Comparative Base Specificity, Stability, and Lectin Activity of Two Lectins from Eggs of Rana catesbeiana and R. japonica and Liver Ribonuclease from R. catesbeiana

Abstract

Two lectins with RNase activity obtained from eggs of Rana catesbeiana and R. japonica and RNase obtained from R. catesbeiana liver show 65-83% protein homology. The base specificity of these frog proteins was studied with 8 dinucleoside phosphates as substrates and 8 nucleotides as inhibitors. The base specificities of the B1 and B2 sites of these proteins are U greater than C and G greater than U greater than A, C, respectively. The three frog proteins are more resistant than RNase A to heat treatment, guanidine-HCl and pH-induced denaturation; i.e., they retain their native conformation up to at least 70 degrees C at pH 7.5. Differences in stability and base specificity among RNase A and the three frog proteins are discussed in relation to the primary structures. Although the two lectins agglutinate tumor cells (e.g., Ehrlich, S-180 and AH109A ascites carcinoma cells), the liver RNase has no such activity. Agglutination of AH109A cells by the two lectins is inhibited by nucleotides. Our results indicate that the agglutination sites are not identical with, but are related to, the active sites of the three frog proteins.

Enzymatic Properties of Sialic Acid Binding Lectin from Rana catesbeiana Modified with a Water-Soluble Carbodiimide in the Presence of Various Nucleophiles.

“…It has been noted that the B2 subsite of cSBL is guanine base preference in contrast to that of RNase A which is adenine base preference. 4) The modified cSBLs indicate a tendency similar (G UϾAϾC) to that of cSBL. However, the B2 site preference for G was apparently reduced while the preference for A and C was slightly increased in the case of the chemically modified cSBL.…”

“…4) The reaction was performed in 50 mM Tris-HCl buffer (pH 7.5) with 2.5 mg/ml RNA at 37°C. (b) RNase activity towards homopolynucleotides, poly U and poly C. The RNase activity was measured using hyperchromicity upon hydrolysis of homopolynucleotides.…”

“…This property differs from those of the other RNase A super family enzymes which show a B2 subsite preference for adenosine. 3,4) It was previously reported that cSBL, which is modified with a water soluble carbodiimide (EDC) in the presence of nucleophiles such as ethylenediamine and glycine methylester, exerted more potent anti-tumor activity than the parent cSBL.…”

Effect of Modification of the Carboxyl Groups of the Sialic Acid Binding Lectin from Bullfrog (Rana catesbeiana) Oocyte on Anti-tumor Activity.

“…3,4) In respect to RNase, cSBL belongs to the RNase A family, and is a very heat stable enzyme. It remained 100% active after heating at 100°C.…”

“…It remained 100% active after heating at 100°C. 3,5) Four RNases belonging to the RNase A family have been isolated from Rana family frogs to date. Their primary structures are shown in Fig.…”