Differential theophylline inhibition of alkaline phosphatase and 5'-nucleotidase of bovine milk fat globule membranes

1. The effects of theophylline (1,3-dimethylxanthine) on alkaline phosphatase and 5'-nucleotidase activities of bovine milk fat globule membranes (MFGM) were examined. 2. Theophylline inhibited MFGM alkaline phosphatase in a concentration-dependent manner with 50% inhibition produced by 99 +/- 28 microM theophylline. 3. The 5'-nucleotidase activity was resistant to theophylline inhibition with 50% inhibition produced by 33.9 +/- 3.1 mM theophylline. 4. Theophylline was an uncompetitive inhibitor of MFGM alkaline phosphatase with a Ki of 126 +/- 15 microM. 5. The extent of theophylline inhibition of alkaline phosphatase activity was independent of the substrate utilized in the assay. 6. The effect of theophylline on bovine MFGM alkaline phosphatase was similar to theophylline effects on other mammalian alkaline phosphatases of liver/bone isoenzyme origin.     

Nucleotide metabolizing ecto-enzymes in Walker 256 tumor cells: molecular identification, kinetic characterization and biochemical properties

In this study we describe the molecular identification, kinetic characterization and biochemical properties of an E-NTPDase and an 5'-nucleotidase in Walker 256 cells. For the ATP, ADP and AMP hydrolysis there were optimum pH in the range 6.5-8.0, and absolute requirement for divalent cations (Mg(2+)>Ca(2+)). A significant inhibition of ATP and ADP hydrolysis was observed in the presence of high concentrations of sodium azide and 0.5 mM of Gadolinium chloride. These activities were insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors. The K(m) values were 464.2+/-86.6 microM (mean+/-SEM, n=4), 137.0+/-31 microM (mean+/-SEM, n=5) and 44.8+/-10.2 microM (mean+/-SEM, n=4), and V(max) values were 655.0+/-94.6 (mean+/-SEM, n=4), 236.3+/-27.2 (mean+/-SEM, n=5) and 177.6+/-13.8 (mean+/-SEM, n=5) nmol of inorganic phosphate min(-1) mg of protein(-1) for ATP, ADP and AMP, respectively. Using RT-PCR analysis we identified the mRNA of two members of the ecto-nucleoside triphosphate diphosphohydrolase family (NTPDase 2 and 5) and a 5'-nucleotidase. The presence of NTPDases and 5'-nucleotidase enzymes in Walker 256 tumor cells may be important to regulate the ratio adenine nucleotides/adenine nucleoside extracellularly, therefore motivating tumor growth.     

Isoelectric-focusing behavior of acid hydrolases in rat kidney lysosomes. Effects of the pH gradient, autolysis and neuraminidase.

Isoelectric focusing was used to study the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase in lysosomes isolated from rat kidney. The isoelectric points of the main protein and hydrolase peaks were 1-1.5 units lower when electrofocusing was done in a pH 3-10 gradient than in a pH 10-3 gradient, apparently because the lysosomal constituents aggregated strongly at their isoelectric points and tended to settle somewhat in the gradient due to gravity. In the extended pH gradient the acidic form of each hydrolase occurred as asingle, relatively discrete peak. However, when pooled acidic fractions were refocused in a restricted pH gradient (pH 6-3 or 3-5) multiple acidic enzyme and protein components were resolved with isoelectric points between 2.7 and 5.1. When autolysis was minimized by extracting lysosomal fractions at alkaline pH (0.2% Triton X-100, 0.1%p-nitrophenyloxamic acid, 0.1 M glycine buffer, pH9) and including 0.1%p-NITROPHENYLOXAMIC ACID, AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND CATHEPSIN D, in the pH gradient, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in two forms, an acidic form with an isoelectric point of about 4.4, and a basic form with an isoelectric point close to 6.2, 6.7 and 8.0, respectively. Acid phosphatase occurred in three forms with isoelectric points of 4.1, 5.6 and 7.4. When some autolytic digestion was permitted by extracting lysosomal fractions in an acidic medium (0.2% Triton X-100, 0.1 M sodium acetate buffer, pH 5.2) AT 0-4DEGREES C and omitting p-nitrophenyloxamic acid from the gradient, the acidic form of beta-glucuronidase and the intermediate form of acid phosphatase were lost, the isoelectric points of the acidic forms of acid phosphatase, arylsulfatase and beta-N-acetylhexosaminidase were increased 0.6-1.2 units, and the isoelectric point of the basic forms of acid phosphatase, arylsulfatase and beta-glucuronidase was increased 0.5 unit. When lysosomal extracts were incubated with bacterial neuraminidase before electrofocusing, the acidic forms of acid phosphatase, arylsulfatase and beta-glucuronidase were largely lost, the isoelectric point of the acidic form of beta-N-acetylhexosaminidase was increased from 4.5 to 6.4, and the isoelectric points of the basic forms of all four hydrolases were increased 0.5-1.5 units. Autoincubation of lysosomal extracts in vitro at pH 5.2 PRODUCED SIMILAR, THOUGH LESS MARKED, effects. cont'd     

Alterations in hepatic 5'-nucleotidase in the tumor-bearing rat

The effect of the growth of the Walker 256 carcinoma on the level of 5'-nucleotidase and alkaline phosphatase in the whole liver and in an isolated hepatocyte membrane preparation of its host was investigated. Alkaline phosphatase activities of whole liver and plasma membrane were increased approximately 5-fold by tumor growth. A 50% decrease in whole liver 5'-nucleotidase activity was observed in tumor-bearing rats while the 5'-nucleotidase activity per milligram membrane protein was unaltered. Tumor growth would therefore appear to affect a pool of 5'-nucleotidase which is not associated with the plasma membrane.     

Effect of calcium concentration on survival, proliferation and activities of alkaline phosphatase, 5'-nucleotidase, gamma-glutamyltransferase and lactate dehydrogenase of adult rat hepatocytes cultured in serum-free medium.

Mature rat hepatocytes were cultured on collagen coated dishes in serum-free alpha-modified Eagle's minimum essential medium containing 0.1 microM insulin, 0.1 microM dexamethasone, 10 mM pyruvate and Ca2+ at concentrations of 0-2 mM. Survival of nondivided cells was best in medium containing 2 mM Ca2+. Proliferation during 5-day culture was greatest with 0.4 mM Ca2+, but DNA synthesis was scarcely affected by the concentration of Ca2+. Both the activities of alkaline phosphatase, 5'-nucleotidase, gamma-glutamyltransferase and lactate dehydrogenase and the number of cell nuclei of cultures in 0.1 mM and 2 mM Ca2+ media were assayed over a 5-day period, and their activities were calculated as enzyme activities per unit number of cell nuclei. Alkaline phosphatase activity increased rapidly during the first day of culture in both media, and its activity in 0.1 mM medium was higher than that in 2 mM medium after culture for 3 days. The activity of 5'-nucleotidase became higher in 0.1 mM medium than in 2 mM medium from day 2 and was maximal on day 3 in both media. gamma-Glutamyltransferase activity increased and lactate dehydrogenase activity decreased with time in culture, both activities showing no appreciable difference in the two media.     

Purification and partial characterization of the soluble low Km 5'-nucleotidase from human seminal plasma

Soluble low Km 5'-nucleotidase from human seminal plasma has been purified to homogeneity by one affinity and two gel-filtration chromatographic steps. The pure enzyme had a specific activity of 2000 nmol min-1 mg-1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified low Km 5'-nucleotidase revealed a single polypeptide band of 40 +/- 7 kDa and a tetrameric structure of 160 +/- 10 kDa has been proposed for the native enzyme. The kinetic properties of low Km 5'-nucleotidase have been determined and rather unique characteristics have been found for this soluble low Km 5'-nucleotidase: the substrate efficiency was slightly higher for IMP with an optimum pH at 7.5; the enzyme showed an absolute dependence on Mg2+ ions. Ca2+ could replace Mg2+ ions for activity while other divalent cations could not substitute for Mg2+; the enzymes were equally activated by ATP and ADP up to 0.1 mM concentrations. At higher concentrations up to 1 mM, ADP was still an activator while ATP caused a gradual decrease of activation to the native activity. This effect could not be related to the Mg-ATP = complexes since the enzymic preparation Mg(2+)-free still showed the same biphasic pattern of activation.     

Purine catabolism in man: characterization of placental microsomal 5'-nucleotidase.

Human placental microsomal 5'-nucleotidase (EC 3.1.3.5) was prepared free of alkaline phosphatase by isoelectric focusing. A total of seven electrophoretic variants were isolated during the preparation of six placentas. Only three to six variants were found in a single placenta. The isoelectric pH's were 6.70, 6.44, 6.23, 6.02, 5.76, 5.63 and 5.44. These were found to be composed of variable quantities of a large, medium and low molecular weight form. The apparent molecular weights of the medium and light form of the enzyme were 86 500 and 43 500, respectively, as estimated from Stokes radius and sedimentation velocity determinations. The electrophoretic variants were not distinguishable with respect to specific activity and Michaelis constants for AMP, GMP or CMP or inhibition by ATP, CTP or adenosine. These electrophoretic variants appeared to be pseudoisozymes based upon different states of aggregation of a common primary sequence. There was a wide range of substrate specificity among nucleoside 5'-monophosphates which included 2-deoxyribose compounds. With AMP as 100, substrate activity was: CMP, 122; NMN, 74; GMP, 68: IMP, 63; XMP, 28 and UDP-glucose, 68. The Michaelis constants for AMP, GMP and CMP ranged from 12-18 muM, from 33-67 muM and from 170-250 muM, respectively. Although 5'-nucleotidase was active in the absence of divalent cation, 5 mM MgCl2 stimulated the enzyme activity to 234% of control and shifted the pH optimum of 9.8 to a plateau from pH 7.4-9.8.     

Regional and Subcellular Distribution in Mammalian Brain of the Enzymes Producing Adenosine

The activities of 5'-nucleotidase, 2'-nucleotidase, alkaline phosphatase, and acid phosphatase were measured in rat and autopsied human brains. The four phosphatases in the rat brain showed little change in activity after death. The activities of adenosine-producing enzymes were compared in various parts of rat and human brains. When phosphatase activity was measured at pH 7.5, 5'-nucleotidase showed the highest activity in the most parts of the brain. The activity of 2'-nucleotidase and that of nonspecific phosphatase were almost the same at pH 7.5. However, higher phosphatase activity was observed in all parts of the brain when nonspecific phosphatase activity was measured at pH 10.0 or 5.5. High specific activity of 5'-nucleotidase in the brain was detected in the membranous components, especially in the synaptic membranes. The activity of 2'-nucleotidase was distributed in the soluble and synaptosomal fractions. The highest activity of both alkaline and acid phosphatases was recovered in the crude mitochondrial fraction, with the highest specific activity in the microsomal fraction. Phosphatase activity was distributed widely in the rat brain. The activity of 5'-nucleotidase was high in the medulla oblongata, thalamus, and hippocampus, but low in the peripheral nerve, spinal cord, and occipital lobe. The activity of 2'-nucleotidase was high in the vermis and frontal lobe. The highest acid and alkaline phosphatase activities were detected in the frontal lobe and in the olfactory bulb, respectively. The distribution of the four phosphatases in the autopsied human brain was similar to that in the rat brain. The highest 5'-nucleotidase activity was observed in the temporal lobe and thalamus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Levamisole inhibition of alkaline phosphatase and 5'-nucleotidase of bovine milk fat globule membranes

1. The effect of levamisole (LMS) on alkaline phosphatase (EC 3.1.3.1) and 5'-nucleotidase (EC 3.1.3.5) activities of bovine milk fat globule membranes (MFGM) was examined. 2. LMS inhibited MFGM alkaline phosphatase activity in a concentration-dependent manner with 50% inhibition produced by 49 +/- 23 microM LMS. 3. 5'-Nucleotidase was resistant to LMS inhibition with 30.9% inhibition produced by 10 mM LMS, the highest concentration tested. 4. LMS was an uncompetitive inhibitor of MFGM alkaline phosphatase with a Ki of 45 +/- 6 microM. 5. The extent of LMS inhibition of alkaline phosphatase was dependent on the substrate utilized in the assay. 6. The effect of LMS on bovine MFGM alkaline phosphatase was similar to LMS effects on other mammalian alkaline phosphatases of liver/kidney/bone/placental isoenzyme origin.     

A family of phosphohydrolases from bovine intestinal mucosa: 5'-nucleotidase

Bovine intestinal 5'-nucleotidase has been partially purified and characterized for comparison with two other phosphohydrolases from the same tissue, alkaline phosphatase and 5'-nucleotide phosphodiesterase, which are closely related structurally and mechanistically. Kinetic studies with a variety of nucleotides and phosphonate analogs show that, although 5'-nucleotidase is a monoesterase like alkaline phosphatase, it more closely resembles 5'-nucleotide phosphodiesterase in its high affinity and specificity for nucleotide binding. 5'-Nucleotidase is bound very strongly by an affinity column containing a bound phosphonate analog of ADP but is not bound by an affinity column containing a non nucleotide phosphonate which selectively binds alkaline phosphatase. 5'-Nucleotidase is strongly bound by immobilized antibodies prepared against 5'-nucleotide phosphodiesterase, and is less strongly bound by immobilized antibodies prepared against alkaline phosphatase. We conclude that 5'-nucleotidase is structurally more similar to 5'-nucleotide phosphodiesterase than to another monoesterase, alkaline phosphatase.     

Enhancement of 5'-nucleotidase activity of human leukemic cells after fractionation: implications for cancer and aging

Previous studies reported that 5'-nucleotidase activity was undetectable or at much lower levels in the homogenate of human chronic lymphocytic leukemic (CCL) cells than in normal lymphocytes. In the present study, 5'-nucleotidase specific activity in acute myelocytic leukemia (AML), which varied in a range from undetectable to 1.4 (nmoles/min.mg protein), was enhanced by cell fractionation, from undetectable in the homogenate, up to 18.8 +/- 1.2, 6.4 +/- 0.7 and 0.68 +/- 0.12 in plasma membranes, microsomes, and cytosol fraction, respectively. In a further fractionation of the cytosol of various leukemic cells with ammonium sulfate, 5'-nucleotidase specific activity increased up to 14-fold in the 60% (NH4)2SO4 fraction, with a recovery of 1266 +/- 115%. These data suggest that 5'-nucleotidase activity in fractionated leukemic cells is higher than reported previously and that the sum of 5'-nucleotidase activity in subcellular compartments is higher than that detected in the homogenate. Furthermore, even when 5'-nucleotidase was undetectable in a homogenate, it became detectable in the plasma membranes, suggesting that its ecto-enzyme function is still active in leukemic cells. The undetectable or low 5'-nucleotidase in the homogenate is indicative of (1) the enzyme itself being in an inactive form but becoming active after the fractionations, or (2) the presence of a factor(s) that prevents the enzyme from being detected but that is separated from the enzyme by the fractionations. In both cases, the rate of nucleotide catabolism by inactive 5'-nucleotidase in rapidly proliferating leukemic cells should be slower than when the enzyme is active. The present finding is consistent with our previous findings that during normal cell aging the high 5'-nucleotidase activity is associated with senescent non-proliferating cells but low or undetectable activity with rapidly proliferating immortal cells. The implications of 5'-nucleotidase for DNA synthesis in aging and cancer are discussed.     

5'-Nucleotidase of human placental trophoblastic microvilli possesses cobalt-stimulated FAD pyrophosphatase activity

An enzyme with FAD pyrophosphatase activity was extracted from human placental syncytiotrophoblast microvilli and purified to near-homogeneity. The enzyme has been identified as 5'-nucleotidase by several criteria. Throughout purification, parallel increases in the specific activities of FAD pyrophosphatase and AMP phosphatase were observed. The enzyme was a glycoprotein with a subunit molecular weight of 74,000. EDTA treatment resulted in a marked decline in both activities, and restoration of FAD pyrophosphatase activity but not 5'-nucleotidase activity was accomplished by the addition of Co2+ or, to a lesser extent, Mn2+. The substrate specificity of the 5'-nucleotidase activity that we observed agreed closely with the results of others. The pyrophosphatase activity was relatively specific for FAD. ADP, ATP, NAD(H), and FMN were not hydrolyzed, and ADP strongly inhibited both activities. For FAD pyrophosphatase activity, a Km of 1.2 x 10(-5) M and a Vmax of 1.1 mumol/min/mg protein were determined in assays performed in the presence of Co2+. In the absence of added Co2+, the Vmax declined but the Km was unchanged. For 5'-nucleotidase (AMP as substrate) the Km was 4.1 x 10(-5) M and the Vmax 109 mumol/min/mg protein. Hydrolysis of FMN to riboflavin was observed in partially purified detergent extracts of microvilli that contained alkaline phosphatase activity and lacked FAD pyrophosphatase and 5'-nucleotidase activity. The presence of both FAD pyrophosphatase and FMN phosphatase activities in syncytiotrophoblast microvilli supports the view that the placental uptake of vitamin B2 involves the hydrolysis of FAD and FMN to riboflavin which is then absorbed, a sequence postulated for intestinal absorption and liver uptake.     

Morphological, histochemical, and biochemical studies on the gut of Haemonchus contortus Rud., 1803).

A study has been made on the structure and chemical composition of the gut of Haemonchus contortus (Rud., 1803). The oesophagus has typically a triradiate, cuticle-lined lumen. The intestinal epithelium is provided with a well-developed brush border which contains periodic acid-Schiff-positive mucoproteins. The intestinal epithelium stores glycogen and lipids. It stains diffusely for phospholipids and general proteins and also for terminal-NH2 group. The presence of Fe2+ and Fe3+ containing pigments and activities of acid and alkaline phosphatases, glucose-6-phosphatase, and 5'-nucleotidase have been observed in the intestinal epithelium. Biochemically pH optimum for intestinal acid phosphatase has been found to be 4.8. The brush border shows positive reactions for acid phosphatase and glucose-6-phosphatase, and negative reactions for alkaline phosphatase and 5'-nucleotidase, and negative reactions for alkaline phosphatase and 5'-nucleotidase. The presence of enzymes in the brush border is related to extracellular digestion and absorption of nutrients.     

Human placental ecto-5'-nucleotidase: isoforms and chemical crosslinking products of the membrane-bound and isolated enzyme

Human placental ecto-5'-nucleotidase is specifically detected on Western blots using poly- or monoclonal antibodies. In two-dimensional electrophoresis 5'-nucleotidase, purified as well as membrane bound, is resolved in up to 13 isoforms distinguished by a different content of neuraminic acid. These forms span a range of molecular masses of about 67-71 kDa and of isoelectric points of 5.8-7.0. Chemical cross-linking of the purified enzyme with either homoor heterobifunctional reagents yields a dimer of about 140 kDa exclusively. On the other hand treatment of plasma membranes with the same reagents results in a crosslinking product of 5'-nucleotidase of about 97 kDa. The partner of the enzyme subunit in this crosslink, a protein of about 30 kDa, is unknown. Using specific antibodies the cytoskeletal components actin and tropomyosin were excluded as possible candidates.     

Mutants of Escherichia coli K-12 "cryptic," or deficient in 5'-nucleotidase (uridine diphosphate-sugar hydrolase) and 3'-nucleotidase (cyclic phosphodiesterase) activity

Mutants of Escherichia coli have been selected for the absence of 5'-nucleotidase (uridine diphosphate-sugar hydrolase) and 3'-nucleotidase (2',3'-cyclic phophodiesterase). Mutants selected for the absence of 5'-nucleotidase are of two kinds: those that lack detectable activity for the enzyme (Ush(-)), and those that possess activity when cell extracts are assayed, but not when intact cells are assayed (cryptic; Crp(-)). The latter class is probably identical to a type of mutant previously reported by Ward and Glaser. When mutants are selected for the absence of 3'-nucleotidase, Crp(-)mutants are also obtained. Thus far, however, mutants totally lacking this enzyme have not been found. The location on the genetic map of one ush mutation is at position 11 min and that of one crp mutation at approximately 67 min. In the crp mutant, 5'-nucleotidase and 3'-nucleotidase remain located in the periplasm. This mutant is also cryptic for alkaline phosphatase but not for acid hexose phosphatase. Treatment of cells with ethylenediamine-tetraacetate substantially alleviated crypticity. These data are discussed in terms of the organization of periplasmic enzymes and of the outer membrane as a permeability barrier.     

Control of the production and partial characterization of repressible extracellular 5'-nucleotidase and alkaline phosphatase in Neurospora crass.

A new species of orthophosphate repressible extracellular 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was found to be released into mycelial culture media when a wild type strain of Neurospora crassa was grown on limiting amounts of phosphate. The production of 5'-nucleotidase and extracellular acid and alkaline phosphatase was inhibited by the addition of rifampicin when it was added at the later stage of mycelial growth, but not when it was added at a very early stage. The 5'-nucleotidase and extracellular alkaline phosphatase were partially purified and characterized. pH optimum of the former was 6.8 and that of the latter was higher than 10.0. The 5'-nucleotidase activity was inhibited by ethylenediaminetetraacetate (EDTA) and ZnCl2 at pH 6.8 and stimulated by MnCl2 and CoCl2 at pH 4.0. Alkaline phosphatase activity was stimulated by EDTA, MgCl2, CoCl2 and MnCl2. 5'-nucleotidase activity was stimulated by EDTA, MgCl2, CoCl2 and MnCl2. 5'-nucleotidase hydrolyzed various 5'-nucletides but not 3'-nucleotides or other various phosphomono- and diester compounds. Alkaline phosphatase hydrolyzed all the phosphomonoester compounds tested. Mutants, nuc-1 and nuc-2, which were originally isolated by the inability to utilize RNA or DNA as a sole source of phosphate, were unable to produce 5'-nucleotidase or six other repressible enzymes reported previously. These mutants showed no or significantly reduced growth on orthophosphate-free nucleotide media depending on the number of conidia inoculated, mainly because of loss of ability to produce these repressible extracellular phosphatases.     

Isoelectric focusing studies on the neutral 5′-nucleotidase from wistar rat hippocampus: Evidence for the absence of isoenzymes

Summary The pattern and some substrates characteristic of the rat brain 5-nucleotidase were studied using the isoelectric focusing technique, which revealed that the enzyme is present in a single form in hippocampus extracts. An alkaline phosphatase, which is also able to split nucleoside monophosphates, is not active at neutral pH values. The isoelectric points were found to be 6.4±0.1 for the specific 5-nucleotidase and 6.8±0.1 for the phosphatase.The present research paper was supported by grants from the Ministerium für Hoch- und Fachschulwesen der DDR     

Comparison of the N-acetylhexosaminidase components in the ram testis and epididymis by isoelectric focusing

1. Several enzymic components, with both N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activity, have been demonstrated in the ram testis and epididymis by isoelectric focusing. 2. The component (I), which predominates in the testis and caput of the epididymis, is isoelectric at pH6.2+/-0.1, whereas the predominant component (II) in the epididymal isthmus and cauda is isoelectric at pH7.0+/-0.1. 3. The total activity and the relative proportions of the enzymic components vary in the different sections of the epididymis. 4. Although their pH optima are slightly different, the appropriate K(m) values for components I and II for the hydrolysis of 4-methylumbelliferyl-N-acetyl-beta-glucosaminide and N-acetyl-beta-galactosaminide and the ratios of the maximal velocities towards the two substrates are very similar.     

Microwave-stimulated brain enzyme incubations are possible at the unphysiological condition of 50 degrees C

Microwave-stimulated enzyme incubations for acetylcholinesterase, 5'-nucleotidase, alkaline phosphatase, lactate dehydrogenase, malate dehydrogenase, succinic dehydrogenase and isocitric dehydrogenase were studied, and compared with incubations in a waterbath. Temperature settings of 37 degrees C and 50 degrees C were used, and the incubation times were varied from 30 seconds to 30 minutes. The desired temperature of the incubation solution was reached in the microwave oven within 1 minute, whilst in the waterbath it took 10 to 25 minutes. The microscopic results for alkaline phosphatase and succinic dehydrogenase at a temperature setting of 50 degrees C were superior in the microwave method for incubation times less than 15 minutes. It is postulated that the increased reaction product of alkaline phosphatase and succinic dehydrogenase is due to a temperature effect, which has to be large enough to be of practical value. For the other enzymes studied, microwave-stimulated incubations were no better than the conventional incubations at corresponding temperatures. For 5'-nucleotidase there were aspecific lead deposits in the microwave method. All enzymes performed at the elevated, unphysiological temperature of 50 degrees C proved to have advantages, except for 5'-nucleotidase, whilst for malate dehydrogenase there was an aspecific reduction of the colour developer at this temperature.     

Enzyme-histochemical identification of lymphatic vessels by light and backscattered image scanning electron microscopy

The walls of lymphatics are characterized by strong 5'-nucleotidase activity, whereas those of blood capillaries reveal significantly lower or no activity. Alkaline phosphatase activity, on the other hand, is markedly higher in blood capillaries than in lymphatic vessels. On the basis of such characteristics, lymphatics and blood capillaries were distinguished histochemically in rat stomach using 5'-nucleotidase-alkaline phosphatase double staining. The distribution and intensity of lead-demonstrated 5'-nucleotidase activity in lymphatic vessels could be determined by comparing the images of the same histochemically stained cryostat section as seen by light and backscattered image scanning electron microscopy. The specificity of the 5'-nucleotidase reaction was obtained by inhibiting nonspecific alkaline phosphatase by including L-tetramisole in the 5'-nucleotidase incubation medium. The products of the 5'-nucleotidase activity were deposited on the outer surface of the plasma membrane of the lymphatic endothelial cells.