New detection method for ribonuclease 2 (RNase 2) using immunoblotting with specific antibody

Mizuta, Keiko; Yasuda, Toshihiro; Ikehara, Yoko; Sato, Wataru; Kishi, Koichiro

doi:10.1007/bf01263035

Cited by 8 publications

(5 citation statements)

References 19 publications

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“…The fact that 0.8 mg of the purified DNase I could be obtained from about 15 litres of urine collected from five rabbits over a few weeks indicates that urine is one of the best source materials for purification of rabbit DNase I. We have already demonstrated that human urine is a good source of several nucleases, such as DNase I [2], DNase II [35] and secretory-type and non-secretory-type RNases [41,51]. Urine has a further advantage, in that its collection is non-invasive.…”

Section: Discussionmentioning

confidence: 99%

Rabbit DNase I: purification from urine, immunological and proteochemical characterization, nucleotide sequence, expression in tissues, relationships with other mammalian DNases I and phylogenetic analysis

Yasuda

Takeshita

Nakajima

et al. 1997

Biochemical Journal

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DNase I from rabbit urine was purified approx. 3600-fold to apparent homogeneity with a 41% yield by affinity chromatography utilizing DNA-cellulose; the purity of the final preparation was assessed by SDS/PAGE, lack of contamination by other nucleases and production of a monospecific antibody against the enzyme. Although the proteochemical and enzymological properties of the purified enzyme resembled those of other mammalian DNases I, the enzymic activity of rabbit DNase I was less efficiently inhibited by monomeric actin than was that of human DNase I, probably due to substitution of an amino acid residue involved in actin binding (Tyr-65 to Phe). The effects of specific antibodies to human, rabbit and rat DNases I on the activities of the corresponding purified enzymes revealed that human DNase I lies between the rat and rabbit enzymes with regard to its immunological properties. An 1158 bp full-length cDNA encoding rabbit DNase I was constructed from the total RNA of rabbit pancreas using a combination of reverse transcriptase-PCR and rapid amplification of cDNA ends, followed by sequencing. This identified a 17- or 21-amino-acid signal sequence, with the mature enzyme containing 260 amino acids and a single N-glycosylation site at Asn-18. The amino acid sequence deduced from the cDNA sequence exactly matched that determined proteochemically from the purified enzyme up to residue 20. A systematic survey of DNase I distribution as measured by both enzymic activity and DNase I gene transcripts in 12 rabbit tissues showed the pancreas and parotid gland to produce equivalent levels, higher than those in other tissues. Enzymic activity and DNase I gene expression levels in each tissue correlated well. The results of phylogenetic and sequence identity analysis, immunological properties and tissue-distribution patterns of DNase I indicated a closer relationship between the rabbit and human enzymes than for other mammalian DNases I. Furthermore, differences between the enzymic activities expressed in mammalian parotid gland and pancreas suggest that the distribution of DNase I in mammalian tissue is species-specific.

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Section: Discussionmentioning

confidence: 99%

Rabbit DNase I: purification from urine, immunological and proteochemical characterization, nucleotide sequence, expression in tissues, relationships with other mammalian DNases I and phylogenetic analysis

Yasuda

Takeshita

Nakajima

et al. 1997

Biochemical Journal

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“…20 times higher than that of RNase I. The molecular weight of RNase 2 as determined by electrophoresis in SDS-PAGE and gel filtration is 32 kDa and 38, respectively: the pH-optimum is in the range of 7.2–7.6 [ 80 ].…”

Section: Dna- and Rna-hydrolyzing Enzymes In The Urinementioning

confidence: 99%

Extracellular Nucleic Acids in Urine: Sources, Structure, Diagnostic Potential

Bryzgunova

Laktionov

2015

Acta Naturae

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Cell-free nucleic acids (cfNA) may reach the urine through cell necrosis or apoptosis, active secretion of nucleic acids by healthy and tumor cells of the urinary tract, and transport of circulating nucleic acids (cir- NA) from the blood into primary urine. Even though urinary DNA and RNA are fragmented, they can be used to detect marker sequences. MicroRNAs are also of interest as diagnostic probes. The stability of cfNA in the urine is determined by their structure and packaging into supramolecular complexes and by nuclease activity in the urine. This review summarizes current data on the sources of urinary cfNA, their structural features, diagnostic potential and factors affecting their stability.

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“…A number of investigators have used either preparative IEF for purifying human RNAases [5,17] or analytical IEF for characterizing human secretory [18][19][20][21] and non-secretory [19,20] RNAases. All of these studies have used conventional CAs in either columns [17,19] or slab gels [18,20,21]. The results of these studies have not been very definitive, and RNAase pl values are given only as a broad range, and in some cases are not given at all.…”

Section: Resultsmentioning

confidence: 99%

Immobilized pH gradient focusing of alkaline proteins: analysis of the isoform composition of purified human non-secretory ribonucleases from kidney, liver and spleen

Coronel

Little

Alhadeff

1993

Biochemical Journal

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Previous studies on the isoform composition of human ribonucleases (RNAases) have resulted in confusing and inconsistent results, presumably due to methodological problems in electrofocusing of alkaline proteins. In the present study, immobilized pH gradient (IPG) carrier ampholyte (CA) isoelectric focusing (IEF) and conventional CA-IEF have been evaluated for the analysis of the isoforms of human non-secretory RNAases purified from kidney, liver and spleen. CA-IEF proved unsuitable since the alkaline RNAase isoforms migrated into the cathode. IPG-CA-IEF, however, resolved the RNAase isoforms and marker proteins in the basic region of the gel matrix. The three RNAases had comparable isoform profiles, each with two protein bands with approximate pI values of 10.3 and 10.4. Western blotting showed that the two protein bands of each RNAase were immunoreactive (with polyclonal antibodies that recognize RNAase), indicating that the protein bands are RNAase isoforms. The present results provide reliable pI data on human RNAase isoforms and suggest that IPG-CA-IEF should be a suitable technique for analysing the isoforms of other alkaline proteins.

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New detection method for ribonuclease 2 (RNase 2) using immunoblotting with specific antibody

Cited by 8 publications

References 19 publications

Rabbit DNase I: purification from urine, immunological and proteochemical characterization, nucleotide sequence, expression in tissues, relationships with other mammalian DNases I and phylogenetic analysis

Rabbit DNase I: purification from urine, immunological and proteochemical characterization, nucleotide sequence, expression in tissues, relationships with other mammalian DNases I and phylogenetic analysis

Extracellular Nucleic Acids in Urine: Sources, Structure, Diagnostic Potential

Immobilized pH gradient focusing of alkaline proteins: analysis of the isoform composition of purified human non-secretory ribonucleases from kidney, liver and spleen

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