Human Deoxyribonucleases

Although mammalian deoxyribonucleases were discovered more than 60 years ago, interest in these enzymes is not weakening. During the last decade, intensive studies of human DNases culminated in discovery of several novel enzymes exhibiting DNase activity. These include an unusual DNase, lactoferrin. For some enzymes, their three-dimensional structure and molecular mechanisms underlying their functioning have been elucidated. In patients with some autoimmune and viral diseases, catalytic antibodies also contribute to alternative pathways of DNA hydrolysis. Some enzymes exhibiting DNase activity play an important role in pathogenesis of various diseases and also in programmed cell death (apoptosis). This review highlights recent achievement in human deoxyribonuclease research. It also considers mechanisms of DNA hydrolysis. The review also summarizes modern data on the biological role of these enzymes in functioning of the human organism, realization of its protective mechanisms, and possible applications of DNases in medicine.     

Deoxyribonucleases in phytohemagglutinin-stimulated lymphocytes

The in situ assay of deoxyribonucleases in DNA-containing polyacrylamide gels following their separation by microdisc electrophoresis was used to determine the deoxyribonuclease pattern of human lymphocytes during stimulation with phytohemagglutinin (PHA). Two additional neutral deoxyribonuclease activities are detectable in stimulated cells, one only active with denatured DNA, the other active with native and denatured DNA as substrate, showing a maximum activity after 36 h and increasing in waves respectively. A group of acid deoxyribonuclease activities also shows a maximum after 36 h of stimulation. A neutral deoxyribonuclease active only with native DNA is missing in stimulated lymphocytes. It is suggested that the acid deoxyribonuclease activities and the neutral deoxyribonuclease active only with denatured DNA are involved in DNA synthesis, whereas the involvement of the neutral deoxyribonuclease active with native and denatured DNA in processing of DNA excreted in stimulated lymphocytes is discussed.     

Deoxyribonucleases of non-pathogenic corynebacteria

Fourteen species of non-pathogenic corynebacteria have been examined for the presence of DNases. Seven of the species were found to be DNase positive when assayed in Toluidine Blue-DNA-containing agar whereas only one, Corynebacterium callunae, could be detectable as DNase positive when the test was performed in DNA-methyl green-containing agar. Electrophoretic patterns obtained from sodium dodecyl sulfate-polyacrylamide gels containing DNA showed the presence of one or two bands of nucleolytic activity in two species of Brevibacterium and of several bands in C. callunae. Degradation of DNases by cellular proteases seem to occur in this species.     

Dinucleosidetriphosphatase from rat brain

Rat brain P1,P3-bis(5'-adenosyl)-triphosphate adenylohydrolase (dinucleosidetriphosphatase, EC 3.6.1.29) was purified 1000-fold. The enzyme hydrolyzed diadenosine and diguanosine triphosphates (Km values 14 and 40 microM, and relative V 100 and 40, respectively) to the corresponding nucleoside di and monophosphates. Dixanthosine triphosphate was hydrolyzed at a residual rate. Diadenosine di and tetraphosphates, NAD+, and artificial phosphodiesterase substrates were not hydrolyzed. Bivalent cations [Mg(II), Mn(II) or Ca(II)] were required for activity, but Zn(II) was a competitive inhibitor (Ki = 5 microM). The optimum pH value was about 7.5. A molecular mass of 34 kdalton (gel filtration) and an isoelectric point of 5.5 (chromatofocusing) were found.     

Serological Investigations of Leptospiral Deoxyribonucleases

Enzymoserological comparison of a selection of leptospira strains tested with sera from rabbits immunized with unpurified DNase of Leptospira interrogans, serotype canicola, indicates the production of DNase of serologically very similar properties by the serotypes canicola, autumnalis, icterohemorrhagiae and pomona. The DNase produced by serotype hyos was serologically different from the others, while the serotypes grippotyphosa and bataviae did not produce DNases at all. The method used made it possible to differentiate between leptospiral DNase and normally occurring DNases in the serum samples. Neither leptospira DNase nor specific leptospira-DNase-antibodies could be detected in dog sera with high agglutinationlysis titres after natural infection.     

Nuclear DNAse from rat brain

Nuclear DNase specific for single-stranded DNA was obtained from the rat brain. The enzyme was isolated from a soluble protein fraction of total cell nuclei using gel filtration and ion-exchange chromatography. Nuclear DNase is Mg2+-dependent exodeoxyribonuclease and hydrolyzes homological, heterological and synthetic substrate at the same rate. It is established that nucleoside-5'-monophosphates are the major product of native and synthetic polydeoxyribonucleotides hydrolysis. It is supposed that the enzyme participates in reparation of neuron DNA.     

Glycopeptides from rat brain glycoproteins

The lipid-free protein residue of rat brain tissue was treated with papain to solubilize the heteropolysaccharide chains of the tissue glycoproteins. The glycopeptides were separated into non-dialyzable and dialyzable glycopeptide preparations. Each preparation was then sorted out into groups of glycopeptides by means of electrophoresis and gel filtration. The quantitatively predominant glycopeptides were the alkali-stable glycopeptides (Group A) which accounted for 64% of the glycopeptide carbohydrate recovered from rat brain. Most of the group A glycopeptides appeared in the non-dialyzable preparation. The molecular weight of the glycopeptides of Group A ranged from approximately 5200–3700. The largest glycopeptide molecule in this mixture possessed the highest electrophoretic mobility and contained one fucose, four N-acetylneuraminic acid (NANA), six N-acetylglucosamine, four galactose, and three mannose residues per molecule. The spectrum of glycopeptides isolated in this group showed a progressive decrease in NANA rsidues, NANA and galactose residues, and NANA, galactose, and N-acetylglucosamine residues which could be correlated with a progressive decline in molecular weight and electrophoretic mobility. Some of the glycopeptides in each fraction recovered from this group of glycopeptides contained sulfate ester groups.A second group of glycopeptides (Group C glycopeptides) accounted for 25% of the total glycoprotein carbohydrate recovered from rat brain. These were recoverd from the dialyzable glycopeptide preparation, and resolved into three fractions by column electrophoresis. These glycopeptides do not contain sulfate, are composed predominately of mannose and N-acetylglucosamine, and possess a molecular weight of approximately 3000.Several minor groups of glycopeptides were detected. Alkali-labile glycopeptides (Group B) appeared in the non-dialyzable glycopeptide preparation. The dialyzable glycopeptide preparation contained glycopeptides (Group E) which contained N-acetylgalactosamine and glucose. These had a molecular weight of approximately 2000. Group D glycopeptides recovered from the dialyzable glycopeptide preparation contained variable amounts of NANA, mannose, galactose, N-acetylglucosamine, and sulfate. These possessed a molecular weight of approximately 2900.     

Activation of Deoxyribonucleases by Divalent Cations

The activation of DNase I by Mg, Mn, Co, Ni, Fe, Cd, Zn, Ba, Sr, Ca, and Cu ions has been studied by several methods, at different pH and salt concentrations. Mg, Mn, and Co are the best activators for initial stages of degradation. A synergistic effect is shown only by the pair Mg-Ca. Optimal pH of action is always situated at 6.5. DNase II is activated to about the same degree by alkaline earths and Mn ions. Cd and Cu are strong inhibitors. Optimal pH is always 4.6. By titration of liberated secondary phosphate groups, two stages in the hydrolysis of DNA by DNase I are evidenced: a rapid phase activated most by Mg and a slow phase activated by Ca. Some possible mechanisms of action of both enzymes are outlined and the general influence of metal ions is discussed.     

Partial Purification and Some Properties of Two Novel Deoxyribonucleases from Bacillus subtilis

Optically active guaiane dienes, 3 and 4, related to sclerosporene 2 were synthesized from (—)-carvone and (—)-α-santonin. The mass spectra of 3 and 4 were not identical with that of natural sclerosporene.     

Splicing-active nuclear extracts from rat brain

In the nervous system, alternative pre-mRNA splicing generates the diverse protein machineries needed for cell excitation and synaptic communication. Yet, many questions remain about how these mechanisms are regulated by RNA binding proteins in the environment of differentiated cells and tissues. Here, we describe the preparation and use of splicing active nuclear extracts derived from the cerebellum and cerebral cortex regions of rat brain as a resource for in vitro studies. These tissue-specific extracts promote the neuron-specific pathway of splicing, and display characteristic changes in hnRNP protein function and expression. These extracts can be used in combination with affinity selection and depletion/complementation assays to identify regulatory factors and to characterize their interactions and effects on spliceosome assembly. The ability to prepare extracts from brain regions at a range of postnatal ages provides opportunities to address related questions as a function of cell differentiation. These neuronal extracts may also be valuable for the development of in vitro assays to elucidate other neuron-specific RNA processing pathways, such as 3' end formation, RNA editing, or miRNA maturation.     

Acetylcholinesterase from Wistar rat brain.

Acetylcholinesterase (E.C.3.1.1.7) was partially purified from rat brains stored in toluene. Extraction was performed using buffers containing non-ionic tensoactive detergents. Some properties of the enzyme were affected by the use of different activity measurement methods, such as the short-time radiometric or the long-time colorimetric method. There were two zones of maximum activity in the range pH 7.5-8.0 and 8.0-8.6, respectively. There seems to be a histidine residue in the enzyme that participates in the catalytic process. Thermal denuration presented first order kinetics and different thermodynamic parameters were obtained on using different incubation periods. On using the short-time activity measurement method there was activation at high substrate concentration, but with the long time method there was a marked inhibition produced by excess of substrate. However, if the enzyme was extracted from fresh rat brain, toluene untreated, these differences dissapeared. Gel filtration and disc electrophoresis showed the presence of multiple and interconvertible forms of the enzyme.     

A mitochondrial phosphoenolpyruvate carboxykinase from rat brain

Phosphoenolpyruvate carboxykinase from the rat brain has been purified approximately 6000-fold. This purified enzyme was stable at −20 °C for several months.