Cloning and expression of rat neutral sphingomyelinase: enzymological characterization and identification of essential histidine residues.
Using cross-species sequence homology, we cloned a cDNA for rat neutral sphingomyelinase (nSMase) composed of 422 amino acids that shares 87.6 and 79.0% identity with the mouse and human forms respectively. The rat nSMase expressed in Escherichia coli catalyzed sphingomyelin hydrolysis at neutral pH in a Mg(2+)-dependent manner, and required Triton X-100, dithiothreitol, and KCl for its full activity. The cloned rat enzyme shares conserved sequences with nSMases from both eukaryotes and prokaryotes. Introduction of single mutations into either of the histidine residues at positions 136 and 272, putative active sites, entirely abolished the activity, supporting a common mechanism for the nSMase family independent of the species. However, mutation in histidine 151, conserved only in eukaryotes, also abolished the activity, suggesting eukaryote-specific control of nSMase linked to this histidine 151. This enzyme also catalyzed the hydrolysis of lyso-platelet activating factor to yield 1-alkylglycerol at a rate that is slightly lower than that with sphingomyelin.
Mizutani
Y
Y
Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanisms and Control, Nagoya University School of Medicine, Japan.
Tamiya-Koizumi
K
K
Irie
F
F
Hirabayashi
Y
Y
Miwa
M
M
Yoshida
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D015870:Gene Expression; D006639:Histidine / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010972:Platelet Activating Factor / Q000031:analogs & derivatives / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; C029271:O-deacetyl platelet activating factor; D010972:Platelet Activating Factor; D006639:Histidine; D013108:Sphingomyelin Phosphodiesterase