rabbit’s ear vein and put a counter on the second ear, it takes a con- 
siderable time for the count to reach maximum. I wonder, therefore, if such short experiments 
can be controlled properly. I think you said you have used intravenous injections, and I wonder 
how long it takes to equilibrate the blood stream completely with respect to the isotope? 
LoFTFIELD: We did this with the rat, and the earliest sample of tissue that we could take was 
about 5 seconds, more or less, after we had done the intravenous injection. At this point the total 
amount of radioactivity in the liver had already reached its maximum. The distance between a 
rat’s tail and its liver is somewhat less than between the rabbit’s ears, in addition to which it has 
a much more vigorous circulation. Experiments, of the type performed by Dintzis and by 
ScHWEET, of course, get away from this problem because a suspension of reticulocytes is used. 
I might say, actually, that Haurowitz and Burnett, in Indiana, even worrying about this, 
found that they could ease the substrate into the cells by cooling them to zero and making the 
medium slightly hypotonic. They would get their substrate in without any other reactions and 
then quickly warm up the cells. This was an effort to achieve this same security against the 
question of how long it takes for things to get in and out. 
