Purification and structure of rat erythroid-specific delta-aminolevulinate synthase.
The existence of erythroid form delta-aminolevulinate synthase (ALAS-E) was historically a matter of some controversy. To obtain direct evidence for a unique ALAS-E, we have purified ALAS-E to homogeneity for the first time, from rat reticulocyte lysate. The papain digestion method was used at the initial step of the purification to overcome the difficulty which repeatedly hampered earlier attempts to purify ALAS-E. The size of the purified papain-resistant core catalytic domain of ALAS-E was estimated electrophoretically to be 49,000 Da. The pH optimum (7.6) and apparent Km values for the substrates, glycine (6.5 mM) and succinyl-CoA (2 microM), were similar to those of the non-specific form of delta-aminolevulinate synthase (ALAS-N); but, in contrast to ALAS-N, the substrate inhibition by succinyl-CoA was not evident in ALAS-E. We then isolated cDNA and genomic DNA clones encoding rat ALAS-E. By combining the nucleotide sequence information of the cDNA and genomic clones, the rat ALAS-E precursor is predicted to be composed of 587 amino acids with a calculated molecular mass of 64,841 Da. All the peptide sequences determined directly from the purified protein agreed with those predicted from the nucleotide data, demonstrating the existence of ALAS-E. Analysis of the papain-resistant core domain further revealed that it overlaps with the evolutionally conserved segment that has been noticed by sequence alignment analysis of ALA synthases from various species.
Munakata
H
H
Department of Biochemistry, Tohoku University School of Medicine, Sendai.
Yamagami
T
T
Nagai
T
T
Yamamoto
M
M
Hayashi
N
N
D000624:5-Aminolevulinate Synthetase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010206:Papain / Q000494:pharmacology; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D012156:Reticulocytes / Q000201:enzymology*; D016415:Sequence Alignment
D018076:DNA, Complementary; D012333:RNA, Messenger; D000624:5-Aminolevulinate Synthetase; D010206:Papain