150 OCCURRENCE OF FREE AMINO ACIDS — INSECTS 
DISCUSSION 
Chairman: NoRMAN H. Horowitz 
GuRoFF: Dr. MITCHELL, | wonder if you could comment on the biosynthesis of tyrosine phos- 
phate and what its function might be? 
MircHELL: I have some evidence that tyrosine phosphate is combined in peptides and perhaps 
a bit in some proteins. We have speculated on the possibility that the substance is concerned in 
blood coagulation as tyrosine sulfate is in mammals. However, there is no evidence for this so far. 
GuRoFF: I wonder if it could have any relation to the serine phosphate found in mammalian 
tissue? 
MircHELL: The tyrosine derivative is much more labile than serine phosphate. This is a phenol 
ester and should have a higher energy bond, so it is, I think, in an entirely different category so far 
as synthesis and reactions are concerned. 
Hatvorson: We have made some observations similar to yours while working with peptides 
attached to yeast ribosomes. These peptides are rapidly labeled during a pulse labeling experiment. 
I wonder if you looked at the microsomal and ribosomal fractions? Is it possible that some of the 
peptides you have observed result from breakdown of such cell structures? 
MITCHELL: We do not know whether the peptides come from direct synthesis or from degrada- 
tion of something more complex. Using 1adioactive amino acids these peptides are labeled much 
faster than proteins. The label in peptides goes into protein, but our present evidence does not 
preclude peptide hydrolysis before protein synthesis. So far as viteosomes are concerned, prepara- 
tion from larvae contain the larger, more complex peptides. However, the procedure for their 
preparations requires a good deal of time and sufficient reactions can occur even at 0° to give a 
misleading picture. 
Hatvorson: Is it feasible to use diisopropylfluorophosphate during rupture of the larvea to 
minimize proteolytic activity ? 
MitTcHELL: I do not think we need to worry about proteolytic action at —80°. Nothing happens 
on incubation of low temperature extracts prepared as I have described. I do not think the use of 
an inhibitor is necessary when making extracts in this way. 
Horowitz: I want to return to the point that Dr. HAtvorson raised; namely, have you done any 
experiments to find out how soon the label appears in ribosomes? 
MitcHELL: No. We have done no labeling experiments with isolated ribosomes. Our intent has 
been to try and find the system which will synthesize protein from peptides but which will not 
incorporate amino acids. If peptides are used directly for making proteins, one should be able to 
find such a system. 
Horowitz: I thought perhaps what Dr. Hatvorson had in mind was the possibility that the 
injected amino acids enter the ribosomes rapidly where they are attached to a template, and that 
your treatment removes them as peptides. 
MitTcHELL: We have not done an experiment directly on this point. I think there would not be 
enough ribosomes to carry all of the peptides that are present. However, I have not made the 
necessary calculations. 
HENDLER: In some of your earlier work you described a large lipid—amino acid pool in larvae 
which seemed to decrease during development. In short-term incubations did you determine 
whether there was isotope uptake in that fraction, and, if so, how did it compare with the uptake 
into the peptide fraction? 
MitcHeELL: The lipid fraction is labeled very quickly but there is no subsequent increase after 
even the earliest samples we have taken. 
HENDLER: You mean ahead of your peptide fraction? 
MITCHELL: Yes, but we have done no work on measuring turnover of label in the lipid fraction. 
WREN has found that a fresh, carefully prepared lipid fraction will firmly bind added amino acids. 
Thus, some artifact of incorporation can be created, but this is a much smaller amount of amino 
acid than is found normally in lipid preparations. I do not know whether the rapid incorporation 
of labeled amino acid into the lipid fraction means anything or not and we have done no work to 
establish its significance. 
CHRISTENSEN: I have a question with regard to the possibility that proteins may be split under 
