A histochemical and immunohistochemical investigation of guanase and nedasin in rat and human tissues.

Human guanase is known as a specific enzyme in the liver, kidney, and brain. However, its functional significance remains poorly understood. In addition, interestingly, a different organ distribution between humans and rats was suggested. Here, we performed immunohistochemical staining with anti-human nedasin (neuronal and endocrine discs large/SAP102 associated protein), whose sequence was identical to that of guanase, antibody and histochemical staining for guanase in normal tissues of rat and human liver, kidney, and small intestine, and compared the results. Guanase activity was observed uniformly in the rat hepatocytes, biliary epithelium and vascular endothelium cells, while it was localized to the hepatocytes and biliary epithelium in the human liver. When the histochemical staining for guanase and the immunohistochemical staining for nedasin were compared, the stained regions were different in the rat liver but were almost consistent in all human tissues. Totally consistent staining results were also obtained between rats and humans in the other organization except the liver. Based upon the research reports to date, the experiments on guanase and nedasin in rat organs performed in this study are considered to have important implications in the investigation of their physiological significance.

Histochemical and immunohistochemical investigation of guanase and nedasin in human tissues.

Guanase is known as an enzyme released from the liver. Recently, cloning and sequencing of the guanase gene were reported. In addition, almost simultaneously, it was reported that an unknown protein that binds to neuronal and endocrine lethal(1)-discs large (NE-dlg), one of the membrane-associated guanylate kinase homologues (MAGUK) family proteins involved in synaptic connection between neurons, was cloned and named nedasin (NE-dlg associated protein), whose sequence was almost identical to that of guanase. We immunostained fresh frozen sections of surgically removed human liver, kidney, and small intestine with anti-nedasin antibody, and simultaneously performed histochemical staining for guanase for comparison. Histochemically, guanase activity was observed in the cytoplasm of hepatocytes and biliary epithelium on the liver, in the mucosal epithelium on the small intestine, and in the proximal tubule on the kidney. Immunohistochemically, a brown discoloration due to DAB oxidation was seen in the cytoplasm of hepatocytes and biliary epithelium on the liver, in the proximal tubule but in the distal tubule a little on the kidney, in the mucosal epithelium on the small intestine. The stained region of the liver and the small intestine were different from that of the kidney. The different staining properties dependent on the organs were considered to be due to different isozymes. The physiological significance of guanase may vary with the isozymes, further studies are considered necessary.

Purification of human liver guanase and characterization of antibody against it by immunoblotting.

Guanase was purified from human liver and its specific antibody was raised in rabbits. The enzyme was purified 1200-fold from the crude liver extract; the specific activity of the purified enzyme was 91.50 units/mg. The purified enzyme gave two distinct peaks on high pressure liquid ion exchange chromatography. The materials in both peaks had a molecular weight of 100,000, and were concluded to be isozymes with different pH optima for guanine. The antiserum completely inhibited the activity of the liver enzyme. It formed a single precipitin line with the human liver extract. On immunoblotting, it bound specifically to the one band with guanase activity, but to no other bands of protein. Thus, this antiserum for human liver guanase should be suitable for use in immunohistochemical demonstration of guanase and determination of this enzyme by radioimmunoassay.

Electron microscopic effects of thallium poisoning on the rat hypothalamus and hippocampus: biochemica changes in the cerebrum.

Rats were given thallous acetate (5 mg elemental thallium/kg body weight) IP daily for 7 days and prepared for electron microscopy (perfusion-fixation) and for biochemical study. Increased incidence of Golgi zones and electron-dense bodies were observed in the anterior hypothalamus and hippocampus. Sequestrated axons were seen in the hypothalamus. Depletion of succinic dehydrogenase and guanine deaminase occurred in the cerebrum.

Cytochemical demonstration of guanase in human liver using yellow tetrazolium.

Histochemical studies of human guanase (guanine deaminase) have seldom been undertaken, in part because of technical difficulties which result in heavy background staining. We reported a modified procedure for histochemical demonstration of guanase in human tissues involving hydrolytic deamination of the substrate guanine to xanthine via guanase, and then oxidation of xanthine to uric acid, with concomitant reduction of nitrotetrazolium blue (NBT). In this report, we describe a modification of this method for cytochemical demonstration of guanase at the fine structural level using yellow tetrazolium in place of NBT for determination of the intracellular distribution of guanase in human hepatocytes. In the hepatocytes, the reaction products were seen to be concentrated in the nucleus, in mitochondria, cisternae of the smooth and/or rough-surfaced endoplasmic reticulum, and lysosomes. The precise locations of the reaction product in the cisternae of the nuclear envelope, chromosomes, and nucleus could not be determined. However, the reaction products in the mitochondria were clearly seen to be located in the spaces of cristae. This information of the intracellular distribution of guanase in normal hepatocytes will be useful in determining the physiological role of this enzyme and in further studies on diseased hepatocytes including those in non-A non-B hepatitis.

The histochemical demonstration of guanase: observations in the human central nervous system.

1. A method is described for the histochemical demonstration of the purine catabolizing enzyme guanase, employing glutaraldehyde fixation and Nitro blue tetrazolium (NBT). Parallel biochemical studies confirm that enzyme activity is not significantly inhibited by exposure to glutaraldehyde. 2. By this procedure guanase activity has been visualized in neurons and glial elements of the human central nervous system (CNS). 3. Controls consisted of direct incubation of cryostat sections with a specific inhibitor of guanase (5-amino-4-imidazole carboxamide) and omission successively of the substrate guanine, of xanthine oxidase and of NBT. Enzyme activity was completely inhibited by the above procedures, and by boiling of tissues for 10 min prior to fixation. 4. Levels of enzyme activity in spinal cord and brain were assessed by a subjective scoring method, and showed close comparability with biochemical assay data in brainstem and cerebral hemispheres; whereas a low correlation for enzyme activity was observed in spinal cord and cerebellum. Differences between biochemical and histochemical assessments of CNS guanase activity are discussed.

A new spectrophotometric assay for enzymes of purine metabolism. II. Determination of guanase activity.

A new method for the determination of guanase is described. Xanthine, the product of the guanase reaction, is oxidized by xanthine oxidase, forming uric acid and hydrogen peroxide. Hydrogen peroxide is further reduced to water by catalase in the presence of ethanol. The acetaldehyde formed in this reaction step is dehydrogenated NAD or NADP dependent by aldehyde dehydrogenase. The NADH or NADPH production is measured and utilized for the calculation of the guanase activity. The sensitivity of the method can be doubled by the addition of uricase, which oxidizes uric acid to permit the formation of another mole of hydrogen peroxide.

Distribution of guanine deaminase in mouse brain.

Guanine deaminase was measured in nearly 100 different areas of mouse brain. The levels are relatively high in all parts of the telencephalon, both gray and white. It is especially active in parts of the olfactory tubercle and amygdala. Levels in the diencephalon range from low to as high as in the telencephalon. Brain areas caudal to the diencephalon, including all parts of the cerebellum, are almost uniformly below the level of detection. The enzyme is also virtually absent from the retina. The extreme range of concentration suggests that guanine deaminase might play a role in the metabolism of a neuroeffector.

New method for guanase activity measurement by high-performance liquid chromatography.

A rapid isocratic high-performance liquid chromatographic method for the determination of guanase (EC 3.5.4.3) activity is proposed. The method is highly reproducible, with a coefficient of variation of less than 1%, and requires only ca. 10 min for a complete chromatographic separation of the enzyme reaction mixture. The method allows the detection of nanomolar changes in the concentrations of both the substrate and the product, and does not require additional reactions or sample pretreatment. Kinetic studies with the proposed method showed the guanase activity to have an apparent Michaelis constant of 13.3 and 8.5 microM, and a maximum rate of 1.95 and 3.84 pmol/min per mg protein at 37 degrees C, in Tris-HCl and phosphate buffer, respectively.

Evaluation of the spectrophotometric assay of guanase with 2,2'-azino-di(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as chromogen.

A simple spectrophotometric assay for serum guanase based on the oxidation of 2,2'-azino-di(3-ethylbenzthiazoline-6-sulphate) (ABTS) using xanthine oxidase, uricase and peroxidase is described and statistically examined through its application to normal and pathological sera. The method is very sensitive, precise (CV below 8.13%) and linear up to 152.5 U/l. Comparison with the methods of Hue & Free ((1965) Clin. Chem. 11, 708-715), and Giusti ((1974) In: Methods of Enzymatic Analysis, Bergmeyer, H. U., ed., p. 1086), and Ito et al. ((1981) Clin. Chim. Acta 115, 135-144) gave a good correlation (r greater than or equal to 0.969). The reference values for the ABTS-method are 2.93 to 23.92 U/l (mean = 13.57 U/l, CV = 22.43%). The mean values of guanase activities determined in sera of patients with different liver diseases (mean = 30.29 U/l), or chronic alcoholics (mean = 35.41 U/l) were significantly higher than normal. The patients with chronic diseases had significantly lower activity (mean = 7.22 U/l, t = 9.25, p less than 0.001).

[Liver and brain guinea-pig guanine aminohydrolase (author's transl)]. / Guanina aminohidrolasa de hígado y de cerebro de cobaya.

Guinea-pig brain guanine aminohydrolase (E.C. 3.5.4.3) (M = 120,000 +/- 5,000 daltons) only exhibited one active electrophoretic band; its mobility is similar with that observed for the guinea-pig liver molecular form III. Both forms appear to have coincident activity with pH, as well as an analogous thermal stability and Km values with different substrates showing a different behaviour with the molecular form I of guinea-pig liver enzyme. Brain guanine aminohydrolase and liver molecular form III have similar pK'a values suggesting the participation of histidine and cysteine (-SH) in the enzymatic catalysis. The competitive inhibition by p-chloromercuribenzoate may corroborate the intervention of the last-one.

A sensitive spectrophotometric assay for guanase activity.

A highly sensitive and accurate spectrophotometric method was developed for determination of guanase activity with guanine as substrate. The assay is based on the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone and N,N-diethylaniline. Xanthine formed from guanine by guanase is oxidized to uric acid and hydrogen peroxide by xanthine oxidase, and the hydrogen peroxide produced is determined by an oxidative-coupling reaction with 3-methyl-2-benzothiazolinone hydrazone and N,N-diethylaniline mediated by peroxidase. Formation of the indamine dye is greatly affected by the superoxide radical ion (O2-) and pH value. These problems can be overcome by separating the two reactions of hydrogen peroxide formation and color production and carrying out that color-producing reaction at pH 3.0. This method is very sensitive and accurate because the indamine dye has a very high molar extinction coefficient of 29,800. It can be used with various kinds of automatic analyzers such as a Hitachi, Olympus, or Technicon analyzer. Comparative studies showed that this method is more sensitive and reproducible than other methods. Furthermore, guanase activities determined by this method correlated well with those determined by the improved Ellis-Goldberg method. This method should be useful for measurement of guanase activity in banked blood for preventing transfusion hepatitis and could be valuable as a liver function test.

Enzymes of purine salvage and catabolism in the mouse preimplantation embryo measured by high performance liquid chromatography.

The activities of five enzymes involved in purine salvage and catabolism--hypoxanthine phosphoribosyl transferase (HPRT), adenine phosphoribosyl transferase (APRT), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and guanase--were measured in mouse embryo extracts, from the one-cell to the blastocyst stage. Xanthine oxidase activity was not detected. The analyses were performed using high performance liquid chromatography and the enzymes showed different patterns of activity during development. Activities of HPRT, APRT and PNP were low before morula formation, and then increased until the blastocyst stage. ADA and guanase showed high activities after fertilization; guanase activity decreased sharply after the two-cell stage and ADA activity decreased sharply after the morula stage. Blastocyst formation was accompanied by a further decline in activity of both enzymes. The methods used may be suitable for measuring these enzymes in single human embryos, or in biopsies derived from them.

Simple, rapid determination of serum guanase activity with the Hitachi 736 automated discrete analyzer.

In this new method for determining serum guanase activity by use of the Hitachi 736-40 automated analyzer, serum is incubated with a mixture of xanthine oxidase, superoxide dismutase, and catalase; a reagent containing KCN, guanine, nitrotetrazolium blue, and Triton X-100 is added; and the increase in absorbance at 570 and 660 nm is measured for 2.4 min. Only 20 microL of sample is required, and results are linearly related to the activity concentration of guanase up to 30 U/L. Within-run and day-to-day precision (CV) was respectively 2.6 to 4.2% and 3.5 to 5.5% over 0-30 U of guanase activity per liter. The normal reference interval, as calculated from data on 40 healthy persons, is 0.1 to 2.2 U/L. Results correlate well (r = 0.997) with those by a kinetic method (Clin Chem 27: 560, 1981). The guanase activity of 150 samples can be measured within 1 h by this method.

Histochemical demonstration of guanase in human liver with guanine in bicine buffer as substrate.

Histochemical studies of human guanase (guanine deaminase) have seldom been undertaken, in part because of technical difficulties which result in heavy background staining. In this report, we describe a modified procedure in which the methodological inadequacies have been overcome. The modified technique has been applied to determine the intracellular and lobular distribution of guanase in normal human liver and in cases of primary biliary cirrhosis and alcoholic cirrhosis. Guanase was present within the cytoplasm of hepatocytes throughout the entire lobule. Enzyme activity was stronger on the sinusoidal side of the hepatocytes and in the periportal area. The reaction was weaker in perivenular hepatocytes. Portal components (bile ducts and veins), fibrous tissue and inflammatory cells were non-reactive, and the enzyme was absent from hepatocyte nuclei and membranes. Sections of skeletal muscle contained no guanase. The specificity of the reaction was confirmed by control tests on liver tissue and by the use of a specific inhibitor of guanase. It is concluded that the modified procedure overcomes the disadvantages inherent in the original method for guanase demonstration, allows the examination of fine cellular detail and should become a valuable histochemical tool with which to study diseases of the liver.