154 J. R. SIMMONS AND H. K. MITCHELL 
at —20°. The extract was washed with water according to Folch’s procedure’? and 
after removal of chloroform and methanol the aqueous phase was retained. The residue 
from the chloroform—methanol extract was dropped into boiling water while still 
frozen and was homogenized for 5 min at 100°. The homogenate was then centrifuged 
at 15 000 rev./min for 20 min in the 30 rotor of the Spinco Model L ultracentrifuge. 
The supernatant was decanted and the water extraction of the residue was then 
twice repeated. The combined water extracts (170 ml) were added to the aqueous 
phase from the wash of the chloroform—methanol extract and the whole was placed on 
an ion-exchange column for fractionation. Fractionation was carried out on a Dowex- 
50-X4, 22mm X 45cm column. The results of the fractionation of the extract are 
shown in Fig. 6. 
As indicated previously! the products of the column fractionations are very complex. 
With respect to radioactive components this is particularly true with glutamic acid. 
Glutamic acid was widely distributed in the column fractions in bound form. When 
radioactive material (not in the region of free glutamic acid) was isolated from column 
fractions by additional purification steps and then hydrolyzed it was found that most 
contained radioactive glutamic acid and other non-radioactive amino acids. It would 
thus seem that glutamic acid was incorporated into a wide variety of peptidic materials. 
The amount of leucine in such material was less and not enough was found in any 
given fraction to do more detailed analysis. This is apparently a reflection of smaller 
pools of leucine-containing material. 
DISCUSSION AND SUMMARY 
The data presented here permit the conclusion that amino acids injected into Dvoso- 
phila larvae are very rapidly incorporated into peptides of various sizes. Subsequently 
they enter into proteins, but the results do not give information as to the mechanisms 
by which the syntheses take place. In initial stages, mixed peptides containing |“C] glu- 
tamic acid are incorporated into proteins at about the same rate as glutamic acid 
itself but whether the peptides are incorporated directly or are first hydrolyzed re- 
mains an open question. 
It is of interest to note that glutamic acid (glycine, alanine and aspartic acid behave 
similarly) goes very rapidly into peptides and relatively slowly into proteins. In 
contrast leucine and valine, which are essential amino acids to Drosophila, appear in 
relatively few peptides and appear to go more directly and rapidly into proteins. 
However, this may be only a reflection of pool sizes for essential and non-essential 
amino acids. Much greater accumulation of peptides would be expected with amino 
acids (such as glutamic acid and glycine) that can be synthesized by the tissues of 
the larvae. 
It is of special interest to note that in this 7m vivo system, chloramphenicol and 
fluorophenylalanine had no influence on the rate of incorporation of amino acids 
into protein even when saturated solutions were injected. So far no inhibitors have 
been found which specifically inhibit protein synthesis in the larvae. 
The methods described here, provide an adequate background for further work 
on isolation of pure peptides and further studies on their metabolism both in vivo 
and in vitro (cf. ref. 1). 
References p. 155 
