Guanine deaminase in rat liver and mouse liver and brain

1. The guanine deaminase in rat liver supernatant preparations was resolved into two fractions, A and B, on DEAE-cellulose columns. The two differed in electrophoretic mobility and in various properties. The most noteworthy distinction between A and B components was that the enzyme A activity showed a sigmoid dependence on substrate concentration whereas the enzyme B showed classical Michaelis-Menten kinetics. The K(m) value of enzyme A for guanine was 5.3mum and that of enzyme B 20mum. 2. The entire guanine deaminase activity of mouse liver was contained in the 15000g supernatant of iso-osmotic homogenates. 3. A reinvestigation of the behaviour of rat brain 15000g supernatant guanine deaminase isoenzymes revealed that one enzyme had sigmoidal kinetics and the other enzyme showed a hyperbolic response. 4. Of the guanine deaminase in mouse brain iso-osmotic sucrose homogenate 80% was recovered in the 15000g supernatant and the rest from the particles. The supernatant guanine deaminase was resolvable into two fractions on DEAE-cellulose columns. One enzyme showed sigmoidal kinetics whereas the other showed a hyperbolic response to increasing substrate concentration; the K(m) values for the reaction with guanine were respectively 5 and 66mum. 5. The particulate fractions of mouse liver and brain were devoid of any overt inhibitory activity.     

Automated enzymatic measurement of adenosine deaminase isoenzyme activities in serum

We developed a simple, rapid, and automated method for simultaneous measurement of adenosine deaminase (ADA, EC 3.5.4.4) isoenzymes in human serum, based on their apparent difference in Ki values for erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) as inhibitor. Serum ADA was partially purified by CM-Sephadex, gel-filtration, and affinity chromatography into two types of isoenzymes, designated ADA1 (300 kDa) and ADA2 (120 kDa). Because ADA2 has a higher Km for adenosine and higher Ki values for EHNA than does ADA1, the activity of ADA1 is almost completely inhibited by EHNA at 0.1 mM (analytical recovery 4.1%), whereas ADA2 is practically unaffected (analytical recovery 94.8%) by that concentration of EHNA. We measured the activities of ADA2 and total ADA in the presence and absence of 0.1 mM EHNA. ADA1 activities were calculated by subtracting the activity of ADA2 from that of total ADA. The mean within-assay CV was 5.7% for ADA1 and 2.7% for ADA2. The interassay CV was 2.8% for ADA1 and 3.1% for ADA2. Results of the present method correlated well (r = 0.9026 for ADA1, 0.9438 for ADA2) with those of the ion-exchange chromatography method. The upper limits of the reference intervals, as calculated from data for 320 healthy donors, are 7.2 U/liter for ADA1, and 14.6 U/liter for ADA2. This method is suitable for analysis of large numbers of samples in clinical laboratories for routine monitoring of the activities of ADA isoenzymes in serum.     

Nuclear and cytoplasmic RNase-activity in regenerating mouse liver

Nuclear and cytoplasmic RNase activities at pH 5.0 and 7.6 were analyzed in regenerating mouse liver at 6, 12, 24, 48, and 72 h after partial hepatectomy. Two different nucleus-isolation methods were used, one in a EDTA-spermidine medium free from divalent cations, and one in a sucrose medium containing these ions. During regeneration, the cytoplasmic alkaline RNase activity in the sucrose medium was unchanged, but in the spermidine medium showed an increase toward the end of the period. Also the cytoplasmic acid RNase activity was unchanged in sucrose medium, whereas in the spermidine it slightly increased during regeneration. The nuclear alkaline RNase activity showed a notable peak 6 h after the operation and later decreased. Also the nuclear acid RNase activity displayed a similar marked peak 6 h after operation, then decreased, but remained high throughout the period. The nuclear RNase activities were about 1% of the corresponding cytoplasmic RNase activities. The absolute activities varied greatly according to the nucleus-isolation methods. In the controls, the absolute activity of nuclear alkaline RNase was slightly above (1.2 times) that of the corresponding acid activity after the spermidine method. After the sucrose method the nuclear alkaline activity was 2.7 times that of the acid activity. The absoluted activity of cytoplasmic alkaline RNase was slightly above (1.2 times) the acid activity after the spermidine method but after the sucrose method it was only 0.25 times that of the acid activity. In sham-operated animals, cytoplasmic acid and alkaline RNase activities generally were fairly similar to the normal value, but corresponding nuclear activities showed marked variations indicating an influence by anesthesia.     

Disulfide reductase activity in mouse regenerating liver

Fourteen hours after partial hepatectomy there was a decrease in basal disulfide reductase and glutathione reductase activity in cytosole fraction of proliferating hepatocytes. In nuclear fraction, the activation effect of cAMP and cGMP on the disulfide recovery was replaced by inhibition. Meanwhile the activity of glutathione reductase noticeably increased. Forty-five hours after operation disulfide reductase activity of cytosole appreciably rose during maximal mitotic activity of the regenerating liver. The data obtained provide evidence in favor of the involvement of disulfide reductase enzymes into reparative regeneration of the liver.     

Lack of adenosine deaminase deficiency in the mutant mouse wasted

The possibility that the mutant mouse wasted (wst/wst) may serve as an animal model for studies of severe combined immunodeficiency disease (SCID) and the role of adenosine deaminase (ADA, EC 3.5.4.4) in adenosine metabolism were investigated. The specific activity of ADA in wst/wst compared with control mice was significantly lower by 26% in thymus, but significantly higher by 18% in spleen and 32% in cerebellum. Vmax values of ADA in spleens were 43% higher in wst/wst mice and no changes were observed in Km values. In contrast, the Vmax of ADA was unchanged in erythrocytes from wst/wst mice, but the Km for adenosine was significantly elevated. Thus, based on ADA measurements alone, it may be premature to consider wst/wst mice as a model for ADA deficiency and SCID in humans.     

The effect of experimental diabetes on the circadian pattern of adenosine deaminase and myeloperoxidase activities in rat liver

This study investigated time-dependent variations in the activities of adenosine deaminase (ADA), an adenosine-metabolizing enzyme, and myeloperoxidase (MPO), an oxidation reaction-catalyzing enzyme, in control and streptozotocin (STZ)-induced diabetic rat liver. The animals were sacrificed at six different times of day (1, 5, 9, 13, 17 and 21 hours after lights on - HALO). The hepatic activity of ADA did not change depending on the STZ treatment whereas MPO activity was significantly higher in the diabetics than in the controls. Hepatic ADA activity was dependent on the time of sacrifice with the lowest activity at 21 HALO and the highest activity at 5 HALO. Both enzyme activities failed to show any significant interaction between STZ treatment and time of sacrifice, which means that diabetes does not influence the 24 h pattern of these activities. Since MPO, a heme protein localized in the leukocytes, is involved in the killing of microorganisms, increased MPO activity in diabetic rat liver may reflect leukocyte infiltration secondary to diabetes. A reduction in ADA activity during the dark (activity/feeding) period will presumably lead to high concentrations of adenosine in the liver, possibly contributing to changes in some metabolic processes, such as glycogen turnover and oxygen supply.     

Localization of adenosine deaminase and adenosine deaminase complexing protein in rabbit brain

Adenosine deaminase and adenosine deaminase complexing protein have been localized in rabbit brain. Brains fixed in paraformaldehyde or in Clarke's solution were blocked coronally. Blocks from brains fixed in paraformaldehyde were either frozen in liquid nitrogen or embedded in paraffin. Tissue fixed in Clarke's solution was embedded in paraffin. Sections from each block were stained by the peroxidase-antiperoxidase method for adenosine deaminase or complexing protein using affinity-purified goat antibodies. Adenosine deaminase and complexing protein did not co-localize. Adenosine deaminase was detected in oligodendroglia and in endothelial cells lining blood vessels, whereas complexing protein was concentrated in neurons. The subcellular location and appearance of the peroxidase reaction product associated with individual cells was also quite distinctive. The cell bodies of adenosine deaminase-positive oligodendroglia were filled with intense deposits of peroxidase reaction product. In contrast to oligodendroglia, the reaction product associated with most neurons stained for complexing protein was concentrated in granular-appearing cytoplasmic deposits. In some instances, these deposits were clustered about the nuclear membrane. Staining of neurons in the granular layer of cerebellum was an exception. Granule cells were lightly outlined by peroxidase reaction product. Cerebellar islands, also referred to as glomeruli, were stained an intense uniform brown. These results raise the possibility that oligodendroglia and blood vessel endothelia, through the action of adenosine deaminase, might play a role in controlling the concentration of extracellular adenosine in brain. They do not, however, support the suggestion that complexing protein aids in adenosine metabolism by positioning adenosine deaminase on the plasma membrane.     

Cytoskeletal reorganization in hepatocytes of the regenerating mouse liver

The intracellular pattern of prekeratin and actin filaments has been studied on sections of mouse livers regenerating after CCl4 injury. Monoclonal antibodies against one of liver prekeratins and monospecific polyclonal actin antibodies were used in the indirect immunofluorescent test. The presence of alpha-fetoprotein and bile canaliculi antigen was also monitored during regeneration. In control livers, prekeratin and actin filaments formed thick bundles adjacent to plasma membranes. The cytoplasmic prekeratin network was unmarked. In contrast to the latter, the bright well developed intracytoplasmic prekeratin network and intracytoplasmic actin fibers were identified in the perinecrotic hepatocytes by the 3d-4th day of regeneration. This rearrangement of the cytoskeleton coincided in time with the appearance of alpha-fetoprotein and the loss of the bile canaliculi antigen in the perinecrotic hepatocytes.     

Glyoxalase I in regenerating mouse liver exposed to carcinogens

Glyoxalase I is the first component of glyoxalase system which is involved in detoxification of alpha-ketoaldehydes and converts them to nontoxic substances. This study reports changes in Glyoxalase I activity in relation to DNA synthesis in regenerating liver treated with two polycyclic aromatic hydrocarbons - 7,12-dimethylbenz (a) anthracene and benzo(a)pyrene. Livers after partial hepatectomy show consistent increase in Gly. I which reaches to its peak at 24 hr after surgery. [3H]Thymidine incorporation into DNA also follows the same trend as does Gly. I in regenerating liver. Both the carcinogens have significantly reduced the activity of Gly. I and DNA biosynthesis when compared with untreated partially hepatectomized control livers. The study reveals that though regenerating liver has been considered as an experimental model for neoplasia, unlike tumors (where Gly. I is either absent or in undetectable quantities) it possesses more Gly. I than in normal liver. On the other hand, preneoplastic liver during initiation (in regenerating liver treated with carcinogens, initiation is expected to occur) has very low activity. This suggests that Gly. I is not only involved in controlling growth but possibly is involved in some other phenomenon which is somehow depressed in preneoplastic and cancerous tissues.