Abstract
Many reports on LAP isozymes in human serum and human organs have been published, since Lawrence and his coworkers confirmed existence of multiple forms of leucine aminopeptidase. But the number of LAP isozymes and their electrolysed position were found different according to the investigators and the methods of electrophoresis, and their clinical significance remanis not clarified. Moreover, it is not clear whether the LAP in multiple forms is a true isozyme controled by gene, or consists of different enzymes which hydrolyze L-leucyl peptide.
In this paper, the authors studied on the LAP isozymes in human serum and human organs with the electro-focussing method of Vesterberg and Svensson, and on the kinetics of the isozymes. LAP of the normal human serum was separated into 3 fractions at pI 3.9, 4.5 and 5.1. The supernatant of the liver was separated into 5 fractions at pI 3.9, 4.5, 5.1, 5.7 and 6.3. The authors call these fractions as LAP1, LAP2, LAP3, LAP4, and LAP5, starting from the anode side. LAP1 and LAP2 were very strongly active in comparison with LAP3, LAP4 and LAP5 in the liver and gall bladder. In the kidney, LAP2 and LAP3 were very strongly active, but LAP1, LAP4 and LAP5 were slightly active. In the pancreas, LAP3 was only strongly active fraction, whereas LAP1, LAP4 and LAP5 were slightly active, and LAP2 was not observed in the pancreas.
Investigation on kinetics of LAP has been hampered on account of lack of the adequate methods, however, in this paper, the method of Goldberg was used which is widely used in the clinical field.
The effects of substrate concentration on the activity of LAP fractions (LAP1, LAP2, LAP3 and LAP4) were shown as similar sigmoid curves and same Km values (=8×10-5M) in 0.1M phosphate buffer pH 7.0. And the activity of these fractions was inhibited by the high substrate concentration (4×10-3M). The effects of different pH were represented in the form of similar curves which showed that the optimal pH of the 5 fractions was 6.5. The effects of metal ions on these 5 fractions were of a similar tendency, that is, Ca+2 and Mn+2 inhibited the LAP activity.
With respect of these results, it is very difficult for the authors to think that these 5 fractions are a complex of different enzymes which hydrolyze L-leucyl peptides.
The multiple forms of LAP in serum of the patients of some diseases were very different from those of normal subjects' serum. The authors separated the LAP isozymes with the electro-focussing method. In the serum from a cholelithiasis patient, LAP1, LAP2, LAP3, LAP4 and LAP5 were remarkably detectable.
LAP2 was more strongly active in comparison with normal serum. 5 fractions were also found in the cases of acute hepatitis and subacute pancreatitis. In liver carcinoma, LAP1 was very strongly active. LAP2 and LAP5 were not observed in serum from patients of liver cirrhosis.
On the ground of these results, it is suggested that more researches on LAP multiple forms may make diagnosis of hepate-biliary disease possible.
