Comparison of brain ribonucleases of rabbit, guinea pig, rat, mouse and gerbil

The brain ribonucleases of rabbit, guinea pig, rat, mouse and gerbil were investigated by histochemical and biochemical methods. For the localization, the ribonucleases were electrophoretically transferred from cryostat sections to polyacrylamide gels. Elevated ribonuclease activities were found in the cortex, the basal ganglia, the hippocampal formation and the ventricles, whereas the corpus callosum and the internal capsule exhibited lower activities. The total RNA degrading activities of the brain extracts of the different species varied in a wide range. However, a pre-requisite for the measurement of acid soluble degradation products in the test system was the inactivation of endogeneous ribonuclease inhibitors, present in all extracts. Molecular weight analysis by means of SDS-polyacrylamide gel electrophoresis revealed a characteristic set of ribonucleases for each species, consisting of enzymes with different pH-optima.     

Comparison of brain ribonucleases of rabbit,guinea pig,rat, mouse and gerbil

Summary The brain ribonucleases of rabbit, guinea pig, rat, mouse and gerbil were investigated by histochemical and biochemical methods. For the localization, the ribonucleases were electrophoretically transferred from cryostat sections to polyacrylamide gels. Elevated ribonuclease activities were found in the cortex, the basal ganglia, the hippocampal formation and the ventricles, whereas the corpus callosum and the internal capsule exhibited lower activities. The total RNA degrading activities of the brain extracts of the different species varied in a wide range. However, a pre-requisite for the measurement of acid soluble degradation products in the test system was the inactivation of endogencous ribonuclease inhibitors, present in all extracts. Molecular weight analysis by means of SDS-polyacrylamide gel electrophoresis revealed a characteristic set of ribonucleases for each species, consisting of enzymes with different pH-optima.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Penetration of various mononuclear ribonucleases into rat experimental granulation-tissue fibroblasts and their intracellular effects

The penetration of five different mononuclear ribonucleases into the subcellular particles of rat experimental granulation-tissue fibroblasts was compared, along with the effects of the enzymes on the fibroblast RNA fractions. Ribonucleases from normal and silica-treated rat peritoneal macrophages have been shown before to regulate the nucleic acid and protein metabolism of rat experimental granulation-tissue fibroblasts. These biologically active enzymes were taken into the fibroblasts in a greater amount than the corresponding human monocyte enzymes and the biologically inactive rat macrophage ribonuclease. The biologically active macrophage enzymes were incorporated mainly into the nuclear fraction. The other three mononuclear ribonucleases were not found particularly in any subcellular compartment. Both biologically active macrophage enzymes degraded the nuclear RNA of fibroblasts and released it to the soluble fraction in contrast to the biologically inactive macrophage enzyme and ribonuclease from normal human monocytes. Instead the ribonuclease from normal human monocytes seemed to degrade RNA in the soluble fraction. There were no marked differences in the subcellular effects of ribonucleases from normal and silica-treated macrophages. However, the treatment of human monocytes with silica changed their ribonuclease so that it split the nuclear RNA of fibroblast and released it to the soluble fraction in the same way as the biologically active macrophage enzymes did.     

Evolutionary trends in 18S ribosomal RNA nucleotide sequences of rat, mouse, hamster and man.

The large T1 ribonuclease fragments of 18S ribosomal RNA from four mammalian species, rat, mouse, hamster and man, were compared by two-dimensional homochromatography fingerprinting. The nucleotide sequences of the large T1 ribonuclease fragments, polypyrimidines and polypurines which were different among the four mammalian species were determined and compared. The method used for determining nucleotide sequences utilizes 32p-labeling of oligonucleotides at their 5'-termini by polynucleotide kinase, partial digestion by ribonucleases and analysis of labeled spots by homochromatography-fingerprinting. Several examples of point mutations were detected. It was of interest that the 18S rRNA of Chinese hamster has more oligonucleotide sequences in common with those of man that rat or mouse.     

Purification and cloning of cytotoxic ribonucleases from Rana catesbeiana (bullfrog)

Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, ~50 and ~25%, respectively.     

The crystal structure of recombinant rat pancreatic RNase A

The three-dimensional structure of rat pancreatic RNase A expressed in Escherichia coli was determined. The backbone conformations of certain critical loops are significantly different in this enzyme compared to its bovine counterpart. However, the core structure of rat RNase A is similar to that of the other members of the pancreatic ribonuclease family. The structural variations within a loop bordering the active site can be correlated with the subtle differences in the enzymatic activities of bovine and rat ribonucleases for different substrates. The most significant difference in the backbone conformation was observed in the loop 15-25. This loop incorporates the subtilisin cleavage site which is responsible for RNase A to RNase S conversion in the bovine enzyme. The rat enzyme does not get cleaved under identical conditions. Molecular docking of this region of the rat enzyme in the active site of subtilisin shows steric incompatibility, although the bovine pancreatic ribonuclease A appropriately fits into this active site. It is therefore inferred that the local conformation of the substrate governs the specificity of subtilisin.     

Responsibility of tRNA(Ile) for spermine stimulation of rat liver Ile-tRNA formation

To determine whether tRNA or aminoacyl-tRNA synthetase is responsible for spermine stimulation of rat liver Ile-tRNA formation, homologous and heterologous Ile-tRNA formations were carried out with Escherichia coli and rat liver tRNA(Ile) and their respective purified Ile-tRNA synthetases. Spermine stimulation was observed only when tRNA from the rat liver was used. Spermine bound to rat liver tRNA(Ile) but not to the purified aminoacyl-tRNA synthetase complex. Kinetic analysis of Ile-tRNA formation revealed that spermine increased the Vmax and Km values for rat liver tRNA(Ile). The Km value for ATP and isoleucine did not change significantly in the presence of spermine. Furthermore, higher concentrations of rat liver tRNA(Ile) tended to inhibit Ile-tRNA formation if spermine was absent. Spermine restored isoleucine-dependent PPi-ATP exchange in the presence of rat liver tRNA(Ile), an inhibitor of this exchange. The nucleotide sequence of rat liver tRNA(Ile) was determined and compared with that of E. coli tRNA(Ile). Differences in nucleotide sequences of the two tRNAs(Ile) were observed mainly in the acceptor and anticodon stems. Limited ribonuclease V1 digestion of the 3'-32P-labeled rat liver tRNA(Ile) showed that both the anticodon and acceptor stems were structurally changed by spermine, and that the structural change by spermine was different from that by Mg2+. The influence of spermine on the ribonuclease V1 digestion of E. coli tRNA(Ile) was different from that of rat liver tRNA(Ile). The results suggest that the interaction of spermine with the acceptor and anticodon stems may be important for spermine stimulation of rat liver Ile-tRNA formation.     

Eosinophil cationic protein cDNA. Comparison with other toxic cationic proteins and ribonucleases

Human eosinophil granules contain several basic proteins including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and major basic protein (MBP). ECP and MBP are potent helminthotoxins while EDN is less so. Both ECP and EDN possess neurotoxic and ribonuclease activities. A clone representing ECP mRNA was isolated from an eosinophil lambda ZAP cDNA library. The cDNA sequence codes for a preprotein of 160 amino acids and a protein of 133 amino acids, the amino terminus of which is identical to the known partial amino acid sequence of ECP. The ECP nucleotide sequence shows similarity to EDN, rat pancreatic ribonuclease, and human angiogenin; all are members of the ribonuclease gene superfamily. Although the deduced amino acid sequence of ECP shares identical active site and substrate binding site residues with EDN, angiogenin, and human pancreatic ribonuclease, the ribonuclease activity of ECP is 50 to 100 times less than that of EDN possibly because of the lack of a positively charged residue at human pancreatic ribonuclease position 122. The calculated isoelectric point (10.8), electronic charge (14.5), and cationic charge distribution of ECP are different from those of EDN but similar to those of MBP, which may account in part for the greater helminthotoxic activity of ECP when compared to EDN. These data suggest that ECP and EDN are derived from a common ancestral ribonuclease gene and that ECP has evolved into a potent helminthotoxin similar in some respects to MBP, while losing much of its ribonuclease activity.     

Isolation of serum albumin-synthesizing polysomes from rat liver

The procedures for the purification of rat liver polysomes synthesizing serum albumin was developed, employing the quantitative precipitin method with rat serum albumin as a carrier and its antibody, and ribonuclease inhibitor from rat liver. The addition of ribonuclease inhibitor to polysomes during the incubation with antibody was found to prevent their degradation. Under these conditions, about 12 % of the membrane-bound polysomes of rat liver was found in the specific precipitate of serum albumin and its antibody, while a negligible amount of free polysomes was precipitated. It is concluded that polysomes synthesizing serum albumin are isolated by this method.     

Binding of adriamycin to sulphated mucopolysaccharides

Two distinct, Mn²⁺- and Mg²⁺-dependent enzymes with ribonuclease H (RNase H) activities, which degrade RNA-DNA hybrid have been purified from rat liver nuclei. These two enzymes were eluted at 0.16 M and 0.37 M of potassium chloride in phosphocellulose chromatography, respectively, and further purified by blue Sepharose. They are distinguished from one another by their ionic requirement, molecular weight, sedimentation coefficient, optimal pH, sensitivity to the -SH reagent and mode of cleavage.     

Alkaline ribonuclease and phosphodiesterase activity in rat liver plasma membranes

The ribonuclease and phosphodiesterase activities of rat liver plasma membranes, purified from the crude nuclear fraction by centrifugation in an A-XII zonal rotor and flotation, were examined and compared. The plasma membrane is responsible for between 65 and 90% of the phosphodiesterase activity of the cell and between 25 and 30% of the particulate ribonuclease activity measured at pH8.7 in the presence of 7.5mm-MgCl(2). Both enzymes were most active between pH8.5 and 8.9. Close to the pH optimum, both enzymes were more active in Tris buffer than in Bicine or glycine buffer. Both plasma-membrane phosphodiesterase and ribonuclease were strongly activated by Mg(2+), there being at least a 12-fold difference between the activity in the presence of Mg(2+) and of EDTA. There is, however, a difference in the response of the enzymes to Mg(2+) and EDTA in that the phosphodiesterase is fully activated by 1.0mm-MgCl(2) and fully inhibited by 1.0mm-EDTA, whereas the ribonuclease requires 7.5mm-MgCl(2) for full activation and 5mm-EDTA for full inhibition. Density-gradient centrifugation has indicated that on solubilization in Triton X-100 most of the ribonuclease activity is released into a small fragment of the same size as that containing the phosphodiesterase activity. The relationship between the two activities is discussed in view of these results.     

Ribonuclease H from rat liver. I. Partial purification and characterization of nuclear ribonuclease H1.

A ribonuclease H, an enzyme that specifically degrades the RNA moiety of RNA-DNA hybrid, has been partially purified from rat liver nuclei and characterized. Neither native or denatured DNA, nor single or double-stranded synthetic polyribonucleotides were degraded by the enzyme. The enzyme possesses a molecular weight of about 36,000 and requires alkaline pH, magnesium ions, and ammonium sulphate for maximum activity. The enzyme acts on the hybrid as an endonuclease, resulting in oligonucleotides with 3'-hydroxyl termini. The properties of this enzyme were distinct from those of the rat liver cytosol enzyme reported by Roewekamp and Sekeris in many respects, such as molecular weight, optimal pH and requirements for divalent cations. Preliminary experiments suggest that the nuclear enzyme is localized in the nucleoplasm and nucleoli. These results indicate that multiple forms of ribonuclease H exist in different regions of rat liver cells.     

Inducible expression of the gene of Zinnia elegans coding for extracellular ribonuclease in Nicotiana tabacum plants

The gene of Zinnia elegans L. coding for S-like extracellular ribonuclease (ZRNase II) was used to produce transgenic tobacco plants with an increased ribonuclease activity. The protein-coding part of ZRNase II included the signal peptide sequence so the transgenic protein was located extracellularly. The cDNA of ZRNase II was cloned under the control of 2′-promoter of the mannopine synthase (MAS 2′) gene from Ti-plasmid of Agrobacterium tumefaciens. It was shown that the resultant transgenic plants had an increased ribonuclease activity of the crude extracts and the induction of MAS 2′ promoter by wounding additionally increased the activity. The plants of two transforming lines characterized by different ribonuclease activities were used to analyze the transgene influence on plant resistance to tobacco mosaic virus. The plants demonstrated either absence of disease symptoms or a significant delay in their appearance, depending on the virus content in the inoculum and ribonuclease activity.     

Poly(A) polymerase from Vigna unguiculata seedlings. A bifunctional enzyme responsible for both poly(A)-polymerizing and poly(A)-hydrolyzing activities

Poly(A)-specific ribonuclease was co-purified with poly(A) polymerase from Vigna unguiculata seedlings. Both activities were separated into two forms (enzymes I and II) by a final hydrophobic column chromatography. The enzyme I preparation, which was homogeneous as examined by SDS/PAGE, had both poly(A) polymerase and poly(A)-specific ribonuclease activities. The antibody raised to the enzyme I preparation precipitated both enzyme activities. These indicate that a single polypeptide (Mr 63,000) is responsible for both poly(A)-polymerizing and poly(A)-hydrolyzing activities. The poly(A)-specific ribonuclease was a 3'-exonuclease specific to single-stranded poly(A), forming 5'AMP as the sole reaction product. The hydrolytic activity required either Mn2+ or Mg2+ with different optimum concentrations, whereas the polymerizing activity required Mn2+ but not Mg2+. ATP and PPi had little or no effect on the poly(A)-specific ribonuclease activity.     

The primary structure of muskrat pancreatic ribonuclease.

Pancreatic ribonuclease from muskrat (Ondatra zibethica) was isolated and its amino acid sequence was determined from tryptic digests of the performic acid-oxidized and the reduced and aminoethylated enzyme. The peptides have been positioned in the sequence by homology with other ribonucleases. This could be done unambiguously for all peptides except Arg-Arg (tentative position 32-33) and Ser-Arg (tentative position 75-76). The amino acid sequences of the peptides were determined by the dansyl-Edman method, with the exception of residues 23-25 and 99-102, which were positioned by homology. The enzyme differs in 38 positions from the enzyme from rat and in 31-42 positions from other mammalian pancreatic ribonucleases, while rat ribonuclease differs at 44-52 positions from the other enzymes. These data point to a common ancestry of the enzymes from muskrat and rat and an increased evolution rate of rat ribonuclease after divergence of the ancestors of both species. Muskrat ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64).     

Hepatic nucleases. 1. Methods for the specific determination and characterization in rat liver.

With a view to the study of the subcellular localization of nucleases, methods ensuring the homogenates. The ribonuclease activity of rat liver is due to the three enzymes with different pH optimun. For acid ribonuclease (pH optimun 5.3), it is possible to avoid interference from the other ribonucleases by performing the incubation at pH 5. Neutral ribonuclease (pH optimum 7.6) is differentiated by relying on its sensitivity to the natural inhibitor from the supernatant of liver homogenate. Comparison of activities before and after pretreatment at 50 degrees C in acid medium permits the specific measurement of alkaline ribonuclease (pH optimum 8.8). The optimal conditions for the determination in liver homogenates of two deoxyribonucleases and of an enzyme acting on polyriboadenylate are also described. The activity of these various nucleases is compared and some of their properties are investigated.     

Purification of rat liver particulate neutral ribonuclease and comparison of properties with pancreas and serum ribonucleases.

Rat liver particulate neutral ribonuclease (EC 3.1.4.22) was extensively purified (up to 40000-fold). It is shown to be an endonuclease, specific for pyrimidine bases, hydrolysing 5'-phosphate ester bonds. The enzyme specificity, Km, pH optimum, stability in acid medium and thermal stability at high temperature are the same as those of rat pancreatic and serum ribonucleases. Like pancreatic and serum neutral ribonucleases, the hepatic enzyme is sensitive to the liver natural inhibitor. This inhibitor was purified 8000-fold; its association with ribonuclease follows zero-order kinetics. These identical properties for ribonuclease of rat liver, pancreas and serum support the hypothesis [Bartholeyns, Peeters-Joris & Baudhuin (1975) Eur. J. Biochem. 60, 385-393] of an extrahepatic origin for the liver enzyme, the plasma ribonuclease of pancreatic origin being taken up by endocytosis in the liver. Neutral ribonuclease activity was detected in all rat organs investigated; its distribution among tissues is different from the distribution of the natural ribonuclear inhibitor.     

Intrinsic ribonuclease activities in ribonuclease and ribosome-inactivating proteins from the seeds of bitter gourd

Alpha- and beta-momorcharins are ribosome-inactivating proteins present in the seeds of the bitter gourd (Momordica charantia). Both of them possess ribonuclease activity which may account for some of their biological properties. However, the activity is weak and hence it is important to confirm that the ribonuclease activity observed is not due to any contamination. To this end, the ribonuclease from the seeds of M. charantia (RNase-MC) was purified and compared with the ribonuclease activity of the momorcharins. Purification was achieved by ion-exchange chromatographies on DEAE-cellulose, SP-Sepharose and Mono-S. RNase-MC had a molecular mass of 22 kDa. It acted on tRNA to release acid-soluble UV-absorbing species with a pH optimum around 6.0-6.5. When polyhomoribonucleotides were used as substrates, it was found that RNase-MC acted preferentially on polyU but exerted much weaker activity on polyC, polyG and polyA. Chromatographic analysis of the reaction product indicated that mono- and oligo-ribonucleotides, but not free base, were generated from polyU, suggesting that the enzymatic action involved ribonucleolytic cleavage. RNase-MC exhibited a much more potent (at least 1000-fold higher) ribonuclease activity than alpha- and beta-momorcharins. RNase-MC, alpha-momorcharin and beta-momorcharin were separable on Mono-S, indicating that the ribonuclease activities present in the three proteins were distinct entities.     

ROLE OF RIBONUCLEASE ACTION IN PHENYLALANINE-INDUCED DISAGGREGATION OF RAT CEREBRAL POLYRIBOSOMES

—l -phenylalanine (1 mg/g body wt) or physiological saline (0.9% NaCl) was given intraperitoneally to infant (7-day old), immature (14-day old), and adult (42-day old) rats. The state of ribosomal aggregation was determined in the cerebral postmitochondrial supernatant and purified polyribosome fractions prepared in the presence of rat liver ribonuclease inhibitor. Polyribosomes isolated from cerebral cortices of infant and immature rats 30 or 60 min after administration of phenylalanine were partially disaggregated, whereas the state of aggregation of polyribosomes from mature cerebrum was unchanged. In contrast, little or no evidence of phenylalanine-induced polyribosome disruption was noted in the postmitochondrial supernatant fractions, from which the cerebral polyribosomes were prepared, in any of the animals. Omission of the ribonuclease inhibitor resulted in polyribosome disaggregation in the postmitochondrial supernatant fractions prepared from saline-treated as well as phenylalanine-treated infant rats, but the disruption was more profound in the latter group. Ribonuclease activities in cerebral postmitochondrial supernatant preparations from infant and immature rats were higher than the corresponding values in preparations from adult animals. In addition, the administration of phenylalanine resulted in increases in ribonuclease activities in cerebral postmitochondrial supernatant preparations from the younger animals, but had no effect on these activities in adult animals. These results suggest that alterations in structure and function of polyribosomes from the infant rat cerebrum following a loading dose of phenylalanine were related to exposure of the polyribosomes during isolation to elevated activities of cerebral ribonucleases resulting from this treatment. This hypothesis was supported by the finding that phenylalanine treatment had no effect on the incorporation in vivo of intracisternally-administered radioactive lysine into total, soluble or ribosomal protein of infant cerebrum. However, when cerebral ribosomal RNA was differentially labelled in phenylalanine-treated and saline-treated infant rats by the intracisternal administration of [³H] or [¹⁴C]uridine, and polyribosome fractions were then prepared from the pooled cerebral cortices of both groups, radioactive ribosomes derived from saline-treated rats were more highly aggregated than those derived from phenylalanine-treated animals. It is concluded that gross alterations in cerebral polyribosome structure and function do not occur in vivo in young rats given a large amount of phenylalanine intraperitoneally. However, this treatment, in addition to increasing ribonuclease activity in cerebral cell-free preparations, also sensitizes cerebral polyribosomes to subsequent breakdown upon exposure to ribonucleases during isolation.     

Ribonuclease activities in rat sympathetic ganglia: Evidence for the presence of an endogenous inhibitor of alkaline ribonuclease

Using³H-labeled rat brain mature RNA as substrate, substantial ribonuclease activity was detected in homogenates of rat superior cervical ganglia with acidic (pH 5.5) and neutral (pH 7.0-7.5) optima. Very little activity could be measured at greater than pH 8. The acidic and neutral activities differed in the optimal conditions required for assay, and showed differential sensitivity to the sulfhydryl blocking agent, N-ethylmaleimide. Only the neutral activity was stimulated, optimally by 2 mM N-ethylmaleimide, and the magnitude of stimulation indicated that the contributing ribonucleases exist largely in a latent form in the ganglion. Ribonucleases in other tissues with neutral pH dependence, known usually as alkaline ribonucleases, are subject to an N-ethylmaleimide-sensitive endogenous inhibitor protein. The existence of a similar inhibitor in rat superior cervical ganglia was indicated by the latency of neutral ribonuclease activity and confirmed by observing the effect of a soluble fraction from the ganglia on the activity of pancreatic ribonuclease A.