الفهرس 
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Abstract
This study presents the purification and characterization of the alanine aminopeptidase from the kidney of the mammalian domestic animals common in Egypt; water buffalo, camel and sheep.
In this study a comparison of alanine, glycine and leucine aminopeptidase specific activities was constructed in the crude extract of the three mammalian kidneys. The comparison revealed that the alanine aminopeptidase exhibited a higher specific activity than glycine and leucine aminopeptidases.
For the study to be more specific, we study the alanine aminopeptidase (AAP) in cortex and medulla of the three mammalian kidneys, the highest specific activity of aminopeptidases were monitored in the cortex of water buffalo, camel and sheep kidneys. So this led to the selection of the cortex of water buffalo, camel and sheep kidneys for the purification of AAP as the richest source of the enzyme.
A simple and reproducible purification procedure is given involved combination of anion exchange chromatography on DEAE-cellulose column followed by gel filtration chromatography on Sephacryl S-300 column.
1- Water buffalo kidney cortex alanine aminopeptidase isoenzymes:
Three alanine aminopeptidases termed AAP1, AAP2 and AAP3 were purified from the water buffalo kidney cortex.
The AAP1, AAP2 and AAP3 isoenzymes were purified from the water buffalo kidney 1.74, 2.36 and 6.83 fold with a yield of 5.18 %, 8.6 % and 51.31 % recovery respectively. The purified water buffalo kidney AAP1, AAP2 and AAP3 isoenzymes turned out to be homogeneous as judged by the native and SDS polyacrylamide gel electrophoresis.
The molecular weights of the purified isoenzymes were confirmed by both gel filtration and SDS-PAGE. The molecular weight of the purified AAP1 isoenzyme was 120 kDa exhibited a homodimeric structure composed of two identical subunits with a subunit of 60 ± 1 kDa, While AAP2 was 400 kDa exhibited a homohexameric structure composed of six identical subunits with a subunit of 67 ± 1 kDa and AAP3 was 350 kDa exhibited a homohexameric structure composed of six identical subunits with a subunit of 58 ± 2 kDa.
AAP1, AAP2 and AAP3 isoenzymes showed an isoelectric point (pI) value at pH 6.4, 6.2 and 6.6 respectively.
AAP1, AAP2 and AAP3 isoenzymes displayed thier optimum activity at pH 8, 7.8 and 7.8 respectively.
The Km values of the purified water buffalo kidney AAP1, AAP2 and AAP3 were found to be 0.15, 0.17 and 0.125 mM of alanine -β-naphthylamide HCl respectively.
The effect of metal ions on the water buffalo kidney AAPs showed that, the activity of AAP3 was increased 112.5 % and 108.2 % in the presence of 0.5 and 1.0 mM MgCl2 respectively and 107.9 % in the presence of 0.5 mM CaCl2. The activity of the three isoenzymes were inactivated by metal ions of Cu2+, Mn2+, Ni2+ and Zn2+, while Co2+ and Fe2+ have no significant effect on AAP3 and at the same time inhibited the activity of AAP1 and AAP2 isoenzymes.
All amino acids increase the activity of AAP2 except L- tyrosine, phenylalnine and L-leucine caused either slight or moderate inhibition of the enzyme, while the activity of AAP3 and AAP1 isoenzymes were inhibited 47.4 % and 32.9 % in the presence of 1 mM tyrosine, 30 % and 17.8 % by 1 mM phenylalnine and 25 % and 7.7 % by 1 mM serine respectively.
The purified enzymes AAP1, AAP2 and AAP3 did not cleave both the synthetic and natural substrates for endopeptidases, trypsin and chymotrypsin