Effects of amines and aminoalcohols on bovine intestine alkaline phosphatase activity

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay (EIA). In this study, we evaluated the effects of various aminoalcohols and amines on the activity of BIALP in the hydrolysis of p-nitrophenyl phosphate (pNPP) at pH 9.8, at 20 °C. The k_cat values at 0.05 M diethanolamine, 0.1 M triethanolamine, and 0.2 M N-methylethanolamine were 190 ± 10, 840 ± 30, and 500 ± 10 s⁻¹, respectively. The k_cat values increased with increasing concentrations of diethanolamine, triethanolamine, and N-methylethanolamine and reached 1240 ± 60, 1450 ± 30, and 2250 ± 80 s⁻¹, respectively, at 1.0 M. On the other hand, the k_cat values at 0.05-1.0 M ethanolamine, ethylamine, methylamine, and dimethylamine were in the range of 100-600 s⁻¹. These results indicate that diethanolamine, triethanolamine and N-methylethanolamine highly activate BIALP and might be suitable as a dilution buffer of BIALP in EIA. Interestingly, the K_m values increased with increasing concentrations of diethanolamine and N-methylethanolamine, but not triethanolamine: the K_m value at 1.0 M diethanolamine (0.83 ± 0.15 mM) was 12-fold higher than that at 0.05 M (0.07 ± 0.01 mM), and that at 1.0 M N-methylethanolamine (2.53 ± 0.20 mM) was 14-fold higher than that at 0.2 M (0.18 ± 0.02 mM), while that at 1.0 M triethanolamine (0.31 ± 0.01 mM) was similar as that at 0.2 M (0.25 ± 0.01 mM), suggesting that the mechanisms of BIALP activation are different between the aminoalcohols.     

Comparison of diurnal feeding rhythms in Temora longicornis and Centropages hamatus with digestive enzyme activity

Zooplankton samples were collected in St. George's Bay In theGulf of St. Lawrence, during July, 1981. The level of chlorophyllplus phaeopigment was measured for Centropages hamatus and Temoralongicornis collected at three depths every 4–6 h fora 36 h sampling period. The results showed that these speciesfeed predominantly at night. Measurements were also made ofthe levels of digestive protease, laminarinase and amylase,in samples of animals with either full or empty guts. No significantdifferences in digestive enzyme acthities could be found suggestingthat digestive enzyme levels are not directly related to instantaneousdigestion rates. Nevertheless it is likely that feeding ratesand digestive enzyme levels are closely linked and a mechanismis proposed here which would explain this apparent inconsistency.     

Ultrastructural localization of alkaline phosphatase activity in the intestine of developingRana Catesbeiana

Summary Alkaline phosphatase activity has been localized at the light and electron microscopic levels in the intestine of developing frog,Rana catesbeiana. The intensity of the histochemical reactivity decreases along the intestinal tract. The intracellular localization of the enzymatic activity shows continuous series of organelles loaded with the reaction product from the Golgi zones to the brush border. These results are in agreement with the biochemical observations made on the same material.This work was supported by grants from the France-Quebec agreements (J. Hourdry) and from the Medical Research Council of Canada (J.S. Hugon)     

Effects of polyethylene glycol on bovine intestine alkaline phosphatase activity and stability

In this study, we evaluated the effects of polyethylene glycol (PEG) on bovine intestine alkaline phosphatase (BIALP) activity and stability. In the hydrolysis of p-nitrophenylphosphate (pNPP) at pH 9.8 at 20 °C, the k(cat)/K(m) values of BIALP plus 5-15% w/v free PEG with molecular masses of 1, 2, 6, and 20 kDa (PEG1000, PEG2000, PEG6000, and PEG20000 respectively) were 120-140%, 180-300%, 130-170%, and 110-140% respectively of that of BIALP without free PEG (1.8 μM(-1) s(-1)), indicating that activation by PEG2000 was the highest. Unmodified BIALP plus 5% PEG2000 and BIALP pegylated with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine exhibited 1.3-fold higher activity on average than that of BIALP without free PEG under various conditions, including pH 7.0-10.0 and 20-65 °C. The temperatures reducing initial activity by 50% in 30-min incubation of unmodified BIALP plus 5% PEG2000 and pegylated BIALP were 51 and 47 °C respectively, similar to that of BIALP without free PEG (49 °C). These results indicate that the addition of PEG2000 and pegylation increase BIALP activity without affecting its stability, suggesting that they can be used in enzyme immunoassay with BIALP to increase sensitivity and rapidity.     

Giardia lamblia: expression of alkaline phosphatase activity in infected rat intestine

Alkaline phosphatase (IAP) is a marker of intestinal microvillus membrane. Changes in IAP activity have been studied as a function of Giardia lamblia (G. lamblia) infection using rat as the experimental model. At day 11 and 15 post-infection, enzyme activity was reduced (p<0.01) compared to controls. The enzyme levels were essentially similar to control values by day 30 post-infection. Analysis of the enzyme activity in cell fractions across crypt-villus axis revealed a marked decrease in enzyme activity in the villus tip and mid villus regions but a considerable increase (p<0.01) in enzyme activity in the crypt base of 11 day post-infected animals compared to that in controls. The observed changes in IAP activity were confirmed by assaying the enzyme activity in acrylamide gels using bromo-chloro-indolyl phosphate staining under non-denaturing conditions. These findings indicate differential changes across the crypt-villus axis, but impaired alkaline phosphatase levels in G. lamblia infected rat intestine.     

Effect of various fish meals on disaccharidases and alkaline phosphatase of the small intestine in rats

Female rats of the Wistar strain, weighing 40-60 g were used to study the effect of fish meals (Coryphaenoides rupestris, Chimaera monstruosa and Merluccius merluccius) on the disaccharidases and alkaline phosphatase in the small intestine in relation to the control group which consumed casein. Fish meal diets diminished lactase and alkaline phosphatase activity, the latter being most remarkable in animals fed Ch. monstruosa meal, while no statistical variations in maltase and sucrase activity were observed. Maltase, sucrase and lactase activity of animals fed Ch. monstruosa meal dropped in comparison with those fed C. rupestris meal, while the alkaline phosphatase activity showed no significant changes.     

Dependence of alkaline phosphatase activity on bioecological parameters of Black Sea fish

The data on alkaline phosphatase activity in the liver of eight dominant species of Black Sea fishes are presented. The enzyme activity varies within a wide range; significant sexual differences have not been registered. The activity of the enzyme is similar in specimens of different age groups, but it decreases in old fishes. The enzyme activity increases in prespawning and spawning, which evidences for its participation in the process of sexual maturation of fish. Based on our and published data on the dependence of alkaline phosphatase activity on the degree of the environmental pollution, the enzyme can be recommended as a biomarker for bioindication, biotesting, and ichthyological monitoring.     

Experimental studies on thermal behaviour and diurnal activity rhythms of juvenile European sturgeon (Acipenser sturio)

Experiments were performed to determine the temperature preference of juvenile sturgeons. The biorhythms of activity and the location of fish in a thermal gradient of an electronic shuttlebox were registered. The fish showed dominant 24 h activity rhythms with its maximum at the nighttime. A temperature preference range or an active thermoregulatory behavior could not be demonstrated by our experiments. The behavior of sturgeons was influenced by temperature: activity and swimming speed were positively correlated to rising temperatures. There are indications that the fish could have been attracted by low light intensities at night.     

Ultrastructural localization of alkaline phosphatase activity in the intestine of developing Rana catesbeiana.

Alkaline phosphatase activity has been localized at the light and electron microscopic levels in the intestine of developing frog, Rana catesbeiana. The intensity of the histochemical reactivity decreases along the intestinal tract. The intracellular localization of the enzymatic activity shows continuous series of organelles loaded with the reaction product from the Golgi zones to the brush border. These results are in agreement with the biochemical observations made on the same material.     

Photoperiodic control of circadian activity rhythms in diurnal rodents

The responses of red squirrels(Tamiasciurus hudsonicus) and eastern chipmunks(Tamias striatus) to complete and skeleton light-dark (LD) cycles were compared. The skeletons, comprised of two 1-h pulses of light per day, effectively simulated the complete photoperiods in the squirrels, but not the chipmunks. Skeleton photoperiods greater than 12-h caused the chipmunks to shift activity from the longer to the shorter of the two intervals between the pulses. To interpret the mechanism of phase control, squirrels and chipmunks were kept in continuous darkness and exposed to 1-h light pulses every 10 days. The time-course of entrainment was also quantified. Both techniques produced light-response curves. The data suggest that the parametric and non-parametric contributions to entrainment are different in these two rodent species.Presented at the Eighth International Congress of Biometeorology, 9–14 September 1979, Shefayim, Israel.     

Stabilization of bovine intestine alkaline phosphatase by sugars

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay. In this study, we evaluated the effects of sugars on the kinetic stability of BIALP in the hydrolysis of p-nitrophenylphosphate (pNPP). The temperatures reducing initial activity by 50% in a 30-min incubation, T(50), of BIALP with 1.0 M disaccharide (sucrose and trehalose) or 2.0 M monosaccharide (glucose and fructose) were 55.0-55.5 °C, 4.7-5.2 °C higher than without sugar (50.3±0.1 °C). The T(50) of BIALP increased to 58.4±0.3 °C when the trehalose concentration was from 1.0 to 1.5 M, but did not change when the glucose concentration was from 2.0 to 3.0 M. Thermodynamic analysis revealed that the stabilization of BIALP by sugars was driven by the increase in the enthalpy change of activation for thermal inactivation of BIALP. No sugars affected the k(cat) of BIALP in the hydrolysis of pNPP. These results suggest that not only trehalose, which is considered the most effective stabilizer of enzymes, but also sucrose, glucose, and fructose can be used as stabilizers of BIALP.     

Affinity-chromatography purification of alkaline phosphatase from calf intestine.

A crude preparation of alkaline phosphatase (EC 3.1.3.1) from calf intestinal mucosa was purified by affinity chromatography on Sepharose-bound derivatives of arsanilic acid, which was found to be a competitive inhibitor of the enzyme. Three biospecific adsorbents were prepared for the chromatography, and the best results were obtained with a tyraminyl-Sepharose derivative coupled with the diazonium salt derived from 4-(p-aminophenylazo)phenylarsonic acid. Alkaline phosphatase was the only enzyme retained by the affinity column in the absence of Pi. The enzyme eluted by phosphate buffer had a specific activity of about 1200 units per mg of protein at pH 10.0, with 5.5mM-p-nitrophenyl phosphate as the substrate.