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. the effect of different inhibitors on the water buffalo kidney AAPs showed that, bestatin was the most potent inhibitor of AAP1, AAP2 and AAP3 since it caused 72 %, 82% and 95% inhibition at a concentration of 1 µM. a linear relationship was observed by constructing the Hill plot for the inhibition of the purified water buffalo kidney AAP3 by bestatin. The slope of the Hill plot was found to be 0.78 indicating the existence of one binding site for bestatin on the water buffalo kidney AAP3. the type of inhibition of water buffalo kidney AAP3 by bestatin was found to be competitive. The Ki value of the AAP3 inhibition by bestatin was determined to be 0.4 µM. 2- camel kidney cortex alanine aminopeptidase isoenzymes: three alanine aminopeptidases termed AAP1, AAP2 and AAP3 were purified from the camel kidney cortex. the isoenzymes AAP1, AAP2 and AAP3 were purified from the camel kidney 1.74, 2.36 and 6.83 fold with a yield of 5.18 %, 8.6 % and 51.31 % recovery respectively. the purified camel 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 118 kDa exhibited a homodimeric structure composed of two identical subunits with a subunit of 60 ± 1 kDa, While AAP2 was 420 kDa exhibited a homohexameric structure composed of six identical subunits with a subunit of 70 ± 1 kDa and AAP3 was 360 kDa exhibited a homohexameric structure composed of six identical subunits with a subunit of 60 ± 2 kDa. AAP1, AAP2 and AAP3 isoenzymes showed an isoelectric point (pI) value at pH 6.2, 5.9 and 6.4 respectively. AAP1, AAP2 and AAP3 isoenzymes displayed thier optimum activity at pH 7.6, 8.0 and 7.8 respectively. the Km values were found to be 0.155, 0.16 and 0.175 mM of alanine -β-naphthylamide HCl for camel kidney AAP1, AAP2 and AAP3 respectively. the effect of metal ions on the camel kidney AAPs showed that, the activity of AAP3 was increased 120.4 % and 110.7 % in the presence of 0.5 and 1.0 mM MgCl2, 120.23 % and 105.79 % in the presence of 0.5 and 1.0 mM CoCl2, 106.74 % in the presence of 1.0 mM CaCl2. The activity of AAP2 was increased 108.05 % in the presence of 1.0 mM FeCl2. The three isoenzymes were inactivated by metal ions of Cu2+, Mn2+, Ni2+ and Zn2+, while Co2+ has inhibitory effect only on AAP1 and AAP2. all amino acids caused either slight or moderate inhibition on the purified camel kidneys AAP1, AAP2 and AAP3 except L- alanine (1 mM) caused slight activation 103.8 % and 101.93 % on the isoenzymes AAP2 and AAP3 respectively and L-serine (1 mM) caused slight activation 105.2 % and 101.69 % on the isoenzymes AAP1 and AAP2 respectively. the purified enzymes AAP1, AAP2 and AAP3 did not cleave both the synthetic and natural substrates for endopeptidases, trypsin and chymotrypsin. the effect of different inhibitors on the camel kidney AAPs showed that, bestatin was the most potent inhibitor of AAP1, AAP2 and AAP3 since it caused 92 %, 78.9% and 94.7% inhibition at a concentration of 1 µM. a linear relationship was observed by constructing the Hill plot for the inhibition of the purified camel kidney AAP3 by bestatin. the slope of the Hill plot was found to be 0.8 indicating the existence of one binding site for bestatin on the camel kidney AAP3. the type of inhibition of camel kidney AAP3 by bestatin was found to be competitive. the Ki value of the AAP3 inhibition by bestatin was determined to be 0.54 µM. 3- Sheep kidney cortex alanine aminopeptidase isoenzymes three alanine aminopeptidases termed AAP1, AAP2 and AAP3 were purified from the sheep kidney cortex. the AAP1, AAP2 and AAP3 isoenzymes were purified from the sheep kidney 0.854, 0.48 and 13.05 fold with a yield of 3.93 %, 3.58 % and 61.2 % recovery respectively. the purified sheep 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 420 kDa exhibited a homohexameric structure composed of six identical subunits with a subunit of 70 ± 1 kDa and AAP3 was 380 kDa exhibited a homohexameric structure composed of six identical subunits with a subunit of 64± 2 kDa. sheep kidney AAP1, AAP2 and AAP3 isoenzymes showed an isoelectric point (pI) value at pH 6.6, 5.4 and 6.2 respectively. AAP1, AAP2 and AAP3 isoenzymes displayed thier optimum activity at pH 7.8, 7.6 and 8.0 respectively. km values were found to be 0.15, 0.17 and 0.185 mM of alanine -β-naphthylamide HCl for sheep kidney AAP1, AAP2 and AAP3 respectively. the effect of metal ions on the sheep kidney AAPs showed that, the activity of AAP3 was increased 106.8 %, 118.5 % and 108.45% in the presence of 0.5 mM MgCl2, MnCl2 and CoCl2 respectively. the activity of AAP1 was increased 113.29 % and 118.42 % in the presence of 0.5 and 1.0 mM FeCl2 respectively and 115.1 % and 109.36 % in the presence of 0.5 and 1.0 mM Mg2+ respectively. The isoenzymes AAP1, AAP2 were inactivated by metal ions of Cu2+, Mn2+, Ni2+, Co2+ and. Ca2+, while the metal ion Zn2+ has a great inhibitory effect on the three isoenzymes AAP1, AAP2 and AAP3. all amino acids caused either slight or moderate inhibition on the purified sheep kidneys AAP1, AAP2 and AAP3 except L- Histidine (1 mM) caused slight activation 100.8 % on the isoenzyme AAP2 and L-Serine (1 mM) caused 102 % on the isoenzymes AAP1. the purified enzymes AAP1, AAP2 and AAP3 did not cleave both the synthetic and natural substrates for endopeptidases, trypsin and chymotrypsin. the effect of different inhibitors on the sheep kidney AAPs showed that, bestatin was the most potent inhibitor of AAP1, AAP2 and AAP3 since it caused 86.8 %, 79% and 93% inhibition at a concentration of 1 µM. a linear relationship was observed by constructing the Hill plot for the inhibition of the purified sheep kidney AAP3 by bestatin. The slope of the Hill plot was found to be 0.82 indicating the existence of one binding site for bestatin on the sheep kidney AAP3. the type of inhibition of sheep kidney cortex AAP3 by bestatin was found to be competitive. The Ki value of the AAP3 inhibition by bestatin was determined to be 0.34 µM. in conclusion, this study presents a simple, convenient and reproducible method for the purification of a well characterized alanine aminopeptidase from the water buffalo, camel and sheep kidney cortex as a locally available source. Production of this enzyme on large scale will make it suitable for various applications such as food industries and investigation of the protein primary structure.