188 A. H. ENNOR AND H. ROSENBERG 
NH, 
HN=C O 
x || 
N-CH,-CH,-O-P-O-CH,-CH-COOH 
H | | 
OH NH, 
II 
Subsequent studies in this laboratory yielded a method’ for the isolation of chem- 
ically pure material in high yield from earthworms and provided ample material for 
chemical examination. Yields (1.1 g/kg) of lombricine in almost quantitative amounts 
are now possible by improvements in the isolation procedure’. Chemical studies under- 
taken with a view to a final determination of the structure of lombricine showed that 
the serine moiety possessed the D-configuration®: !4 and provided the first unequivocal 
proof for the presence of a D-amino acid in animal tissue. Chemical synthesis of DL- 
and L-lombricine was then achieved" and was followed by synthesis of the D-isomer 
which was shown to be identical with p-lombricine isolated from natural sources!!. 
Confirmation of the proposed structure of the compound was thus provided and was 
followed by a search for the biological precursor which, it had been suggested, might 
berSEP)\ (reie12): 
The presence of SEP was detected’ in earthworm extracts but the amounts obtained 
were sufficient only to permit chromatographic examination of the products of acid 
hydrolysis. A re-examination® of the problem resulted in the almost quantitative 
isolation of SEP from earthworms and permitted positive identification of the serine 
component. It was found that this had the p-configuration. The presence of D-SEP 
in the earthworm, although consistent with the hypothesis! that it might be the 
biological precursor of lombricine, could not be regarded as proof of it. Convincing 
evidence that D-SEP was indeed the precursor of D-lombricine was however afforded 
by the demonstration!’ that the oral administration of [!4C]amidine-labelled arginine 
to an earthworm was followed by the appearance of 94°% of the label in the guanidino- 
ethanol moiety of D-lombricine. It would seem, then, that the amidine group of 
lombricine is derived from arginine as a consequence of a transamidinase reaction. 
The unequivocal demonstration of such a reaction has not yet been achieved in an 
in vitro system because of the large amounts of arginase which are present in extracts 
of earthworm tissue and the impossibility of separating this enzyme from trans- 
amidinase. However such a demonstration would appear unnecessary in view of the 
appearance in lombricine of the C-labelled amidine group from arginine. This result 
also makes it unnecessary to postulate any other biosynthetic pathway for the for- 
mation of lombricine as THOAT?® has done particularly as this was based on a failure 
to find SEP in earthworms. 
Some attention has been paid to the nature of the possible precursors of D-SEP and 
D-lombricine in the earthworm. *2P; when given orally is incorporated into both pD- 
SEP and p-lombricine at a rate which is about equal to that at which the isotope 
appears in phospholipids!®. The oral administration of [1,2-4C]ethanolamine, DL- 
[3-4C]serine, L-[*H|serine or p-{#H|serine is also followed by incorporation of isotope 
into both D-SEP and p-lombricine!®. In these experiments the specific radioactivity 
of both p-SEP and p-lombricine was greater in the viscera than in the muscle, in- 
dicating that biosynthesis occurred in some internal organ. The incorporation of 
References p. 192/193 
