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We have isolated a full-length cDNA (HPAsn.6) for human placenta glycosylasparaginase using a 221-bp PCR amplified fragment containing rat liver asparaginase gene sequences. The deduced amino acid sequence from the human clone showed sequence identity to both the alpha and beta subunits of the rat enzyme. The human enzyme is encoded as a 34.6 kDa polypeptide that is post-translationally processed to generate two subunits of approx. 19.5 (alpha) and 15 (beta) kDa. A charge enriched region is present at the predicted site where cleavage occurs. Using polyclonal antibodies against the alpha and beta subunits of rat liver asparaginase, we have shown that the human enzyme is similar in structure to the rat enzyme.
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1. Activities of asparagine synthetase, asparaginase, glutamine synthetase and glutaminase have been determined in red muscle, white muscle, brain, kidney, liver and gills of goldfish. 2. Muscle and brain show a capacity for net amide synthesis, while liver and gills are capable of both amide synthesis and degradation. 3. These results are consistent with the hypothesis that amide synthesis and degradation functions as a mechanism controlling tissue ammonia levels and ammonia excretion rates.
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Anandamide loses its cannabimimetic activities upon hydrolysis to arachidonic acid and ethanolamine. So far the anandamide hydrolyzing activity widely distributed in mammalian organs has been attributed exclusively to an enzyme referred to as anandamide amidohydrolase with an optimum pH around 9. We found another enzyme hydrolyzing anandamide in a human megakaryoblastic cell line (CMK). The enzyme present in the 12,000 x g pellet of the cell homogenate was solubilized by freeze-thaw. The solubilized enzyme showed an optimal pH around 5, and was almost inactive at alkaline pH. The enzyme activity was increased by the addition of dithiothreitol. In contrast, anandamide amidohydrolase of RBL-1 cells was mostly insoluble even after freeze-thaw, showed an optimal pH at 9, and was not affected by dithiothreitol. Furthermore, the enzyme of CMK cells was much less sensitive to phenylmethylsulfonyl fluoride and methyl arachidonoyl fluorophosphonate potently inhibiting anandamide amidohydrolase, and effectively hydrolyzed palmitoylethanolamide, which was a poor substrate for anandamide amidohydrolase. Thus, the enzyme of CMK cells is distinguishable from anandamide amidohydrolase.
Acylamidohydrolases from higher plants have not been characterized or cloned so far. AtAMI1 is the first member of this enzyme family from a higher plant and was identified in the genome of Arabidopsis thaliana based on sequence homology with the catalytic-domain sequence of bacterial acylamidohydrolases, particularly those that exhibit indole-3-acetamide amidohydrolase activity. AtAMI1 polypeptide and mRNA are present in leaf tissues, as shown by immunoblotting and RT-PCR, respectively. AtAMI1 was expressed from its cDNA in enzymatically active form and exhibits substrate specificity for indole-3-acetamide, but also some activity against L-asparagine. The recombinant enzyme was characterized further. The results show that higher plants have acylamidohydrolases with properties similar to the enzymes of certain plant-associated bacteria such as Agrobacterium-, Pseudomonas- and Rhodococcus-species, in which these enzymes serve to synthesize the plant growth hormone, indole-3-acetic acid, utilized by the bacteria to colonize their host plants. As indole-3-acetamide is a native metabolite in Arabidopsis thaliana, it can no longer be ruled out that one pathway for the biosynthesis of indole-3-acetic acid involves indole-3-acetamide-hydrolysis by AtAMI1.
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