Multiple ribonucleases of human urine

Abstract

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.

Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

“…The finding that there was no detectable extraneous RNA contamination was not surprising, because urine contains ribonucleases. 15 The fact that microvesicles can resist RNase and DNase digestion and still protect the nucleic acids contained within them is quite remarkable, and adds further support to the previously reported stable nature of urinary exosomes. 9 …”

“…In contrast, no obvious extraneous RNA could be detected (Figure 2b), consistent with the presence of ribonucleases in urine. 15 Analysis of the nucleic acids within microvesicles revealed some striking features, including the presence of prominent 18S and 28S rRNA peaks similar to those seen for RNA isolated from rat kidney tissue (Figure 2c). Such rRNA was not prevalent in previously reported serum-derived or cell culture-derived microvesicles, although they may be detectable depending on isolation technique as shown in the serum RNA isolation profile in Supplementary Figure S1d.…”

Human extracellular ribonucleases: multiplicity, molecular diversity and catalytic properties of the major RNase types

“…During the late 1970s, in addition to an inherent interest in the enzymes themselves, the study of human ribonucleases was stimulated by suggestions that these enzymes, when released into circulation or excreted in urine, might serve as diagnostic markers for a variety of diseases [1]. But the multiplicity of these enzymes [5] made it difficult to prove any correlation between RNase activity and disease states. In particular, in the early 1980s, the validity of any conclusions based on enzymatic determinations of RNase activity in body fluids was questioned [6].…”

Short Communication

“…3 Electrophoretic Analysis in a Poly(C) Cast SDS-Polyacrylamide Gel of the Active RNAase Fractions after Re-Chromatography in a C 4 Reversed-Phase Column was Carried out as Described (Sugiyama et a/., 1981) with Some Modification (Bravo efa/.,1992). 3 Electrophoretic Analysis in a Poly(C) Cast SDS-Polyacrylamide Gel of the Active RNAase Fractions after Re-Chromatography in a C 4 Reversed-Phase Column was Carried out as Described (Sugiyama et a/., 1981) with Some Modification (Bravo efa/.,1992).…”