caa a22 4500 397557612 CHVBK 20180308164807.0 cr unu---uuuuu 161202e199605 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021337 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021337 Pyridoxal 5′-Phosphate Binding of a Recombinant Rat Serine: Pyruvate/Alanine: Glyoxylate Aminotransferase [Elektronische Daten] [Kuniko Ishikawa, Eizo aneko, Arata Ichiyama] Serine: pyruvate/alanine: glyoxylate aminotransferase in the liver is a class IV amino-transferase. The present study was undertaken to characterize the pyridoxal 5′-phosphate (PLP) binding to a recombinant rat serine:pyruvate/alanine:glyoxylate aminotransferase (SPT10), which is a homodimer of 44.4 kDa subunits. Purified SPT10 exhibited absorption maxima at ˜330 nm in addition to a 278 nm protein peak and a ˜420 nm peak of PLP bound via Schiff base, and contained 0.56-0.69 mol of PLP per mol of subunit. Apo-SPT10 without measurable bound PLP did not exhibit the absorbance at ˜420 nm, but still showed the ˜330 nm peak. Upon reconstitution, 0.73-0.79 mol of PLP per mol of subunit was bound to apo-SPT10 with an apparent Kd of ˜0.1 μM, resulting in a holo-SPT10 preparation whose specific activity and A−420A−330 absorbance ratio were higher than those of the original SPT10. On SDS/PAGE of BrCN-cleavage peptides of NaBH4-reduced SPT10, 22-23 kDa fragments migrated as a pair of bands. On amino acid sequencing, the ˜22 and ˜23 kDa pair gave the same sequence, except that Lys was released only from the ˜22 kDa band material in the cycle corresponding to Lys209. NaB3H4 -treated SPT10 also migrated as a pair of 44-45 kDa bands and 3H was incorporated only into the ˜45 kDa band. It appears that SPT10 has the capacity to bind 1 mol of PLP to Lys209 of every subunit, but usually binds less PLP in a Schiff base structure, probably due to the presence of a 330 nm-absorbing chromophore. © by the Journal of Biochemistry Regular Papers nationallicence absorption spectrum of a B6 enzyme nationallicence pyridoxal 5′-phosphate nationallicence pyridoxal 5′-phosphate binding Lys residue nationallicence SDS/PAGE after NaBH4 nationallicence reductionserine:pyruvate/alanine: glyoxylate aminotransferase nationallicence Ishikawa Kuniko First Department of Biochemistry Hamamatsu University School of Medicine 3600 Handa-cho, Hamamatsu, Shizuoka 431-31 aut aneko Eizo First Department of Internal Medicine, Hamamatsu University School of Medicine 3600 Handa-cho, Hamamatsu, Shizuoka 431-31 aut Ichiyama Arata First Department of Biochemistry Hamamatsu University School of Medicine 3600 Handa-cho, Hamamatsu, Shizuoka 431-31 aut The Journal of Biochemistry Oxford University Press 119/5(1996-05), 970-978 0021-924X 119:5<970 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021337 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021337 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ishikawa Kuniko First Department of Biochemistry Hamamatsu University School of Medicine 3600 Handa-cho, Hamamatsu, Shizuoka 431-31 aut NATIONALLICENCE 700 1- aneko Eizo First Department of Internal Medicine, Hamamatsu University School of Medicine 3600 Handa-cho, Hamamatsu, Shizuoka 431-31 aut NATIONALLICENCE 700 1- Ichiyama Arata First Department of Biochemistry Hamamatsu University School of Medicine 3600 Handa-cho, Hamamatsu, Shizuoka 431-31 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/5(1996-05), 970-978 0021-924X 119:5<970 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford