Purification and properties of urinary alkaline ribonucleases from patients with nephrotic syndrome

Yamanaka, Masayoshi; Akagi, Kenzo; Murai, K; Hirao, N.; Fujimi, Satoshi; Omae, Teruo

doi:10.1016/0009-8981(77)90306-0

Cited by 9 publications

(12 citation statements)

References 11 publications

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“…For the preparation of "urine RNase concentrate", 2.6 L of urine was cooled to 4 °C, filtered, dialyzed against 5 mM Tris-HCl buffer (pH 7.4), and concentrated 10-fold by dialysis against Carbowax 6000 (Union Carbide). Following further dialysis against 10 mM sodium phosphate (pH 6.7), the RNase solution was loaded onto four phosphocellulose columns (Brown Co., Selectacel type 40, 1.02 mequiv/g), each with dimensions of 0.55 X 6.0 cm, prepared according to Yamanaka et al (1977). After each column was washed with 2 column volumes of equilibrating buffer, RNase activity was eluted with 3 mL of 10 mM sodium phosphate-2 M NaCl (pH 6.7); eluate containing activity from the first column was used to elute activity from the second column, ad seriatim.…”

Section: Methodsmentioning

confidence: 99%

“…fraction II 25.0-22.3 b 7.8-8.0 alkaline (major) fraction III 15.0b 6.4-6.9 acid (minor) Rabin & Weinberger (1975) urine RNase 33.0" 6.5d novel Bardon et al (1976a) fraction A 30.0" 8.5 secretory (major) fraction B 7.0 nonsecretory (minor) Yamanaka et al (1977) RNase 1 45.0b 8.5 alkaline Reddi (1977) urine RNase 22.0f 6.5d pancreatic 0 Determined by equilibrium sedimentation and amino acid analysis. b Determined by gel filtration.…”

Section: Methodsmentioning

confidence: 99%

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Multiple ribonucleases of human urine

Sugiyama¹,

Blank²,

Dekker³

1981

Biochemistry

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Four major urine ribonuclease (RNase) activities, designated bands A-D, were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and activity staining. Bands A, B, and C have alkaline pH optima and display molecular weights of 31 000, 23 000, and 20 000, respectively, upon sodium dodecyl sulfate (NaDodSO4) gel electrophoresis and weights of 44 000, 28 000, 22 000 upon gel filtration. Band D, with a pH optimum slightly below neutrality, has a molecular weight of 16 000 or 15 000, respectively, determined by the above methods. Band A, the most abundant activity in urine, is heterogeneous and resembles serum RNase 1 on electrophoresis and on phosphocellulose and Sephadex chromatography. Band B is similar to a minor, unnamed component of serum RNase activity while band C resembles serum RNase 3. Band D is similar to the leukocyte RNase-like activity of serum [Blank, A., & Dekker, C.A. (1981) Biochemistry (preceding paper in this issue)]. Band A is present in urine at a concentration high than that of RNase 1 in serum. In contrast, urine counterparts of serum RNases 2, 4, and 5 are not apparent upon either phosphocellulose chromatography [see also Yamanaka, M., Akagi, K., Murai, K., Hirao, N., Fujimi, S.,& Omae, T. (1977) Clin. Chim. Acta 78, 191-201] or NaDodSO4 get electrophoresis; a urine counterpart of serum RNase 3 can be detected only by the more sensitive electrophoretic method. These results indicate that RNase 2-5 are processed differently by the kidney than RNase 1. After reconciliation of reported differences in their pH optima and molecular weights, five apparently diverse RNase preparations described in the literature can be related to band A activity and three preparations to band D. However, we are unable to confirm a previous report of a human urine enzyme indistinguishable from bovine pancreatic RNase A.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Multiple ribonucleases of human urine

Sugiyama¹,

Blank²,

Dekker³

1981

Biochemistry

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“…Purification of human serum (Schmuckler et al, 1975;Reddi, 1975;Akagi et al, 1976Akagi et al, , 1978aBardon et al, 1976a), urine (Delaney, 1963;Naskalski, 1972a,b;Rabin & Weinberger, 1975;Bardon et al, 1976a;Yamanaka et al, 1977;Reddi, 1977), andCSF RNases (Rabin et al, 1977) has been undertaken in several laboratories. The RNase activity of all three fluids is chromatographically heterogeneous, though the full extent and physical bases of the heterogeneity are unknown.…”

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confidence: 99%

Ribonucleases of human serum, urine, cerebrospinal fluid, and leukocytes. Activity staining following electrophoresis in sodium dodecyl sulfate-polyacrylamide gels

Blank¹,

Dekker²

1981

Biochemistry

103

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The ribonucleases (RNases) of human blood serum, urine, cerebrospinal fluid (CSF), and leukocytes were visualized by activity staining after electrophoresis in RNA-case sodium dodecyl sulfate-polyacrylamide gels. Samples were prepared for electrophoresis by heating for 2 min at 100 degrees C in 2% sodium dodecyl sulfate (NaDodSO4) and 5% mercaptoethanol, conditions which dissociate proteins into their constituent polypeptide chains and permit estimation of molecular weight. It was found that each of the five peaks of serum alkaline RNase activity separable on phosphocellulose columns, i.e., RNases 1-5 of Akagi et al. [Akagi, K., Murai, K., Hirao, N., & Yamanaka, M. (1976) Biochim. Biophys. Acta 442, 368-378], is associated with electrophoretically distinct enzymes. The molecular weights exhibited by these enzymes in NaDodSO4 gels are 31 000 and 28 000 (major species of RNase 1), 25 000 (RNase 2), 20 000 (RNase 3), 16 000 (RNase 4), and 14 000 (RNase 5). The RNase activity of leukocytes displays a molecular weight of 17 000 and exhibits a characteristic dependence of its Rf on the temperature at which samples (in 2% NaDodSO4 without mercaptoethanol) are prepared for electrophoresis. An RNase activity like that of leukocytes, distinct from RNases 1-5, is found in serum. Urine RNase activity is less heterogeneous than that of serum, consisting mainly of species like serum RNase 1 and an enzyme similar to leukocyte RNase. Conversely, CSF RNase activity is more complex and includes enzymes resembling serum RNases 1-5 as well as additional species either not observed in serum or detected in serum as minor components following chromatography. The analytical methods described herein are particularly useful for assessment of heterogeneity of RNase preparations and for direct comparison of the RNases of crude and purified samples.

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“…Although protein purification techniques, such as different types of column chromatography, can be used to study multiple nucleases (Naskalski, 1972; Abbreviations used: %T denotes the total percentage concentration of both acrylamide (a) and NN'-methylenebisacrylamide (b) and is calculated by using the following formula: (a+b) x 100/c, where a and b are in grams and c, the total volume, is in ml; %C is the percentage concentration of the cross-linker relative to the total concentration: b(100)/(a + b); when NN'-diallyltartardiamide (DATD) is substituted for NN'-methylenebisacrylamide, the following notation is used: %C (DATD); endo-H, endo-f-N-acetylglucosaminidase, SDS, sodium dodecyl sulphate. Yamanaka et al, 1977), such methods are timeconsuming and do not lend themselves easily to examination of many samples.…”

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confidence: 99%

Isoelectric focusing—polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities

Karpetsky¹,

Brown²,

McFarland³

et al. 1984

Biochemical Journal

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Individual native nuclease activities from human leucocytes are separated by using two-dimensional gel electrophoresis in an apparatus that allows the simultaneous running of 28 gels. Proteins are separated by isoelectric focusing in a disc gel, followed by electrophoresis into a slab gel containing DNA. Protein denaturants are avoided in the second dimension by the use of a running pH well above the optimal pH for DNAase (deoxyribonuclease) activity. Electrophoresed gels are incubated in appropriate buffers to activate nuclease activity. After staining for intact DNA, the positions of active enzymes, unobscured by the presence of other proteins, are revealed as colourless spots in a reddish-purple field. The technique is easy to use and is sensitive to 50pg of DNAase I. Versatility is provided by the use of either acidic or basic electrophoresis running buffers and by the use of specific gel incubation conditions to reveal different sets of enzyme activities. Two DNAases active at pH 7.4 in the presence of Mg2+ and Ca2+, and sixteen DNAases active at acidic pH and not requiring metals, are detected. Treatment of the human enzymes with specific glycosidases reveals that many of the human DNAases are glycoproteins containing negatively charged moieties and may be derived from modification of parent activities.

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Purification and properties of urinary alkaline ribonucleases from patients with nephrotic syndrome

Cited by 9 publications

References 11 publications

Multiple ribonucleases of human urine

Multiple ribonucleases of human urine

Ribonucleases of human serum, urine, cerebrospinal fluid, and leukocytes. Activity staining following electrophoresis in sodium dodecyl sulfate-polyacrylamide gels

Isoelectric focusing—polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities

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