Distribution of a Kidney Acid-ribonuclease-like Enzyme and the Other Ribonucleases in Bovine Organs and Body Fluids

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 BSPJ, an alkaline RNase (RNase BL₄), and another alkaline RNase in serum.     

Nuclear and cytoplasmic RNase-activity in regenerating mouse liver

Nuclear and cytoplasmic RNase activities at pH 5.0 and 7.6 were analyzed in regenerating mouse liver at 6, 12, 24, 48, and 72 h after partial hepatectomy. Two different nucleus-isolation methods were used, one in a EDTA-spermidine medium free from divalent cations, and one in a sucrose medium containing these ions. During regeneration, the cytoplasmic alkaline RNase activity in the sucrose medium was unchanged, but in the spermidine medium showed an increase toward the end of the period. Also the cytoplasmic acid RNase activity was unchanged in sucrose medium, whereas in the spermidine it slightly increased during regeneration. The nuclear alkaline RNase activity showed a notable peak 6 h after the operation and later decreased. Also the nuclear acid RNase activity displayed a similar marked peak 6 h after operation, then decreased, but remained high throughout the period. The nuclear RNase activities were about 1% of the corresponding cytoplasmic RNase activities. The absolute activities varied greatly according to the nucleus-isolation methods. In the controls, the absolute activity of nuclear alkaline RNase was slightly above (1.2 times) that of the corresponding acid activity after the spermidine method. After the sucrose method the nuclear alkaline activity was 2.7 times that of the acid activity. The absoluted activity of cytoplasmic alkaline RNase was slightly above (1.2 times) the acid activity after the spermidine method but after the sucrose method it was only 0.25 times that of the acid activity. In sham-operated animals, cytoplasmic acid and alkaline RNase activities generally were fairly similar to the normal value, but corresponding nuclear activities showed marked variations indicating an influence by anesthesia.     

Ribonuclease deficiency in lymphocytes of patients with chronic lymphocytic leukemia (CLL)

Ribonuclease (RNase) activity in the lymphocytes of 20 chronic lymphocytic leukemia (CLL) patients and 10 normal subjects was studied. It was found that in the lymphocytes of the control subjects the RNase activity could be detected in the pH range 4.5 to 8.6, inclusive. The RNase activity versus pH profile of normal lymphocytes consists of an acid RNase peak at pH 6.5 and alkaline RNase peak at pH 7.8. When treated with pCMB an inhibitor-bound RNase activity was revealed. The peak of this activity lay between pH 6.7 to 7.0. Liberating the inhibitor-bound RNase activity changed the RNase activity-pH profile, yielding one peak curve with a maximum at pH 7.0. RNase activity in CLL lymphocytes was remarkably lower than that in normal lymphocytes. The acid RNase in 80% of the CLL patients was lower by a factor of ten. Likewise, a many fold decrease in alkaline RNase activity (in some cases down to the zero level) was observed in CLL lymphocytes. However, in 70% of CLL patients, a level of the inhibitor-bound RNase activity was similar to that found in normal lymphocytes. In 20% of the studied CLL patients, a remarkable decrease in both free alkaline and inhibitor-bound RNase activity was observed. When poly-C was used as a substrate for determining RNase activity, a decrease to approximately 15% in CLL lymphocytes was observed, when poly-U was used instead of poly-C, a decrease to 65% was found only as compared with normal lymphocytes. This may suggest that CLL lymphocytes are deficient in a poly-C specific RNase which displays its activity within a neutral and alkaline pH range.     

An alkaline phosphatase-linked assay for ribonucleases

A convenient and rapid assay for ribonucleases has been developed using commerical unlabeled materials. This assay detected less than 1 ng of RNase A. The assay was also applied to RNase T₁ and micrococcal nuclease. The phosphate end groups generated at the cleavage sites of the RNA substrate were measured by incubating with excess alkaline phosphatase and determining the phosphate released. Initial reaction rates were measured and accurate units of activity established, which is not possible with most RNase assays. Commercial preparations of alkaline phosphatase from E. coli are contaminated with RNase. A procedure was described for removal of RNase from the alkaline phosphatase preparations.     

Evidence for a high molecular weight factor(s) in serum which increases alkaline phosphatase specific activity in HeLa.

HeLa (substrain Ho) grown in serum free medium showed an increase in the specific activity of alkaline phosphatase when fetal calf serum (10%) was added to the medium (9.7 nmoles/sec/mg protein to 86.8). Under the same conditions, eight intracellular enzymes showed no increase in activity. Similar results were obtained using a different serum or medium, and with a second strain of HeLa (substrain ATC). For a given set of growth conditions, the effect of serum was dependent on its concentration and required one or more culture generations to develop. The type of isozyme expressed did not change. Neither zinc nor a total serum lipid extract would substitute for serum. The enzyme expressed by HeLaHo was not induced by prednisolone, while that in HeLaATC was. However, for cells grown in excess prednisolone without serum, the specific activity was 25% of that found for cells grown with prednisolone and serum. Cortexolone, an antagonist of prednisolone, was without effect for HeLaHo grown in A3 medium with or without serum. The serum factor had the following characteristics. It was not lost on dialysis, treatment with DNase and RNase, or removal of lipoproteins. It was reduced after heating by 65% and after treatment with Pronase by 82%. The data are interpreted to indicate the presence of a factor (s) in serum, probably a protein, which is involved in stimulating alkaline phosphatase specific activity.     

Serum ribonuclease levels in patients with cystic fibrosis

The levels of serum pancreatic-type ribonuclease (RNase) activity in normal individuals and in individuals homozygous and heterozygous for the cystic fibrosis (CF) gene have been studied. In 21 CF patients, ages 3–24 years, RNase levels of 459 ± 95 units/ml did not differ significantly from the RNase levels of 467 ± 105 units/ml in 17 normal individuals of the same age group nor from those of 490 ± 52 units/ml in 5 individuals heterozygous for the CF gene.     

Expression of Soluble Bovine Pancreatic Ribonuclease A in Pichia pastoris and its Purification and Characterization

A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased k _cat, 60-70% of the activity of wild-type RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since T _m of the glycosylated RNase A was decreased by 6°C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.     

Heat labile ribonuclease HI from a psychrotrophic bacterium: gene cloning, characterization and site-directed mutagenesis.

The rnhA gene encoding RNase HI from a psychrotrophic bacterium, Shewanella sp. SIB1, was cloned, sequenced and overexpressed in an rnh mutant strain of Escherichia coli. SIB1 RNase HI is composed of 157 amino acid residues and shows 63% amino acid sequence identity to E.coli RNase HI. Upon induction, the recombinant protein accumulated in the cells in an insoluble form. This protein was solubilized and purified in the presence of 7 M urea and refolded by removing urea. Determination of the enzymatic activity using M13 DNA-RNA hybrid as a substrate revealed that the enzymatic properties of SIB1 RNase HI, such as divalent cation requirement, pH optimum and cleavage mode of a substrate, are similar to those of E.coli RNase HI. However, SIB1 RNase HI was much less stable than E.coli RNase HI and the temperature (T(1/2)) at which the enzyme loses half of its activity upon incubation for 10 min was approximately 25 degrees C for SIB1 RNase HI and approximately 60 degrees C for E.coli RNase HI. The optimum temperature for the SIB1 RNase HI activity was also shifted downward by 20 degrees C compared with that of E.coli RNase HI. Nevertheless, SIB1 RNase HI was less active than E.coli RNase HI even at low temperatures. The specific activity determined at 10 degrees C was 0.29 units/mg for SIB1 RNase HI and 1.3 units/mg for E.coli RNase HI. Site-directed mutagenesis studies suggest that the amino acid substitution in the middle of the alphaI-helix (Pro52 for SIB1 RNase HI and Ala52 for E.coli RNase HI) partly accounts for the difference in the stability and activity between SIB1 and E.coli RNases HI.     

RNase A及其链亲和素融合蛋白在pET系统中的表达

在大肠杆菌中用pET28a表达载体表达重组RNaseA。变性条件下,利用His-Resin亲和纯化,得到60mg／L电泳纯的RNaseA。纯化的RNaseA复性后,利用含大量RNA分子的碱法抽提质粒为底物,测定重组RNaseA活性,与商品化的RNaseA活性相当。同时在RNaseA活性测定体系中加入4mol／L尿素会使RNA分子切割效率提高10倍左右。在此基础上,成功表达RNaseA与链亲和素(streptavidjn)的融合蛋白,经纯化复性后,该融合蛋白同时具有核酸酶、biotin结合活性,在分子生物学中具有重要的应用价值。     

Heterogeneity of alkaline ribonuclease in the mouse and Ehrlich ascites cells.

The specific activity of alkaline RNase II was l00 to 1800 times higher in mouse pancreas than in mouse liver, serum, ascites fluid, and Ehrlich ascites cell grown intraperitoneally. Ehrlich ascites cells grown in cell culture medium had a much lower alkaline RNase II activity than cells grown intraperitoneally. Chromatography on CM-52 cellulose of acid- and heat-treated preparations showned a considerable heterogeneity of the mouse enzymes. Depending on the source of the extract, two to six forms fo alkaline RNase were eluted. Pancreatic extract contained two RNase forms. These also seemed to be present as minor components in preparations from other sources except Ehrlich ascites cells grown in vitro. Ehrlich ascites cells grown in vivo contained forms of the RNase which were not present in other extracts. Possible reasons for this heterogeneity were investigated. In addition to their stability to acid and heat the different RNase forms were similar in that they were much more active at alkaline pH than at acidic pH, they did not require divalent metal ions for activity, and they degraded RNA 'endonucleolytically.' Also, native DNA, denatured DNA, and poly A were poor substrates compared with RNA. Some differences seemed to exist, however, with respect to their abilities to degrade poly U and poly C and their sensitivities to the endogenous RNase inhibitor.     

The Na/K/2Cl cotransporter is increased in hypertrophied vascular smooth muscle cells.

Hypertrophy of vascular smooth muscle cells (VSMC) is a pathogenic feature of hypertension which may contribute to abnormal vessel tone and function. As a consequence of the increase in cell size associated with hypertrophy, it is likely that alterations in the mechanisms that regulate VSMC intracellular volume occur. Because the Na+/H+ exchanger plays an important role in volume regulation and because we previously observed long term alterations in Na+/H+ exchange and pHi in response to angiotensin-II-induced (ang II) hypertrophy, we studied cell-acidifying mechanisms. To do this, we measured alkaline recovery from NH4Cl-mediated alkalinization, using the fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. VSMC were growth-arrested (0.4% calf serum for 24 h) or hypertrophied (100 nM ang II in 0.4% calf serum for 24 h). Ang II-treated cells exhibited a 107% increase in alkaline recovery over control cells (13.86 +/- 1.87 versus 6.68 +/- 1.01 mmol H+/min/liter cells). The increase in alkaline recovery was not a result of increased Cl-/HCO-3 exchange becaue it was not HCO-3 dependent nor inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid. Studies with bumetanide and the sterically inhibited substrate N(CH3)4+ showed that the alkaline recovery was mediated by NH4+ transport via the Na/K/2Cl cotransporter. Ang II-treated cells exhibited a 334% increase in bumetanide-sensitive alkaline recovery over control cells (9.16 +/- 1.90 versus 2.11 +/- 1.46 mmol H+/min/liter cells). Ang II-treated cells also exhibited a 90% increase in bumetanide-sensitive 86Rb uptake over control cells. These findings demonstrate that Na/K/2Cl cotransport activity is specifically induced in ang II-hypertrophied VSMC and establish this transporter as a component of the hypertrophic growth response.     

Regulation of the RNAse activity of the saliva in healthy subjects and in stomach cancer

Acidic and alkaline RNase activity from healthy humans and gastric cancer patients has been studied. A decrease in daily saliva production and an increase in RNase activity was detected saliva of cancer patients. This suggests the existence of RNase inhibitors in healthy humans. This supposition is further confirmed by comparative analysis of total, joint fractional and reconstituted RNase activity. A considerable increase in acidic RNase inhibitors and disappearance of alkaline RNase inhibitor was observed in cancer patients. The specificity, mechanisms and clinical significance of this phenomena has been discussed.     

A mutation in the rnh-locus of Escherichia coli affects the structural gene for RNase H. Examination of the mutant and wild type protein

Ribonuclease H (RNase H, EC 3.1.26.4) was purified to homogeneity from Escherichia coli wild type strain KS 351 and the RNase H mutant strain FB 2. The specific activity of the wild type enzyme was 43,200 units/mg, while that of the mutant enzyme was 3,430 units/mg, less than 8% of the wild type activity. Isoelectric focusing also revealed differences in the protein from mutant and wild type. The activity of the wild type enzyme was separated into two peaks with isoelectric points of 9.6 and 9.0. In contrast, the activity of the mutant enzyme focused in a single peak with a pI of 9.4. These results indicate that the mutation in the FB2 strain affects the structural gene for RNase H. The molecular weight of both enzymes was determined by gel filtration as well as NaDodSO4-polyacrylamide gel electrophoresis and found to be identical. Both enzymes are very sensitive to increased temperatures and show indistinguishable rates of inactivation. The basis for the heterogeneity of the isoelectric point and the altered activity of the mutant enzyme is still unknown.     

The major birch pollen allergen, Bet v 1, shows ribonuclease activity

The major birch (Betula alba L.) pollen allergen, Bet v 1, has been shown to be homologous to pathogenesis-related proteins in a number of plants. Recently, it was demonstrated that a ginseng protein with high homology to an intracellular pathogenesis-related protein of parsley and to Bet v 1 is a ribonuclease (RNase). Birch pollen extract was separated in an RNase activity gel. Four major RNase bands were excised from the gel, reseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting with a specific Bet v 1 monoclonal antibody and patient's serum. Thus the monomer and the dimer of Bet v 1 showed RNase activity. Purified recombinant Bet v 1 was shown to degrade plant RNA. The RNase activity of recombinant Bet v 1 was 180 units · mg^?1.     

Purification of plasmid DNA by tangential flow filtration

A simple, scalable method for purification of plasmid DNA is described. The method includes modification of the classical alkaline-lysis-based plasmid extraction method by extending the solubilization step from less than 30 min to 24 h. The extraction is followed by the novel use of tangential flow filtration (TFF) for purification of the remaining contaminants. The method does not include the use of any organic solvents, RNase, high-speed centrifugation, or column chromatography steps. The method typically yields 15 to 20 mg of plasmid DNA per liter of bacterial culture and results in removal of >99% of RNA and >95% of the protein that remains after the modified alkaline lysis procedure. The procedure has been demonstrated to be effective in the isolation of seven different plasmids. Plasmids isolated using this method had comparable transfection capability relative to plasmid isolated using a classical, cesium chloride gradient-based method.     

A thermoresistant mutant of ribonuclease T1 having three disulfide bonds

Molecular-dynamic calculations predict that, if Tyr24 and Asn84 are each replaced by a Cys residue, it should be possible to form a third disulfide bond in ribonuclease T1 (RNase T1) between these residues, with only minimal conformational changes at the catalytic site. The gene encoding such a mutant variant of RNase T1 (Tyr24----Cys24, Asn84----Cys84) was constructed by the cassette mutagenesis method using a chemically synthesized gene. In order to reduce the toxic effect of the mutant enzyme (RNase T1S) on an Escherichia coli host, we arranged for the protein to be secreted into the periplasmic space by using a vector that harbors a gene for an alkaline phosphatase signal peptide under the control of the trp promoter. The nucleolytic activity of RNase T1S toward pGpC was approximately the same as that of RNase T1 at 37 degrees C (pH 7.5). Moreover, at 55 degrees C, RNase T1S retained nearly 70% of its activity while the activity of the wild-type enzyme was reduced to less than 10%. RNase T1S was also more resistant to denaturation by urea than the wild-type enzyme. However, unlike RNase T1, RNase T1S was irreversibly and almost totally inactivated by boiling at 100 degrees C for 15 min.     

Direct assay for creatine kinase by reversed-phase high-performance liquid chromatography

A direct assay for creatine kinase (CK) activity was developed based on the separation and quantitation of adenosine triphosphate (ATP) by high-performance liquid chromatography. The total incubation time is 13 min and the elution time for ATP is 16 min. Using lyophilized CK as the sample, a sensitivity in the range of 8 U/l (units/liter) was obtained. The method presented also has clinical significance in that CK levels in serum can easily be determined with minimal sample preparation. Using serum samples from a healthy patient and a heart attack victim, activities of 26.6 U/l and 609.0 U/l, respectively, were obtained. Because of the direct measurement of ATP, this method eliminates the coupled reactions encountered in the common spectrophotometric and colorimetric methods of analysis resulting in a simpler and inexpensive assay.     

Acid ribonuclease from HeLa cell lysosomes.

An acid ribonuclease has been purified from HeLa cell lysosomes. The specific activity of the RNase in lysosomes is 8-fold higher than that in nuclei and 15-fold higher than that in the postlysosomal fraction. The purified enzyme showed no detectable DNase, phosphodiesterase, phosphatase, or alkaline RNase activity. The acid RNase binds to Con A-agarose and is inferred to be a glycoprotein. It has a low isoelectric point at pH 3.0 to 3.5, and the optimal pH for activity is between 5.0 and 5.5. The enzyme requires no divalent cation for optimal activity and is totally inhibited by 1 mM Cu2+ or Hg2+. Monovalent cations including Na+, K+, and NH4+ stimulate the activity in low ionic strength buffer. The enzyme degrades rRNA faster than tRNA, and tRNA faster than poly(U); poly(A) and poly(C) are highly resistant. The products from rRNA are mostly oligonucleotides with 3'-phosphate ends. An acid RNase is also present in the lysosomes of L-cells grown in a medium free of serum; it is probably identical to the one described here.     

Ozone inactivation of cell-associated viruses

The inactivation of HEp-2 cell-associated poliovirus (Sabin 1) and coxsackievirus A9 was investigated in three experimental systems, using ozone as a disinfectant. The cell-associated viral samples were adjusted to a turbidity of 5 nephelometric turbidity units. The cell-associated poliovirus and coxsackievirus samples demonstrated survival in a continuous-flow ozonation system at applied ozone dosages of 4.06 and 4.68 mg/liter, respectively, for 30 s. Unassociated viral controls were inactivated by the application of 0.081 mg of ozone per liter for 10 s. Ultrasonic treatment of cell-associated enteric viruses did not increase inactivation of the cell-associated viruses. The batch reactor with a declining ozone residual did not effect total inactivation of either cell-associated enteric virus. These cell-associated viruses were completely inactivated after exposure to ozone in a batch reactor using continuous ozonation. Inactivation of cell-associated poliovirus required a 2-min contact period with an applied ozone dosage of 6.82 mg/liter and a residual ozone concentration of 4.70 mg/liter, whereas the coxsackievirus was completely inactivated after a 5-min exposure to an applied ozone dosage of 4.81 mg/liter with an ozone residual of 2.18 mg/liter. These data indicate that viruses associated with cells or cell fragments are protected from inactivation by ozone concentrations that readily inactivate purified virus. The cell-associated viral samples used in this research contained particles that were 10 to 15 microns in size. Use of a filtration system before ozonation would remove these particles, thereby facilitating inactivation of any remaining viruses associated with cellular fragments.     

Expression of soluble bovine pancreatic ribonuclease A in Pichia pastoris and its purification and characterization

A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased kcat, 60-70% of the activity of wildtype RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since Tm of the glycosylated RNase A was decreased by 6 degrees C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.