Enzyme kinetics of RNase present in Testes (original) (raw)
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ISOLATION AND IDENTIFICATION OF RNASE A FROM TESTIS THROUGH HPLC AND CHROMATOFOCUSSING
From the previous studies it was clear that RNase A is present in pancreas and our new Finding includes that RNase that is similar to RNase A was also present in testis along with other RNases in the tissue. In chromatofocussing the peak was observed in fraction 5 of PI 9.6 and in HPLC it was present in all fractions of the column. The aim of present work is to isolate and identify RNase A through HPLC and chromatofocussing. From the results we conclude that RNase A is present in testis in addition to its presence in epidydymis .Future perspectives include finding of other RNases present in the testis and also further evaluation by other methods. Please cite this article in press as EswariBeeram et al. Isolation and Identification of Rnase A from Testis Through HPLC and Chromatofocussing. Indo American Journal of Pharmaceutical Research.2017:7(03).
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1985
Human seminal ribonuclease (a basic protein occurring in a glycosylated and in a non-glycosylated form) is very active against double-stranded RNAs (De Prisco, R., Sorrentino, S., Leone, E. and Libonati, M. (1984) Biochim. Biophys. Acta 788, 356-363). The action of the two enzyme forms on single-stranded and double-stranded substrates was studied as a function of pH and ionic strength. Results indicate (1) that glycosylation of the RNAase molecule does not affect enzyme action on single-stranded RNAs, while (2) degradation of double-stranded RNAs is moderately increased by the presence of carbohydrates in the enzyme molecule. Human seminal RNAase shows a marked helix-destabilizing activity on poly(dA-dT) X poly(dA-dT). Under various conditions, this action (1) is definitely stronger than that of bovine RNAase A, and (2) seems to be less dependent on the glycosylation than on the basicity of the enzyme protein. The remarkable activity of human seminal RNAase on double-stranded RNA may, at least partly, be related to the enzyme properties mentioned above.
Bull Semen Ribonucleases. 1. Purification and Physico-Chemical Properties of the Major Component
European Journal of Biochemistry, 1972
A high ribonuclease activity has been detected in bull seminal plasma; two major fractions (RNAase BS-1 and RNAase BS-2) have been identified, which are responsible for such activity and one of the two, RNAase BS-I, has been purified and crystallized. It has a molecular weight of 29000, an isoelectric point a t pH 10.3 and A:'&, a t 278 nm is 4.65, The amino acid composition has been determined, its main features being a high content of basic residues, the absence of tryptophan and cysteine, and the presence of 18 half-cystine residues. The enzyme is produced by the seminal vesicles, and occurs in seminal plasma as a free, soluble component. For some years we have been studying the ribonuclease activity which we have found to occur in high concentration in the seminal plasma of the bull, with general properties similar to those of bovine pancreatic ribonuclease. We have seen that such activity is the result of a t least two enzymes and our research has been focused on the major component. We wish to give here a full report of the purification procedure of this enzyme (RNAase BS-I), with its amino acid composition and the relevant physicochemical properties, while the following paper [l] will deal with the mechanism of action. Partial results of our research have already been published [2-61. MATERIALS AND METHODS Bull-Seminal Plasma Bull semen, obtained from the insemination tenters in the frozen state, was centrifuged to remove the spermatozoa. The seminal plasma was either immediately processed or stored frozen a t-20 "C with no loss of ribonuclease activity for periods of storage up to six months. Unless otherwise stated, all purification steps were effected a t 4 "C. Enzymes. Adenosine deaminase (EC 3.5.4.4) ; dinucleotide nucleotidohydrolase (EC 3.6.1.9) ; XADase or NAD nucleosidase (EC 3.2.2.5); 5'-nucleotidase (EC 3.1.3.5); phosphodiesterase (EC 3.1.4.1) ; purine-nucleoside phosphorylase (EC 2.4.2.1); RNAase A or bovine pancreatic ribonuclease or polyribonucleotide 2-oligonucleotidotransferase (cyclizing) (EC 2.7.7.16); xanthine oxidase (EC 1.2.3.2).
Characterization of the ribonuclease activity on the skin surface
Genetic vaccines and therapy, 2006
The rapid degradation of ribonucleic acids (RNA) by ubiquitous ribonucleases limits the efficacy of new therapies based on RNA molecules. Therefore, our aim was to characterize the natural ribonuclease activities on the skin and in blood plasma i.e. at sites where many drugs in development are applied. On the skin surfaces of Homo sapiens and Mus musculus we observed dominant pyrimidine-specific ribonuclease activity. This activity is not prevented by a cap structure at the 5'-end of messenger RNA (mRNA) and is not primarily of a 5'- or 3'-exonuclease type. Moreover, the ribonuclease activity on the skin or in blood plasma is not inhibited by chemical modifications introduced at the 2'OH group of cytidine or uridine residues. It is, however, inhibited by the ribonuclease inhibitor RNasin although not by the ribonuclease inhibitor SUPERase* In. The application of our findings in the field of medical science may result in an improved efficiency of RNA-based therapies t...
Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 1989
The distribution of secretory-type ribonuclease in human serum, urine and seminal plasma has been studied by immunological measurements. Inhibition of enzyme activity by antibodies against pure human seminal RNAase shows that a cross-reactive enzyme is predominant (90%) in seminal plasma and is a significant component (70-80%) in urine and serum. A competitive binding radioimmunoassay has been developed by using specific antibodies and 125I-labelled RNAase as radioligand. The procedure, very sensitive, reproducible and specific, has been used to determine seminal RNAase levels in seminal plasma samples from 48 healthy individuals (age range, 20-58 years). The mean concentration of the enzyme was found to be 6.6 micrograms/ml (S.D. +/- 1.9).
DNA damage by ribose: inhibition at high ribose concentrations
Indian journal of biochemistry & biophysics, 2010
Nucleobases and DNA react non-enzymatically with sugars, and generate DNA-advanced glycation end products (DNA-AGEs). Incubation of plasmid pBr322 with ribose for 3-7 days caused the transformation of the supercoiled plasmid to the open circular and linear forms. Removal of sugar after an initial 24 h incubation resulted in marked enhancement in the transformation rate. Enhancement in transformation was also observed when bovine serum albumin (BSA) exposed to ribose for short durations was incubated with plasmid in absence of the sugar. The effect on DNA was attenuated when excess ribose remained in the incubation mixture of ribose and protein. The data suggested that an increase in ribose concentration in the vicinity of DNA could be damaging and the damage exacerbated, if sugar levels fell subsequently. Incubation of herring sperm DNA with ribose resulted in a concentration and time-dependent increase of N2-carboxyethyl-2'-deoxyguanosine (CEdGA,B). The concentration of CEdGA,B...
DNA and RNA enzymes with peroxidase activity An investigation into the mechanism of action
Canadian Journal of Chemistry, 2006
A DNAhemin complex (PS2.Mhemin), and its RNA counterpart (rPS2.Mhemin), have previously been reported, in the presence of nitrogenous buffers such as HEPES, to show enhanced peroxidative activity relative to both uncomplexed hemin and a control DNAhemin complex (Chem. Biol. 5, 505, 1998). A kinetic analysis of these two hemin-utilizing nucleic acid enzymes provides key insights into the mechanisms for their catalyzed peroxidation reactions. First, control experiments indicate that charge on the added detergent, required for solubility reasons, has little effect on the efficiency of the nucleic-acid-catalyzed reactions. Second, the key functional impact of the two nucleic acid frameworks, either DNA or RNA, appears to be a reduction in the acidity of a water molecule coordinated to the iron atom of the hemin that is bound to the ribozyme and DNAzyme scaffolds. This effect could result from a polar environment and possibly hydrogen bond(s) at the axial position of the hemin, along...
Ribonuclease-RNAase inhibitor complex from rat testis
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1988
The RNAase inhibitor from rat testis has been purified to homogeneity. The purified protein appeared as a single spot after two-dimensional electrophoresis. The calculated M r value is 48 000 which coincides with that obtained for the native protein on gel filtration chromatography, thus indicating a single polypeptide chain. The amino acid composition and the characteristics of the inhibitor activity are reported and compared to those of other RNAase inhibitors from mammalian tissues. The naturally occurring ribonuclease-RNAase inhibitor complex from rat testis has also been studied and compared with the rat testis inhibitor-RNAase A as model complex. The ribonuclease released from the natural rat testis complex showed heterogeneity of size. The significance of the rat testis ribonuclease/RNAase inhibitor system is discussed in terms of the important functionality of this organ.
Theriogenology, 2005
Sperm cell membranes are susceptible to peroxidative damage by an excess of reactive oxygen species (ROS). Antioxidative defence systems consisting of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) physiologically control the balance between ROS production and neutralization. In the present study the hypothesis was tested that lipid peroxidation occurs during storage of semen at 5 8C and that semen extender has positive effects on the antioxidative potential of equine semen. The aim of the study was to determine the activity of GSH-Px, SOD and CAT and the concentration of thiobarbituric acid reactive substances (TBARS) as an indicator of lipid peroxidation in native semen and after addition of extender, cooling and storage. Semen was collected from fertile Shetland stallions. In experiment 1, activity of antioxidative enzymes was determined immediately after semen collection and after 24 h storage at 5 8C. Enzyme activities were measured in native semen, semen diluted with semen extender, spermatozoa resuspended after centrifugation in extender and 0.9% NaCl as well as in undiluted and extender-diluted seminal plasma. In experiment 2, TBARS concentrations were analysed during storage of semen at 5 8C for 24 h. Semen storage for 24 h at 5 8C did not change activity of the examined enzymes. Antioxidative activity was significantly higher in extended than in native semen as well as www.journals.elsevierhealth.com/periodicals/the