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.
A guanine nucleotide-specific RNase (RNase Po1) was isolated from caps of the fruit bodies of Pleurotus ostreatus. RNase Po1 is most active towards RNA at pH 8.0. The effect of heating on the molar ellipticity at 210 nm of RNase Po1 showed that RNase Po1 is more stable than RNase T1. The primary structure of RNase Po1 was determined to be < ETGVRSCNCAGRSFTGTDVTNAIRSARAGGSGNYPHVYNNFEGFSFSCTPTFFEFPVFRGSVYSGGSPG ADRVIYD- QSGRFCACLTHTGAPSTNGFVECRF. It consisted of 101 amino acid residues, with a molecular weight of 10,760. RNase Po1 has relatively higher sequence homology with RNase T1 family RNase. It contains 6 half cystine residues. The locations of four of them are superimposable on those of RNase U1 and RNase U2. The amino acid residues forming the active site of RNase T1 were well conserved in this RNase. Therefore, RNase Po1 is a unique member of the RNase T1 family in respect of the location of one disulfide bridge, and its stability.
1. A major glucoamylase [EC 3.2.1.3] of Aspergillus saitoi was purified by ultrafiltration followed by successive chromatography on DEAE-Sephadex, Ultrogel AcA 44 and SP-Sephadex. The purification achieved was 23-fold from crude extract with a yield of 21%. The purified enzyme, named Gluc M1, was proved homogeneous as judged by polyacrylamide gel electrophoresis, isoelectric focusing, ultracentrifugation, and also from the absence of the glycosidase activities detected in crude extract. 2. Gluc M1 was a glycoprotein containing 18% neutral sugar and 0.77% glucosamine, and its molecular weight was estimated to be about 90,000 by SDS-polyacrylamide gel electrophoresis and amino acid composition. The N-terminal amino acid was identified as alanine. 3. The pH optimum of Gluc M1 was 4.5 with soluble starch as a substrate. The enzyme was stable between pH 2.5 and 7.5 and retained full activity at temperatures up to 50 degrees C. The enzyme activity was inhibited by Hg2+ and, to a lesser extent, by Pb2+ and Mn2+. 4. The Km value for malto-oligomer markedly decreased with increasing chain length of substrate in glucose unit (n) and the Vmax value increased with n, thus resulting in the increase in the Vmax/Km value with n. The kinetic parameters for other substrates such as soluble starch, glycogen and isomaltose as well as the K1 values for some saccharides were also determined.
RNase He1 from Hericium erinaceus, a member of the RNase T1 family, has high identity with RNase Po1 from Pleurotus ostreatus with complete conservation of the catalytic sequence. However, the optimal pH for RNase He1 activity is lower than that of RNase Po1, and the enzyme shows little inhibition of human tumor cell proliferation. Hence, to investigate the potential antitumor activity of recombinant RNase He1 and to possibly enhance its optimum pH, we generated RNase He1 mutants by replacing 12 Asn/Gln residues with Asp/Glu residues; the amino acid sequence of RNase Po1 was taken as reference. These mutants were then expressed in Escherichia coli. Using site-directed mutagenesis, we successfully modified the optimal pH for enzyme activity and generated a recombinant RNase He1 that inhibited the proliferation of cells in the human leukemia cell line. These properties are extremely important in the production of anticancer biologics that are based on RNase activity.
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