Purification and characterization of a non-S-RNase and S-RNases from styles of Japanese pear (Pyrus pyrifolia).
In this study we biochemically characterized stylar ribonucleases (RNases) of Japanese pear (Pyrus pyrifolia), which exhibits S-RNase-based gametophytic self-incompatibility. We separated the RNase fractions NS-1, NS-2, and NS-3 from stylar extracts of the cultivar Nijisseiki (S(2)S(4)). The RNase in each fraction was purified to homogeneity through a series of chromatographic steps. Chemical analysis of the proteins revealed that the basic RNases in the NS-2 and NS-3 fractions were the S(4)- and S(2)-RNases, respectively. Five additional S-RNases were purified from other cultivars. An acidic RNase in the NS-1 fraction was also purified from other cultivars, and identified as a non-S-allele-associated RNase (non-S-RNase). The non-S-RNase is composed of 203 amino acids, is non-glycosylated and is a N-terminal-pyroglutamylated enzyme of the RNase T(2) family. The substrate specificities and optimum pH levels of the non-S-RNase and S-RNases were similar. Interestingly, the specific activity of the non-S-RNase was 7.5-221-fold higher than those of the S-RNases when tolura yeast RNA was used as the substrate. The specific activity of the S(2)-RNase was 8.8-28.6-fold lower than those of the other S-RNases. These differences in specific activities among the stylar RNases are discussed.
Norioka
Shigemi
S
Division of Protein Chemistry, Institute for Protein Research, Osaka University, Suita, Osaka, Japan.
Oneyama
Chitose
C
Takuma
Seiji
S
Shinkawa
Toyohide
T
Ishimizu
Takeshi
T
Nakanishi
Tetsu
T
Sakiyama
Fumio
F
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D031989:Pyrus / Q000201:enzymology* / Q000235:genetics; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D012260:Ribonucleases