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.     

Age-dependent changes are observed in the levels of an enzyme mediator of interferon action: a (2'-5')A(n)-dependent endoribonuclease

A (2'-5')oligoadenylate-dependent endoribonuclease (RNase L) is an important mediator of interferon's antiviral actions. Levels of this enzyme were determined in spleen, lung, liver, and kidney of mice at different times after birth. The levels of RNase L were found to be relatively low in newborn kidney, lung, and spleen. RNase L levels rise 2- to 10-fold in these three tissues as mice approach 5 days of age. In the spleen, levels of RNase L remain high as mice reach adult life. In the lung and kidney, however, RNase L levels decrease after 14 days. RNase L levels in the liver are highest from birth to 5-7 days and then decrease subsequently and remain low in adult mice. These changes in RNase L levels with postnatal development may be important with regard to age-specific susceptibility to some virus infections.     

On the mechanism of ribonuclease secretion in the murine exocrine pancreas

Summary In the rat pancreas, normal diurnal and pilocarpine-induced variations in the protein, ribonuclease (RNase), and water content were studied. The results show a considerable variation in the dirunal content of RNase, whereas the protein and water concentrations remained constant. Furthermore, the experiments provide evidence that RNase is secreted by means of a mechanism partly independent of other proteins and that every zymogen granule does not contain the same amount of the enzyme. The earlier presumption that there are at least two intracellular storage forms of the enzyme is confirmed and the intracellular variability in the capacity to attract anti-RNase antibodies is explained.     

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.     

9 alpha,11 beta-prostaglandin F2 formation in various bovine tissues. Different isozymes of prostaglandin D2 11-ketoreductase, contribution of prostaglandin F synthetase and its cellular localization

9 alpha,11 beta-prostaglandin F2 was formed from prostaglandin D2 by its 11-ketoreductases in 100,000 x g supernatants of various bovine tissues in the presence of an NADPH-generating system. The reductase activities were high in liver (51.09 nmol/h/mg of protein), lung (24.99), and spleen (14.20); moderate in heart and pancreas (3.09-3.61); weak in stomach, intestine, colon, kidney, uterus, adrenal gland, and thymus (0.11-2.63); and undetectable in brain, retina, carotid artery, and blood (less than 0.10). No formation of prostaglandin F2 alpha from prostaglandin D2 was detected in all tissues. In immunotitration analyses with a polyclonal antibody specific for prostaglandin F synthetase, the reductase activities in lung and spleen showed identical titration curves to that of the purified synthetase and decreased to less than 15% of the initial activity under the condition of antibody excess. Prostaglandin F synthetase-immunoreactive protein in these two tissues showed peptide fingerprints identical to that of the purified enzyme after partial digestion with Staphylococcus aureus V8 protease. The antibody was partially cross-reactive to the reductase in liver (about 20% of that to the synthetase) but not to the reductase(s) in other tissues. The Km value for prostaglandin D2 of the reductase activity was the same in lung and spleen as that of the purified prostaglandin F synthetase (120 microM) but differed in liver (6 microM), heart, and pancreas (15 microM). The predominant distribution of prostaglandin F synthetase in lung and spleen was confirmed by radioimmunoassay (2.8 and 1.0 micrograms/mg protein, respectively) and Northern blot analyses. In immunoperoxidase staining, this enzyme was localized in alveolar interstitial cells and nonciliated epithelial cells in lung, histiocytes and/or dendritic cells in spleen, and a few interstitial cells in kidney and adrenal cortex.     

Studies on the state of tyrosyl residues in a ribonuclease from seminal vesicles.

In order to study the state of tyrosyl residues in a ribouuclease from bovine semina vesicles [EC 3.1.4.22, RNase Vs1] several lines of experiments were carried out. Spectrophotometric titration of RNase Vs1 indicated that two out of 8 tyrosine residues were titrated very easily and their apparent pKa values were about 9.8. Next, about 4 residues were titrated at pH up to 13.5. The remaining 2 residues were titrated time-dependently at pH 13.5. In 8 M urea, about 6 tyrosine residues were titrated with apparent pK4 values of about 11.2 and about 2 residues were titrated time-dependently at pH 13.5. Acetylation of RNase Vs1 with N-acetylimidazole was studied at pH 7.5. In aqueous solution, about 1.1-3.5 tyrosine residues were acetylated, depending on the experimental conditions, and in 8 M urea, 5.3 tyrosine residues were modified. RNase Vs1 was nitrated with tetranitromethane at pH 7.5. In aqueous solution, about 2.5 tyrosine residues were nitrated very easily; the enzymatic activity of the modified enzymes was 130-200% of that of the native enzyme. In 8 M urea, the reactivity of the tyrosine residues increased and about 4-5.5 residues were modified. The results of chemical modification and spectrophotometric titration indicated that about two tyrosine residues in RNase Vs1 were exposed to the solvent and were more reactive to various reagents, and 3-4 tyrosine residues were less reactive. The final 2 residues were not accessible to the reagent even in the presence of urea, but were titraten at pH 13.5. The solvent perturbation difference spectrum using ethylene glycol as a perturbant indicated that about 4 tyrosine residues were perturbed. When the pH of the enzyme solution was changed from 7.0 to 1.0, the change in optical density of RNase Vs1 due to denaturation blue shift was about 1,600 at 287nm. The optical density change at 287 nm of native RNase Vs1 on exposure to 8 M urea and 6 M guanidine-HCl indicated that the environments of 2-3 and 4 tyrosine residues were changed by the addition of the denaturants, urea and guanidine-HCl, respectively. In RNase Vs1 having about four nitrotyrosine residues, the two most inaccessible tyrosine residues remained resistant to titration with alkali. On adding nucleotide, nitrated RNase Vs1 gave a difference spectrum in the ultraviolet region but not in 320-460 nm region, where nitrotyrosine residues absorb light. This may indicate that tyrosine residues located relatively near the surface of the molecule are not perturbed directly by nucleotide binding.     

Occurrence and subcellular location of NADH- and NADPH-linked aquacobalamin reductases in human liver

1. Both activities of NADH- and NADPH-linked aquacobalamin reductases were found in some human tissues, liver, kidney pancreas, heart, spleen, lung, cerebrum, cerebellum, adrenal glands, stomach, duodenum, jejunum, ileum, colon and bone marrow. 2. Human liver contained both enzymes with higher specific activities than any other tissues. 3. The liver NADH-linked enzyme was distributed in both mitochondrial (approx. 60%) and microsomal (40%) fractions; similar to the distribution of the NADPH-linked enzyme, but of which 40% activity was found in the mitochondria and the remaining activity was recovered in the microsomes. 4. The results suggest that the synthetic systems of the cobalamin coenzymes occur in both mitochondria and microsomes of human liver.     

Tissue and cellular distribution of alpha-L-iduronidase in the pig

We investigated the alpha-L-iduronidase activity of various pig tissues. Furthermore, we examined the tissues using antibody, enzyme immunoassay (EIA), and immunohistochemical methods. The amounts of enzyme measured by the EIA method in the various tissues were proportional to their enzyme activities and also to their immunohistochemical characteristics. The tissues could thus be classified into three groups: a high enzyme activity group composed of the liver, kidney, and spleen; a moderate activity group comprising the lung, lymph nodes, stomach, ileum, colon, and pancreas; and a low activity group consisting of the heart, diaphragm, iliopsoas muscle, cerebrum, cerebellum, and skin. The molecular weight of the enzyme in each tissue did not reveal any heterogeneity, having two components of 70 KD and 62 KD by Western blot analysis. Immunohistochemically, alpha-L-iduronidase was strongly detected in the lysosomal membranes of cells of the mononuclear phagocyte system, epithelial cells of the proximal tubules in the kidney, and some blastic cells, whereas hepatocytes revealed weak positive reactions. The tissue and cellular distribution of the enzyme appeared to have a close relation to tissues that manifest or are affected by alpha-L-iduronidase deficiency.     

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.     

Catalytic properties of human platelet 12-lipoxygenase as compared with the enzymes of other origins

Arachidonate 12-lipoxygenases of porcine and bovine leukocytes were different in substrate specificity and immunogenicity from the enzyme of bovine platelets (Arch. Biochem. Biophys. (1988) 266, 613). In order to extend the comparative studies on the two types of 12-lipoxygenase, we purified the enzyme from the cytosol of human platelets by immunoaffinity chromatography to a specific activity of about 0.3 mumol/min per mg protein at 37 degrees C. The purified enzyme was active with eicosapolyenoic acids and docosahexaenoic acid. Linoleic and linolenic acids were poor substrates in contrast to the high reactivity of the leukocyte enzymes with these octadecapolyenoic acids. The finding that the human platelet enzyme catalyzed 15-oxygenation of 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, raised a question if lipoxins were produced by incubation of the enzyme with leukotriene A4. However, the leukotriene A4 was scarcely transformed to lipoxin isomers by 12-lipoxygenases of human and bovine platelets. In sharp contrast, the porcine and bovine leukocyte enzymes converted leukotriene A4 to various lipoxin isomers by the reaction rates of 3% and 2% of the arachidonate 12-oxygenation. Thus, 12-lipoxygenases of human and bovine platelets were catalytically distinct from the porcine and bovine leukocyte enzymes in terms of their reactivities not only with linoleic and linolenic acids, but also with leukotriene A4 as lipoxin precursor.     

牛泡沫病毒中国株感染兔的研究

将牛泡沫病毒(BFV3026)感染的细胞经耳缘静脉注射兔子,并以正常细胞注射的兔为对照.1年后处死,病毒挽救实验及PCR检测显示兔经一次注射即可被BFV3026感染,病毒广泛分布于感染兔的多种脏器中,通过共培养可从感染兔血、肝、脾、肺、肾中拯救出相应感染性病毒颗粒,并在脑、骨髓、心、胰、肠系膜中检到高拷贝BFV原病毒DNA存在.同时,血清学检测表明感染兔在接受注射一个月后即产生高滴度抗病毒蛋白抗体,并维持该滴度水平直至实验终止,兔未表现任何可观病变.     

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.     

Differential expression of phospholipases D1 and D2 in mouse tissues

The differential expression of phospholipase D (PLD) isozymes, which include PLD1 and PLD2, was examined in various murine tissues, including the cerebrum, cerebellum, heart, lung, liver, spleen, stomach, pancreas, ileum, colon, adrenal gland, kidneys, testes, ovaries, and uterus. In Western blot analysis, only PLD1 was detected in the heart and ovary, while only PLD2 was detected in the pancreas and ileum. Both PLD1 and PLD2 were strongly expressed in the cerebrum, cerebellum, and lung, and both were also expressed in the liver, spleen, stomach, colon, kidney, testes, and uterus. Immunohistochemistry showed intense PLD immunostaining in the cerebrum, cerebellum, lungs, intestines, and testis, and weak PLD immunostaining in the liver, kidneys, spleen, and heart. These findings suggest that PLD1 and PLD2 are differentially expressed in the various organs of mice, and that each PLD isozyme plays a distinct role in each organ.     

Abundance of RNase4 and RNase5 mRNA and protein in host defence related tissues and secretions in cattle

Members of the RNaseA family are present in various tissues and secretions but their function is not well understood. Some of the RNases are proposed to participate in host defence. RNase4 and RNase5 are present in cows' milk and have antimicrobial activity. However, their presence in many tissues and secretions has not been characterised. We hypothesised that these two RNases are present in a range of tissues and secretions where they could contribute to host defence. We therefore, determined the relative abundance of RNase4 and RNase5 mRNA as well as protein levels in a range of host defence related and other tissues as well as a range of secretions in cattle, using real time PCR and western blotting. The two RNases were found to be expressed in liver, lung, pancreas, mammary gland, placenta, endometrium, small intestine, seminal vesicle, salivary gland, kidney, spleen, lymph node, skin as well as testes. Corresponding proteins were also detected in many of the above tissues, as well as in seminal fluid, mammary secretions and saliva. This study provides evidence for the presence of RNase4 and RNase5 in a range of tissues and secretions, as well as some major organs in cattle. The data are consistent with the idea that these proteins could contribute to host defence in these locations. This work contributes to growing body of data suggesting that these proteins contribute to the physiology of the organism in a more complex way than acting merely as digestive enzymes.     

Tissue-dependent expression of two (alpha 1----2)-fucosyltransferases specified by the H and Se genes

Human blood group H substance is produced from its percursor by the action of (alpha 1----2)-fucosyltransferase. In a classical model, the Se gene that determines secretor status is a regulatory gene controlling the expression of H gene-specified fucosyltransferase in the secretory tissues. However, recent biochemical evidence supports a new genetic model in which the H and Se genes are both structural genes encoding two fucosyltransferases with different characteristics. The H gene-specified enzyme (H enzyme) is absent in secretory tissues and secretory fluids, while the Se gene-specified enzyme (Se enzyme) is missing in hematopoietic tissues. The H enzyme has a much lower Km for phenyl-beta-galactoside than does the Se enzyme. The tissue-dependent expression of the two enzymes was examined according to this criteria. The H enzyme was found to be predominant in the tissues of secretors examined (lung, liver, kidney, stomach, and skeletal muscle). While the Se enzyme exists in the secretor's lung, liver and kidney, very little or none is found in the stomach and skeletal muscle tissues.     

SD大鼠主要脏器间的相关性分析

目的测定成年SD大鼠体重与各脏器重量,并对不同脏器之间及体重与各脏器重量的相关性进行分析。方法选用三月龄SD大鼠雄性共24只,进行人道处死,解剖后分别测定心、肝、肺、肾等脏器重量,并作相关性分析。结果 SD大鼠的心、肝、肺、脾、肾与胃空体的相关性分析中,SD大鼠的心脏及肝脏重量之间不相关(P>0.05),且分别与其他各脏器及胃空体之间也无明显相关(P>0.05);而脾脏的重量与肾脏重量之间则相关极为显著(P<0.01);胃空体与肺、脾呈现极显著相关(P<0.01),与肾脏呈显著相关(P<0.05)。结论通过解剖比较SD大鼠各脏器之间的关系,对动物体内各脏器的大小及相互关系有了初步的认知,为今后进行的动物相关实验研究打下基础。     

Immunochemical characteristics of the peroxidases of several mouse tissues]

Enzymes catalyzing peroxidase reaction of a lysosomal fraction in bone marrow, leucocytes, spleen, thyroid gland, stomach, kidney, heart, lungs, brain and skeletal muscle of mice were investigated by immunochemical methods. A high level of peroxidase activity was discovered in leucocytes, bone marrow, spleen, heart and lung, a lower activity appeared to be characteristic of liver, thyroid gland and kidney. The peroxidase activities in brain, skeletal muscle and stomach were low. The reaction of immunoprecipitation with myeloperoxidase-specific antiserum revealed considerable antigenic distinctions between the enzymes catalysing peroxidase reaction in various tissues of mice.     

Two immunologically and catalytically distinct arachidonate 12-lipoxygenases of bovine platelets and leukocytes

12-Lipoxygenases were found in the cytosol fraction of bovine leukocytes and platelets. The bovine leukocyte enzyme was immunoprecipitable by a monoclonal antibody directed to 12-lipoxygenase of porcine leukocytes, but not by a monoclonal antibody against the human platelet enzyme. In contrast, the bovine platelet enzyme cross-reacted only with antibody against the human platelet enzyme. The leukocyte and platelet enzymes were partially purified to final specific enzyme activities of 1.1 and 0.3 mumol/min/mg protein, respectively, by immunoaffinity chromatography using each cross-reacting antibody as a ligand. The leukocyte enzyme reacted with various octadecapolyenoic acids as well as eicosapolyenoic and docosapolyenoic acids, whereas the platelet enzyme was almost inactive with octadecapolyenoic acids. Moreover, the two enzymes showed different heat-instabilities and reaction time courses. Thus, the 12-lipoxygenases of bovine leukocytes and platelets were immunologically and catalytically distinct enzymes.     

Model to evaluate the toxic effect of drugs: vincristine effect in the mass of organs and in the distribution of radiopharmaceuticals in mice

There are evidences that the biodistribution of radiopharmaceuticals can be modified by some drugs. As chemotherapeutic drugs present important toxic effects, we studied the vincristine effect in the mass of organs and are trying to develop a model to evaluate the action of chemotherapeutic drug using the biodistribution of radiopharmaceuticals. Vincristine was administered (n=15) into female Balb/c mice, the organs isolated and their mass determined. To study the vincristine effect in the biodistribution of technetium-99m-dimercaptosuccinic acid (99mTc-DMSA) or technetium-99m-diethylenetriaminepentaacetic acid (99mTc-DTPA), vincristine (0.03 mg) was administered in the animals (n=15) in three doses. 99mTc-DMSA or 99mTc-DTPA was injected 1h after the last dose. After 0.5h, the animals were sacrificed and the percentage of radioactivity (%ATI) and the percentage of radioactivity per gram of tissue (%ATI/g) in each organ were calculated. The results have shown that the mass decreased significantly (Wilcoxon test, P<0.05) in thymus, spleen, ovary, uterus, kidneys, pancreas. The %ATI to 99mTc-DMSA increased in lung, pancreas, heart, thyroid, brain, and bone, and the %ATI/g increased in uterus, ovary, spleen, thymus, kidney, lung, liver, pancreas, heart, thyroid, brain and bone. To 99mTc-DTPA, the %ATI increased in uterus, ovary, spleen, thymus, kidney, lung, liver, stomach, heart and bone, and the %ATI/g increased in uterus, ovary, spleen, thymus, kidney, lung, liver, stomach, heart and bone. The results were statistically significant (Wilcoxon test). The results can be explained by the metabolization, therapeutic, toxicological or immunosupressive action of the vincristine. This model, probably, should be used to evaluate the toxic effect of various drugs.