Enzymes RNase

Cell lines and tissue samples yield far greater amounts of miRNA than plasma or urine, which contain high levels of endogenous ribonuclease (RNase) enzymes that degrade RNA molecules even in very small quantities... 

The two RNase enzymes that will be used in this experiment are RNase A and RNase Ti. RNase A is a heat-stable nuclease that will hydrolyze the phosphodiester bonds linking the pyrimidine bases (U or C) in the molecule. RNase A will cleave both the mRNA and the tRNA present in the reaction. 

Prepn. 3, 9 (1953). Bovine pancreatic RNase A is a single-chain peptide of 124 amino add residues. Primary structure C. H. W. Hits et al J. Biol. Chem. 235, 633 (I960). Amino acid sequence D. H. Spackman et al.. Ibid. 648. Pictorial representation of entire structure Stein, Moore, Sci. Am. 204, no. 2, 81-92 (Feb. 1961). Ribonuclease from plant leaves has slightly different characteristics Markham, Strominger, Biochem. J. 64, 46p (1956). Can be obtained as a by-product of microbial erythromydn production Japan, pat. 263>38( 63) (to Shionogi). Chemical synthesis of materials possessing partial RNase enzyme activity Gutte,... 

RNase Bovine ribonnclease A, an enzyme 124 amino acid residues. Note diat RNase lacks tryptophan. 

Fike most enzymes (see Chapter 14), nucleases exhibit selectivity or specificity for the nature of the substance on which they act. That is, some nucleases act only on DNA (DNases), while others are specific for RNA (the RNases). Still... 

Dicer represents the key enzyme in the RNAi pathway. Dicer is also known as Helicase with RNAse motif, heRNA, Helicase-moi, K12H4.8-like, or KIAA0928. Dicer produces cleaves long double-stranded RNA into small pieces of about 21-23 nucleotides. These so-called siRNA duplexes produced by the action of Dicer contain 5 -phosphates and free 3 -hydroxylgroups... 

RNAse is an enzyme that catalyses the breakdown of RNA molecules into their component nucleotides. RNAses are extremely common in the modern world, resulting in very short life spans for any RNA that is not in a protected environment. 

The association between RNase A and 3 -UMP or 3 -dUMP has been studied by P n.m.r. and kinetic methods, respectively. In both cases the participation of two dissociable groups at the active site of the enzyme was demonstrated, in agreement with n.m.r. and A -ray - studies on the binding of 3 -CMP to RNase. In the binding of Tj RNase to purine nucleotide monophosphates, the phosphate group appears to have an important effect while the ribose ring is relatively unimportant. ... 

Hydrolysis of RNA by alkali or pancreatic RNase leads initially to fragments which terminate in 2, 3 -cyclic phosphodiesters. Micrococcal nuclease, on the other hand, gives rise to fragments terminating in 3 -phos-phomonoester groups which facilitate their isolation, and this enzymic hydrolysis has been used to prepare 3 -ribodinucleotides. ... 

However, there are a number of other miscellaneous biological roles played by this complex. The [Co(NH3)6]3+ ion has been shown to inhibit the hammerhead ribozyme by displacing a Mn2+ ion from the active site.576 However, [Co(NH3)6]3+ does not inhibit ribonuclease H (RNase),577 topoisomerase I,578 or hairpin ribozyme,579 which require activation by Mg2+ ions. The conclusions from these studies were that an outer sphere complex formation between the enzyme and Mgaq2+ is occuring rather than specific coordination of the divalent ion to the protein. These results are in contrast to DNase I inhibition by the same hexaammine complex. Inhibition of glucose-induced insulin secretion from pancreatic cells by [Co(NH3)6]3+ has been found.580 Intracellular injection of [Co(NH3)6]3+ into a neurone has been found to cause characteristic changes to the structure of its mitochondria, and this offers a simple technique to label neuronal profiles for examination of their ultrastructures.581... 

This enzyme is associated with the virions of RNA tumor viruses such as the Rous sarcoma virus (RSV). The enzyme has remarkable enzymatic activity in that it can catalyze several seemingly diverse steps in the synthesis of double-stranded DNA from the single-stranded RNA viral genome. The enzyme uses a tRNA for tryp-tophan as a primer to make a copy of DNA that is complementary to the viral RNA. The resulting RNA-DNA hybrid is converted to a double-stranded DNA molecule by ribon-uclease (RNase)H and DNA-dependent DNA polymerase activities that are intrinsic to reverse transcriptase. 

Fig. 9.4 The enzyme RNase can be unfolded by reduction agents (urea and mercaptoethanol). Oxidative removal of the reduction agents causes the molecule to take up its three-dimensional structure again it regains almost its full enzymatic activity...

Heparin inhibits many enzymes, including all RNases, therefore, it is criticalfor this step. It also inhibits the labeling step therefore it needs to be removed later (by a LiCl precipitation). We did not find any comparable alternative (in efficiency and cost) to heparin. The composition and concentrations of other ingredients can be changed depending on the cell type or experiment. 

The successful of recovery of RNase A functional activity by a heat-induced AR method suggested the possibility of recovering RNase A immunoreactivity as well. The immunoreactivity of native RNase A and RNase A that was incubated at a concentration of 4 mg/mL in 10% neutral buffered formalin for 1 day and then freed of formaldehyde by dialysis against PBS was compared using capture enzyme-linked immunosorbent assay (ELISA). Selected fractions that... 

The structural changes that occurred upon incubation at 65°C resulted in the partial restoration of immunoreactivity of the samples as shown in Figure 15.11a for the 5% formalin oligomers and Figure 15.11b for 10% formalin oligomers. In both figures, curve 1 corresponds to unheated native enzyme, curve 2 corresponds to unheated formalin-treated RNase A, and curve 3 corresponds to samples heated at 65°C for 4h in TAE buffer (pH 4). The... 

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