Genetic polymorphism of human serum ribonuclease I (RNase I).

One of the human urinary ribonucleases (RNases) was isolated and purified to homogeneity (SDS-PAGE) by means of a series of column chromatographies. The enzyme, designated RNase 1, is a glycoprotein with a molecular weight of approximately 16,000. Rabbit antibody to the purified RNase 1 reacted with human urine and sera, as well as with the purified RNase 1. The genetic polymorphism of serum RNase 1 was studied by polyacrylamide gel isoelectric focusing (IEF-PAGE) in a pH range of 5-8, followed by immunoblotting with antisera specific for RNase 1. Two common phenotypes, RNASE1 1 and RNASE1 1-2, were easily recognized. The homogeneous phenotype, RNASE1 1, consisted of four major bands with different pI values, and the heterogeneous phenotype, RNASE1 1-2, was presumed to represent a mixture of each of the homogeneous phenotypes 1 and 2; however, the other homogeneous phenotype, RNASE1 2, was not detected in our samples. Family studies are in agreement with an autosomal codominant transmission of the two alleles. Population studies indicate that the frequencies of the RNASE 1 and RNASE1 2 alleles are .988 and .012, respectively.     

Human ribonucleases. Quantitation of pancreatic-like enzymes in serum, urine, and organ preparations

Antibodies against pure human pancreatic ribonuclease (RNase) were used to study ribonuclease levels in human tissues and body fluids. The antibodies completely inhibit the activity of purified RNase as well as ribonuclease activity in crude pancreatic extracts. RNase activity is inhibited by 70-80% in serum and urine, indicating that a significant proportion of the RNases in these preparations are structurally like the pancreatic enzyme. In contrast, inhibition of RNase activities from spleen (8%) and liver (30%) was inefficient suggesting that most of the RNases in these tissues are structurally unlike the pancreatic enzyme. A competitive binding radioimmunoassay (RIA), sensitive in the range of 1-100 ng of RNase, was developed to quantitate the pancreatic like enzymes. The RIA of crude tissue preparations and samples fractionated by gel filtration was compatible with inhibition results. Enzymes structurally like pancreatic RNase could be quantitated despite the presence of other RNase activities. Immunological quantitation of pancreatic like RNases was also found to be much more simple and precise than enzymatic assays comparing RNA and polycytidylate substrates. We suggest the immunological assays will be useful in the quantitation and definition of tissue of origin of RNases in serum of patients with pancreatic carcinoma.     

Purification and characterization of three ribonucleases from human kidney: comparison with urine ribonucleases

Three ribonucleases (RNases) with different molecular masses were isolated from human kidney. The enzymes were purified to an electrophoretically homogeneous state, and their respective molecular masses were found to be 18,000 (tentatively named RNase HK-1), 20,000 (RNase HK-2A), and 22,000 (RNase HK-2B) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the amino acid compositions, amino-terminal sequences, and enzymological properties of the enzymes indicate that RNase HK-1 is related to "nonsecretory" RNase, and that RNases HK-2A and HK-2B are both related to "secretory" RNase. Furthermore, RNase HK-1 showed cross-reactivity with an antibody specific to nonsecretory RNase from human urine, whereas RNases HK-2A and HK-2B showed cross-reactivity with another antibody specific to human urine secretory RNase. However, the carbohydrate compositions of RNases HK-2A and HK-2B were markedly different from that of the secretory urine RNase. This finding seems to indicate that the kidney is not the origin of the urine enzyme.     

Isolation, characterization, and primary structure of a base non-specific and adenylic acid preferential ribonuclease with higher specific activity from Trichoderma viride.

In order to elucidate the structure-function relationship of RNases belonging to the RNase T2 family (base non-specific and adenylic acid-preferential RNase), an RNase of this family was purified from Trichoderma viride (RNase Trv) to give three closely adjacent bands with RNase activity on slab-gel electrophoresis in a yield of 20%. The three RNases gave single band with the same mobility on slab-gel electrophoresis after endoglycosidase F digestion. The enzymatic properties including base specificity of RNase Trv were very similar to those of typical T2-family RNases such as RNase T2 from Aspergillus oryzae and RNase M from A. saitoi. The specific activity of RNase Trv towards yeast RNA was about 13-fold higher than that of RNase M. The complete primary structure of RNase Trv was determined by analyses of the peptides generated by digestion of reduced and carboxymethylated RNase Trv with Staphylococcus aureus V8 protease, lysylendopeptidase and alpha-chymotrypsin. The molecular weight of the protein moiety deduced from the sequence was 25,883. The locations of 10 half-cystine residues were almost superimposable upon those of other RNases of this family. The homologies between RNase Trv and RNase T2, RNase M, and RNase Rh (Rhizopus niveus) were 124, 132, and 92 residues, respectively. The sequences around three histidine residues, His52, His109, and His114, were highly conserved in these 4 RNases.     

Primary structure of a ribonuclease from porcine liver, a new member of the ribonuclease superfamily

In most tissues, ribonucleases (RNases) are found in a latent form complexed with ribonuclease inhibitor (RI). To examine whether these so-called cytoplasmic RNases belong to the same superfamily as pancreatic RNases, we have purified from porcine liver two such RNases (PL1 and PL3) and examined their primary structures. It was found that RNase PL1 belonged to the same family as human RNase Us [Beintema et al. (1988) Biochemistry 27, 4530-4538] and bovine RNase K2 [Irie et al. (1988) J. Biochem. (Tokyo) 104, 289-296]. RNase PL3 was found to be a hitherto structurally uncharacterized type of RNase. Its polypeptide chain of 119 amino acid residues was N-terminally blocked with pyroglutamic acid, and its sequence differed at 63 positions with that of the pancreatic enzyme. All residues important for catalysis and substrate binding have been conserved. Comparison of the primary structure of RNase PL3 with that of its bovine counterpart (RNase BL4; M. Irie, personal communication) revealed an unusual conservation for this class of enzymes; the 2 enzymes were identical at 112 positions. Moreover, comparison of the amino acid compositions of these RNases with that of a human colon carcinoma-derived RNase, RNase HT-29 [Shapiro et al. (1986) Biochemistry 25, 7255-7264], suggested that these three proteins are orthologous gene products. The structural characteristics of RNases PL1 and PL3 were typical of secreted RNases, and this observation questions the proposed cytoplasmic origin of these RI-associated enzymes.     

Purification and properties of magnesium- and manganese-dependent ribonucleases H from chick embryo

Two RNases H, Mg2+- and Mn2+-dependent RNases H, are present in extracts of chick embryo. These RNases H can be separated by phosphocellulose column chromatography. Mg2+-dependent RNase H was purified over 900-fold and Mn2+-dependent RNase H over 1,700-fold from chick embryo extracts. The molecular weight of the purified Mg2+-dependent RNase H was about 40,000 and of the Mn2+-dependent RNase H about 120,000, when estimated by gel filtration. Mg2+-dependent RNase H exhibits maximal activity at pH 9.5, and requires 15 to 20 mM Mg2+ for maximal activity, whereas Mn2+-dependent RNase H is most active at pH 8.5, and is maximally active at the concentration of 0.4 mM Mn2+, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. Mn2+-dependent RNase H was inhibited by o-phenanthroline, pyrophosphate, and those polyamines tested, whereas Mg2+-dependent enzyme was not, although it was inhibited by NaF. Both RNases H liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini endonucleolytically.     

Toxic Metabolites of an Unidentified Filamentous Fungus Isolated from Zinnia Leaves

Two ribonucleases (RNases) designated RNase I and RNase II were found in Euphausia superba and isolated by (NH₄)₂SO₄ fractionation, 2 cycles of CM-cellulose chromatography and gel filtration on Sephadex G-100. This procedure resulted in a 2,116-fold purification of RNase I and a 130-fold purification of RNase II. The molecular weight of both purified enzymes was estimated by gel filtration to be 31,500. The isoelectric points were 6.0 (RNase I) and 7.0 (RNase II). Each enzyme hydrolyzed poly A-U, poly U but did not degrade poly G, poly C and DNA. Both enzymes were classified as endonuclease from the hydrolysis product of yeast RNA and poly A. The enzymes were located mainly in the cardiac and pyloric portion of the stomach.     

Purification and properties of bovine kidney ribonucleases

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.     

An affinity adsorbent, 5'-adenylate-aminohexyl-sepharose. II. Purification and characterization of multi-forms of RNase T2

RNase T2 bound to an affinity adsorbent, 5'-adenylate-aminohexyl-Sepharose 4B, specifically at pH 4.5. The colorless enzyme was eluted only by the simultaneous addition of 2'(3')-AMP (1 mM) and NaCl (greater than 1 M) at pH 4.5. By applying this affinity chromatography to the purification of RNase T2, pure enzyme with a specific activity of 60 was obtained in only four steps and the yield was about 10 times higher than that of the previous purification method. This enzyme preparation was found to be heterogeneous in molecular weight and was separated into two fractions on Sephadex G-75 gel filtration. As the smaller enzyme with a molecular weight of 36,000 was identical with RNase T2 in every property examined, we tentatively designated the larger one with an apparent molecular weight of 80,000 as high molecular weight RNase T2 (RNase T2-L). RNase T2-L was still heterogeneous and was separated into five fractions, RNases T2-L 1-5, by repeated Sephadex G-150 gel filtration. The amino acid and carbohydrate analyses revealed that each of these fractions has a protein moiety in common with RNase T2 and the heterogeneities were due to the carbohydrate content, mainly galactose content.     

Genetic analysis of human deoxyribonuclease I by immunoblotting and the zymogram method following isoelectric focusing

We have devised two independent detection methods for investigating possible molecular heterogeneity and genetic polymorphism in human DNase I, in terms of both its antigenicity and enzymatic activity. One was an immunoblotting method using an antibody specific to DNase I following polyacrylamide gel isoelectric focusing (IEF-PAGE). The DNase I-specific antibody was raised in a rabbit using purified enzyme from human urine as the immunogen. DNase I in urine was found to exist in multiple forms with different pI values separable by IEF-PAGE within a pH range of 3.5-4.0. This method was able to detect as little as 0.1 micrograms of the purified DNase I and facilitated classification of desialylated urine samples from different individuals into several groups according to differences in DNase I isozyme patterns. About 0.5 ml of the original urine was sufficient for analysis of the isozyme patterns. The other method was the zymogram method, which had a high sensitivity and resolution almost identical to those of the immunoblotting method for analysis of DNase I patterns. It was easier to perform, more time-saving, and more useful since it did not require antibody specific to DNase I. These two methods should prove valuable for biochemical and genetic analysis of DNase I isozymes.     

Extracellular RNase produced by Yarrowia lipolytica

Production of extracellular RNase(s) by Yarrowia lipolytica CX161-1B was examined in media between pHs 5 and 7. RNase production occurred during the exponential growth phase. High-molecular-weight nitrogen compounds supported the highest levels of RNase production. Several RNases were detected in the supernatant medium. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the RNases had estimated molecular weights of 45,000, 43,000, and 34,000. It was found that Y. lipolytica secretes only one RNase (the 45,000-molecular-weight RNase) and that the 43,000 and 34,000-molecular-weight RNases are degradation products of this RNase. The alkaline extracellular protease secreted by Y. lipolytica was shown to have a major role in the 45,000- to 43,000-molecular-weight conversion, and it was demonstrated that the 45,000-molecular-weight RNase could be purified from a mutant which does not produce the alkaline extracellular protease. Purification of the RNase from a wild-type strain resulted in purification of the 43,000-molecular-weight RNase. This RNase was a glycoprotein with a molecular weight of 44,000 as estimated by gel filtration, an isoelectric point of pH 4.8, and a pH optimum between 6.5 and 7.0.     

Characterization of Ribonucleases from Culture Medium of Lentinus edodes

Lentinus edodes (shiitake) cultivated in potato dextrose medium produced five RNases in the culture filtrate. The two major RNases (RNase Le37 and RNase Le45) were highly purified and their molecular masses, base specificities, N-terminal amino acid sequences, and amino acid compositions were analyzed and compared to RNase Le2 isolated from the fruit bodies of the same mushroom. RNase Le37 and RNase Le45 are base non-specific and adenylic acid preferential RNases like RNase Le2 and their N-terminal sequences are very similar to RNase Le2, but they are glycoproteins and their amino acid compositions are significantly different from that of RNase Le2. In addition to these enzymes, a guanylic acid-specific RNase with a molecular mass 13 kDa was partially purified. Since RNase Le2, which has very similar N-terminal sequence to RNase Le 37 and RNase Le 45, was not excreted from the mycelia, the analysis of the structures of these two excreted RNase may shade a light on the mechanism of excretion of RNases in this organism.     

RNase T affects Escherichia coli growth and recovery from metabolic stress.

To determine the essentiality and role of RNase T in RNA metabolism, we constructed an Escherichia coli chromosomal rnt::kan mutation by using gene replacement with a disrupted, plasmid-borne copy of the rnt gene. Cell extracts of a strain with mutations in RNases BN, D, II, and I and an interuppted rnt gene were devoid of RNase T activity, although they retained a low level (less than 10%) of exonucleolytic activity on tRNA-C-C-[14C]A due to two other unidentified RNases. A mutant lacking tRNA nucleotidyltransferase in addition to the aforementioned RNases accumulated only about 5% as much defective tRNA as did RNase T-positive cells, indicating that this RNase is responsible for essentially all tRNA end turnover in E. coli. tRNA from rnt::kan strains displayed a slightly reduced capacity to be aminoacylated, raising the possibility that RNase T may also participate in tRNA processing. Strains devoid of RNase T displayed slower growth rates than did the wild type, and this phenotype was accentuated by the absence of the other exoribonucleases. A strain lacking RNase T and other RNases displayed a normal response to UV irradiation and to the growth of bacteriophages but was severely affected in its ability to recover from a starvation regimen. The data demonstrate that the absence of RNase T affects the normal functioning of E. coli, but it can be compensated for to some degree by the presence of other RNases. Possible roles of RNase T in RNA metabolism are discussed.     

Ribonuclease from the hepatopancreas of the red king crab <Emphasis Type="Italic">Paralithodes camtschatica</Emphasis>

Three enzyme preparations, two acid and one alkaline RNases, were isolated from the hepatopancreas of the red king crab Paralithodes camtschatica using DEAE-cellulose chromatography and gel chromatography. The alkaline RNase was activated by Mg²⁺ ions and had a pH optimum of 7.2; the acid RNases, a pH optimum of 5.5. The molecular weight of the alkaline RNase was 19 kDa; two acid RNases, 33 and 70 kDa, respectively. The enzymes exhibited a sufficiently high thermostability (IT₅₀ = 53–55°C) and were strongly inhibited by NaCl (IC₅₀, 0.1–0.25 M). The alkaline RNase exhibited no specificity for heterocyclic bases, whereas the acid RNases hydrolyzed poly(U) and poly(A) at maximum rates.     

Partial Purification and Properties of Tea Leaf Ribonuclease

Four fractions with ribonuclease activity have been isolated from tea leaves by DEAE-cellulose column chromatography and designated as RNase Tf-1, RNase Tf-2, RNase Tf-3 and RNase Tf-4. The bigger fractions of both RNase Tf-3 and RNase Tf-4 have been partially purified by Sephadex G-100 column chromatography.

RNase Tf-3 and RNase Tf-4 were respectively found to have their optimum pH at 4.75 and 4.9 and molecular weights of approximately 13,000 and 16,000, as determined by gel filtration. Both enzymes were inhibited by Cu²⁺ and Hg²⁺, and inactivated by heating at over 50°C. By addition of yeast RNA to the two enzymes, however, their thermostabilities increased. The activities of the enzymes were stable in a pH range of 4.5 to 6.5. Like other plant RNases, RNase Tf-3 and RNase Tf-4 appeared to have no preference for base in RNA.     

Natural and engineered ribonucleases as potential cancer therapeutics

By reason of their cytotoxicity, ribonucleases (RNases) are potential anti-tumor drugs. Particularly members from the RNase A and RNase T1 superfamilies have shown promising results. Among these enzymes, Onconase, an RNase from the Northern Leopard frog, is furthest along in clinical trials. A general model for the mechanism of the cytotoxic action of RNases includes the interaction of the enzyme with the cellular membrane, internalization, translocation to the cytosol, and degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the capability of the RNase to evade the intracellular RNase inhibitor has not yet been fully elucidated. This paper discusses the various approaches to exploit RNases as cytotoxic agents.     

Purification from normal human plasma and biochemical characterization of a ribonuclease specific for poly(C) and poly(U)

A new specific ribonuclease from normal human plasma has been purified to homogeneity, following a five-step purification protocol that included DEAE-Sepharose, CM-Sepharose, and Heparin-Sepharose chromatographies. The purified enzyme was found to be glycosylated and appeared as a single 25-kDa band on a SDS polyacrylamide gel. This RNase is poly(C) preferential, degrading poly(U) at a lower rate. Activity of this RNase toward cleavage of native substrates such as ribosomal RNA was also detected. The human plasma ribonuclease is a thermolabile molecule, exhibiting maximum activity at pH 6.5. Comparison between other known plasma RNases and the human plasma ribonuclease described here indicated a variety of differences in their biochemical and catalytic properties.     

Specificity of guanylic RNases to polynucleotide substrates

Kinetic parameters kcat and KM were measured for cleavage of poly I, poly A, poly U, poly C and poly I poly C by guanyl-specific RNases Sa, Pb1 and T1 and compared with that of guanyl-preferential RNase Bi. Catalytic efficiencies of the investigated enzymes to polynucleotide substrates vary considerably. The structural basis for specificity of these RNases is discussed. A hypothesis is suggested that Ser-56 plays an important role in recognition of poly A by RNase Bi.     

Characterization of a unique nonsecretory ribonuclease from urine of pregnant women.

We have reported previously [Sakakibara, et al. (1991) Chem. Pharm. Bull. 39, 146-149] that a protein purified from a partially purified pharmaceutical preparation of human chorionic gonadotropin (a urinary protein preparation from pregnant women) is a unique nonsecretory ribonuclease (RNase)-like protein on the basis of its amino terminal sequence homology. We purified the protein further from the same materials by gel filtration and reversed-phase column chromatographies with RNase activity as an index. The purified protein was designated RNase UpI-2. The catalytic activity and its sensitivity to inhibition by divalent cations suggest that the protein is related to nonsecretory RNase. The estimated molecular weight of RNase UpI-2 (38 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was significantly higher than that of urinary nonsecretory RNases (13 to 19 kDa) reported so far. After trifluoromethanesulfonic acid treatment, the molecular weight of RNase UpI-2 was reduced and approached that of nonsecretory RNase, which indicated that the protein contains a significant amount of carbohydrate (approximately 50%). RNase UpI-2 was immunoreactive with antibodies to a nonsecretory RNase, RNAase 1 [Yasuda et al. (1988) Biochim. Biophys. Acta 965, 185-194]. By immunoblot analysis of the protein freshly prepared from various urine samples, it was shown that a considerable amount of RNase UpI-2 is present in urine of pregnant women, but only a trace of RNase UpI-2, if any, was detected in urine of nonpregnant women and men. These results suggest the possibility that RNase UpI-2 may have been formed via a specific protein modification in pregnant women.     

Oligoribonuclease is distinct from the other known exoribonucleases of Escherichia coli.

Oligoribonuclease, an exoribonuclease specific for small oligoribonucleotides, was initially characterized 20 years ago (S. K. Niyogi and A. K. Datta, J. Biol. Chem. 250:7307-7312, 1975) and shown to be different from RNase II and polynucleotide phosphorylase. Here we demonstrate, using mutant strains and purified enzymes, that oligoribonuclease is not a manifestation of RNases D, BN, T, PH, and R, exoribonucleases discovered subsequently. Thus, oligoribonuclease is the eighth distinct exoribonuclease discovered in Escherichia coli. We also show that oligoribonuclease copurifies with polynucleotide phosphorylase.