Amino Acid Sequence and Characterization of a Rice Bran Ribonuclease

Abstract

RNase T2 family enzymes which are base non-specific and have a molecular mass of about 24 kDa, distributed widely from viruses to microbes, plants and animals. 1)They share two well conserved active site sequences, CAS I and CAS II. Therefore, they are a good model with which to study the evolution of RNases. In the plant kingdom, members of the RNase T2 family are classified into two classes, S-RNases which are the products of S-genes and function as self-incompatibility factors, 2) and S-like RNases.3) The latter enzymes are known to be induced by phosphate deprivation [Jost et al.,4) Bariola et al. 5)], in the case of senescence [Lers et al. 6) ], and exist in the seeds probably for defense [Ide et al.,7) Rojo et al. 8,9) ]. In terms of the evolutional change of plant RNases, knowledge is limited to those plants belonging to Dicotyledoneae, except for barley. 10) These include several plants of Solanaceae [tomato,4,11) and tobacco, 3,12) petunia 13) and a kind of potato 14) ], Cucurbitaceae [bitter gourd, 7) cucumber, 18) and others. In this report, we will describe the amino acid sequence of an RNase from rice bran (Oryza sativa) which is a member of Monocotyledoneae, and its enzymatic properties. MATERIALS AND METHODSEnzymes Lysylendopeptidase and g-pyroglutamine aminopeptidase were obtained from Wako Pure Chemical Ind., Ltd. (Osaka, Japan) and Calbiochem-Novabiochem. Co., Ltd., (LaJolla, LA, U.S.A.), respectively.Other Reagents Yeast RNA was a product of Marin Bio (Tokyo, Japan). Sixteen dinucleoside phosphates were purchased from Sigma (St. Louis, MO, U.S.A.). Phospho-cellulose and DE-52 were obtained from Whatman (Clifton, NJ, U.S.A.). A Mono Q column was obtained from Amersham Pharmcia Biotech (Buckinghamshire, England).Enzyme Assay (i) RNA as substrate: RNase assay was carried out as described 19) with RNA as substrate at pH 5.5 and 37°C. (ii) Dinucleosidephosphates as substrate: The rates of hydrolysis of 16 dinucleoside phosphates (XpYs), where X and Y were one of A, G, C and U, were measured spectrophotometrically as described by Imazawa et al. 20) in 0.1 M sodium acetate buffer (pH 5.5) at 22°C.Protein Concentration The protein concentration was measured spectrophotometrically assuming the absorbancy at 280 nm of a 0.1% protein solution to be 1.0.Purification of an RNase from Rice Bran Purification of an RNase from rice bran was performed by modification of the procedure reported by Yokoyama et al. 21)Step 1. Four kilograms of rice bran (product from a strain of Oryza sativa, Koshihikari) was suspended in 12 l of 20 mM sodium phosphate buffer (pH 7.0) and extracted by vigorous stirring at 4°C. The extract was filtered through a layer of cotton sheet, and the filtrate was centrifuged at 10000 rpm for 10 min. The pH of the supernatant was adjusted to 5.0 by addition of dil. HCl. The supernatant was kept at 4°C overnight, and the precipitate formed was eliminated by centrifugation at 10000 rpm for 10 min (crude extract).Step 2. The crude extract was fractionated by ammonium sulfate, and the pr...

RNase T2 genes from rice and the evolution of secretory ribonucleases in plants

“…Five proteins (OsRNS2, OsRNS6, OsRNS8, OsRNS1, OsRNS3) contain the two histidines that are present in the conserved active site (CAS) I and II motifs, and are essential for ribonuclease activity (Irie 1999). OsRNS3 corresponds to the previously described RNase Os, an active RNase purified from rice bran (Iwama et al 2001). On the other hand, OsRNS4 and OsRNS5 have lost most conserved residues in the two CAS.…”

“…Two genes belonging to the RNase T2 family have been described in rice. The first, RNase Os, encodes an active enzyme that was purified from rice bran (Iwama et al 2001). A barley enzyme, GAR-RNase, also accumulates in the aleurone layer, and GAR-RNase expression is induced by gibberellins and repressed by abscisic acid (Rogers and Rogers 1999).…”