DISCUSSION 761 
AXELROD: There is no acyl activation involved here at all. There is no requirement for coen- 
zymes; you do not need anything but the free fatty acid and the free amino acid. 
LoFTFIELD: I was interested in comparing Dr. AXELROD’s experiences with the work that OrREKo- 
vicH has done in Russia on GREENSTEIN’s acylase which he has purified extensively. He finds 
that it does do a very nice job of reversing the hydrolysis of acylamino acid. The equilibrium 
constants have been determined, and at reasonable physiological concentrations there is a quite 
substantial (1 or 2 per cent) synthesis of the acyl compound, and of course, the very compounds 
that are synthesized are those that would be hydrolyzed by carboxypeptidase again. I just wonder 
Dr. AXELROD, if you have found a GREENSTEIN enzyme in the liver, so to speak? Do you know if it 
has other comparable properties and have you done kinetics to measure the relative rates of 
reaction? 
AXELROD: We have not done any kinetics at all. We have only shown that we could split the 
product that was being formed, and we have not determined the equilibrium constant for the 
reaction. We have tried to block the reaction by using inhibitors for esterases. DFP inhibits it to 
a rather good degree, as does physostigmine. We also tried hydrocinnamic acid, which is a specific 
inhibitor for carboxypeptidase, but found that it had no influence on the formation of the com- 
pound. 
Winitz: If I might add to Dr. LorrFreLp’s statement with regard to OREKOVICH’s data, it is 
indeed perfectly valid data. However, OREKOVICH uses extremely high concentrations of sub- 
strate, in the vicinity of about 2 or 3 MW, whereas in the GREENSTEIN resolution procedure, con- 
centrations of about 0.1 to 0.2 M are employed in order to ensure complete hydrolysis. With 
concentrations of one molar or higher, the hydrolysis of an acylamino acid is in the vicinity of 
about 95 per cent or less. With regard to the distribution of acylase I, it is present in various 
tissues such as liver, brain and heart. However, the highest concentrations are found in kidney, 
and that is the tissue we generally use for its isolation. 
