Dependent P-nitrophenylphosphatase Research Articles

Stimulation of p‐nitrophenylphosphatase activity of Na+/K+‐ATPase by NaCl with oligomycin or ATP

It is known that the addition of NaCl with oligomycin or ATP stimulates ouabain-sensitive and K+-dependent p-nitrophenylphosphatase (pNPPase) activity of Na+/K+-ATPase. We investigated the mechanism of the stimulation. The combination of oligomycin and NaCl increased the affinity of pNPPase activity for K+. When the ratio of Na+ to Rb+ was 10 in the presence of oligomycin, Rb+-binding and pNPPase activity reached a maximal level and Na+ was occluded. Phosphorylation of Na+/K+-ATPase by p-nitrophenylphosphate (pNPP) was not affected by oligomycin. Because oligomycin stabilizes the Na+-occluded E1 state of Na+/K+-ATPase, it seemed that the Na+-occluded E1 state increased the affinity of the phosphoenzyme formed from pNPP for K+. On the other hand, the combination of ATP and NaCl also increased the affinity of pNPPase for K+ and activated ATPase activity. Both activities were affected by the ligand conditions. Oligomycin noncompetitively affected the activation of pNPPase by NaCl and ATP. Nonhydrolyzable ATP analogues could not substitute for ATP. As NaE1P, which is the high-energy phosphoenzyme formed from ATP with Na+, is also the Na+-occluded E1 state, it is suggested that the Na+-occluded E1 state increases the affinity of the phosphoenzyme from pNPP for K+ through the interaction between alpha subunits. Therefore, membrane-bound Na+/K+-ATPase would function as at least an (alphabeta)2-diprotomer with interacting alpha subunits at the phosphorylation step.

Inhibition of Na +,K +-ATPase by the extract of Stephania cephararantha H ayata and bisbenzylisoquinoline alkaloid cycleanine, a major constituent

The Stephania cephararantha H ayata extract, and its constituent bisbenzylisoquinoline alkaloids, such as cycleanine, cepharanthine, isotetrandrine, berbamine, homoaromoline, and cepharanoline were studied for effects on Na +,K +-ATPase activity. The S. cephararantha H ayata extract inhibited Na +,K +-ATPase activity with an apparent ic 50 value of 540 μg/mL. Cycleanine markedly inhibited Na +,K +-ATPase activity with an ic 50 value of 6.2×10 −4 M. It slightly inhibited Mg 2+-ATPase, H +-ATPase, and Ca 2+-ATPase. No effects on alkaline and acid phosphatase activities were observed. The inhibition by isotetrandrine, homoaromoline, cepharanthine, and berbamine was less marked, and cepharanoline showed no effect. Five synthetic analogues of cepharanthine slightly inhibited the activity. The mechanism of inhibition by cycleanine on Na +,K +-ATPase activity was examined in detail, and the following results were obtained in the overall reaction: (1) the mode of inhibition was noncompetitive with respect to ATP; (2) the degree of inhibition was decreased with a decrease of K + concentration; (3) it was not affected by Na + concentration; (4) the inhibition mechanism was different from that of ouabain. The activity of K +-dependent p-nitrophenyl phosphatase, a partial reaction of Na +,K +-ATPase, did not appear to have been inhibited by cycleanine in the reaction mixture containing 15 mM K + (optimum condition). However, cycleanine increased the K 0.5 value for K + and reduced the K i values for Na + and ATP, in K +-dependent p-nitrophenyl phosphatase. Cycleanine might interact with the enzyme in Na·E 1-P form and prevents the reaction step from Na·E 1-P to E 2-P.

The pyridoindole antioxidant stobadine attenuates histochemical changes in kidney of streptozotocin-induced diabetic rats

The aim of the present study was to investigate the effects of dietary supplementation with the pyridoindole antioxidant stobadine on histochemical parameters in kidney of streptozotocin-induced diabetic rats. Diabetic male Wistar rats were fed a standard diet or a diet supplemented with stobadine (0.05% w/w) for 24 weeks. The diabetic state was characterized by significantly elevated plasma levels of glucose and glycated hemoglobin, severe reduction of total body weight and relatively enlarged kidneys. Kidney alkaline phosphatase activity was not changed by diabetes. Activity of 5'-nucleotidase, K(+)-dependent p-nitrophenylphosphatase, ATPase and mitochondrial succinic dehydrogenase were markedly decreased in kidneys of diabetic rats. In contrast, activity of beta-hydroxybutyrate dehydrogenase was moderately increased in kidney of diabetic rats as compared to controls. Long-term treatment of diabetic animals with stobadine attenuated histochemical changes in kidney tissue.

Cytochemical localization of Na+/K+-ATPase activity in cochlear strial marginal cells after various catecholamine administrations.

Sodium/potassium-activated adenosine triphosphatase (Na+/K+-ATPase) activity in the kidney and brain is high, and is regulated by catecholamines. Na+/K+-ATPase activity is also high in the basolateral infoldings of the strial marginal cells, where it aids in maintaining the characteristic electrolyte composition of the endolymph. To clarify the involvement of humoral control in strial function, particularly the role of catecholamines, the K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity of strial marginal cells was investigated in guinea pigs using a cerium-based cytochemical method. The effects of reserpine, serotonin (5-HT), norepinephrine (NE), epinephrine (EP), both alone and in combination, were studied. High doses of reserpine cause depletion of sympathetic substances. Strial K+-NPPase activity was decreased after reserpine or dopamine treatment, and was increased after 5-HT, NE, and EP treatment. After reserpinization, repeated treatment with 5-HT, NE, or EP led to detectable strial enzyme activity. Thus, exogenous 5-HT, NE, and EP were able to restore strial K+-NPPase activity in the reserpine-treated animals. These results suggested that biogenic amines regulate strial K+-NPPase activity. Thus, the function of the stria vascularis may be regulated by the opposing actions of these catecholamines, and 5-HT.

Inhibition of H +,K +-ATPase by hinesol, a major component of So-jutsu, by interaction with enzyme in the E 1 state

Hinesol, a major component of the crude drug “So-jutsu” (Atractylodis Lanceae Rhizoma), strongly inhibited H +,K +-ATPase activity with a ic 50 value of 5.8 × 10 −5 M. It also inhibited Na +,K +-ATPase, Mg 2+-ATPase, Ca 2+-ATPase, and H +-ATPase activities, although the inhibition rate was lower. No effects on alkaline or acid phosphatase activities were observed. The mechanism by which hinesol inhibited H +,K +-ATPase activity was studied in detail. The inhibition was uncompetitive with respect to ATP, and it increased as the Mg 2+ concentration was raised, whereas it was not affected by the K + concentration. The activity of K +-dependent p-nitrophenyl phosphatase (K +-pNPPase), a partial reaction of H +,K +-ATPase, was inhibited by hinesol noncompetitively with respect to pNPP ( ic 50 value of 1.6 × 10 -4 M), and competitively with respect to K +, whereas it was not affected by the Mg 2+ concentration. These results suggest that hinesol is a relatively specific inhibitor of H +,K +-ATPase. It appears that hinesol reacts with enzyme in the E 1 state in the presence of ATP and Mg 2+ and forms the complex hinesol-H + · E 1-ATP or hinesol · E 1-P, blocking the conformational change to the E 2 state. Furthermore, hinesol enhanced the inhibitory effect of omeprazole on H +,K +-ATPase, and the inhibitory site of hinesol was different from that of omeprazole. The effect of So-jutsu as an anti-gastric ulcer agent may be ascribed to the inhibitory effect of hinesol on H +,K +-ATPase activity.

Correlation between acid secretion and proton pump activity during inhibition by the proton pump inhibitors omeprazole and pantoprazole

Omeprazole and pantoprazole are known to be irreversible, SH-acting inhibitors of gastric H +,K +-adenosine triphosphatase (H +,K +-ATPase). Both drugs concentration-dependently and pH-dependently inhibited K +-dependent p-nitrophenyl phosphatase (K +-pNPPase) activity in purified rabbit gastric microsomes. The potency of omeprazole was about three times that of pantoprazole in the pH ranges tested. Both drugs also inhibited acid secretion, as determined by [ 14C]aminopyrine accumulation in isolated rabbit gastric glands, with the potency ratio being about 5 (omeprazole over that of pantoprazole). Under conditions in which acid secretion was inhibited completely by the drugs, the total K +-pNPPase activity in the digitonin-permeabilized glands was scarcely reduced, showing an apparent discrepancy between the acid secretion and the proton pump activity. The isolated glands were stimulated with secretagogues for 30 min in the presence of the inhibitors, homogenized, and then separated into fractions in which K +-pNPPase activity was measured. Omeprazole exclusively inhibited the activity in the low-speed fraction, which was rich in the apical membranes, whereas pantoprazole did not inhibit activity in any fraction. When the time of treatment with the inhibitors was increased up to 5 hr, the inhibition of the total K +-pNPPase activity in the glands reached a plateau at an inhibition rate lower than 50% within 2 hr. This suggested that no continuous recycling of the proton pump was occurring during stimulation. The inhibitory effect of both drugs on the permeabilized gland preparation was less potent than that on the purified enzyme, especially at the higher pH, and it appeared to be partially reversible. The extent of the reduction in potency was more prominent for pantoprazole. It is concluded that a lower amount of proton pump activity needs to be inhibited by pantoprazole than by omeprazole to achieve the same extent of acid secretion inhibition. This appears to be due to the nature of pantoprazole, i.e. the requirement of low pH for activation and the partial reversibility of the inhibition.

Effects of Epinephrine on Ouabain-Sensitive, K+-DependentP-Nitrophenylphosphatase Activity in Strial Marginal Cells of Guinea Pigs

In strial marginal cells, Na+/K(+)-ATPase activity is abundant, and contributes to maintain the characteristic electrolyte composition of the cochlear endolymph. In the present study, to clarify the relationship between epinephrine and strial Na+/K(+)-ATPase activity, the ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K(+)-NPPase) activity of strial marginal cells was investigated with a cerium-based method in normal guinea pigs and guinea pigs treated with reserpine, epinephrine, and reserpine plus epinephrine. In our previous study, K(+)-NPPase activity had almost completely decreased 3 to 20 days after reserpine administration. In the present study, at 10 days after reserpinization and following repeated epinephrine treatment, enzyme activity was detectable. These results suggest that exogenous epinephrine was able to restore strial K(+)-NPPase activity in the reserpine-treated animals, and that epinephrine might increase strial Na+/K(+)-ATPase activity.

Localization of Na,K-ATPase activity in developing rat distal colon: role of corticosteroids

The role of corticosteroids in the development of Na,K-ATPase and its distribution along the crypt base-colonic surface was investigated in suckling, weanling and adult rats using a biochemical and a histochemical approach. The cerium-based histochemical method for detection of ouabain-sensitive K +-dependent p-nitrophenylphosphatase (K-NPPase) component of the Na,K-ATPase complex was used to localize Na,K-ATPase in the epithelium. The activity of Na,K-ATPase was very low 2 days after birth and increased to a maximum in adulthood. Mature surface colonocytes and immature cells at the crypt base were similarly reactive and the reaction product was decreased by the addition of ouabain and inhibited by omission of K +. Adrenalectomy decreased colonic Na,K-ATPase activity in surface and deep crypt cells of suckling, weanling and adult animals. Mineralocorticoids (deoxycorticosterone acetate, DOCA) restored the Na,K-ATPase activity both in surface and crypt cells of adrenalectomized weanling rats and the effect of DOCA was inhibited by the mineralocorticoid receptor antagonist, spironolactone. Physiological doses of glucocorticoids (dexamethasone) stimulated Na,K-ATPase activity in surface colonocytes of adrenalectomized weanling rats; supraphysiological doses restored Na,K-ATPase probably via cross-over into mineralocorticoid receptors both in surface and crypt cells. High dietary Na + intake during the weaning period reduced the reaction product to the level detected in adrenalectomized rats. The distribution of Na, K-ATPase activity in the epithelium of adrenalectomized rats with substitutional replacement hormone therapy was the same as in control animals or, in some animals, the surface absorptive epithelium exhibited a stronger reaction than the crypt cells. Similarly, the surface colonocytes of adult rats kept on a low-salt diet showed a stronger reaction than the crypt cells. These data indicate that postnatal development of Na,K-ATPase is regulated predominantly by aldosterone and that both surface and crypt cells are responsive to mineralocorticoids. Surface cells are also responsive to glucocorticoids.

Effect of Norepinephrine on Ouabain-sensitive, K+-dependent p-Nitrophenylphosphatase Activity in Strial Marginal Cells of the Cochlea in Normal and Reserpinized Guinea Pigs

On the basolateral infoldings of the strial marginal cells in the cochlea, Na-K ATPase activity is abundant. To clarify the humoral control by norepinephrine, K-NPPase activity of strial marginal cells in the cochlea was investigated in normal, reserpine, norepinephrine (NE), reserpine plus NE-treated guinea pigs using a cerium-based method. K-NPPase activity was almost completely decreased 3-20 days after reserpine administration. At 10 days after reserpinization and following NE repeated treatment, enzyme activity was detectable. These results suggested that norepinephrine might restore and regulate strial K-NPPase activity.

Cytochemical Localization of Ouabain-sensitive, K+-dependentp-nitrophenylphosphatase Activity in the Facial Nerve of Reserpinized Guinea Pigs

Ion-transporting Na,K-ATPase plays an essential role in nerve conduction. To clarify the cytochemical effects of reserpine on transport Na,K-ATPase activity, the localization of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase (K-NPPase) activity was investigated in the facial nerves of normal and reserpinized guinea pigs using a cerium-based method. In the normal facial nerve, the reaction product of K-NPPase activity was observed on the internodal axolemma and Schmidt-Lanterman incisures. In the Ranvier nodes, enzyme activity was localized to the paranodal and nodal axolemma. In the reserpinized nerves, reaction product was detectable on the nodal axolemma but was undetectable on the other parts of the axolemma. Nodal K-NPPase was not affected by reserpine treatment. Therefore, the transport Na,K-ATPase on the nodal axolemma might differ from that on the other parts of the axolemma. Allowing reserpinized animals to survive. Two different ouabain-sensitive K-NPPase reactivities, "reserpine-sensitive" and "reserpine-resistant," might be present in the facial nerve of guinea pigs.

K+ supplementation increases muscle [Na+-K+-ATPase] and improves extrarenal K+ homeostasis in rats.

Effects of K+ supplementation (approximately 200 mmol KCl/100 g chow) on plasma K+, K+ content, and Na+-K+-adeonsinetriphosphatase (ATPase) concentration ([Na+-K+-ATPase]) in skeletal muscles as well as on extrarenal K+ clearance were evaluated in rats. After 2 days of K+ supplementation, hyperkalemia prevailed (K+-supplemented vs. weight-matched control animals) [5.1 +/- 0.2 (SE) vs. 3.2 +/- 0.1 mmol/l, P < 0.05, n = 5-6], and after 4 days a significant increase in K+ content was observed in gastrocnemius muscle (104 +/- 2 vs. 97 +/- 1 micromol/g wet wt, P < 0.05, n = 5-6). After 7 days of K+ supplementation, a significant increase in [3H] ouabain binding site concentration (344 +/- 5 vs. 239 +/- 8 pmol/g wet wt, P < 0.05, n = 4) was observed in gastrocnemius muscle. After 2 wk, increases in plasma K+, K+ content, and [3H]ouabain binding site concentration in gastrocnemius muscle amounted to 40, 8, and 68% (P < 0.05) above values observed in weight-matched control animals, respectively. The latter change was confirmed by K+-dependent p-nitrophenyl phosphatase activity measurements. Fasting for 1 day reduced plasma K+ and K+ content in gastrocnemius muscle in rats that had been K+ supplemented for 2 wk by 3.1 +/- 0.3 mmol/l (P < 0.05, n = 5) and 15 +/- 2 micromol/g wet wt (P < 0.05, n = 5), respectively. After induction of anesthesia, arterial plasma K+ was measured during intravenous KCl infusion (0.75 mmol KCl x 100 g body wt(-1) x h(-1)). The K+-supplemented fasted group demonstrated a 42% (P < 0.05) lower plasma K+ rise, associated with a significantly higher increase in K+ content in gastrocnemius muscle of 7 micromol/g wet wt (P < 0.05, n = 5) compared with their control animals. In conclusion, K+ supplementation increases plasma K+, K+ content, and [Na+-K+-ATPase] in skeletal muscles and improves extrarenal K+ clearance capacity.

Inhibition of Na +, K +-ATPase by 1,2,3,4,6-Penta-O-galloyl-β-D-glucose, a major constituent of both moutan cortex and paeoniae radix

The inhibition of Na∗,K +-ATPase activity by various constituents of Moutan Cortex and Paeoniae Radix was studied. 1,2,3,4,6-Penta-O-galloyl-β-D-glucose (PGG), a major component of both crude drugs, strongly inhibited Na +,K +-ATPase activity (lC 50 = 2.5 x 10 -6 M), whereas galloylpaeoniflorin, benzoic acid, and catechin were weakly inhibitory, and albiflorin, oxypaeoniflorin, paeoniflorin, paeonol, and phenol were ineffective. The inhibition of Na +,K +-ATPase activity by PGG was decreased in the presence of BSA or phospholipids. The inhibition mode of PGG was noncompetitive with respect to ATP. The K 0.5 value for Na + was increased by the addition of PGG from 9.1 to 12.3 mM, whereas that for K + was not altered. PGG also inhibited K +-dependent p-nitrophenyl phosphatase activity with an IC 50 value of 5.3 x 10 -6 M, and the extent of the inhibition increased at higher concentrations of K +. The K 0.5 value for K + was decreased by the addition of PGG from 3.3 to 2.0 mM. These results suggested that the inhibition of Na +,K +-ATPase activity is caused by interaction of PGG with the enzyme in the E 2 state. The inhibitory effect of Moutan Cortex or Paeoniae Radix is considered to be mainly attributable to PGG.

Reduced concentration of myocardial Na+,K(+)-ATPase in human aortic valve disease as well as of Na+,K(+)- and Ca(2+)-ATPase in rodents with hypertrophy.

Myocardial Na+,K(+)-ATPase was studied in patients with aortic valve disease, and myocardial Na+,K(+)- and Ca(2+)-ATPase were assessed in spontaneously hypertensive rats (SHR) and hereditary cardiomyopathic hamsters using methods ensuring high enzyme recovery. Na+,K(+)-ATPase was quantified by [3H]ouabain binding to intact myocardial biopsies from patients with aortic valve disease. Aortic stenosis, regurgitation and a combination hereof were compared with normal human heart and were associated with reductions of left ventricular [3H]ouabain binding site concentration (pmol/g wet weight) of 56, 46 and 60%, respectively (p < 0.01). Na+,K(+)- and Ca(2+)-ATPases were quantified by K(+)- and Ca(2+)-dependent p-nitrophenyl phosphatase (pNPPase) activity determinations in crude myocardial homogenates from SHR and hereditary cardiomyopathic hamsters. When SHR were compared to age-matched Wistar Kyoto (WKY) rats an increase in heart-body weight ratio of 75% (p < 0.001) was associated with reductions of K(+)- and Ca(2+)-dependent pNPPase activities (mumol/min/g wet weight) of 42 (p < 0.01) and 27% (p < 0.05), respectively. When hereditary cardiomyopathic hamsters were compared to age-matched Syrian hamsters an increase in heart-body weight ratio of 69% (p < 0.001) was found to be associated with reductions in K(+)- and Ca(2+)-dependent pNPPase activities of 50 (p < 0.001) and 26% (p = 0.05), respectively. The reductions in Na+,K(+)- and Ca(2+)-ATPases were selective in relation to overall protein content and were not merely the outcome of increased myocardial mass relative to Na+,K(+)- and Ca(2+)-pumps. In conclusion, myocardial hypertrophy is in patients associated with reduced Na+,K(+)-ATPase concentration and in rodents with reduced Na+,K(+)- and Ca(2+)-ATPase concentrations. This may be of importance for development of heart failure and arrhythmia in hypertrophic heart disease.

Corticosteroid induction of renal and intestinal K(+)-dependent p-nitrophenylphosphatase in young and adult rats.

The post-natal development of the K(+)-dependent p-nitrophenylphosphatase (K-NPPase) activity of the Na, K-ATPase complex and its regulation by corticosteroids was studied in renal and intestinal epithelia of the rat using the p-nitrophenylphosphatecerium capture method. The distribution of the phosphatase was analysed in detail in the renal epithelia of the medullary thick ascending limb of Henle's loop and distal convoluted tubule and in the surface epithelial cells of the distal colon. The convoluted tubule and Henle's loop segments showed a stronger reaction for K-NPPase than the colon epithelium both in adult and young animals (suckling and weanling pups). The intensity of staining rose progressively in all three epithelia during early postnatal development and reached the highest levels during the weaning period and in adulthood. The most distinct change was observed between days 10 and 16. Adrenalectomy significantly reduced the density of the final reaction product in weanling and adult rats. Replacement hormone therapy of adrenalectomized weanling rats with the glucocorticoid dexamethasone restored the K-NPPase activity in the two renal epithelia, whereas the mineralocorticoid deoxycorticosterone acetate had no effect on the activity in the medullary thick ascending limb, a very slight effect in distal convoluted tubules, and a strong effect on the distal colon epithelial activity. The observed small effect of the mineralocorticoid in distal convoluted tubule activity may reflect a cross-over into glucocorticoid receptors. We conclude that the postnatal development of Na, K-ATPase is regulated by glucocorticoids in nephron epithelia and predominantly by mineralocorticoids in the surface enterocytes of the distal colon.

Impaired Ranvier node sodium-potassium adenosine triphosphatase may induce facial palsy.

To clarify the part of the neuron essential for myelinated nerve conduction, the cytochemical localization of potassium ion (K+)-dependent p-nitrophenylphosphatase (K-NPPase) activity was investigated in the normal and reserpine-treated facial nerve of guinea pigs. In the normal animals, K-NPPase activity was localized to the internodal axolemma and Schmidt-Lanterman incisures. In the Ranvier nodes, enzyme activity was observed along the paranodal and nodal axolemma. In reserpinized nerves, K-NPPase activity was absent along the internodal axolemma and Schmidt-Lanterman incisures. In the Ranvier nodes, however, enzyme activity was detectable only in the nodal axolemma. The reserpinized animals demonstrated no evidence of facial palsy. Because K-NPPase is essential for nerve conduction, these results indicate that the location of enzyme activity in reserpinized animals, namely the nodal axolemma, may be of prime importance in saltatory nerve conduction.

Specific inhibition of Na +, K +-ATPase activity by atractylon, a major component of Byaku-jutsu, by interaction with enzyme in the E 2 state

Atractylon, a major component of the crude drug “Byaku-jutsu” (rhizomes of Atractylodes japonica), strongly inhibited Na +,K +-ATPase activity with an I 50 value of 8.9 × 10 −6 M. It also inhibited Mg 2+ ATPase, H +,K +-ATPase, H +-ATPase and Ca 2+-ATPase activities, but less potently. No effects on alkaline and acid phosphatase activities were observed. The inhibition of Na +,K +-ATPase activity by atractylon was non-competitive with respect to ATP and was greater with increasing K + concentration, whereas it was not affected by Na + concentration. The activity of K +-dependent p-nitrophenyl phosphatase, a partial reaction of Na +,K +-ATPase, was inhibited noncompetitively with respect to substrate (I 50 value of 1.8 × 10 −5 M), and the inhibition rate was independent of the K + concentration. Furthermore, atractylon increased the K i value for Na + from 130 to 190 mM, but did not alter the K i value for ATP. Inhibition of the phosphoenzyme formation by atractylon was greater at 0.1 M than at 1 M NaCl. K +-dependent dephosphorylation ( E 2- P to K · E 2) was inhibited by atractylon, whereas ADP-sensitive ( Na · E 1- P to Na · E 1) and non-specific dephosphorylation steps were not affected. These results suggest that atractylon, a specific inhibitor of Na +,K +-ATPase, interacts with enzyme in the E 2 state and inhibits the reaction step from E 2- P to K · E 2.

Developmental changes of K+-dependent para-nitrophenylphosphatase (Na+-K+-ATPase) distribution in the synaptic regions in the cerebral cortex of rats

Using Mayahara's method, the distribution of K+-dependent p-nitrophenylphosphatase activity was examined electron microscopically in the synaptic regions of the cerebral cortex of 10, 15 and 60-day-old Wistar rats. The enzyme achieved gradually its characteristic localization and uniform distribution. The main developmental changes were associated with the establishment of the postsynaptic density's activity. The controls with ouabain revealed activity only on the postsynaptic densities.

Zinc-ion-dependent acid phosphatase exhibits magnesium-ion-dependent myo-inositol-1-phosphatase activity.

We have purified bovine brain Zn(2+)-dependent acid phosphatase (Zn(2+)-APase), which requires Zn2+ ions to hydrolyze the substrate p-nitrophenyl phosphate (pNPP) in an acidic environment. The substrate specificity and metal requirement of Zn(2+)-APase at a physiological pH was also studied. The enzyme exhibited hydrolytic activity on myo-inositol-1- and -2-monophosphates, 2'-adenosine monophosphate, 2'-guanosine monophosphate, and the alpha- and beta-glycerophosphates, glucose-1-phosphate, and fructose-6-phosphate in 50 mM Tris-HCl buffer (pH 7.4) in the presence of Mg2+ ions, but not on pNPP and phosphotyrosine. Zn2+, Mn2+ and Co2+ ions were less effective for activation. Among the above substrates, myo-inositol-1-phosphate was the most susceptible to hydrolysis by the enzyme in the presence of 3 mM Mg2+ ions. The enzyme exhibited an optimum pH at around 8 for myo-inositol-1-phosphate in the presence of 3 mM Mg2+ ions. The Mg(2+)-dependent myo-inositol-1-phosphatase activity of the enzyme was significantly inhibited by Li+ ions. The Zn(2+)-dependent p-nitrophenyl phosphatase activity and Mg(2+)-dependent myo-inositol-1-phosphatase activity of the purified enzyme fraction exhibited similar behavior on Sephadex G-100 and Mono Q colomns. These findings suggest that Zn(2+)-APase also exhibits Mg(2+)-dependent myo-inositol-1-phosphatase activity under physiological conditions.

Quantification in crude homogenates of rat myocardial Na+, K(+)- and Ca(2+)-ATPase by K+ and Ca(2+)-dependent pNPPase. Age-dependent changes.

Assays for complete quantification of Na+, K(+)- and Ca(2+)-ATPase in crude homogenates of rat ventricular myocardium by determination of K(+)- and Ca(2+)-dependent p-nitrophenyl phosphatase (pNPPase) activities were evaluated and optimized. Using these assays the total K(+)- and Ca(2+)-dependent pNPPase activities in ventricular myocardium of 11-12 week-old rats were found to be 2.98 +/- 0.10 and 0.29 +/- 0.02 mumol x min-1 x g-1 wet wt. (mean +/- SEM) (n = 5), respectively. Coefficient of variance of interindividual determinations was 7 and 12%, respectively. The total Na+, K(+)- and Ca(2+)-ATPase concentrations were estimated to 2 and 10 nmol x g-1 wet wt., respectively. Evaluation of a putative developmental variation revealed a biphasic age-related change in the rat myocardial Ca(2+)-dependent pNPPase activity with an increase from birth to around the third week of life followed by a decrease. By contrast, the K(+)-dependent pNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes were simple outcome of variations in water and protein content of myocardium. Expressed per heart, the K(+)- and Ca(2+)-dependent pNPPase activity gradually increased to a plateau. The present assay for Na+, K(+)-ATPase quantification has the advantage over [3H] ouabain binding of being applicable on the ouabain-resistant rat myocardium, and is more simple and rapid than measurements of K(+)-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase) in crude tissue homogenates. Furthermore, with few modifications the pNPPase assay allows quantification of Ca(2+)-ATPase on crude myocardial homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochemical study of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase activity in guinea pig facial nerve.

The localization of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase (K-NPPase) activity, the second dephosphorylative property of the Na-K adenosine triphosphatase complex, was cytochemically studied in the intra-temporal portion of the facial nerve in normal guinea pigs using a cerium-based method. A fine-granular reaction product of the K-NPPase activity was observed on the cytoplasmic side of the axolemma of the axon cylinder, of the incisures of Schmidt-Lanterman, and of the nodes of Ranvier. No reaction product was detected on the periaxonal and outermost plasma membrane of Schwann cells and in the myelin sheath. In control tissue samples, the enzyme activity was almost completely inhibited by 10 mM ouabain, and no reaction was noted in medium without K+.