The effects of polyamines on a residue-specific human plasma ribonuclease.

Schmukler, Morton; Jewett, P B; Levy, C.

doi:10.1016/s0021-9258(19)41703-1

Cited by 74 publications

(7 citation statements)

References 26 publications

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“…Akagi et al addressed this question, stating that RNases 1-5 each rechromatographed true on phosphocellulose and that treatment of serum with 7 M urea prior to chromatography did not reduce the number of peaks. Nonetheless, the pronounced tendency of serum RNase activity to aggregate (Schmuckler et al, 1975;Akagi et al, 1976), and the paucity of data documenting the distinctness of RNases 1-4, suggested reexamination of the nature of the putatively high molecular weight activities described by these authors.…”

Section: Discussionmentioning

confidence: 99%

“…The resolving power of polyacrylamide gel electrophoresis and the sensitivity of activity staining are sufficiently high to permit examination of very crude samples. Inclusion of NaDodS04 in the electrophoresis buffers eliminates the aggregation to which serum RNases are subject (Schmuckler et al, 1975;Akagi et al, 1976), disrupts noncovalent complexes of RNases with other proteins [including, presumably, RNase inhibitors; e.g., see Blackburn et al (1977)], causes anodal migration of both acidic and basic RNases, and permits estimation of molecular weight. Thus, the individual RNase species of unfractionated fluids and homogenates can be identified, their molecular weights estimated, their catalytic properties examined, and their response to various physical and chemical treatments ascertained-all without separation of individual RNases from one another or from the bulk of contaminating protein.…”

Section: Discussionmentioning

confidence: 99%

“…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%

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

Blank¹,

Dekker²

1981

Biochemistry

104

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Blank¹,

Dekker²

1981

Biochemistry

104

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“…shown for human serum RNase activity (Schmuckler et al, 1975;Akagi et al, 1976).] Whatever the cause(s), the molecular weight estimated by Sephadex is probably high.…”

Section: Discussionmentioning

confidence: 99%

“…The apparent discrepancy was resolved when we assayed band A (Sephadex G-75 fraction eluting at 47 mL, Figure 2) with poly(C) and phosphate buffer as did Rabin & Weinberger: The pH optimum of band A does indeed shift to 6.6 when assayed under these conditions. RNases from other human sources likewise display pH optima of 6.5 when both poly(C) and phosphate buffer are employed in assays, but have alkaline optima when RNA is substituted for poly(C) or when Tris-HCl is substituted for sodium phosphate [e.g., Schmuckler et al (1975)].…”

Section: Discussionmentioning

confidence: 99%

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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Anamalous electrophoresis behavior of rebonuclease from human urine and a novel treatment method for reliable analysis

Thomas

Hodes

1981

Electrophoresis

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Since desialylated ribonuclease (RNase) from human urine has a highly alkaline isoelectric point (PI 9.5-10.5), an acidic buffer system is required for electrophoretic analysis of different urine specimens. In a common acid disc electrophoresis system in thin-layer polyacrylamide gel, the cathodal mobility of the RNase enzyme(s) was markedly reduced from that expected on the basis of the estimated PI. Dialysis of urine against water had a very detrimental effect on the separation of the isozymes, and the addition of buffer to the dialyzed urine further exacerbated this effect. The urine RNase exhibited anomalous electrophoretic behavior in the form of artifactual variation in migration and mobility of the RNase from different urine specimens or from the same urine specimen adjusted to different concentrations. Treatment of urine with various cations had a significant, positive effect on the migration and resolution of the isozymes. This and other experimental evidence is consistent with the hypothesis that the anomalous behavior was due to the binding of anions from the buffer. A standardized protocol was developed for processing urine specimens, including treatment of urine with myoglobin, which abolished the artifactual variation observed previously and enabled reliable electrophoretic separation of the urine RNase isozymes. In addition, myoglobin treatment had a similar positive effect on the electrophoretic behavior of RNase enzymes from some other sources.

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The effects of polyamines on a residue-specific human plasma ribonuclease.

Cited by 74 publications

References 26 publications

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

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

Multiple ribonucleases of human urine

Anamalous electrophoresis behavior of rebonuclease from human urine and a novel treatment method for reliable analysis

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