Purification and characterization of a ribonuclease from human spleen

Abstract

A ribonuclease has been isolated from human spleen (RNase HS) by means of acid extraction, ammonium sulphate fractionation, successive column chromatographies on CM-cellulose, heparin-actigel, and poly(G)-agarose, and double gel-filtration on Sephadex G-75. The purified preparation was homogeneous as judged by SDSIPAGE. RNase HS was found to be a glycoprotein, containing three fucose, one mannose and five glucosamine residues/molecule, with a molecular mass of 17 kDa as determined by both SDS/PAGE and gel filtration. The catalytic properties and structural features, including its amino acid composition and the amino acid sequence of the N-terminal 35 residues, indicated that the enzyme was strictly related to nonsecretory RNase isolated from human urine and liver. In particular, the amino acid sequence of the N-terminal was identical with that of urine nonsecretory RNase and eosinophil-derived neurotoxin. Furthermore, analyses using three different antibodies specific to RNase HS, urine nonsecretory RNase and urine secretory RNase, indicated that RNase HS was not immunologically distinguishable from urine nonsecretory RNase, but clearly so from urine secretory RNase. However, the carbohydrate compositions of RNase HS and urine nonsecretory RNase were found to differ. It therefore remains to be resolved whether or not the tissue of origin of nonsecretory RNase in urine is the spleen.Sierakowska and Shugar [ 11 classified the pyrimidinespecific neutral and alkaline ribonuclease (RNase) of mammalian origin into two distinct classes : secretory and nonsecretory RNases. The former enzyme shows a pH optimum of around 8.5 and a greater preference for poly(C) than poly(U) and RNA. The latter cleaves RNA most effectively at pH 7.0, and its preference for poly(C) is less than the former.

Structure–function relationships in human ribonucleases: main distinctive features of the major RNase types

“…This may support the suggestion already advanced [53,54] that ribonuclease (presently classified as hydrolase) has indeed evolved primarily to catalyze RNA transphosphorylation. If so, it should be reclassified as transferase.…”

“…One of the most important difference consists in the fact that although nptRNases efficiently catalyze the depolymerization of RNA, they do not show any detectable (catalytically significant) hydrolytic activity towards 2',3'-cyclic phosphodiesters [12,17,36], which are usually considered intermediates in the RNase A-catalyzed cleavage of RNA. Regarding this point, work from different laboratories [46,53,54] demonstrated that bovine RNase A and other pancreatictype RNases release into solution cyclic phosphodiesters as true products of the transphosphorylation of RNA, which can be hydrolysed in a separate slower reaction. It is worth noticing here that (Table 1) while the ribonucleases capable of hydrolysing cyclic nucleotides have a phenylalanine at position 120, those lacking this residue are inactive on the same substrates.…”

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

“…tivity ratio of 4 and 12, respectively [17,51], pt/nptRNase 4 degrades poly(U) at least 1000-fold faster than poly(C) [21,22]. This preference [17,21,22,32,35,42,51] seems to be due to a significant reduction of their affinity for poly(C), rather than to an increased preference for poly(U) and could be attributed to the different microenvironment of the B1 subsite of both nptRNases and pt/nptRNase 4. In nptRNases 2 and 3 ( fig.…”

“…This enzyme [41] (also named EDN [19]) occurs predominantly in spleen [42], eosinophils [43], liver [8] and placenta [44], but has been also isolated from urine [32] and kidney [35]. All these proteins (with quite similar glycosylation patterns [45]) are products of the same Multi-author Review Article gene, which was cloned [19,46] and located on chromosome 14 (region q24 -q31) [31].…”

The amino acid sequence of human ribonuclease 4, a highly conserved ribonuclease that cleaves specifically on the 3′ side of uridine

“…This enzyme exists in many sizes and with a range of properties, due in part to the covalent attachmcnt of carbohydrates (Beintema et al, 1988a(Beintema et al, , 1988b. The 'non-secretory' RNase (EDN) which is found in spleen (Delaney, 1963;Yasuda et al, 1990), kidney and urine (Delaney, 1963;lwama et al, 1981;Cranston et al, 1980), eosinophils , liver (Sorrentino et al, 1988) and placenta (Shapiro and Vallee, 1991), has also been sequenced (Beintema ct al., 1988b) and is a very different, though homologous, enzyme. Two additional RNase family members whosc sequences havc been determined are ECP , Rosenberg et al, 1989a, which has been isolated thus far only from granulocytes, and angio- genin (Strydom et al, 1985) which is found in tumor-cellconditioned medium (Fett et al, 1985) as well as in plasma (Shapiro et al, 1987).…”