Specific Ribonuclease Activity

Drought also has a profound effect on protein synthesis. In many plant tissues, a reduced water potential causes a reduction of total protein synthesis and a rapid dissociation of polyribosomes. The latter has been shown not to be the consequence of increase in ribonuclease activity (Hsiao, 1973 Dhindsa Bewley, 1976). For a specific protein, Jacobsen, Hanson Chandler (1986) have shown in barley leaves that water stress enhances the synthesis of one of the a-amylase isozymes. Using a cDNA probe they found that water-stressed leaves contained much more a-amylase mRNA than unstressed plants. 

In recent years, several papers appeared in the literature concerning NMR and X-ray studies of thermostable ribonucleases. Among archaebacteria, several histone-like proteins from Sulfolobus strains have been identified and grouped into molecular mass classes. From the 7 kDa class, Sac7e from S. acidocaldarius and Sso7d from S. solfataricus, possessing DNA binding activity in combination with non-specific RNase activity, were identified. ... 

A series of carboxyl derivatized polyglucoses were studied as inhibitors of ribonuclease activity, in an attempt to relate charge density to inhibitory activity.202 In comparison with other factors, it was concluded that coulombic forces probably play a major role in complex-formation between enzyme and substrate, and between enzyme and inhibitor. However, other specific, nonelectrostatic forces were shown to participate in the binding of bovine pancreatic ribonuclease to ribonucleic acid.204... 

The addition of ferrie heme chloride at 5 /ig/ml of medium did not alfect general synthesis of protein, RNA, or DNA as determined with tracers [128]. Hemin did not directly affect the activity or lifetime of ALA-synthetase either in isolated mitochondria or in cell culture. When cells were cultured with AIA for 12 hours to increase the level of ALA-synthetase, the medium was changed, and both actinomycin D and hemin were added, the decay rate for ALA-synthetase was increased from i/2=5.2 hours (for actinomycin D alone) to t,/2=3.6 hours (Table III). These results suggest that hemin either shortened the lifetime of mRNA for ALA-synthetase or interfered specifically with the synthesis of ALA-synthetase. Previous studies had shown that heme did not stimulate ribonuclease activity. The inhibitory effect of hemin on the transport of soluble ALA-synthetase into the mitochondria has been discussed in Section V. 

The regulation of mRNA translation occurs only during the inducible phases and is due to the labile posttranscriptional repressor, R. The mechanism of action of the repressor R is unknown, and, as in the lac (for example, references [63,64]) system, discovery of its mechanism must await the development of a cell-free system in which it functions. It is easy to speculate on a mechanism by which the attachment of repressor to the initiation region of the messenger RNA would prevent ribosome attachment and permit a nuclease to begin a hydrolysis of the inactive template [65]. Possibly related are the reports of decreased liver ribonuclease activities in the presence of different inducers of specific protein synthesis [66,67]. 

Many difficulties have to be faced during the purification of tRNA methyltransferases. The major ones are ii) enzyme multiplicity, which makes it cumbersome to isolate each specific reaction product, and then difficult to evaluate specific activity and extent of purification (ii) enzyme instability (Hi) frequent contamination by ribonuclease activity (iv) possible presence of complexes with endogenous tRNA (v) unavailability of a proper tRNA substrate, as we have already discussed. To point out the problem of... 

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... 

Among the specific enzymes whose activity has been reported to be decreased after in vitro ozone exposure are papain, glyceraldehyde-3-phosphate dehydrogenase, lysozyme, ribonuclease, and acetylcholinesterase. The latter enzyme appears to be particulady susceptible to free-radical and oxidative states. A loss in acetylcholinesterase activity has been reported in the red cells of humans and mice that inhaled ozone. However, there are only minimal amounts of this enzyme in lupg tissue, and, although it has been suggested that acetylcholinesterase is important in bronchial tract ciliary activity, there is no direct evidence to support this conjecture. 

In addition to the enzymes that catalyse the formation of nucleotides and polynucleotides, a large number of catabolic systems exist which operate at all levels of the internucleotide pathways. The ribonucleases and deoxyribonucleases that degrade polynucleotides are probably not significantly involved in purine analogue metabolism, but the enzymes which dephosphorylate nucleoside 5 -monophosphates are known to attack analogue nucleotides and may be of some importance to their in vivo activity. Phosphatases of low specificity are abundant in many tissues [38], particularly the intestine [29]. Purified mammalian 5-nucleotidases hydrolyse only the nucleoside 5 monophosphates [28] and... 

Ribonuclease A was the first enzyme to be synthesized in the laboratory. Fully active ribonuclease has been synthesized,752 as have new modified enzymes. For example a 63-residue peptide made up of five segments of the native RNase sequence retained measurable catalytic activity.753 Using total synthesis, unnatural amino acids, such as 4-fluorohistidine, have been incorporated at specific positions in RNAse.752... 

A very different ribonuclease participates in the biosynthesis of all of the transfer RNAs of E. coli. Ribonuclease P cuts a 5 leader sequence from precursor RNAs to form the final 5 termini of the tRNAs. Sidney Altman and coworkers in 1980 showed that the enzyme consists of a 13.7-kDa protein together with a specific 377-nucleotide RNA component (designated Ml RNA) that is about five times more massive than the protein.779 Amazingly, the Ml RNA alone is able to catalyze the ribonuclease reaction with the proper substrate specificity.780 7823 The protein apparently accelerates the reaction only about twofold for some substrates but much more for certain natural substrates. The catalytic center is in the RNA, which functions well only in a high salt concentration. A major role of the small protein subunit may be to provide counterions to screen the negative charges on the RNA and permit rapid binding of substrate and release of products.783 Eukaryotes, as well as other prokaryotes, have enzymes similar to the E. coli RNase R However, the eukaryotic enzymes require the protein part as well as the RNA for activity.784... 

Bovine pancreatic RNase A is a member of a homologous superfamily. In addition, there is a separate family of guanine-specific microbial RNases that have evolved to have a similar active site.192,193 Ribonuclease T1 from Aspergillus oryzae and the 110-residue bamase from Bacillus amyloliquefaciens of Mr 12 392 (see Chapter 19) are the best known examples. One of the histidine residues is replaced by a glutamate in these enzymes. The microbial enzymes are much more amenable to study by protein engineering. 

Ribonuclease N, has properties similar to those of RNase Ti in regard to base specificity and molecular size (6). However, it has a higher phosphotransferase activity and far less hydrolase activity than RNase Tl As suggested from this finding, RNase N, is more advantageous than RNase T for the synthesis of oligonucleotides with 5 -terminal... 

A new RNase activity, tentatively named RNase V, was found in cell-free extracts of E. coli. Ribonuclease V is an exoribonuclease attacking mRNA from 5 to 3 terminal producing 5 -mononucleotides. It is characterized by the requirements of ribosomes, G and T factors, tRNA, K+ or NH4+, Mg2+, GTP and a sulfhydryl compound by its specificity and by the fact that it degrades poly U, poly A, T4 phage mRNA or E. coli mRNA, but not ribosomal RNA (132). 

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