Cloning, bacterial expression, and unique structure of adenosylhomocysteine hydrolase-like protein 1, or inositol 1,4,5-triphosphate receptor-binding protein from mouse kidney.
Adenosylhomocysteine hydrolase (SAHase)-like protein 1 (SAH-L), also called inositol 1,4,5-triphosphate receptor-binding protein (IRBIT) is a novel protein involved in fish embryo development and calcium release in mammalian cells through protein-protein interactions. To better understand its reaction mechanism, purified protein is indispensable. Here we describe a simple purification procedure and the unique properties of SAH-L. The cDNA was isolated from mouse kidney by RT-PCR and inserted into various pETtrade mark vectors. Escherichia coli harboring a plasmid coding for SAH-L with a C-terminal His-tag could solely produce a soluble protein. SAH-L purified through a Ni(2+) column gave M(r)s of 59,000 and 190,000 by SDS-PAGE and gel filtration, respectively, which is suggestive of a trimer, but chemical cross-linking experiments demonstrated a dimer. The incompatible M(r) values implicate an irregular structure of SAH-L. In fact, SAH-L was partially purified in a form lacking the 31 N-terminal residues, and was found to be extremely susceptible to proteases in the region around residue 70. The N-terminal polypeptide (residues 1-98) was also expressed as a soluble form and was trypsin-sensitive. Circular dichroism revealed a low alpha-helix content but not a randomly extended structure. Interestingly, SAH-L contained tightly bound NAD(+) despite showing no SAHase activity. The characterized properties of SAH-L and its N-terminal fragment present the notion that the structure of the protease-sensitive N-terminal region is relatively loose and flexible rather than compact, and which protrudes from the major SAHase-like domain. This structure is supposed to be favorable to interact with the IP(3) receptor.
Gomi
Tomoharu
T
Life Science Research Center, University of Toyama, Toyama 930-0194, Japan.
Takusagawa
Fusao
F
Nishizawa
Mikio
M
Agussalim
Bukhari
B
Usui
Isao
I
Sugiyama
Eiji
E
Taki
Hirofumi
H
Shinoda
Kouichiro
K
Hounoki
Hiroyuki
H
Miwa
Toshiro
T
Tobe
Kazuyuki
K
Kobayashi
Masashi
M
Ishimoto
Tetsuya
T
Ogawa
Hirofumi
H
Mori
Hisashi
H
D043383:Adenosylhomocysteinase / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D005786:Gene Expression Regulation / Q000187:drug effects; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007668:Kidney / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis; D014169:Transformation, Bacterial
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C534112:adenosylhomocysteine hydrolase-like protein 1, mouse; D007544:Isopropyl Thiogalactoside; D043383:Adenosylhomocysteinase