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A pyrimidine base specific and most basic alkaline RNase named RNase BL4 was isolated from bovine liver as a protein showing a single band on slab gel-electrophoresis. The enzyme is most active at pH 7.5. The enzyme was immunologically distinguishable from the known bovine RNases such as pancreatic RNase (RNase A), seminal RNase, kidney non-secretory RNase (RNase K2), and brain RNase (RNase BRb). The primary structure of this pyrimidine base-specific RNase was determined to be less than EDRMYQRFLRQHVDPDETG- GNDSYCNLMMQRRKMTSHQCKRFNTFIHEDLWNIRSICSTTNIQCKNGQMNCHEGVVRV- TDCRETGSSRAPNCRYRAKASTRRVVIACEGNPEVPVHFDK. It consists of 119 amino acid residues, and is 5 amino acid residues shorter than RNase A. The sequence homology of RNase BL4 with RNase A is 46.2%, and optimal alignment of RNase A and RNase BL4 requires five deletions, one at the 24th position, two at the 75th and 76th positions, and two at the C-terminus in RNase BL4. The RNase BL4 was highly homologous with a porcine liver RNase (RNase PL3, 94.1% homology) studied by Hofsteenge et al. (personal communication from Hofsteenge, J., Matthies, R., and Stones, S.R.).

In order to investigate the nature of the amino acid residues involved in the active site of a ribonuclease from Rhizopus sp. (RNase Rh), carboxymethylation of RNase Rh with iodoacetate was performed. RNase Rh was found to be inactivated markedly at pH 3-5 by iodoacetate. From the pH profile of the rate of inactivation of RNase Rh, it was suggested that functional groups having pKa values of ca. 7.3 and 4.3 might be involved in this inactivation reaction. The determination of the amino acid composition of RNase Rh inactivated by iodoacetate at pH 5.0 indicated that the formation of about one residue of N3-carboxymethylhistidine was responsible for the loss of enzymatic activity. The results were very similar to those for an RNase from Asp. saitoi having very similar base specificity. The carboxymethylation of RNase Rh was inhibited by competitive inhibitors. Thus, the histidine residue modified might be involved in the active site of the enzyme.

The primary structure of a pyrimidine base-specific ribonuclease from bovine brain was determined. The sequence determined is (sequence; see text). Although the sequence homology of this RNase with bovine pancreatic RNase A is 78.2%, it consists of 140 amino acid residues, and it is 16 amino acid residues longer than RNase A at the carboxyl-terminal. In addition to an N-glycosylated long carbohydrate chain, the bovine brain RNase has two short O-glycosylated carbohydrate chains at the 129th and the 133rd serine residues. The additional C-terminal tail of the bovine brain RNase has a unique composition: 6 proline, 5 hydrophobic amino acids, and two basic amino acids, arginine and histidine.

To determine the distribution of a kidney acid RNase (RNase K2) and other RNases, the levels of RNase K2, RNase A, and seminal RNase (RNase Vs1) in bovine tissues and body fluids were measured by enzyme immunoassay. The crude extracts of several tissues and body fluids were fractionated by phospho-cellulose column chromatography. The enzymatic activities at pH 7.5 and 6.0 and enzyme contents of each tube were measured by enzyme assay and enzyme immunoassay, respectively. In the pancreas, parotid gland, and heart, most RNase activity was due to a single peak of RNase A, but a small amount of RNase K2 was always observed. In the kidney, there was about 5 times as much RNase K2 as RNase A. In the lung, although RNase K2 and RNase A were the major components, there are another two alkaline RNase peaks. In the spleen and liver, there are four RNases, two acid RNases, one of which is RNase K2, and two alkaline RNases including RNase A. A new acid RNase (non RNase K2-acid RNase) from both organs was immunologically the same. In serum, there are at least four RNases. By partial purification of serum RNases by phosphocellulose and heparin-Sepharose column chromatographies, at least 4 RNases, RNase A, RNase K2 and the other two alkaline RNases, one of which is immunologically indistinguishable from liver alkaline RNase, were confirmed. The other serum alkaline RNase was immunologically related to lung and spleen alkaline RNases. In conclusion, in bovine tissues and body fluids there are at least 7 types of pyrimidine-base-specific RNases: brain RNase, seminal RNase, RNase A, RNase K2, an acid RNase (RNase BSP1), an alkaline RNase (RNase BL4), and another alkaline RNase in serum.

The primary structure of a non-secretory ribonuclease from bovine kidney (RNase K2) was determined. The sequence determined was VPKGLTKARWFEIQHIQPRLLQCNKAMSGV NNYTQHCKPENTFLHNVFQDVTAVCDMPNIICKNGRHNCHQSPKPVNLTQCNFIAGRYPDC RYHDDAQYKFFIVACDPPQKTDPPYHLVPVHLDKYF. The sequence homology with human non-secretory RNase, bovine pancreatic RNase, and human secretory RNase are 46, 34.6, and 32.3%, respectively. The bovine kidney RNase has two inserted sequences, a tripeptide at the N-terminus and a heptapeptide between the 113th and 114th position of bovine pancreatic RNase; on the other hand, it is deleted of the hexapeptide consisting of the 17th to the 22nd amino acid residue of RNase A. The amino acid residues assumed to be the constituents of the bovine pancreatic RNase active site are all conserved except F120 (L in RNase K2).

The carboxyl group in a ribonuclease from Rhizopus sp. (RNase Rh) was modified by a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate (CMC). From the relation between the extent of modification and the enzymatic activity, it was concluded that at least the modification of two carboxyl groups seemed to induce the loss in enzymatic activity. In the presence of 1 M cytidine, RNase Rh activity was protected from the CMC-modification. Under conditions in which the enzyme was inactivated to 20% activity, about 70% of the enzymatic activity was retained in the presence of cytidine. The inactivation of the RNase Rh pre-treated with CMC in the presence of cytidine with [14C]CMC indicated that the RNase Rh lost its enzymatic activity with the incorporation of about one [14C]CMC. Therefore, it could be concluded that one carboxyl group is involved in the active site of RNase Rh. The binding of the CMC-modified RNase Rh with 2'-AMP was studied spectrophotometrically. The affinity of the modified RNase Rh towards 2'-AMP decreased markedly upon CMC modification.

In order to clarify the reason for the variation in specific activities of ribonuclease preparations from Rhizopus sp. ribonuclease (RNase Rh), low specific activity species (RNase Rh') were separated from native RNase Rh by DEAE Toyopearl 650 column chromatography and characterized. When RNase Rh' was subjected to gel electrophoresis in the absence of 2- mercaptoethanol, it gave a 24 kilodalton (kDa) protein band, but in the presence of the reducing agent it gave 17 and 7 kDa bands. These two peptides were separated by gel filtration and their NH2-terminal amino acid sequences were determined. The results indicated that RNase Rh' was an enzyme species cleaved at about the 50th residue of native RNase Rh by proteases during the course of purification, but the two fragments were still covalently joined by S-S bridges. RNase Rh' retained about 70% of the native activity and has a similar conformation to the native enzyme.

Two acid RNases were purified from bovine spleen by means of ammonium sulfate fractionation, chromatographies on-phospho-cellulose, heparin-Sepharose CL-6B, poly G-Sepharose, and 2', 5'-ADP-Sepharose, and gel filtration on Toyopearl HW 55F. Both purified preparations were homogeneous as judged by disc electrophoresis at pH 4.3. They were designated as RNase BSP1 and RNase BSP2 in the order of elution from a phospho-cellulose column. RNase BSP2 was immunologically indistinguishable from RNase K2 from bovine kidney. RNase BSP1 was a typical pyrimidine base-specific, uridylic acid-preferential RNase and had very sharp pH optimum at 6.5. RNase BSP1 thus obtained was a glycoprotein giving two major bands on SDS-slap electrophoresis. Although the apparent molecular weight of RNase BSP1 was distributed in the range of 27,000-20,000, it decreased to about 17,000-18,000 after endoglycosidase F digestion. The N-terminal amino acid sequence up to the 20th amino acid had no homology to those of RNase K2 and RNase A.

Two RNases (RNases K1 and K2) were purified from bovine kidney by means of column chromatography on phospho-cellulose, Sephadex G-50, CM-cellulose, heparin-Sepharose, nd agarose-APUP. They were named RNase K1 and RNase K2 in order of elution from the heparin-Sepharose column. The purity of RNase K1 thus obtained was about 90% by SDS-disc electrophoresis. RNase K2 was purified to homogeneity by SDS- and pH 4.3 disc electrophoresis. The yield of RNase K2 was 3.4 mg from 11 kg of kidneys. The antigenic properties of the two bovine renal RNases were studied by Ouchterlony's double diffusion analysis. RNase K1 and RNase A were serologically indistinguishable. RNase K2 did not cross-react immunologically with RNase K1 or RNase A. The molecular weights of these RNases determined by gel-filtration on Sephadex G-50 were 13,400 and 14,600 for RNase K1 and RNase K2, respectively. The pH optima for RNase K1 and RNase K2 were 8.5 and 6.5, respectively. Both RNase K1 and RNase K2 were as acid stable as RNase A. RNase K2 was less heat-stable than RNase K1 and RNase A. Although both renal RNases were pyrimidine nucleotide-specific enzymes, RNase K1 and RNase A were more preferential or cytidylic acid than RNase K2. The chemical composition of RNase K2 was determined. RNase K2, like human urinary RNase Us, contained one tryptophan residue. The N-terminal sequences of RNase K2 and RNase Us were determined by Edman degradation. Rnase K2 had a homologous sequence of about 10 amino acid residues with the sequence of RNase Us, a typical non-secretory RNase, within the N-terminal 30 residues.